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Immunological dysfunction has been suggested to play a major role in the pathogenesis of idiopathic granulomatous mastitis (IGM). We recently showed that ozone therapy was effective in patients with steroid-resistant IGM. This study assessed alterations in intracellular cytokine expression patterns in different T-lymphocyte subsets after ozone therapy in refractory IGM. Peripheral blood T lymphocyte subsets (CD8(+), CD4(+), CD4(+)CD25(+) CD127(-)) were analyzed via flow-cytometry for intracellular cytokine expressions IFN-gamma, TNF-alpha, IL-10, and TGF-beta before and after completion of 4-month systemic ozone therapy. Ozone therapy significantly increased the CD4(+)IFN-gamma(+) (p = 0.032), CD4(+)TNF-alpha(+) (p = 0.028), and the CD8(+) TNF-alpha(+) (p =0.012) T cells. In contrast, significant decreases in CD4(+) IL-10(+) (p = 0.047) and CD8(+)IL-10(+) T cells (p = 0.022) and CD4(+)CD25(+) CD127(-//low) Treg cells secreting TGF-beta (p = 0.005) were found after ozone therapy. When patients were analyzed according to the response to ozone therapy, patients with a complete remission were more likely to have increased CD3(-)CD16(+)CD56(+) natural killer cells (p = 0.0027) and decreased CD19(+)B lymphocytes (p = 0.046) following ozone therapy. Our results suggest that ozone therapy stimulated a T-helper-1 response associated with IFN-gamma production and downregulation of TGF-beta expression in CD4(+)CD25(+)CD127(-) Treg cells. These alterations in the immune system following ozone therapy can improve wound healing and restore immune dysfunction in patients with refractory IGM. Istanbul Breast Society

The main role of the host immune system is to identify and eliminate cancer cells, which is a complex process, but it is not a fail-safe mechanism. Many sarcoma patients succumb to this disease despite treatments rendered. The aim of this pilot study was to compare the levels of CD4+ T-cells, T-regulatory (Treg) cells, and cytokines such as tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-17A (IL-17A), and transforming growth factor-beta-1 (TGF-β1) in peripheral blood leukocytes of sarcoma patients and healthy controls. For gene expression studies, total ribonucleic acid (RNA) was extracted from peripheral blood leukocytes and genes that were differentially regulated in peripheral blood leukocytes of sarcoma patients compared with healthy controls were determined using a commercial T-helper cell differentiation quantitative polymerase chain reaction (qPCR) array. Flow cytometer analysis was performed on blood samples from 26 sarcoma patients and 10 healthy controls to identify the levels of CD4+ T-cells and T-reg cells. The level of cytokines in plasma and culture supernatant were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits. A marked reduction in the percentage of CD4+ T-cells (p = 0.037) and levels of TNF-α (p = 0.004) and IFN-γ (0.010) was observed in sarcoma patients. Gene expression analysis showed five genes (homeobox A10 (HOXA10), GATA binding protein 3 (GATA3), prostaglandin D2 receptor 2 (PTGDR2), thymocyte selection associated high mobility group box (TOX), and C-C motif chemokine receptor 3 (CCR3)) were dysregulated (p < 0.05) in sarcoma patients. This study suggests that T-helper-1 immune responses are reduced in sarcoma patients.

Preeclampsia is a "disease of theory" because of the cause and pathophysiology of preeclampsia is still uncertain. In preeclampsia invasion of trophoblast cells does not occur completely, the spiral arteries not undergo remodeling so it still has a component of muscle and elastic tissue, in addition to endothelial dysfunction characterized by reduced relaxation factor and increased contraction factor resulting in ischemia of the placenta by The end result of hypoxia, hypoxia is the condition that causes the production of free radicals in large quantities. Free radicals can damage all the cellular biochemical components of lipids by taking lipid electrons in a cell membrane called lipid peroxidation. Lipid peroxidation process produces several products including Malondialdehyde (MDA) also mediate proinflammatory cytokines such expenditure Interferon Gamma (IFN-γ). In this study, researchers took the form of placental tissue paraffin blocks which have been made, on each of the paraffin block containing a number of 6 groups of placental tissue (30 blocks of paraffin). The method used is double staining with the reading of 3 field trophoblast cells with 200x magnification. On examination Malondialdehyde done by TBARS and analyzed by immunoflourence by staining rhodamine and DAPI, while on inspection of IFN-γ of the preparations that has been staining with antibodies PE anti-rat IFN-γ, and DAPI were analyzed using immunoflourence, then interpreted using software imunoflow. Provision of Phycocyanin in this study proved to reduce levels of MDA and Interferon-γ. Phycocyanin dose 40 ng / 100 gramss BB which has an effective dose decreases MDA and Interferon-γ levels, Because of phycocyanin has a role as an anti-inflammatory, antioxidant and immunomodulatory. In giving of Phycocyanin which in large doses can trigger an increase in proinflammatory cytokines. Further research is needed on the protective effect of Phycocyanin on human placental tissue.

(1) Background: SARS-CoV-2 T cell immunity is rapidly activated following SARS-CoV-2 infection and vaccination and is crucial for controlling infection progression and severity. The aim of the present study was to compare the levels of T cell responses to SARS-CoV-2 between cohorts of subjects with hybrid immunity (convalescent and vaccinated), vaccinated naïve (non-exposed) and convalescent unvaccinated subjects. (2) Methods: We performed a retrospective descriptive analysis of data collected from the medical records of adult individuals who were consecutively examined at a large, private Medical Center of Attica from September 2021 to September 2022 in order to be examined on their own initiative for SARS-CoV-2 T cell immunity response. They were divided into three groups: Group A: SARS-CoV-2 convalescent and vaccinated subjects; Group B: SARS-CoV-2 naïve vaccinated subjects; Group C: SARS-CoV-2 convalescent unvaccinated subjects. The SARS-CoV-2 T cell response was estimated against spike (S) and nucleocapsid (N) structural proteins by performing the methodology T-SPOT.COVID test. (3) Results: A total of 530 subjects were retrospectively included in the study, 252 females (47.5%) and 278 (52.5%) males ranging from 13 to 92 years old (mean 55.68 ± 17.0 years). Among them, 66 (12.5%) were included in Group A, 284 (53.6%) in Group B and 180 (34.0%) in Group C. Among the three groups, a reaction against S antigen was reported in 58/66 (87.8%) of Group A, 175/284 (61.6%) of Group B and 146/180 (81.1%) of Group C (chi-square, p < 0.001). Reaction against N antigen was present in 49/66 (74.2%) of Group A and in 140/180 (77.7%) of Group C (chi-square, p =0.841). The median SFC count for S antigen was 24 (range from 0–218) in Group A, 12 (range from 0–275) in Group B and 18 (range from 0–160) in Group C (Kruskal–Wallis test, p < 0.001; pairwise comparisons: groups A–B, p < 0.001; groups A–C, p = 0.147; groups B–C, p < 0.001). The median SFCs count for N antigen was 13 (range 0–82) for Group A and 18 (range 0–168) for Group C (Kruskal–Wallis test, p = 0.27 for A–C groups). (4) Conclusions: Our findings suggest that natural cellular immunity, either alone or combined with vaccination, confers stronger and more durable protection compared to vaccine-induced cellular immunity.

Objective: The kinetics of immune responses to various SARS-CoV-2 vaccines in cancer patients were investigated. Methods: In total, 57 cancer patients who received BNT162b2-RNA or BBIBP-CorV vaccines were enrolled. Cellular and humoral immunity were assessed at three-time points, before the first vaccine dose and 14–21 days after the first and second doses. Chemiluminescent microparticle immunoassay was used to evaluate SARS-CoV-2 anti-spike IgG response, and QuantiFERON® SARS-CoV-2 kit assessed T-cell response. Results: Data showed that cancer patients’ CD4+ and CD8+ T cell-median IFN-γ secretion of SARS-CoV-2 antigens increased after the first and second vaccine doses (p = 0.027 and p = 0.042). BNT162b2 vaccinees had significantly higher IFN-γ levels to CD4+ and CD8+ T cell epitopes than BBIBP-CorV vaccinees (p = 0.028). There was a positive correlation between IgG antibody titer and T cell response regardless of vaccine type (p < 0.05). Conclusions: This study is one of the first to investigate cellular and humoral immune responses to SARS-CoV-2 immunization in cancer patients on active therapy after each vaccine dose. COVID-19 immunizations helped cancer patients develop an effective immune response. Understanding the cellular and humoral immune response to COVID-19 in cancer patients undergoing active treatment is necessary to improve vaccines and avoid future SARS pandemics.

The peste des petits ruminant (PPR) virus is a transboundary virus found in small domestic ruminants that causes high morbidity and mortality in naive herds. PPR can be effectively controlled and eradicated by vaccinating small domestic ruminants with a live-attenuated peste des petits ruminant virus (PPRV) vaccine, which provides long-lasting immunity. We studied the potency and safety of a live-attenuated vaccine in goats by detecting their cellular and humoral immune responses. Six goats were subcutaneously vaccinated with a live-attenuated PPRV vaccine according to the manufacturer’s instructions, and two goats were kept in contact. Following vaccination, the goats were monitored daily, and we recorded their body temperature and clinical score. Heparinized blood and serum were collected for a serological analysis, and swab samples and EDTA blood were collected to detect the PPRV genome. The safety of the used PPRV vaccine was confirmed by the absence of PPR-related clinical signs, a negative pen-side test, a low virus genome load as detected with RT-qPCR on the vaccinated goats, and the lack horizontal transmission between the in-contact goats. The strong humoral and cellular immune responses detected in the vaccinated goats showed that the live-attenuated PPRV vaccine has a strong potency in goats. Therefore, live-attenuated vaccines against PPR can be used to control and eradicate PRR.

Cellular and humoral immunity are both required for SARS-CoV-2 infection recovery and vaccine efficacy. The factors affecting mRNA vaccination-induced immune responses, in healthy and fragile subjects, are still under investigation. Thus, we monitored the vaccine-induced cellular and humoral immunity in healthy subjects and cancer patients after vaccination to define whether a different antibody titer reflected similar rates of cellular immune responses and if cancer has an impact on vaccination efficacy. We found that higher titers of antibodies were associated with a higher probability of positive cellular immunity and that this greater immune response was correlated with an increased number of vaccination side effects. Moreover, active T-cell immunity after vaccination was associated with reduced antibody decay. The vaccine-induced cellular immunity appeared more likely in healthy subjects rather than in cancer patients. Lastly, after boosting, we observed a cellular immune conversion in 20% of subjects, and a strong correlation between pre- and post-boosting IFN-γ levels, while antibody levels did not display a similar association. Finally, our data suggested that integrating humoral and cellular immune responses could allow the identification of SARS-CoV-2 vaccine responders and that T-cell responses seem more stable over time compared to antibodies, especially in cancer patients.

In Italy, from January 2021, the Ministry of Health indicated a vaccination plan against COVID for frail patients and physicians based on a three-dose scheme. However, conflicting results have been reported on which biomarkers permit immunization assessment. We used several laboratory approaches (i.e., antibodies serum levels, flow cytometry analysis, and cytokines release by stimulated cells) to investigate the immune response in a cohort of 53 family pediatricians (FPs) at different times after the vaccine. We observed that the BNT162b2-mRNA vaccine induced a significant increase of specific antibodies after the third (booster) dose; however, the antibody titer was not predictive of the risk of developing the infection in the six months following the booster dose. The antigen stimulation of PBMC cells from subjects vaccinated with the third booster jab induced the increase of the activated T cells (i.e., CD4+ CD154+); the frequency of CD4+ CD154+ TNF-α+ cells, as well as the TNF-α secretion, was not modified, while we observed a trend of increase of IFN-γ secretion. Interestingly, the level of CD8+ IFN-γ+ (independently from antibody titer) was significantly increased after the third dose and predicts the risk of developing the infection in the six months following the booster jab. Such results may impact also other virus vaccinations.

