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Low-level laser therapy (LLLT) has been a treatment modality by many androgenetic alopecia (AGA) patients in recent years. It remained unclear as to how long the treatment regime should be maintained, and which characteristics of patients should this be recommended. A real-world study was carried out with an FDA-cleared low-level laser helmet for 1383 patients. Ordinal logistic regression analysis with propensity score matching (PSM) was used to investigate the factors related to efficacy assessment. More than 80% of users were between 18 and 40 years old. The median use times were 133 for mild AGA patients and 142 for moderate-to-severe AGA patients, which equated to 38 weeks and 40 weeks, respectively. The overall clinical effectiveness was nearly 80%. PSM analysis revealed that gender (P = 0.002), use period (P = 0.068), scalp conditions with dandruff, rash, and itchy symptoms were associated with the grading of efficacy assessment. Male users (ordinal OR: 1.35, CI: (1.01, 1.79)); use for more than 180 times or use period for 1 year (ordinal OR: 1.40, CI: (1.11, 1.96)); and those with scalp dandruff (ordinal OR: 1.34, CI: (1.01, 1.87)), rash (ordinal OR: 1.47, CI: (1.04, 2.07)), and itchy symptoms (ordinal OR: 1.51, CI: (1.12, 2.03)) had better efficacy assessments. The recommended treatment regime with low-level laser helmet was more than 1 year or 180 use times. Male patients with dandruff, rash, and itchy symptoms in scalps tended to have a better efficacy assessment.

Survival plots of mice with s.c./s.c. melanomas treated with PBS or wildtype (WT) CD8+ T cells injected into the s.c. Tumor 1. PBS: n=8; WT CTLs: n=5. ns: not significant (p > 0.05). log-rank test.

A, Quantification of the tumor bioluminescence in the mice from the indicated treatment groups. D24-RGDOX: Delta-24-RGDOX. B, Waterfall plots showing tumor burden change from baseline in mice of indicated treatment groups base on the bioluminescence from the tumor. Baseline time point: Day 8 for s.c. tumor, Day 15 for i.c. tumor. Evaluation time point: Day 57 or the last time point before euthanasia.

gp100 and ovalbumin (OVA) expression in indicated mouse melanoma cell lines. Cell lysates were analyzed with immunoblotting. GAPDH levels are shown as a protein loading control.

Purpose:Intratumoral injection of oncolytic adenovirus Delta-24-RGDOX induces efficacious antiglioma immunity in syngeneic glioma mouse models. We hypothesized that localized treatment with the virus is effective against disseminated melanomas.Experimental Design:We tested the therapeutic effect of injecting Delta-24-RGDOX into primary subcutaneous (s.c.) B16-Red-FLuc tumors in s.c./s.c. and s.c./intracranial (i.c.) melanoma models in C57BL/6 mice. Tumor growth and in vivo luciferase-expressing ovalbumin-specific (OT-I/Luc) T cells were monitored with bioluminescence imaging. Cells were profiled for surface markers with flow cytometry.Results:In both s.c./s.c. and s.c./i.c. models, 3 injections of Delta-24-RGDOX significantly inhibited the growth of both the virus-injected s.c. tumor and untreated distant s.c. and i.c. tumors, thereby prolonging survival. The surviving mice were protected from rechallenging with the same tumor cells. The virus treatment increased the presence of T cells and the frequency of effector T cells in the virus-injected tumor and mediated the same changes in T cells from peripheral blood, spleen, and brain hemispheres with untreated tumor. Moreover, Delta-24-RGDOX decreased the numbers of exhausted T cells and regulatory T cells in the virus-injected and untreated tumors. Consequently, the virus promoted the in situ expansion of tumor-specific T cells and their migration to tumors expressing the target antigen.Conclusions:Localized intratumoral injection of Delta-24-RGDOX induces an in situ antovaccination of the treated melanoma, the effect of which changes the immune landscape of the treated mice, resulting in systemic immunity against disseminated s.c. and i.c. tumors.

Expression of TCRs in CD8+ T cells from pmel-1 (A, gp100-Tet+) or OT-I (B, OVA-Tet+) transgenic mice. Splenocytes from the mice were stained with tetramers binding with TCR specific for gp100 (pmel-1) or OVA (OT-I), and antibody against CD8. Stained cells were analyzed with flow cytometry. The numbers in upright corner of the dot plots indicate the frequency (% of splenocytes) of gp100-targeting (A) or OVA-targeting (B) in CD8+ T cells.

Antibody information

The effect of combining pmel-1 T cells and Delta-24-RGDOX. A, A cartoon depiction of the treatment scheme for schedule (left) and positions of tumor implantation, T-cell and virus injection (right). s.c.: subcutaneously; i.t.: intratumorally. B, Plots of the bioluminescence of the tumors from the indicated treatment groups. C, Survival plots of the treatment groups. n = 9 except n = 8 for combination group. RGDOX: Delta-24-RGDOX. ns: not significant (p > 0.05); **: p

Cancer cell heterogeneity and immunosuppressive tumor microenvironment (TME) pose a challenge in treating solid tumors with adoptive cell therapies targeting limited tumor-associated antigens (TAAs), such as CAR T-cell therapy. We hypothesize that oncolytic adenovirus Delta-24-RGDOX activates the TME and promote antigen spread to potentiate the abscopal effect of adoptive TAA-targeting T-cells in localized intratumoral treatment. Herein, we used C57BL/6 mouse models with disseminated tumors derived from B16 melanoma cell lines to assess therapeutic effects and antitumor immunity. gp100-specific pmel-1 or OVA-specific OT-I T-cells were injected into the first subcutaneous (s.c.) tumor, followed by three injections of Delta-24-RGDOX. We found TAA-targeting T-cells injected into one s.c. tumor showed tumor tropism. Delta-24-RGDOX sustained the systemic tumor regression mediated by the T-cells, leading to improved survival rate. Further analysis revealed that, in mice with disseminated B16-OVA tumors, Delta-24-RGDOX increased CD8+ leukocyte density within treated and untreated tumors. Importantly, Delta-24-RGDOX significantly reduced the immunosuppression of endogenous OVA-specific cytotoxic T lymphocytes (CTLs) while increasing that of CD8+ leukocytes and, to a lesser extent, adoptive pmel-1 T-cells. Consequently, Delta-24-RGDOX drastically increased the density of the OVA-specific CTLs in both tumors, and the combination synergistically enhanced the effect. Consistently, the splenocytes from the combination group showed a significantly stronger response against other TAAs (OVA and TRP2) than gp100, resulted in higher activity against tumor cells. Therefore, our data demonstrate that, as an adjuvant therapy followed TAA-targeting T-cells in localized treatment, Delta-24-RGDOX activates TME and promotes antigen spread, leading to efficacious systemic antitumor immunity to overcome tumor relapse.

Cancer cell heterogeneity and immunosuppressive tumor microenvironment (TME) pose a challenge in treating solid tumors with adoptive cell therapies targeting limited tumor-associated antigens (TAAs), such as CAR T-cell therapy. We hypothesize that oncolytic adenovirus Delta-24-RGDOX activates the TME and promote antigen spread to potentiate the abscopal effect of adoptive TAA-targeting T-cells in localized intratumoral treatment. Herein, we used C57BL/6 mouse models with disseminated tumors derived from B16 melanoma cell lines to assess therapeutic effects and antitumor immunity. gp100-specific pmel-1 or OVA-specific OT-I T-cells were injected into the first subcutaneous (s.c.) tumor, followed by three injections of Delta-24-RGDOX. We found TAA-targeting T-cells injected into one s.c. tumor showed tumor tropism. Delta-24-RGDOX sustained the systemic tumor regression mediated by the T-cells, leading to improved survival rate. Further analysis revealed that, in mice with disseminated B16-OVA tumors, Delta-24-RGDOX increased CD8+ leukocyte density within treated and untreated tumors. Importantly, Delta-24-RGDOX significantly reduced the immunosuppression of endogenous OVA-specific cytotoxic T lymphocytes (CTLs) while increasing that of CD8+ leukocytes and, to a lesser extent, adoptive pmel-1 T-cells. Consequently, Delta-24-RGDOX drastically increased the density of the OVA-specific CTLs in both tumors, and the combination synergistically enhanced the effect. Consistently, the splenocytes from the combination group showed a significantly stronger response against other TAAs (OVA and TRP2) than gp100, resulted in higher activity against tumor cells. Therefore, our data demonstrate that, as an adjuvant therapy followed TAA-targeting T-cells in localized treatment, Delta-24-RGDOX activates TME and promotes antigen spread, leading to efficacious systemic antitumor immunity to overcome tumor relapse.