Background: The PCSK3 gene encodes for the protease enzyme Furin, which promotes proteolytic maturation of important regulators of the immune response, and also enhances the secretion of interferon-γ (IFN). Several studies have suggested its possible involvement in the pathogenesis of chronic inflammatory diseases. Methods: We investigated the PCSK3 gene expression level in peripheral blood mononuclear cells isolated from Sjögren’s Syndrome (SS) patients and healthy controls and we evaluated a possible correlation with IFN-γ gene expression. Moreover, we also explored the variability of two PCSK3 genetic polymorphisms (rs4932178 and rs4702) to evaluate a possible association between these polymorphisms and the expression levels of this gene. Results: We observed, by RT-qPCR, that the PCSK3 expression level was significantly higher in SS patients compared to the controls (p = 0.028), and we confirmed a positive correlation between PCSK3 and IFN-γ expression levels (p < 0.001). Moreover, we reported that the variant homozygous genotype of rs4932178 SNP is associated with a higher expression of the PCSK3 gene (p = 0.038) and with the SS susceptibility (p = 0.016). Conclusions: Our data suggest that Furin could play a role in SS development, also promoting IFN-γ secretion.

Mycobacterium (M.) bovis BCG vaccination is recommended for healthy babies after birth in several countries with a high prevalence of tuberculosis, including Ghana. Previous studies showed that BCG vaccination prevents individuals from developing severe clinical manifestations of tuberculosis, but BCG vaccination effects on the induction of IFN-γ after M. tuberculosis infection have hardly been investigated. Here, we performed IFN-γ-based T-cell assays (i.e., IFN-γ Release Assay, IGRA; T-cell activation and maturation marker assay, TAM-TB) in children who had contact with index tuberculosis patients (contacts). These contacts were classified as either being BCG vaccinated at birth (n = 77) or non-BCG-vaccinated (n = 17) and were followed up at three timepoints for a period of one year to determine immune conversion after M. tuberculosis exposure and potential infection. At baseline and month 3, BCG-vaccinated contacts had significantly lower IFN-γ levels after stimulation with M. tuberculosis-specific proteins as compared to non-BCG-vaccinated contacts. This resulted in decreased proportions of positive IGRA results (BCG-vaccinated: 60% at baseline, 57% at month 3; non-BCG-vaccinated: 77% and 88%, respectively) at month 3. However, until month 12, immune conversion in BCG-vaccinated contacts led to balanced proportions in IGRA responders and IFN-γ expression between the study groups. TAM-TB assay analyses confirmed higher proportions of IFN-γ-positive T-cells in non-BCG-vaccinated contacts. Low proportions of CD38-positive M. tuberculosis-specific T-cells were only detected in non-BCG-vaccinated contacts at baseline. These results suggest that BCG vaccination causes delayed immune conversion as well as differences in the phenotype of M. tuberculosis-specific T-cells in BCG-vaccinated contacts of tuberculosis patients. These differences are immune biomarker candidates for protection against the development of severe clinical tuberculosis manifestations.

Introduction Liver cancer is a malignant tumor with high incidence and short survival time. In order to increase the cure rate and disease-free survival rate of liver cancer, it is necessary to seek effective treatment methods. Objectives The objective of this study is to evaluate the therapeutic effects of IL-21 and IFN-γ inducing the formation of CD4+CXCR5+CD57+T cells on liver cancer. Methods The methods of analyze the relationship between CD4+CXCR5+CD57+T cells and the survival time of hepatocellular carcinoma (HCC), and study the effect of IL-21 combined with IFN-γ in inducing stem cells to differentiate into CD4+CXCR5+CD57+T cells. The effects of IL-21 combined with IFN-γ induced CD4+CXCR5+CD57+T cells on liver cancer were studied through animal experiments, and the regulatory mechanism, and the effect of hepatitis B virus (HBV) on it. Results The study found that the number of CD4+CXCR5+CD57+T cells in serum of liver cancer patients with prolonged survival time increased significantly, the expression of CD4, CD57, and CXCR5 in the tumor microenvironment increased, and the serum IL-21 and IFN-γ concentrations increased. IL-21 and IFN-γ induce stem cells to differentiate into CD4+CXCR5+CD57+T cells and induce HepG2 cells apoptosis. HBV leads to a decrease in the number of CD4+CXCR5+CD57+T cells and a chronic inflammatory response. Treg cells can regulate CD4+CXCR5+CD57+T cells. IL-21 combined with IFN-γ induced an increase in the number of CD4+CXCR5+CD57+T cells in hepatocarcinoma-bearing mice, which has an inhibitory effect on H22 liver cancer. Conclusion The conclusion of the study is that IL-21 combined with IFN-γ induces stem cells to differentiate into CD4+CXCR5+CD57+T cells, Treg can control the increase in their number, and HBV can cause their number to decrease, which can control the growth of liver cancer.

Hongyu Wang,1,&ast; Guixiong Zhang,1,&ast; Wenzhe Fan,1 Yanqin Wu,1 Jiang Zhang,2 Miao Xue,1 Yue Zhao,1 Wang Yao,1 Jiaping Li1 1Department of Interventional Oncology, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, 510080, People’s Republic of China; 2Department of Clinical Laboratory, The first Affiliated Hospital of Sun Yat-Sen University, Guangzhou, 510080, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Jiaping LiDepartment of Interventional Oncology, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, 510080, People’s Republic of China, Tel +86-20-13352890908, Fax +86-20-87755766, Email lijiap@mail.sysu.edu.cnPurpose: To examine the clinical significance of circulating lymphocyte subtypes and cytokines in patients with hepatocellular carcinoma (HCC) undergoing transarterial chemoembolization (TACE).Patients and Methods: We enrolled 53 patients and assessed lymphocyte subsets (CD4+ T cells, CD8+ T cells, CD4+/CD8+ cell ratio, natural killer cells, and regulatory T cells) and cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, and interferon [IFN]-γ) on the day before TACE, and 1 day and 4– 8 weeks thereafter. The correlation between baseline inflammatory markers and IL-6, the differences between clinical subgroups, and the prognostic baseline inflammatory values and changes therein were analyzed.Results: We found that baseline IL-6 was positively correlated with IL-10 and IFN-γ. Univariate analysis revealed that patients with higher baseline IL-6, IL-10, and IFN-γ levels had worse liver function and greater tumor burden, suggesting a poor prognosis. Multivariate analysis showed that higher baseline IFN-γ levels were associated with a shorter time to tumor progression (hazard ratio [HR] = 1.912; 95% confidence interval [CI] = 1.013– 3.607; p = 0.045), and higher IL-10 levels were associated with poor overall survival (HR = 2.576; 95% CI = 1.237– 5.364; p = 0.011). Baseline lymphocyte subtypes and changes therein did not have any significant association with prognosis.Conclusion: This study confirmed the association between inflammation and poor prognosis. The prognostic significance of IFN-γ was different from previous studies, while the prognostic significance of lymphocyte subtypes remains to be verified.Keywords: hepatocellular carcinoma, TACE, IFN-γ, cytokines, lymphocyte subtypes

PurposeMalaria is a life-threatening mosquito-borne disease caused by Plasmodium parasites, mainly in tropical and subtropical countries. Plasmodium falciparum (P. falciparum) is the most prevalent cause on the African continent and responsible for most malaria-related deaths globally. Important medical needs are biomarkers for disease severity or disease outcome. A potential source of easily accessible biomarkers are blood-borne small extracellular vesicles (sEVs).MethodsWe performed an EV Array to find proteins on plasma sEVs that are differentially expressed in malaria patients. Plasma samples from 21 healthy subjects and 15 malaria patients were analyzed. The EV array contained 40 antibodies to capture sEVs, which were then visualized with a cocktail of biotin-conjugated CD9, CD63, and CD81 antibodies.ResultsWe detected significant differences in the protein decoration of sEVs between healthy subjects and malaria patients. We found CD106 to be the best discrimination marker based on receiver operating characteristic (ROC) analysis with an area under the curve of > 0.974. Additional ensemble feature selection revealed CD106, Osteopontin, CD81, major histocompatibility complex class II DR (HLA-DR), and heparin binding EGF like growth factor (HBEGF) together with thrombocytes to be a feature panel for discrimination between healthy and malaria. TNF-R-II correlated with HLA-A/B/C as well as CD9 with CD81, whereas Osteopontin negatively correlated with CD81 and CD9. Pathway analysis linked the herein identified proteins to IFN-γ signaling.ConclusionsEV-associated proteins can discriminate between healthy individuals and malaria patients and are candidates for future predictive biomarkers. Purpose: Malaria is a life-threatening mosquito-borne disease caused by Plasmodium parasites, mainly in tropical and subtropical countries. Plasmodium falciparum (P. falciparum) is the most prevalent cause on the African continent and responsible for most malaria-related deaths globally. Important medical needs are biomarkers for disease severity or disease outcome. A potential source of easily accessible biomarkers are blood-borne small extracellular vesicles (sEVs). Methods: We performed an EV Array to find proteins on plasma sEVs that are differentially expressed in malaria patients. Plasma samples from 21 healthy subjects and 15 malaria patients were analyzed. The EV array contained 40 antibodies to capture sEVs, which were then visualized with a cocktail of biotin-conjugated CD9, CD63, and CD81 antibodies. Results: We detected significant differences in the protein decoration of sEVs between healthy subjects and malaria patients. We found CD106 to be the best discrimination marker based on receiver operating characteristic (ROC) analysis with an area under the curve of > 0.974. Additional ensemble feature selection revealed CD106, Osteopontin, CD81, major histocompatibility complex class II DR (HLA-DR), and heparin binding EGF like growth factor (HBEGF) together with thrombocytes to be a feature panel for discrimination between healthy and malaria. TNF-R-II correlated with HLA-A/B/C as well as CD9 with CD81, whereas Osteopontin negatively correlated with CD81 and CD9. Pathway analysis linked the herein identified proteins to IFN-γ signaling. Conclusion: sEV-associated proteins can discriminate between healthy individuals and malaria patients and are candidates for future predictive biomarkers. Trial registration: The trial was registered in the Deutsches Register Klinischer Studien (DRKS-ID: DRKS00012518).

In equine medicine, the use of regenerative therapeutics has gained growing attention, but is still a new and complex field with room for improvement. Platelet lysate (PL) can be used as therapeutic agent but is also a promising supplement for the culture of multipotent mesenchymal stromal cells. To enable a targeted use of PL both in clinic and laboratory, it is crucial to learn more details on its effective ingredients. While so far, mainly growth factor components have been analyzed in platelet-based products such as PL, the current study focuses on the content of cytokines in serum, plasma, platelet concentrate and PL. Blood was harvested from 20 clinically healthy horses and subjected to blood count and chemistry analysis, as well as to further processing to PL. Plasma and platelet concentrate were produced by a buffy-coat-based method and PL was produced from the concentrate by freeze-thawing. Samples from each horse were analyzed regarding interleukin (IL)-1β, −4, −6 and −10, interferon-γ and tumor necrosis factor-α concentrations using sandwich ELISAs. Cytokine concentrations in serum, plasma, concentrate and PL were similar and correlated significantly. However, there was a large inter-individual variability in cytokine concentrations between the different donor horses. The samples from some donor animals had overall very high cytokine concentrations, while samples from other donors had no measurable cytokine ingredient. This pattern was observed for all cytokines. There was a noticeable link between high cytokine concentrations in the blood products and abnormal findings in blood chemistry. Cytokine concentrations in samples from horses with abnormal findings were significantly higher than in samples from the remaining horses. The interindividual differences in cytokine concentrations could be highly relevant when using PL for therapy and cell culture, as the mode of action of the PL is likely changed depending on the presence of pro- and anti-inflammatory cytokines. Blood chemistry might be useful to predict cytokine concentrations in blood products.

Canine leishmaniosis is a complex infection that ranges from the apparent absence of disease to a severe fatal clinical illness. Numerous epidemiological serosurveys have been carried out in Europe, although these studies often lack assessment of clinical health status. The aim of this study was to evaluate signalment, immunological and parasitological status correlated with clinicopathological findings of L. infantum -seropositive apparently healthy dogs living in endemic areas of Europe. Routine laboratory tests, endpoint in-house ELISA to quantify the anti- Leishmania antibodies, blood Leishmania quantitative PCR (qPCR) and IFN-γ ELISA were performed. Dogs were classified as healthy or sick depending on the results of routine laboratory tests. Seropositive sick dogs presented a worse clinical status that included a higher proportion of medium to high antibody levels and positive qPCR, lower IFN-γ concentration and several clinicopathological findings compared to truly healthy seropositive dogs. In conclusion, further investigation of apparently healthy L. infantum -seropositive dogs is necessary in the clinical setting, as a significant proportion of seropositive dogs that are sick with clinicopathological findings remain undetected if routine laboratory tests are not undertaken. Performing these routine laboratory tests with the combination of a thorough physical examination will improve early disease diagnosis, treatment and prognosis of clinical leishmaniosis. Canine leishmaniosis caused by Leishmania infantum is a disease with a wide range of clinical manifestations. Epidemiological serosurveys performed in Europe often lack a thorough assessment of clinical health status of studied dogs. The aim of this study was to evaluate signalment, immunological and parasitological status and clinicopathological findings of L. infantum -seropositive apparently healthy dogs (n = 212) living in endemic areas. Routine laboratory tests, endpoint in-house ELISA to quantify the anti- Leishmania antibodies, blood Leishmania qPCR and IFN-γ ELISA were performed. All dogs enrolled were L. infantum -seropositive and were classified as healthy (n = 105) or sick (n = 107) according to LeishVet guidelines. The sick group presented a higher proportion of medium to high antibody levels and positive qPCR and lower IFN-γ concentration compared to the healthy group. Sick dogs were mostly classified in LeishVet stage IIa. Biochemical alterations (98%) were the most common clinicopathological findings, with fewer urinary tract (46%) and hematological (40%) alterations. Apparently healthy L. infantum -seropositive dogs can be classified between truly healthy dogs and sick dogs with clinicopathological findings. Sick dogs presented medium to high seropositivity and parasitemia and low IFN-γ concentrations, and their most common clinicopathological abnormalities were serum protein alterations followed by proteinuria and lymphopenia.

This study was designed to prepare and evaluate the specific Mycobacterium bovis (M. bovis) Bacillus Calmette-Guerin (BCG) transfer factor (TF) from spleen cells of two lambs vaccinated subcutaneously S/C with BCG vaccine twice 2 weeks’ intervals, similarly one lamb injected with normal saline used as a donor of non-specific TF. The lambs were clinically examined at zero time and along 3 days post each immunization, then, delayed type hypersensitivity (DTH) was checked post 4 weeks. TF was obtained from spleen cells of the lambs, inspected for sterility; safety.TF was evaluated in 4 groups (five in each) of lambs 3-4 months old. The first group (recipient) was injected S/C with 2 ml of specific BCG-TF, similarly the 2nd, 3rd and 4th groups were injected with non-specific TF, BCG and normal saline respectively. The clinical signs and DTH test were checked as described above, only the specific TF recipient and BCG vaccinated lambs gave a positive DTH test. The serum concentration of inerlukin-17 (IL-17), interferon-gamma (IFN-γ) and migration inhibitory factor (MIF) cytokines were measured by Enzyme-linked immunosorbent assay (ELISA), which revealed a highly significant increase in the TF recipient lambs in comparison with the control and vaccinated groups. It concluded that the prepared specific M. bovis-BCG transfer factor was effective to induce cell mediated immunity via DTH and increasing the serum concentration of cytokines in recipient lambs.

Chang-He Liu,1 Na Hua,2 Huai-Yu Yang1 1Department of Cardiology, Affiliated Zhongshan Hospital of Dalian University, Dalian, People’s Republic of China; 2Department of Otolaryngology, Affiliated Zhongshan Hospital of Dalian University, Dalian, People’s Republic of ChinaCorrespondence: Chang-He LiuDepartment of Cardiology, Affiliated Zhong Shan Hospital of Dalian University, 6 Jiefang Street, Zhongshan District, Dalian, People’s Republic of ChinaTel +86-13478661433Fax +86-041162893555Email liuchanghe159951@163.comBackground: Accumulating evidence has shown the important role of the inflammatory process in the pathophysiology of mental disorders. However, the relative levels of inflammatory markers in patients with panic disorder (PD) have rarely been evaluated. The aim of the present study was to conduct a systematic review to determine the correlation of peripheral C-reactive protein (CRP) and inflammatory cytokine profiles with PD.Methods: This study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. We searched for quantitative research studies published up to July 31, 2021 that measured peripheral levels of CRP and inflammatory cytokines in people with PD compared with controls. Meta-analysis using a random-effects model was performed for the levels of CRP and inflammatory cytokines with data from three or more studies.Results: Fourteen identified studies met the inclusion criteria. In total, 18 cytokines were evaluated. Markers that were reported in more than 3 studies were included in this meta-analysis. The results showed that peripheral levels of CRP, IL-6, IL-2 and TNF-α were significantly higher in PD patients than in healthy controls, while there was no significant difference in peripheral levels of IL-1β, IL-10 and IFN-γ between groups. Notably, the relevant studies involving IL-6, IL-1β, IL-10 and IFN-γ in PD patients were highly heterogeneous. Similar to meta-analyses of other inflammatory factors in mental disorders, our meta-analysis also reflected differences in participant medication use, comorbid anxiety or depression, sampling methods and detection methods. Eight inflammatory cytokines were reported in only one study, and their expression levels were higher, lower, or unchanged compared with those in healthy controls.Conclusion: There is preliminary evidence to suggest a significant inflammatory response in PD patients, but the role of inflammatory markers in PD remains unclear. Studying inflammatory markers in PD will help to clarify the etiology and pathophysiological mechanisms of the disorder.Keywords: CRP, interleukin, TNF-α, IFN-γ, panic disorder

Guohong Hu, Xiansheng Zhou Dermatology Hospital of Jiangxi Province, Nanchang, 330001, Jiangxi, People’s Republic of ChinaCorrespondence: Guohong HuDermatology Hospital of Jiangxi Province, No. 388 Yingbin North Road, Nanchang, 330001, Jiangxi, People’s Republic of ChinaTel +86-791-85221034Email 2009020212@st.btbu.edu.cnBackground: Gallic acid (GA) has an anti-inflammatory effect by regulating inflammatory molecules. This study aimed to investigate the effect of GA on atopic dermatitis (AD)-like skin inflammation.Methods: 4-dinitrochlorobenzene (DNCB) was used to induce an AD-like skin inflammation model. The effect of GA on DNCB-induced inflammation was assessed by measuring the thickness and histopathological examination of the ear. Serum IgE and TNF-α levels were detected. The effect of GA on lymph nodes was determined by measuring the weights and mRNA/protein expression levels of TNF-α, IL-4, IFN-γ and IL-17. Ratio of Treg cells and Th17 cells was also analyzed.Results: It was found that the thickness and pathology of the ear were significantly improved by GA in the DNCB-induced mice. Serum IgE and TNF-α levels were significantly reduced in GA-treated model mice compared to the model group. GA treatment lowered the weight of lymph node and the expression of mRNAs of TNF-α, IL-4, IFN-γ, and IL-17 of lymph node. In the ear, inflammatory factors (IL-4, IL-5, IL-17, or IL-23) showed a significant decrease in GA-treated model mice versus model mice, while the expression levels of IL-10 and TGF-β showed a great increase in GA-treated model mice. ROR-γt showed a decrease in GA-treated model group, along with an increase expression of SOCS3.Conclusion: GA could ameliorate AD-like skin inflammation possibly through Th17 mediated immune regulation in a DNCB-induced mouse model.Keywords: 4-dinitrochlorobenzene, DNCB, Th17, IL-10, TGF-β, IFN-&#x03B3

Hepatocellular carcinoma (HCC) is one of the commonest lethal malignancies worldwide, and often diagnosed at an advanced stage, without any curative therapy. Immune checkpoint blockers targeting the programmed death receptor 1 (PD‐1) have shown impressive antitumor activity in patients with advanced‐stage HCC, while the response rate is only 30%. Inducible PD‐L1 overexpression may result in a lack of response to cancer immunotherapy, which is attributed to a mechanism of adaptive immune resistance. Our study investigated that the overexpression of PD‐L1 promoted the invasion and migration of liver cancer cells in vitro, and the induced overexpression of PD‐L1 in the tumor microenvironment could weaken the effects of anti‐PD‐1 immunotherapy in a BALB/c mouse model of liver cancer. CPI‐203, a small‐molecule bromodomain‐containing protein 4 (BRD4) inhibitor, which can potently inhibit PD‐L1 expression in vitro and in vivo, combined with PD‐1 antibody improved the response to immunotherapy in a liver cancer model. Cell transfection and chromatin immunoprecipitation assay manifested that BRD4 plays a key role in PD‐L1 expression; CPI‐203 can inhibit PD‐L1 expression by inhibiting the BRD4 occupation of the PD‐L1 promoter region. This study indicates a potential clinical immunotherapy method to reduce the incidence of clinical resistance to immunotherapy in patients with HCC., Antibodies that block the PD‐1/PD‐L1 inhibition axis activate tumor‐specific T cells producing IFN‐γ, but continuous IFN‐γ leads to a PD‐L1 expression increase in tumor cells, which can engage the PD‐1 receptor on T cells to suppress antitumor immunity. CPI‐203, which can inhibit immunotherapy‐induced PD‐L1 expression, provides the conditions for efficacy of anti‐PD‐1 immunotherapy.