Oncolytic virotherapy shows great potential for treating brain tumors and other solid tumors that metastasize to the brain. A phase I clinical trial testing the oncolytic adenovirus Delta-24-RGD (DNX-2401) in patients with recurrent malignant gliomas demonstrated significant clinical benefits for a subset of patients, and another phase I trial testing Delta-24-RGD in patients with pediatric brain tumors also indicated similar clinical benefits. However, virus injection in tumors initiates both anti-virus and anti-tumor immune responses by activating respective clones of CD8+ T-cells, which compete for limited resources for clonal expansion. Expansion of T-cells against highly immunogenic viral antigens might limit the expansion of subdominant clones against tumor antigens. We hypothesized that inducing immune tolerance for the dominant viral antigens will allow the expansion of previously subdominant tumor-specific T-cells. In this work, we observed that nanoparticles encapsulating adenoviral antigens successfully induced immune tolerance for viral antigens. These nanoparticles were taken up by liver resident macrophages, which are involved in maintaining peripheral immune tolerance. RNA-sequencing revealed that T-cells recognizing the encapsulated antigens had unique transcriptome signatures with upregulation of genes specifically involved in peripheral immune tolerance, including immune coinhibitory molecules. Virus-specific immune tolerance using nanoparticles redirected the focus of the immune response towards antigens for melanoma as measured by interferon-gamma secretion (P < 0.0001). Reduction of virus-specific T-cells and simultaneous expansion of tumor-specific T-cell clones were confirmed with tetramer staining (P < 0.05). Importantly, virotherapy in combination with nanoparticle-induced immune tolerance towards viral antigens increased the percentage of long-term survivors compared to virus treatment alone (100% versus 50%). Our data provide the basis to develop future clinical trials that aim to maximize the potential of cancer virotherapy in solid tumors.

Oncolytic viruses are considered part of immunotherapy and have shown promise in preclinical experiments and clinical trials in a small fraction of patients. To improve the response rate, it is necessary to reshape the tumor microenvironment that shields the tumor from the immune system of the patient. We engineered Delta-24-RGDOX (DNX-2440), an oncolytic adenovirus to express the T-cell activator OX40L, which triggered, in murine glioblastoma models, a dramatic reshaping of the tumor microenvironment dominated by changes in immune processes in an RNA-seq analysis of ingenuity pathways. Paradoxically, network analyses also revealed that Delta-24-RGDOX induced robust activation of the cytokine-driven immunosuppressive IDO-Kynurenine-AhR circuitry, indicating a potential mechanism of resistance of gliomas to oncolytic virotherapy. To reverse this immunosuppression, we combined Delta-24-RGDOX with clinically relevant IDO inhibitors to treat glioma-bearing mice. The combined therapy decreased the activation of the IDO network without affecting viral activity. Flow cytometry assays revealed a decrease in the presence of the immunosuppressive cell populations, MDSCs and Tregs, which presence was higher in Delta-24-RGDOX-treated tumors. These data were further confirmed in gene set enrichment analyses. Functional co-culture studies showed that the combined therapy activated splenocytes against tumor antigens and that this activation was reversed by Kynurenine. Importantly, the combination treatment eradicated the tumors in a CD4-dependent manner and significantly prolonged the survival of glioma-bearing mice. Altogether, these studies indicate that the combination treatment promotes an adaptive immune response while decreasing the immunosuppression caused by the virus-induced IDO activation. Furthermore, our data identified the striking role of immunosuppressive pathways in the resistance of gliomas to oncolytic virotherapy. Specifically, the activity of the tumor microenvironment IDO circuitry was responsible, at least partially, for the remodeling of local immunosuppression after tumor infection. Combining molecular and immune-related therapies may improve outcomes in human gliomas treated with virotherapy.

The median survival for GBM patients is 15 months with a five-year survival rate of less than 10%. Current conventional and experimental treatments, including immune checkpoint blockade, have not improved outcomes in the vast majority of glioma patients. Oncolytic viruses (OVs) are designed to specifically infect and lyse cancer cells to elicit an anti-tumor immune response. In a clinical trial developed by the MDACC team, 20% of GBM patients treated with the oncolytic adenovirus Delta-24-RGD showed complete response and survival longer than three years (NCT00805376). Although these studies have shown that robust immune responses can be invoked using oncolytic virotherapy, the molecular mechanisms underlying OV-mediated anti-tumor immunity remain elusive. Exposure to viruses by innate immune sensors induces protective antiviral immunity. Using bulk RNA sequencing of cells infected with adenovirus we identified the main upstream regulators of the innate immune response including IFN gamma, Interferon responsive genes 3 and 7, and STAT3. However, RNAseq unexpectedly uncovered NONO (Non-POU Domain Containing Octamer Binding) as the most relevant upstream regulator. In agreement with the RNAseq data, Protein analyses demonstrated the upregulation of NONO after adenovirus infection. In addition, immunofluorescence and confocal microscopy examination of the infected cells showed the overexpression and translocation to the nucleus of NONO upon virus infection. We are the first laboratory that has identified NONO as a sensor of adenovirus infection, which may have great significance for the future development of oncolytic viruses. Importantly, we discovered the network of proteins that are upregulated after NONO activation and, finally, we have established that changes in the expression level and subcellular localization of NONO are coincident in time during the infection of cultured cells. Future work is focused on examining the immunomodulatory functions of NONO in adenoviral infected tumors and subsequent generation of NONO-resistant oncolytic adenovirus.