Guohong Hu, Xiansheng Zhou Dermatology Hospital of Jiangxi Province, Nanchang, 330001, Jiangxi, People’s Republic of ChinaCorrespondence: Guohong HuDermatology Hospital of Jiangxi Province, No. 388 Yingbin North Road, Nanchang, 330001, Jiangxi, People’s Republic of ChinaTel +86-791-85221034Email 2009020212@st.btbu.edu.cnBackground: Gallic acid (GA) has an anti-inflammatory effect by regulating inflammatory molecules. This study aimed to investigate the effect of GA on atopic dermatitis (AD)-like skin inflammation.Methods: 4-dinitrochlorobenzene (DNCB) was used to induce an AD-like skin inflammation model. The effect of GA on DNCB-induced inflammation was assessed by measuring the thickness and histopathological examination of the ear. Serum IgE and TNF-α levels were detected. The effect of GA on lymph nodes was determined by measuring the weights and mRNA/protein expression levels of TNF-α, IL-4, IFN-γ and IL-17. Ratio of Treg cells and Th17 cells was also analyzed.Results: It was found that the thickness and pathology of the ear were significantly improved by GA in the DNCB-induced mice. Serum IgE and TNF-α levels were significantly reduced in GA-treated model mice compared to the model group. GA treatment lowered the weight of lymph node and the expression of mRNAs of TNF-α, IL-4, IFN-γ, and IL-17 of lymph node. In the ear, inflammatory factors (IL-4, IL-5, IL-17, or IL-23) showed a significant decrease in GA-treated model mice versus model mice, while the expression levels of IL-10 and TGF-β showed a great increase in GA-treated model mice. ROR-γt showed a decrease in GA-treated model group, along with an increase expression of SOCS3.Conclusion: GA could ameliorate AD-like skin inflammation possibly through Th17 mediated immune regulation in a DNCB-induced mouse model.Keywords: 4-dinitrochlorobenzene, DNCB, Th17, IL-10, TGF-β, IFN-&#x03B3

There is significant interest and debate regarding the best mouse model of human disease, since studies in wild-type mice may not always recapitulate human diseases. The NSG mouse model has been one of the most commonly used mouse models to study cancer; however, this mouse model, even though it has several advantages in regard to the ease of tumor implantation and financial feasibility, does not represent human disease due to the immunodeficient nature of this model. In this study, we performed oral and pancreatic tumor studies in NSG and hu-BLT mice and found several distinguishing features that make hu-BLT model more suitable for studying human cancer. In addition, we compared the immune function of humans to hu-BLT mice to understand the differences and similarities of the models. Oral and pancreatic cancer stem cells were implanted in NSG and hu-BLT mice. Both tumors grew robustly in NSG mice and killed them within a short period of time. On the contrary, unlike NSG mice, tumor-bearing hu-BLT mice survived longer, grew smaller tumors, and the grown tumors exhibited lower rates of expansion, with a higher surface expression of MHC-class I and lower NK cell-mediated cytotoxicity that was previously shown to have more of a differentiated phenotype. Although the peripheral blood of hu-BLT mice in comparison to that of humans had lower percentages of NK cells and cytotoxic function, it mediated a higher secretion of IFN-γ, likely contributing to the differentiation of the tumor cells and subsequent decrease in the tumor size in the hu-BLT mice in comparison to the NSG mice. Spleen-derived hu-BLT mouse NK cells were able to expand in the presence of autologous osteoclasts and substantially increase both cytotoxicity and secretion of IFN-γ, similar to those seen in peripheral blood-derived human NK cells, indicating that NK cells from hu-BLT mice are capable of expansion and functional activation when activating signals are given. Thus, the many similarities between human and hu-BLT mouse immune systems make this mouse model more appropriate to study human cancer. In particular, it is well-suited for studies of allogeneic NK cell-based immunotherapy in cancer treatment. The advantages and challenges of hu-BLT mice in cancer studies are also discussed in this report.

Sepsis remains a major cause of death worldwide. The immune system plays a central role in the response to infection. The initial state of inflammation, triggered by the innate immune response, can progress to uncontrolled self-amplification and organ failure. At the same time, in order to control it, a negative anti-inflammatory response is established which can lead to a state of immunosuppression, resulting in secondary infections and mortality in intensive care. This state is characterised by a quantitative and qualitative deficit in the immune response. Monocyte expression of HLA-DR (mHLA-DR), a MHC class II molecule presenting antigen to T cells, is a relevant marker of the sepsis-induced immunosuppression state to date. Its expression is regulated primarily by the class II transactivator, CIITA, which is transcriptionally controlled by a highly conserved region located in the promoter. The identification of genetic polymorphisms, or SNPs (Single Nucleotide Polymorphism), in this promoter region that have been implicated in inflammatory diseases, as well as evidence of positive selection in the 5' region, allowed us to target the SNP G-286A*rs3087456.In the first part of our work, we demonstrated the role of the SNP in the decrease of CIITA promoter III activity, the recovery of CIITA promoter III activity after IFN-γ stimulation, and its role in the mortality of patients in septic shock in relation to the decrease of mHLA-DR.In the following, we presented safety and efficacy data of IFN-γ treatment in patients with sepsis-induced immunosuppression. In addition, we validated a flow cytometric technique for measuring mHLA-DR at the patient's bedside on small volumes of fresh blood. We then described the myeloid phenotypes and their role in immunosuppression in a severe case series of patients with COVID-19 pneumonia complicated by bacterial sepsis. Finally, we conducted a study on immunological biomarkers of healthcare-associated infections in paediatric liver transplant patients, for which some of the experiments and analyses are still ongoing.; Le sepsis reste une cause importante de mortalité dans le monde. Le système immunitaire joue un rôle central dans la réponse à l'infection. L'état d'inflammation initial, déclenché par la réponse immunitaire innée, peut évoluer vers une auto-amplification incontrôlée et des défaillances d'organes. Parallèlement, afin d'en assurer le contrôle, une réponse négative anti-inflammatoire se met en place pouvant conduire à un état d'immunosuppression, pourvoyeur d'infections secondaires et de mortalité en soins intensifs. Cet état se caractérise par un déficit quantitatif et qualitatif de la réponse immunitaire. L'expression monocytaire de HLA-DR (mHLA-DR), molécule du CMH de classe II présentant l'antigène aux lymphocytes T, est à ce jour un marqueur pertinent de l'état d'immunosuppression induite par le sepsis. Son expression est régulée principalement par le transactivateur de classe II, CIITA, lui-même contrôlé au niveau transcriptionnel par une région hautement conservée située dans le promoteur. La mise en évidence dans cette région promotrice de polymorphismes génétiques, ou SNPs (Single Nucleotide Polymorphism), impliqués dans des maladies inflammatoires, ainsi que des preuves d'une sélection positive dans la région 5' du gène, nous ont permis de cibler le SNP G-286A*rs3087456.Dans la première partie de notre travail, nous avons démontré le rôle du SNP dans la diminution de l'activité du promoteur III de CIITA, la récupération de celle-ci après stimulation par IFN-γ, et son rôle dans la mortalité des patients en choc septique en lien avec la diminution de mHLA-DR.Dans la suite de notre travail, nous avons présenté des données de sécurité et d'efficacité du traitement par IFN-γ chez des patients atteints d'immunosuppression induite par le sepsis. De plus, nous avons validé une technique de mesure par cytométrie de flux de mHLA-DR au lit du malade sur des faibles volumes de sang frais. Nous avons ensuite décrit les phénotypes myéloïdes et leurs rôles dans l'immunosuppression chez une série de cas graves de patients atteints de pneumopathie à COVID-19 se compliquant de sepsis bactérien. Enfin, nous avons réalisé une étude sur les biomarqueurs immunologiques d'infections associées aux soins chez des patients pédiatriques transplantés du foie, dont une partie des expériences et les analyses sont encore en cours.

Acute respiratory distress syndrome (ARDS) is characterized by severe hypoxemia and high-permeability pulmonary edema. A hallmark of the disease is the presence of lung inflammation with features of diffuse alveolar damage. The molecular pathogenetic mechanisms of COVID-19-associated ARDS (CARDS), secondary to SARS-CoV-2 infection, are still not fully understood. Here, we investigate the effects of a cytokine-enriched conditioned medium from Spike S1-activated macrophage on alveolar epithelial A549 cells in terms of cell proliferation, induction of autophagy, and expression of genes related to protein degradation. The protective effect of baricitinib, employed as an inhibitor of JAK-STAT, has been also tested. The results obtained indicate that A549 exhibits profound changes in cell morphology associated to a proliferative arrest in the G0/G1 phase. Other alterations occur, such as a blockade of protein synthesis and the activation of autophagy, along with an increase of the intracellular amino acids content, which is likely ascribable to the activation of protein degradation. These changes correlate to the induction of IFN-regulatory factor 1 (IRF-1) due to an increased secretion of IFN-γ in the conditioned medium from S1-activated macrophages. The addition of baricitinib prevents the observed effects. In conclusion, our findings suggest that the IFN-γ-IRF-1 signaling pathway may play a role in the alveolar epithelial damage observed in COVID-19-related ARDS.

CONTEXTELe syndrome de susceptibilité Mendélienne aux infections mycobactériennes (SSMIM) est un groupe de maladies génétiques entraînant une susceptibilité accrue aux infections à des mycobactéries peu virulentes comme le bacille vaccinal de Calmette et Guérin (BCG). Parmi les 19 formes génétiques de SSMIM, les mutations mono-alléliques pertes de fonction de STAT1 confèrent une déficience partielle (AD) de réponse cellulaire à l’IFN-γ. Ce déficit immunitaire rare reste mal caractérisé avec seulement 26 individus rapportées depuis 2001.OBJECTIFDécrire sur le plan génétique, clinique, et immunologique les individus avec une déficience partielle dominante (PD) en STAT1.MÉTHODEÉtude rétrospective internationale incluant l’ensemble cas de déficience AD en STAT1 publiés et nouvellement diagnostiqués dans le laboratoire de Génétique Humaine des Maladies infectieuses (GHMI) ayant un variant délétère mono-allélique dans STAT1. Le recueil des données génétiques, cliniques et immunologiques s’effectue à l’aide d’un questionnaire.RÉSULTATSSoixante-quatre individus issus de 28 familles et 13 pays, avec 24 différents variants délétères (12 non rapportées) ont été inclus. Cinquante-huit épisodes d’infections mycobactériennes (IM) ont été identifiés chez 42 patients (M. bovis-BCG, 77,5 %, M. avium, 10,3 %, M. tuberculosis (12,0 %). L’âge médian au premier épisode était 4,2 ans (intervalle 0,1-18). Parmi les 41 individus vaccinés par le BCG, 29 ont développé une infection disséminée (n = 23), régionale (n = 4) ou localisée à la peau (n = 2). La prévalence des localisations des IM osseuses (n = 24) et cutanées (n = 18) était particulièrement élevée. Le bilan immunologique de première intention était normal. La pénétrance des IM est estimée à 45 % à 55 ans avec 22 individus asymptomatiques. La survie est excellente (97 %) avec 11 rechutes (temps médian (± SD) après le premier épisode de 6 ± 3,6 ans) et huit récurrences (18 ± 20 ans).CONCLUSIONLa déficience AD en STAT1 est une maladie cliniquement hétérogène, allant des formes asymptomatiques aux formes sévère et disséminées d’IM à M. bovis-BCG et M. avium. Un dépistage de cette maladie devrait être réalisé devant une IM aux localisations osseuses et/ou cutanées. Il n’existe pas un marqueur biologique pour le dépistage et le bilan immunologique de routine est normal. Un diagnostic précoce est nécessaire pour fournir un conseil génétique (y compris intrafamilial) et au traitement préventif ou curatif adapté. Le vaccin BCG est contre-indiqué dans cette maladie.

The present work aimed to study the impact of infection with a locally isolated vNDV genotype VII in commercial broiler chickens of different ages from a histopathological point of view with related changes in pulmonary cytokine expression. 100 commercial broiler chickens were allocated into 4 groups as follows; group 1 served as control non-infected group and three infected groups, were infected with 106 EID50 NDV at ages of 10,20, and 30 days. Tissue samples from different organs were collected at the 1st, 3rd, 5th, and 7th days post-inoculation for histopathological and immunohistochemical examination. Quantitative real-time RT-PCR for virus re-isolation as well as expression of IL-6 and IFN-γ genes estimation were performed on lung tissue collected at 3 dpi. RT-PCR for virus revealed increased virus load in the lungs of older birds and cytokines profiling revealed significant up-regulation of both IL-6 and IFN-γ genes in the groups that received infection at older ages. The severity and distribution of pathological lesions were directly proportioned to the age of infection and the time interval post-infection. Immunostaining of the viral antigen in the different tissues confirmed the pantropic nature of the virus.