Tumor-targeting biological therapies, including adoptive T cell therapy (ACT) and oncolytic viruses, are being tested clinically to address the challenges in treating advanced solid tumors with heterogeneity and immune suppressive microenvironment (TME). Since complementary properties of these two approaches have been observed, we hypothesize that localized intratumoral treatment with combination of ACT and Delta-24-RGDOX (third generation oncolytic adenovirus developed by our group) induces not only potent therapeutic effect in treated tumor but also abscopal benefit to eliminate distant disseminated tumors. Using a B16-C57BL/6 melanoma model with disseminated tumors in C57BL/6 mice, we found gp100-specific CD8+ T cells injected into the first subcutaneous (s.c.) tumor showed tropism for distant s.c. and intracranial tumors, tumor draining lymph nodes, compared to spleen, blood and normal brain hemisphere. In a B16-OVA-C57BL/6 s.c./s.c. tumor model, Delta-24-RGDOX injections following gp100-specific T cells in the first tumor increased total cytotoxic T cell presence within both tumors, including gp100- and OVA- specific T cells. Further analysis revealed that Delta-24-RGDOX reduced the exhaustion of OVA-specific T cells while that of gp100-specific T cells kept at a low level. Moreover, Delta-24-RGDOX augmented the activity of the splenocytes against the tumor cells and other tumor-associated antigens (OVA, TRP2) than gp100. Consequentially, the combination of OVA-specific T cells and Delta-24-RGDOX was more potent to inhibit the injected and untreated distant tumor growth and caused improved survival rate than either of the agent alone (p < 0.05). Importantly, we observed relapse of the regressed tumors in the T cell treatment group, but not in the combination group. The survivors developed immune memory against rechallenging with B16-OVA cells. Collectively, our data demonstrate that localized treatment with intratumoral ACT followed by Delta-24-RGDOX induces global immune activation and antigen spread to expand antitumor T cell repertoire, leading to efficacious systemic anti-tumor immunity and sustainable tumor regression.

BACKGROUND To evaluate the effect of the hepatitis B virus (HBV) on the coagulation parameters in patients with hepatitis B virus-related decompensated cirrhosis (HBV-DeCi). METHODS A retrospective analysis was conducted on the medical records of 112 patients with HBV-DeCi. Baseline clinical and laboratory characteristics were retrieved. Subjects were subdivided into 3 groups. Group I: 22 cases of hepatitis B were HBsAg, HBeAg, and HbcAb positive; Group II: 67 patients were HBsAg, HBeAb, and HbcAb positive; Group III: 23 patients were HBsAb, HBeAb and HbcAb positive. The coagulation indicators, such as prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (FIB), and international normalized ratio (INR, a method to standardize reporting of the PT, using the formula, INR = (PTpatient/PTcontrol)ISI) of each groups were analyzed, The correlation between the characteristics of coagulation function and the type of hepatitis infection were studied. RESULTS The FIB values of Group I and II were lower than those of Group III, and Group I had significantly longer TT compared to Group III. CONCLUSIONS In patients with HBV-DeCi, hepatitis B virus has an effect on coagulation parameters; therefore, antiviral treatment must be carried out as soon as possible.