PROPOSE The purpose of this study was to investigate panels of enzyme-linked immunospot assays (ELISpot) to detect drug-specific mediator releasing cells for confirming culprit drugs in severe cutaneous adverse reactions (SCARs). METHODS Frequencies of drug-induced interleukin-22 (IL-22)-, interferon-gamma (IFN-γ)-, and granzyme-B (GrB)-releasing cells were measured by incubating peripheral blood mononuclear cells (PBMCs) from SCAR patients with the culprit drugs. Potential immunoadjuvants were supplemented to enhance drug-induced mediator responses. RESULTS Twenty-seven patients, including 9 acute generalized exanthematous pustulosis (AGEP), 10 drug reactions with eosinophilia and systemic symptoms, and 8 Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) were recruited. The average frequencies of drug-induced IL-22-, IFN-γ-, and GrB-releasing cells were 35.5±16.3, 33.0±7.1, and 164.8±43.1 cells/million PBMCs, respectively. The sensitivity of combined IFN-γ/IL-22/GrB ELISpot was higher than that of IFN-γ ELISpot alone for culprit drug detection in all SCAR subjects (77.8% vs 51.9%, P < 0.01). The measurement of drug-induced IL-22- and IFN-γ releasing cells confirmed the culprit drugs in 77.8% of AGEP. The measurement of drug-induced IFN-γ- and GrB-releasing cells confirmed the culprit drugs in 62.5% of SJS/TEN. Alpha-galactosylceramide supplementation significantly increased the frequencies of drug-induced IFN-γ releasing cells. CONCLUSION The measurement of drug-induced IFN-γ-releasing cells is the key for identifying culprit drugs. The additional measurement of drug-induced IL-22-releasing cells enhances ELISpot sensitivity to identify drug-induced AGEP, while the measurement of drug-induced GrB-releasing cells could have a role in SJS/TEN. ELISpot sensitivity might be improved by supplementary alpha-galactosylceramide. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT02574988.

Qin Li,1,&ast; Jie Ren,2,&ast; Wei Liu,3 Guoqin Jiang,2 Rongkuan Hu1 1GenePharma Co., Ltd., Suzhou, 215125, Jiangsu, People’s Republic of China; 2Department of General Surgery, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, 215004, People’s Republic of China; 3Department of Pathology, The First Affiliated Hospital of Soochow University, Suzhou, 215006, Jiangsu, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Guoqin JiangDepartment of General Surgery, The Second Affiliated Hospital, Soochow University, 1055 Sanxiang Road, Suzhou, 215004, Jiangsu, People’s Republic of ChinaTel +86-512-67784797Email jiang_guoqin@163.comRongkuan HuGenePharma Co., Ltd., 199 Dongping Street, Suzhou, 215125, Jiangsu, People’s Republic of ChinaTel +86-512-86668828Email rkhu@mail.ustc.edu.cnPurpose: The purpose of our research was to identify and evaluate synthetic phosphorothioate-modified CPG oligodeoxynucleotides (CPG-ODNs) activating innate and adaptive immune responses. Furthermore, combined treatment with CpG and an mRNA cancer vaccine was evaluated in melanoma models as a therapeutic approach.Methods: A molecular assay was used to screen new CpG molecules; mouse modeling and pathological analysis were used to confirm the antitumor effect of CpG alone or in combination with an mRNA vaccine. Finally, safety was assessed by monitoring blood biochemistry.Results: We first screened and identified a new CpG-B class ODN (CpG2018B) that effectively stimulated type II interferons in both mouse plasmacytoid dendritic cells (pDCs) and human peripheral blood mononuclear cells (PBMCs). In addition, CpG2018B promoted cytokine production mainly via toll-like receptor 9 (TLR9) pathways. We further demonstrated that intratumoral (IT) injection of CpG2018B inhibited melanoma growth in syngeneic models and could turn “cold” tumors into “hot” tumors. Then, CpG2018B and an mRNA-based neoantigen cancer vaccine were encapsulated in lipid nanoparticles (LNPs) and intratumorally injected into melanoma mouse models. Interestingly, vaccination with CpG or the mRNA vaccine alone could inhibit tumor growth, while combination of CpG with the mRNA vaccine enhanced the antitumor effect. Finally, we described the long-term safety and tolerability of CpG2018B and mRNA therapy in mice model.Conclusion: We identified a novel CpG-B class ODN to promote the immune response, and CpG combined with mRNA cancer vaccines is an attractive candidate approach for immunostimulatory sequence (ISS)-based therapeutic strategies.Keywords: CpG, IFN-γ, TLR9, mRNA vaccine, neoantigen

Cryptosporidiosis has been considered as a serious diarrheal disease, especially in immunodeficient patients, where they failed to clear the infection leading to several consequences of infection (i.e death). The role of cell mediated immunity in clearing the infection was demonstrated by the increased susceptibility of HIV/AIDS patients to infection. To date, no specific treatment has been proven for cryptosporidiosis in immunodeficient patients. The study aimed to evaluate the efficacy of Aloe vera gel for the treatment of cryptosporidiosis in immunocompetent and dexamethasone immunosuppressed mice in comparison to that of nitazoxanide. Mice were orally administrated with Aloe vera gel, in a daily dose of 250 mg/L in drinking water, for 14 consecutive days post infection. Parasitological, molecular and immunological measurements were recorded on the 7th, 14th, 21st and 32nd days post infection. Our in vitro results showed that 250 mg/L of prepared gel achieved the highest parasitic reduction. The body weights of Aloe vera treated mice on the 21st and 32nd day post infection, either in immunocompetent or immunosuppressed groups, were nearly the same as those of their corresponding control groups. Aloe vera gel succeeded in clearing cryptosporidiosis with a percent reduction of 100% in immunocompetent mice and 99.67% in immunosuppressed mice. The anti-inflammatory effect of Aloe vera reduced the levels of IFN-γ, IL-4, -6 and -17. The success of Aloe vera gel, in clearing cryptosporidiosis in immunosuppressed mice, was obvious either from the reduction of Cryptosporidium DNA or the oocysts in stool samples; and from the improvement of histopathological sections., Graphical abstract Image 1

Objective(s): Outer inflammatory protein A (OipA) is an essential adhesin of Helicobacter pylori. We aimed to evaluate the effects of a recombinant OipA in the induction of crucial cytokines as a vaccine candidate and propolis as an adjuvant in C57BL/6 mice.Materials and Methods: C57BL/6 mice were divided into nine groups according to the disposition of antigen and adjuvant and route of administration: subcutaneous (sc) or gavage. The administrated recombinant purified OipA and propolis concentrations were 10 μg/ml and 40 μg/ml, respectively. After vaccination, we measured expression levels of IFN-γ and IL-4 cytokine genes in the spleen cells of mice by real-time PCR. Results: All results were contrasted with the negative sample. By sc injection, the expression of INF-γ was increased 3.5 and 2.9-fold for OipA and OipA plus propolis, respectively. By gavage 4.4 and 11-fold increase was found for OipA and OipA plus propolis, respectively. The administration of propolis by gavage showed more increase than Sc injection concerning the production of INF-γ. The 11-fold increase for injection of OipA plus propolis by gavage was comparable OipA plus Freund’s adjuvant injected subcutaneously. This result suggested an excellent immunological response toward OipA concerning the production of INF-γ in mice. In all cases there were no notable IL-4 production increases.Conclusion: The results confirm the efficiency of OipA in induction of IFN-γ production, and thereby the cellular immune response. Propolis could be a suitable adjuvant.

Background Myelomatous pleural effusion (MPE), as a presentation of extramedullary infiltration of multiple myeloma (MM), is rare and currently associated with poor outcomes without effective therapy. The potential value of cytokine detection in pleural effusion to MPE has not been reported to date. Case presentation We herein report a case of refractory and relapsed multiple myeloma that developed bilateral MPE due to disease progression caused by intolerance to various chemotherapy regimens. Cytomorphology and flow cytometry were adopted for diagnosis confirmation. Chemotherapy containing immunomodulators combined with thoracic catheterization drainage was applied to the patient, showing a certain therapeutic effect. During the course of disease, the change of cytokine profile in pleural effusion was monitored by cytometric bead array (CBA) technology, revealing that cytokines related to tumor load such as interleukin 6 (IL-6) and interleukin 10 (IL-10) in pleural effusion decreased with the improvement of disease, while other cytokines such as interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 17A (IL-17A), tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), granzyme A, granzyme B, perforin and granulysin increased with the improvement of disease. Conclusion There is a prospect that cytokine level in pleural effusion may indicate treatment response of MPE, and in light of this case, immunomodulators may be utilized in treating patients suffering MPE. Due to limitations of our single case, we urge more groups to evaluate the potential role of cytokine profile in MPE.

The levels of messenger RNA (mRNA) transcription of FOXP3, IFN-γ, TNF, IL-6 and COX-2 from both COVID-19 infected and control subjects were evaluated using SYBRTM green real-time polymerase chain reaction (RT-PCR). Severe/critical cases showed significantly lower lymphocyte counts and higher neutrophil counts than the mild or moderate cases. There were significantly lower levels of mRNA expressions of IFN-γ, TNFα and FOXP3 in COVID-19 patients than in the control group. On the other hand, IL-6 and COX-2 expressions were significantly higher in patients suffering from severe disease. FOXP3 expressions were correlated with the severities of hypoxia and were excellent in predicting the disease severity. This was followed by the IL-6, COX-2 and TNFα expressions. FOXP3 expression was the only biomarker to show a significant correlation with patient mortality. It was concluded that SARS-CoV-2 infection is associated with the downregulation of FOXP3 and upregulations of IL-6 and COX-2.

Junhui Xu,1,2,&ast; Liang Gao,1,&ast; Miao Yan,1 Bingjie Wang,1 Zhengyang Song,1 Huihui Liu,1 Kunyan Sun,3 Lin Nong,4 Mangju Wang,1 Xinan Cen1 1Department of Hematology, Peking University First Hospital, Beijing, People’s Republic of China; 2Xiamen Cardiovascular Hospital, Xiamen University, Xiamen, People’s Republic of China; 3Department of Respiration, Peking University First Hospital, Beijing, People’s Republic of China; 4Department of Pathology, Peking University First Hospital, Beijing, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Mangju Wang; Xinan CenDepartment of Hematology, Peking University First Hospital, No. 8 Xi Shi Ku Street, Xi Cheng District, Beijing, 100034, People’s Republic of ChinaTel +86-10-83575746Email wang_m_j@163.com; cenxn@bjmu.edu.cnBackground: Myelomatous pleural effusion (MPE), as a presentation of extramedullary infiltration of multiple myeloma (MM), is rare and currently associated with poor outcomes without effective therapy. The potential value of cytokine detection in pleural effusion to MPE has not been reported to date.Case Presentation: We herein report a case of refractory and relapsed multiple myeloma that developed bilateral MPE due to disease progression caused by intolerance to various chemotherapy regimens. Cytomorphology and flow cytometry were adopted for diagnosis confirmation. Chemotherapy containing immunomodulators combined with thoracic catheterization drainage was applied to the patient, showing a certain therapeutic effect. During the course of disease, the change of cytokine profile in pleural effusion was monitored by cytometric bead array (CBA) technology, revealing that cytokines related to tumor load such as interleukin 6 (IL-6) and interleukin 10 (IL-10) in pleural effusion decreased with the improvement of disease, while other cytokines such as interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 17A (IL-17A), tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), granzyme A, granzyme B, perforin and granulysin increased with the improvement of disease.Conclusion: There is a prospect that cytokine level in pleural effusion may indicate treatment response of MPE, and in light of this case, immunomodulators may be utilized in treating patients suffering MPE. Due to limitations of our single case, we urge more groups to evaluate the potential role of cytokine profile in MPE.Keywords: myelomatous pleural effusions, cytokine, IFN-γ, IL-2, immunomodulator

Zijiang Yang,1,&ast; Aiqin Gao,2,&ast; Wenjing Shi,1 Jingnan Wang,1 Xianchao Zhang,3 Zhengyan Xu,3 Tingting Xu,3 Yan Zheng,4 Yuping Sun,5 Fei Yang3 1Department of Oncology, Jinan Central Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, 250013, People’s Republic of China; 2Department of Thoracic Radiation Oncology, Shandong Cancer Hospital and Institute, Jinan, Shandong, 250117, People’s Republic of China; 3Department of Pathology, Jinan Central Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, 250013, People’s Republic of China; 4Research Center of Translational Medicine, Jinan Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, 250013, People’s Republic of China; 5Proton Center, Shandong Cancer Hospital and Institute, Jinan, Shandong, 250117, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Fei YangDepartment of Pathology, Jinan Central Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, 250013, People’s Republic of ChinaTel +86 13370586609Email yangf-2008@163.comPurpose: Immune checkpoint blockade (ICB) therapy shows little or no clinical benefit in most colorectal cancer (CRC) patients, due to the immunosuppressive T cell contexture in the tumor microenvironment (TME). Immunoglobulin-like transcript (ILT) 4 is an immunosuppressive molecule in myeloid cells. ILT4 is enriched in solid tumor cells, facilitating their proliferation, invasion, and metastasis. However, the regulatory role of ILT4 in T cell immunity of CRC is still undetermined. Here, we aimed to explore how tumor cell-derived ILT4 orchestrates T cell infiltration, subset distribution, and function in CRC.Methods: A total of 145 paraffin-embedded cancer tissues and the corresponding clinicopathological information were collected from CRC patients. Immunohistochemical (IHC) staining and public database analyses determined the correlation of ILT4 expression with different T cell subset densities, IFN-γ levels, and patient outcomes. Paired Ig-like receptor B (PIR-B, ILT4 mouse ortholog)-overexpressing/-downregulated MC38 cells were subcutaneously injected into C57BL/6 mice as a CRC transplantation model. The frequencies, subsets, and IFN-γ levels of T cells in mouse blood and spleens were determined using flow cytometry and immunohistochemistry, respectively.Results: High ILT4 expression in CRC cells was associated with decreased T cell infiltration, disease progression, and poor patient survival. T cell subset analyses indicated that ILT4-high patients showed reduced CD8+ T cell but elevated FOXP3+ regulatory T (Treg) cell frequencies in the TME. High ILT4 levels predicted lower IFN-γ production by tumor-infiltrating lymphocytes (TILs), especially by CD8+T cells in human CRC tissues. Moreover, PIR-B overexpression accelerated MC38 growth in mice, decreased CD3+/CD8+/IFN-γ+ T cell densities, and elevated Treg infiltration in the TME, blood, and spleens. PIR-B knockdown had the opposite effects.Conclusion: ILT4 in CRC cells induced immunosuppressive T cell subset infiltration and impaired IFN-γ production in TILs, suggesting that ILT4 might be a potential immunotherapeutic target and prognostic biomarker.Keywords: immunoglobulin-like transcript 4, colorectal cancer, T cell subsets, IFN-γ, immunosuppression

Nicotinamide adenine dinucleotide (NAD)+-biosynthetic and consuming enzymes are involved in various intracellular events through the regulation of NAD+ metabolism. Recently, it has become clear that alterations in the expression of NAD+-biosynthetic and consuming enzymes contribute to the axonal stability of neurons. We explored soluble bioactive factor(s) that alter the expression of NAD+-metabolizing enzymes and found that cytokine interferon (IFN)-γ increased the expression of nicotinamide nucleotide adenylyltransferase 2 (NMNAT2), an NAD+-biosynthetic enzyme. IFN-γ activated signal transducers and activators of transcription 1 and 3 (STAT1/3) followed by c-Jun N-terminal kinase (JNK) suppression. As a result, STAT1/3 increased the expression of NMNAT2 at both mRNA and protein levels in a dose- and time-dependent manner and, at the same time, suppressed activation of sterile alpha and Toll/interleukin receptor motif-containing 1 (SARM1), an NAD+-consuming enzyme, and increased intracellular NAD+ levels. We examined the protective effect of STAT1/3 signaling against vincristine-mediated cell injury as a model of chemotherapy-induced peripheral neuropathy (CIPN), in which axonal degeneration is involved in disease progression. We found that IFN-γ-mediated STAT1/3 activation inhibited vincristine-induced downregulation of NMNAT2 and upregulation of SARM1 phosphorylation, resulting in modest suppression of subsequent neurite degradation and cell death. These results indicate that STAT1/3 signaling induces NMNAT2 expression while simultaneously suppressing SARM1 phosphorylation, and that both these actions contribute to suppression of axonal degeneration and cell death.

Background This study investigated the mechanisms underlying the preventive effect of dihydromyricetin (DHM) against liver fibrosis involving hepatic stellate cells (HSCs) and hepatic natural killer (NK) cells. Methods A carbon tetrachloride (CCl4)-induced liver fibrosis model was established in C57BL/6 mice to study the antifibrotic effect of DHM based on serum biochemical parameters, histological and immunofluorescence stainings, and the expression of several fibrosis-related markers. Based on the immunoregulatory role of DHM, the effect of DHM on NK cell activation ex vivo was evaluated by flow cytometry. Then, we investigated whether DHM-induced autophagy was involved in HSCs inactivation using enzyme-linked immunosorbent assays, transmission electron microscopy, and western blot analysis. Thereafter, the role of DHM in NK cell-mediated killing was studied by in vitro coculture of NK cells and HSCs, with subsequent analysis by flow cytometry. Finally, the mechanism by which DHM regulates NK cells was studied by western blot analysis. Results DHM ameliorated liver fibrosis in C57BL/6 mice, as characterized by decreased serum alanine transaminase and aspartate transaminase levels, decreased expressions of collagen I alpha 1 (CoL-1α1), collagen I alpha 2 (CoL-1α2), tissue inhibitor of metalloproteinases 1 (TIMP-1), α-smooth muscle actin (α-SMA) and desmin, as well as increased expression of matrix metalloproteinase 1 (MMP1). Interestingly, HSCs activation was significantly inhibited by DHM in vivo and in vitro. As expected, DHM also upregulated autophagy-related indicators in liver from CCl4-treated mice. DHM also prevented TGF-β1-induced activation of HSCs in vitro by initiating autophagic flux. In contrast, the autophagy inhibitor 3-methyladenine markedly abolished the antifibrotic effect of DHM. Surprisingly, the frequency of activated intrahepatic NK cells was significantly elevated by DHM ex vivo. Furthermore, DHM enhanced NK cell-mediated killing of HSCs by increasing IFN-γ expression, which was abolished by an anti-IFN-γ neutralizing antibody. Mechanistically, DHM-induced IFN-γ expression was through AhR-NF-κB/STAT3 pathway in NK cells. Conclusion These results demonstrated that DHM can ameliorate the progression of liver fibrosis and inhibition of HSCs activation by inducing autophagy and enhancing NK cell-mediated killing through the AhR-NF-κB/STAT3-IFN-γ signaling pathway, providing new insights into the preventive role of DHM in liver fibrosis.

Background and aims Several oncolytic viruses applications have been approved in the clinic or in different phases of clinical trials. However, these methods have some rudimentary problems. Therefore, to enhance the delivery and quality of treatment, considering the advantage of cell carrier-based methods such as Mesenchymal Stem Cells (MSC) have been proposed. This study was designed to evaluate the performance and quality of cancer treatment based on MSCs loaded by oncolytic reovirus in the cancerous C57BL/6 mouse model. Also, we evaluated MSCs migration potency in vitro and in vivo following the oncolytic reovirus infection. Methods C57BL/6 mice were inoculated with TC-1 cell lines and tumors were established in the right flank. Mice were systemically treated with reovirus, MSCs-loaded with reovirus, MSCs, and PBS as a control in separated groups. Effects of infected AD-MSCs with reovirus on tumor growth and penetration in the tumor site were monitored. All groups of mice were monitored for two months in order to therapeutic and anticancer potential. After treatments, tumor size alteration and apoptosis rate, as well as cytokine release pattern was assessed. Results The results of the current study indicated that the effect of reovirus infection on AD-MSCs is not devastating the migration capacity especially in MOI 1 and 5 while intact cells remain. On the other hand, MSCs play an efficient role as a carrier to deliver oncolytic virus into the tumor site in comparison with systemic administration of reovirus alone. Apoptosis intensity relies on viral titration and passing time. Followed by systemic administration, treatment with oncolytic reovirus-infected AD-MSCs and MSCs alone had shown significant inhibition in tumor growth. Also, treatment by reovirus causes an increase in IFN-γ secretion. Conclusion The results of in vitro and in vivo study confirmed the tumor-homing properties of infected AD-MSCs and the significant antitumor activity of this platform. Hence, our results showed that the cell carrier strategy using oncolytic reovirus-loaded AD-MSCs enhanced virus delivery, infiltration, and antitumor activity can be effectively applied in most cancers.

Background and aims: Congenital toxoplasmosis is assumed to play a role in developing schizophrenia in human. This study aimed to estimate the relationship between Toxoplasma infection and schizophrenia by using enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Methods: In this case-control study, 46 schizophrenic patients forming our case group as well as 40 healthy relatives of schizophrenic patients forming our control group were selected from among 456 inpatients with schizophrenia in Ibn Sina and Hejazi hospitals in Mashhad from June 2016 to February 2017. The blood samples from both groups were collected for serological and molecular tests. Different statistical methods such as Chi-square, independent t test, and logistic regression models were used in the present study. Results: In the present study, the seroprevalence of T. gondii was 54% in case group and 45% in control (OR=1.44, 95% CI: 062- 3.40, P = 0.38). There was no significant association between the seroprevalence of T. gondii infection and age, gender, and season as the risk factors in the case group. Furthermore, a poor agreement was observed between the microscopy and PCR methods. Non-significant differences were found between the mean levels of interferon gamma (IFN-γ), C-reactive protein (CRP), and white blood cell (WBC) in two groups. Conclusion: Contrary to the reports from some studies, no association was found between Toxoplasma infection and the schizophrenia. In order to better understand the effect of Toxoplasma on schizophrenia, it is necessary to develop laboratory methods to differentiate acquired toxoplasmosis from congenital one.