Background: This study aims to survey the demographic status of aesthetic plastic surgeons in an economically developed region and to investigate their continuing professional education needs in contents, training methods, and barriers in private and public hospitals. Methods: A cross-sectional survey with a self-administered questionnaire was conducted from January to December, 2020 in Guangdong, China. An in-depth key informant interviews were conducted to gain insights on the current demographic status. Demographic data and training needs assessment were collected and compared with the chi-square test, Fisher’s test, Mann-Whitney U-test using R software. Results: The disparity of surgeons’ demographic data between private and public hospitals was small. Over 60% of practicing aesthetic plastic surgeons transferred from other specialties, and one third of them had less than three years of working experience. Half surgeons attended training less than three times with an affordable expense of 1000-5000RMB. Almost 80% of them had strong willingness to attend a continuing professional education program. They prefer to attending further study in a tertiary hospital, short topic-focused training course with operation demonstrations. Their favorable training contents were rhinoplasty, eye surgery, and new technologies. Questionnaire survey revealed the likelihood that the surgeons had a graduate degree in public hospitals outweighed those in private hospitals (PConclusion: It would be favorable to take the demographic status of aesthetic plastic surgeons and organizational needs into consideration in designing a continuing professional education program in plastic surgery.

Recent clinical observations that some coronavirus infections induced complete remissions in lymphoma patients emphasized again the potential of cancer virotherapy. Infection of cancer cells with oncolytic viruses reshapes the tumor microenvironment by activating anti-viral and anti-tumor immunity. A phase 1 clinical trial using oncolytic adenovirus Delta-24-RGD (DNX-2401) to treat recurrent malignant gliomas demonstrated activation of CD8+ T-cells and significant clinical benefits for a subset of patients. However, both anti-virus and anti-tumor immune responses are contingent on the activation of respective clones of CD8+ T-cells, which compete for clonal expansion. Thus, overexpansion of T-cells against viral antigens reduces the frequency of subdominant clones against tumor antigens. We hypothesized that inducing immune tolerance for viral antigens will decrease anti-viral immunity and in turn derepress anti-tumor immunity, resulting in enhanced efficacy of cancer virotherapy. In this work, we used nanoparticles encapsulating adenoviral antigens E1A, E1B and hexon that distributed to liver resident macrophages (P Citation Format: Dong Ho Shin, Hong Jiang, Debora Kim, Teresa T. Nguyen, Sagar Sohoni, Xuejun Fan, Yanhua Yi, Arie C. Van Wieren, Gheath Al-Atrash, Chantale Bernatchez, Ramon Alemany, Marta Alonso, Frederick Lang, Candelaria Gomez-Manzano, Bulent Ozpolat, Juan Fueyo. Redirection of anti-virus immunity towards anti-tumor immunity using nanoparticle-induced immune tolerance for oncolytic adenovirus therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3559.

CAR T therapy greatly improves the survival of patients with hematological malignancies. But its effect in solid tumors is suboptimal. Intratumoral injections of oncolytic viruses, including Delta-24-RGDOX (DNX-2440) from our group, directly lyse cancer cells and activate tumor microenvironment, leading to adaptive antitumor immunity. To take advantage of instant antitumor activity of T cells and virus-mediated in situ autovaccination against heterogeneous cancer cells, we hypothesize that intratumorally injections of tumor-targeting T cells followed by Delta-24-RGDOX overcome antigen escape of CAR T therapy, leading to more sustainable systemic anti-cancer immunity. Thus, we used subcutaneous (s.c.)/s.c. B16 melanoma mouse models to assess the effect of localized treatment in disseminated tumors. OVA (or gp100)-specific CD8+ T cells or CD19 CAR T cells were injected into the first tumor, followed by three injections of Delta-24-RGDOX into the same tumor. T cells and tumor cells from the mice were profiled for surface markers with flow cytometry and immune staining. Activity of splenocytes against tumor cells and specific tumor-associated-antigens (TAAs) was measured with ELISA. Tumor growth was monitored through measuring tumor size. The animal survival curves were plotted according to the Kaplan-Meier method. We found TAA-specific T cells injected into the first s.c. tumor showed tropism for disseminated s.c. and intracranial tumors, tumor draining lymph nodes, but not for spleen, peripheral blood and normal brain. Unlike untreated and Delta-24-RGDOX-treated tumor, T cell-treated tumor showed decreased expression of the target TAA which was depleted in the recurrent tumor. Moreover, Delta-24-RGDOX increased total T cell presence within the tumors, and the activity of the splenocytes against the tumor cells and other antigens than the one targeted by injected T cells. Consequentially, the combination of OVA-specific T cells and Delta-24-RGDOX was more potent to inhibit the injected and untreated disseminated tumor growth and caused improved survival rate than either of the agent alone (p < 0.05). Importantly, we observed relapse of the regressed tumors in the group treated by T cells alone, but not in the combination group. The survivors from the combination therapy were protected from rechallenging with B16-OVA cells but not lung carcinoma cells, suggesting the development of immune memory. In summary, our study indicates the virus induces antigen spread, resulting in expansion of antitumor T cell repertoire to prevent cancer relapse in adoptive T cell therapy (ACT). Our data demonstrate that Delta-24-RGDOX collaborates with ACT to induce more potent systemic immunity against the tumors, leading to sustainable tumor regression. Citation Format: Hong Jiang, Dong Ho Shin, Yanhua Yi, Xuejun Fan, Joy Gumin, Marta M. Alonso, Frederick F. Lang, Candelaria Gomez-Manzano, Juan Fueyo. Combining oncolytic adenovirus Delta-24-RGDOX with adoptive T cell therapy in localized treatment induces sustainable regression of disseminated solid tumors through antigen spreading [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3569.