Background: Hyperfibrinolysis and pro/anti-inflammatory imbalance usually occur in the early stage of severe burns. Soluble urokinase-type plasminogen activator receptor (suPAR) is involved in fibrinolysis and inflammation. To date, the levels of circulating suPAR in non-survivors with severe burns remain unknown. This study aimed to investigate the early association between circulating suPAR levels and biomarkers of fibrinolysis, pro/anti-inflammatory, and prognosis. Methods: Sixty-four consecutive Chinese patients with severe burns and 26 healthy volunteers were enrolled in a prospective observational cohort. Clinical characteristics and laboratory data were collected prospectively. Blood samples were collected at 48 h post-burn, and suPAR and biomarkers of pro/anti-inflammatory and fibrinolysis were detected by enzyme-linked immunosorbent assays. Important indicators between non-survivors and survivors were compared. Linear regression analysis was performed to screen variables associated with suPAR. Logistic regression analysis and receiver operating characteristic curve (ROC) analysis were performed to evaluate the prognostic value of suPAR. Result: Compared with the control group, the circulating suPAR levels in the survivors (P

Background Influenza is a highly contagious, acute, febrile respiratory infection caused by a negative-sense, single-stranded RNA virus, which belongs in the Orthomyxoviridae family. Cigarette smoke (CS) exposure worsens influenza infection in terms of frequency and severity in both human and animal models. Methods C57BL/6 mice with or without CS exposure for 6 weeks were inoculated intranasally with a single, non-lethal dose of the influenza A virus (IAV) A/Puerto Rico/8/1934 (PR8) strain. At 7 and 10 days after infection, lung and mediastinal lymph nodes (MLN) cells were collected to determine the numbers of total CD4 + and CD8 + T cells, and IAV-specific CD4 + and CD8 + T cells, using flow cytometry. Bronchoalveolar lavage fluid (BALF) was also collected to determine IFN-γ levels and total protein concentration. Results Although long-term CS exposure suppressed early pulmonary IAV-antigen specific CD8 + and CD4 + T cell numbers and IFN-γ production in response to IAV infection on day 7 post-infection, CS enhanced numbers of these cells and IFN-γ production on day 10. The changes of total protein concentration in BALF are consistent with the changes in the IFN-γ amounts between day 7 and 10, which suggested that excessive IFN-γ impaired barrier function and caused lung injury at the later stage of infection. Conclusions Our results demonstrated that prior CS exposure caused a biphasic T cell and IFN-γ response to subsequent infection with influenza in the lung. Specifically, the number of IAV antigen-specific T cells on day 10 was greatly increased by CS exposure even though CS decreased the number of the same group of cells on day 7. The result suggested that CS affected the kinetics of the T cell response to IAV, which was suppressed at an early stage and exaggerated at a later stage. This study is the first to describe the different effect of long-term CS on T cell responses to IAV at early and late stages of infection in vivo.

The measurement of specific T-cell responses can be a useful tool for COVID-19 diagnostics and clinical management. In this study, we evaluated the IFN-γ T-cell response against the main SARS-CoV-2 antigens (spike, nucleocapsid and membrane) in acute and convalescent individuals classified according to severity, and in vaccinated and unvaccinated controls. IgG against spike and nucleocapsid were also measured. Spike antigen triggered the highest number of T-cell responses. Acute patients showed a low percentage of positive responses when compared to convalescent (71.6% vs. 91.7%, respectively), but increased during hospitalization and with severity. Some convalescent patients showed an IFN-γ T-cell response more than 200 days after diagnosis. Only half of the vaccinated individuals displayed an IFN-γ T-cell response after the second dose. IgG response was found in a higher percentage of individuals compared to IFN-γ T-cell responses, and moderate correlations between both responses were seen. However, in some acute COVID-19 patients specific T-cell response was detected, but not IgG production. We found that the chances of an IFN-γ T-cell response against SARS-CoV-2 is low during acute phase, but may increase over time, and that only half of the vaccinated individuals had an IFN-γ T-cell response after the second dose.

Mucosal leishmaniasis (ML) is a severe form of tegumentary leishmaniasis associated with a persistent inflammatory response. High levels of TNF, IFN-γ, CXCL9 and CXCL10 are found in ML patients, and the association of pentoxifylline with antimony is more effective in decreasing the healing time in ML patients when compared to antimony alone. The present study aimed to investigate the existence of a correlation between cytokine and chemokine production and ML severity and evaluate the potential value of cytokine and chemokine production as marker of therapeutic response in ML patients. This prospective study included 86 subjects in an area of endemic Leishmania braziliensis transmission. Patients diagnosed with ML were classified into clinical stages ranging from I to V according to disease severity. TNF, IFN-γ, CXCL9 and CXCL10 levels were quantified in the supernatant of the mononuclear cell cultures by ELISA before and after treatment with antimony alone or antimony plus pentoxifylline. The median TNF level in the group with mild disease (Stages I–II) was 1064 pg/mL (142–3738 pg/mL), while, in the group with moderate or severe disease (Stages III–V), it was 1941 pg/mL (529–5294 pg/mL) (p = 0.008). A direct correlation was observed between ML clinical severity and levels of TNF production (r = 0.44, p = 0.007). Patients who were treated with antimony and pentoxifylline healed significantly faster than those treated with antimony alone (52 vs. 77 days, hazard ratio = 0.60; 95% confidence interval = 0.38–0.95, p = 0.013). Therapeutic failure was higher in the group that received antimony alone (25% vs. 7%; p = 0.041). There was a significant decrease in CXCL9 after therapy of ML in both groups (p = 0.013; p = 0.043). TNF levels are associated with the severity of mucosal diseases, and pentoxifylline associated with antimony should be the recommended therapy for ML in countries where liposomal amphotericin B is not available.

(1) Background: CMV and EBV co-infections can affect the HIV disease progression by modulating the immune system. The disease dynamics can differ in HIV-positive adults and children. In Pakistan, HIV is rapidly expanding, especially in children; however, the prevalence of CMV and EBV co-infection and the effect on immune modulation in HIV-positive children are not known. This study aimed to bridge this gap by estimating the rate of active CMV and EBV co-infection in HIV-positive children, followed by the analysis of differential expression of cytokines in HIV mono- and HIV/CMV/EBV co-infected children. (2) Methods: DNA samples from 319 HIV-positive children, previously recruited as part of a study to investigate the HIV outbreak in Larkana, Pakistan, in 2019, were screened for CMV and EBV through qPCR. Subsequently, differences in HIV viral loads and CD4 counts were analyzed between the HIV mono- and HIV/CMV/EBV co-infected groups. The RNA samples were used to determine the differential expression of both pro- and anti-inflammatory cytokines in the mono- and co-infected groups using RT-qPCR, while unpaired T-test and Pearson correlation test were applied to, respectively, analyze the differential cytokine expression and correlation between cytokine in the two groups. (3) Results: Of 319 samples, the rate of active EBV and CMV co-infection in HIV-positive children was observed in 79.9% and 38.9%, respectively. A significant difference was observed in HIV viral load between HIV mono- and co-infected groups. IFN-γ expression was found to be lower in the HIV mono-infected group, while higher in all other three co-infected groups. Meanwhile, mRNA expression of TGF-β1 was found to be lower in HIV mono- and HIV–CMV–EBV co-infected groups, while higher in HIV–CMV and HIV–EBV co-infected groups. IFN-γ and IL-2 exhibited a significant positive correlation in all except HIV–CMV co-infected group. (4) Conclusions: The study suggests that the presence of EBV/CMV co-infection can affect the HIV viral loads and expression of certain cytokines (IFN-γ and TGF-β1), which may affect the HIV disease dynamics in infected children.

Metoda ELISPOT (angl. Enzyme-Linked ImmunoSpot) se zaradi preproste izvedbe in visoke občutljivosti pogosto uporablja v diagnostiki in raziskavah na različnih področjih. Test interferon (IFN)-γ ELISPOT omogoča štetje antigensko (Ag)-specifičnih limfocitov T po Ag-specifični stimulaciji. Glavni namen magistrskega dela je bil vzpostaviti test na osnovi metode ELISPOT za določanje Ag-specifičnega imunskega odziva CD8+ citotoksičnih limfocitov T (CTL). Iz levkocitnih pripravkov zdravih krvodajalcev smo izolirali enojedrne celice periferne krvi (PBMC), ki smo jih uporabili za različne poskuse. Izvedli smo metodo mešane levkocitne reakcije (MLR), PBMC ex vivo stimulirali z različnimi peptidnimi mešanicami ali iz PBMC z imunomagnetno selekcijo izolirali celokupne CD8+ limfocite T in monocite. Slednje smo diferencirali v dendritične celice (DC), ki smo jih nadalje uporabili za in vitro stimulacijo avtolognih CD8+ CTL. Na podlagi rezultatov optimizacije metode ELISPOT smo določali Ag-specifičnost limfocitov T. Pokazali smo, da lahko z metodo ELISPOT zaznamo alo-Ag-specifične limfocite T in virusno-specifične limfocite T v populaciji PBMC po 24-urni ex vivo stimulaciji z mešanico peptidov citomegalovirusa (CMV), virusa Eppstein-Barr in virusa gripe A (CEF) ter mešanico peptidov CMV fosfoproteina 65 (pp65). Dokazali smo tudi, da lahko z metodo ELISPOT zaznamo redke klone melanomsko-specifičnih CD8+ CTL po in vitro stimulaciji z zrelimi avtolognimi DC (αDC in zDC), pulziranimi s peptidi, značilnimi za melanom, gp100, Melan-A in tyr. V sklopu in vitro stimulacije CD8+ CTL z DC smo dokazali tudi, da imajo zrele zDC ojačano sposobnost prožitve melanomsko-specifičnega imunskega odziva CTL v primerjavi z αDC. The ELISPOT (angl. Enzyme-Linked ImmunoSpot) method is a highly sensitive and user-friendly method, commonly applied in different areas of research and diagnostics. The interferon (IFN)-γ ELISPOT assay is used to enumerate antigen (Ag)-specific T cells upon Ag-specific stimulation. The principal aim of our study was to establish an ELISPOT assay for the determination of Ag-specific immune response of CD8+ cytotoxic T cells (CTL). Peripheral blood mononuclear cells (PBMC) were isolated from blood samples obtained from healthy human blood donors. PBMCs were used to run the mixed lymphocyte reaction (MLR) and were ex vivo stimulated with different peptide pools. Whole CD8+ T cells and monocytes were isolated from PBMC by positive immunomagnetic selection and monocytes were used for dendritic cell (DC) differentiation. The latter were used for in vitro stimulation of autologous CD8+ CTL. After its optimization, the ELISPOT assay was applied to determine the Ag-specific immune response of T cells. Our results show that the ELISPOT assay enables the detection of allo-Ag-specific T cells and can be used to determine virus-specific T cells in PBMCs upon 24 h ex vivo stimulation with peptide pool from cytomegalovirus (CMV), Epstein-Barr virus and influenza A virus (CEF) and peptide pool of the phospohoprotein 65 (pp65) from CMV. The ELISPOT method also enables the detection of rare clones of melanoma-specific CTL upon in vitro stimulation with autologous mature DC (αDC and zDC), pulsated with melanoma-associated peptides gp100, Melan-A and tyr. Finally, our results demonstrate zDC are superior in their capacity to induce melanoma-specific CTL compared to αDC.

Francisella tularensis je gram-negativna unutarstanična bakterija. Pripada porodici Francisellaceae i uključuje 3 podvrste: tularensis, holarctica i mediasiatica koje se međusobno razlikuju u virulenciji, geografskoj rasprostranjenosti i ekologiji. Francisella je uzročnik tularemije, re-emergentne bolesti ljudi, domaćih i divljih životinja. Francisella ima sposobnost inficirati makrofage, nakon čega različitim mehanizmima koji trenutno nisu u potpunosti definirani, bježi iz fagosoma te se opsežno replicira u citosolu. Živi atenuirani soj LVS (engl. Live Vaccine Strain), koji je dobiven iz podvrste holarctica, nije virulentan za ljude no vrlo je virulentan za miševe stoga je idealan za proučavanje mehanizma virulencije roda Francisella. U stanicama sisavaca veliku ulogu za preživljavanje i replikaciju te posljedično virulenciju Francisella imaju geni koji se nalaze unutar Francisella patogenog otoka (engl. Francisella pathogenicity island, FPI). Gen iglI neophodan je za virulenciju, citotoksičnost i bijeg iz fagosoma. Cilj ovog diplomskog rada bio je utvrditi ulogu iglI gena in vivo praćenjem preživljavanja i određivanjem broja bakterija u organima intradermalno inficiranih miševa. Također, ispitivao se i imunološki odgovor domaćina sintezom interferon gamma (IFN-γ) i interleukin 10 (IL-10) citokina, in vivo na C57BL/6 miševima nakon intradermalne infekcije s F. tularensis subsp. holarctica LVS, mutantom iglI i njenom komplementantom iglI/iglI. Kod testa preživljavanja miševa, tijekom 15 dana praćeno je 10 miševa po skupini koji su intradermalno inficirani. Za određivanje broja bakterija u tkivu jetre, slezene i pluća, 3 miša po skupini inficirani su s LD50 (5 x 104 bakterija/mišu), te su 6, 24, 48, 72 sata nakon infekcije organi odstranjeni i dalje analizirani. qRT-PCR-om određena je razina IFN-γ i IL-10 citokina u tkivu jetre, slezene i pluća 72 sata nakon intradermalne infekcije miševa kako bi se ispitao odnos između mutante iglI, divljeg soja LVS te komplementante iglI/iglI. Miševi inficirani iglI mutantom imali su 100% preživljavanje kod svih ispitivanih doza što se ne može reći za miševe inficirane s F. tularensis subsp. holarctica LVS. Također, broj bakterija mutante iglI u svim ispitivanim organima bio je statistički značajno smanjen u odnosu na soj LVS. Zaključno možemo reći kako je iglI gen važan za unutarstaničnu replikaciju F. tularensis subsp. holarctica LVS i da ima utjecaj na sintezu IFN-γ i IL-10 citokina u tkivu jetre, slezene i pluća., Francisella tularensis is a gram-negative intracellular bacteria. It belongs to the family Francisellaceae and includes three subspecies: tularensis, holarctica and mediasiatica which differ in virulence, geographical distribution and ecology. Francisella is the causative agent of tularemia, a re-emerging disease of humans, domestic, and wild animals. Francisella has the ability to infect macrophages, whereupon escapes from phagosomes by various mechanisms that are not currently fully defined, and replicates extensively in the cytosol. The live attenuated strain LVS (Live Vaccine Strain), produced from the subspecies holarctica, is not virulent to humans but is very virulent to mice and is therefore ideal for studying the virulence mechanism of the genus Francisella. In mammalian cells, Francisella pathogenicity island (FPI) genes play a major role in the survival, replication, and consequent virulence of Francisella. The iglI gene is essential for virulence, cytotoxicity, and phagosome escape. The aim of this thesis was to determine the role of iglI gene in vivo by monitoring survival and determining the number of bacteria in organs of intradermally infected mice. The immune response of the host was also examined by synthesis of interferon gamma (IFN-γ) and interleukin 10 (IL-10) cytokines, in vivo in C57BL/6 mice after intradermal infection with F. tularensis subsp. holarctica LVS, iglI mutant and its complement iglI/iglI. In the mice survival test, 10 mice per group of intradermally infected were followed for 15 days. To determine the number of bacteria in the liver, spleen and lungs tissue, 3 mice per group were infected with LD50 (5 x 104 bacteria/mouse), and the organs were removed 6, 24, 48, 72 hours after infection and further analyzed. The levels of IFN-γ and IL-10 cytokines in liver, spleen and lungs tissue were determined by qRT-PCR, 72 hours after intradermal infection of mice to examine the relationship between iglI mutant, wild-type LVS and complement iglI/iglI. Mice infected with the iglI mutant had 100% survival at all tested doses, which cannot be said for mice infected with F. tularensis subsp. holarctica LVS. Also, the number of iglI mutant bacteria in all examined organs was statistically significantly reduced compared to the LVS strain. In conclusion, the iglI gene is important for intracellular replication of F. tularensis subsp. holarctica LVS and that it affects the synthesis of IFN-γ and IL-10 cytokines in liver, spleen and lungs tissues.

Aim: to assess peripheral blood NK cell (pNK) counts in women with recurrent pregnancy loss.Materials and Methods. There were examined 102 women: 28 non-pregnant (immediately after repeatedly terminated pregnancy, group IA) and 34 pregnant women with history of previous recurrent pregnancy loss (group IB). Control and comparison groups were consisted of healthy women lacking any recorded miscarriage: 20 non-pregnant (group IIA) and 20 pregnant women (group IIB), respectively. Along with assessing complete blood count range, all subjects were analyzed percentage of pNK cell CD16bright(+) CD56dim(-) cytotoxic phenotype by using fluorescence microscopy as well as level of cytokines IL-2, IL-6, IL-10, tumor necrosis factor (TNF-α), vascular endothelial growth factor (VEGF) and interferon-gamma (IFN-γ) using ELISA.Results. It was found that the level of NK cell cytotoxic phenotype - percentage of CD16+CD56- was elevated in both groups: groups: group IA - 36.5 % collared with the control group IIA - 27.5% (р < 0.05); in group IB up to 37.0 %, in comparison group IIB - 27.4% (р < 0.01). It was further corroborated by increased level of serum cytokine IL-6 IL-6 comprising in group IA vs. IIA - 28.5 pg/ml vs. 14.2 pg/ml (р < 0.01) as well as in group IB vs. IIB up to 16.7 pg/ml vs. 12.2 pg/ml (р < 0.01), respectively. However, level of serum IL-2 and IL-10 did not significantly differ in all groups examined. Interestingly, level of VEGF in subjects aged 21-30 years was elevated: in non-pregnant (group IA) vs. control (group IIA) - up to 548.5 pg/ml vs. 310.8 pg/ml (р < 0.01); pregnant women (IB) vs. comparison group (IIB) - insignificantly elevated up to 476.1 pg/ml vs. 381.5 pg/ml, respectively. In contrast, no significant changes were observed in 31-40-year group. Concentration of IFN-γ in group IA vs. control group (IIA) was increased up to 9.2 pg/ml vs.8.64 pg/ml; group IB vs. comparison group - up to 8.36 pg/ml vs. 7.56 pg/ml, respectively.Conclusion. Elevated percentage of cytotoxic CD16+CD56- NK cells in peripheral blood results in imbalance of immune-related parameters that directly correlated with increased serum level of IL-2, IL-6, TNF-α that may alter maternal immune tolerance to the fetus and subsequently resulting in recurrent pregnancy loss. Hence, the aforementioned data may be used as diagnostic and prognostic criteria in recurrent pregnancy losses.

Symposium mini review

Multidrug-Resistant Tuberculosis (MDR TB) is caused by an organism that is resistant to at least isoniazid and rifampisin, the two most potent TB drug. Immune response to against Mycobacterum tuberculosis infection is related to the function of immunity. The function of interferon-γ (pro-inflammatory) is to activate macrophages, to stimulate antimicrobial molecules (to reactive oxygen species and nitric oxide), and to inhibits interleukin-10. Interleukin-10 function is to triggers humoral immunity, to inhibit IFN- γ. This study aimed to analyze level changes and the correlation with clinical data, also months of MDR TB patients who received standard OAT therapy. This was an observational study using cross sectional design. There were 29 patients who received standard MDR TB OAT therapy from 1-24 months, who met the inclusion criteria. Then, the patients were divided based on duration of the therapy, which are the initial/intensive and advanced phase. The initial phase divided into 2: first one is for 1-4 months therapy’s time (5 patients) and the second one is for more than 4-8 months (6 patients). Then, the advanced group divided into two groups again, which are third group with more than 8-16 months (13 patients) and fouth group with more than 16-24 months (5 patients). Then, measured serum concentration IFN-γ, IL-10 at the start of the study and 4 weeks later with the ELISA method. This research during the period July-December (6 months). IFN-γ post concentrations were decreased by 39.14 ± 139.12 pg/mL (p > 0.05). The concentration of IL-10 was decreased by 33.93 ± 109.20pg/mL (p>0.05). Based on the TB score bandim method during pre and posts results were 1 patient experienced severity change from severity class 1 to 2, 1 patient from severity class 2 to 1, 1 patient remained in severity 2 and 26 patient remained in severity 1. The results showed that serum IFN-γ and IL-10 levels in initial/intensive and advanced phase patients who received MDR TB regiment after four weeks did not changed

Background: Immunotherapy for wart employs ability of immune system to recognize certain viral, bacterial, and fungal antigens in previously sensitized individual inducing Type IV delayed-type hypersensitivity reaction (up-regulated Th1 cytokines IL-1, TNF-α, IFN-γ; down-regulated Th2 cytokines IL-10), not only to injected antigen but also against wart virus. Aims: To evaluate and compare the pattern of production of Th1 cytokines (IL-1, TNF-α, IFN-γ) and Th2 cytokines (IL-10) in patients receiving immunotherapy with purified-protein-derivative (PPD), Mycobacterium w (Mw), or mumps-measles-rubella (MMR) vaccine. Methods: The cohort study conducted on patients receiving immunotherapy with PPD, Mw, or MMR which was injected intradermally at baseline, repeated every 2 weeks for 6 doses?. Five-millilit?e?r blood was collected for evaluation of cytokines at baseline and 12 weeks of treatment. Blood was centrifuged to separate serum, stored at -80°C. Cytokines were measured by ELISA using a standard kit. Results: Nine participants in PPD group, 11 in Mw group, and 12 in MMR group completed the study. IL-1 was raised from baseline in all study arms and was significant in PPD group (P = 0.008). There was a predicted increase in IFN-γ in Mw and MMR groups but not in the PPD group. In the PPD group, IFN-γ was found to be down regulated. IL-10, a Th 2 cytokine was down regulated in all the groups at the study end from baseline, significantly so in the PPD group (P = 0.027) and MMR group (P = 0.001). TNF-α, being a Th1 cytokine was down regulated in all groups instead of an increase. In PPD group, IL-10 was significantly low at study end in patients who had complete resolution of warts. Limitations: Longer follow-up could not be done due to logistic issues. Conclusion: IL-1, TNF-α upregulation and IL-10 downregulation confirm that cytokine milieu plays an important role in wart immunotherapy. TNF-α has no contributory role. IL-10 can be used as a biomarker of complete response in PPD therapy.

Background & Aims Immune checkpoint inhibitors have shed light on the importance of antitumor immunity as a therapeutic strategy for hepatocellular carcinoma (HCC). The altered glucose metabolism known as the Warburg effect recently has gained attention as a cancer immune-resistance mechanism. Considering glycolysis inhibitors as therapeutic agents, their specific delivery to cancer cells is critical not to induce adverse effects. Thus, we investigated antitumor effects of a glycolysis inhibitor, consisting of 2-deoxy-D-glucose (2DG)-encapsulated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (2DG-PLGA-NPs), against hepatocellular carcinoma in mice. Methods The antitumor effects of 2DG-PLGA-NPs were examined using hepatoma cell lines, xenograft tumors, and hepatocarcinogenic and syngeneic mouse models. Results The 2DG-PLGA-NPs induced cytotoxic effects and antitumor immunity through enhanced T-cell trafficking. In addition, 2DG-PLGA-NPs induced decreased lactate production and increased interferon-γ–positive T cells in liver tumors. Human CD8+ T cells cocultured with 2DG-PLGA-NP–treated Huh7 cells showed their increased interferon-γ production and glucose uptake compared with the CD8+ T cells co-cultured with PLGA-NP–treated Huh7 cells. Chemotaxis of CD8+ T cells was suppressed by lactate and enhanced by glucose. Interferon-γ enhanced CD8+ T-cell chemotaxis in both an autocrine and paracrine manner. Notably, the 2DG-PLGA-NPs augmented chemokine (CXCL9/CXCL10) production in liver tumors via interferon-γ–Janus kinase–signal transducers and activator of transcription pathway and 5' adenosine monophosphate-activated protein kinase–mediated suppression of histone H3 lysine 27 trimethylation. These 2DG-PLGA-NPs not only amplified antitumor effects induced by sorafenib or an anti–programmed death-1 antibody, but also suppressed anti–programmed death-1–resistant tumors. Conclusions The newly developed 2DG-PLGA-NPs showed antitumor immunity and cytotoxicity in liver tumors in mice, suggesting the potential of 2DG-PLGA-NPs for future clinical applications., Graphical abstract