Oncolytic viruses are a promising experimental treatment for solid tumors. Recently, several phase 1 clinical trials have reported encouraging therapeutic effects of oncolytic viruses in adult and pediatric patients with malignant gliomas. To further improve the therapeutic outcome of viroimmunotherapy, we have developed Delta-24-RGDOX (DNX-2440), a replication competent adenovirus encompassing the T-cell activator OX40L in the genetic backbone of Delta-24-RGD. We have previously reported the effect of Delta-24-RGDOX in murine brain tumors supporting the translation of this new agent to treat patients with recurrent malignant gliomas (NCT03714334). In the work presented here, we have tested the therapeutic effect of Delta-24-RGDOX in murine syngeneic models of breast (4T1), gastric (M12) and lung cancer (LLC and CMT 167). We found that Delta-24-RGDOX infected all cancer cell lines efficiently. In addition, infection of cells was followed by the expression of the ectopic ligand in vitro and in vivo. Because the elicitation of an anti-tumor immunity is part of the mechanisms underlying the therapeutic effect of oncolytic viruses, we examined whether infection of tumors led to the reshaping of the tumor microenvironment. We observed that Delta-24-RGDOX infection was followed by increased frequencies of tumor infiltrating lymphocytes, particularly CD8+ T cells and NK cells. In addition, the CD8+/CD4+ ratio was increased in Delta-24-RGDOX-treated tumor versus PBS-treated tumors. Interestingly, abscopal modifications were observed in breast cancer brain metastases with increased frequency of CD8+ T cells at the distal, untreated site. Delta-24-RGDOX treatment induced an anti-cancer effect in orthotopically implanted breast cancer and subcutaneously implanted lung and gastric tumors, as well as in metastatic niches. In summary, our data showed that treatment of solid tumors with Delta-24-RGDOX induces robust remodeling of the tumor microenvironment and produces anti-tumor effects leading to decrease in tumor volume, along with a delay in the development and in the reduction of the number of metastases. These data suggest that Delta-24-RGDOX should be tested in the clinical setting in patients with metastatic breast, gastric and lung cancers. Citation Format: Arie C. Van Wieren, Sagar Sahoni, Teresa Nguyen, Ashley Ossimetha, Yisel Rivera-Molina, Hong Jiang, Dong Ho Shin, Debora Kim, Xuejun Fan, Yanhua Yi, Natalie M. Melendez-Vazquez, Filipa Godoy-Vitorino, Joy Gumin, Frederick F. Lang, Marta M. Alonso, Juan Fueyo, Candelaria Gomez-Manzano. Viroimmunotherapy for solid tumors results in local and abscopal anti-cancer effects and the remodeling of tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3565.

ObjectiveCoronary heart disease (CHD) is currently one of the major causes of death with high morbidity. Due to the increasing heterogeneity and complexity in the CHDs progression, biomarkers for specific diagnosis and monitoring of disease progression need to be developed. The study was aimed to investigate the roles of serum exosomal miR-23a and miR-23b in diagnosis of CHDs.Methods16 healthy individuals and 56 patients with CHDs were enrolled in this study, including the CHDs of stable angina, unstable angina, non-ST elevation myocardial infarction (NSTEMI), ST elevation myocardial infarction (STEMI) and acute myocardial infarction (AMI). Serum exosomal miR-23a and miR-23b were quantified by Q-PCR. The associations of miR-23a/b with multiple clinical parameters were analyzed.ResultsSerum exosomal miR-23a was downregulated in the 56 CHD patients. In the specific prediction of stable angina and AMI, miR-23a achieved the area under the receiver operating characteristic (AUC-ROC) of 0.809 and 0.783, respectively. The levels of serum creatinine (CREA) and miR-23a were associated, which was consistent with risk of kidney injury in CHDs patients. Exosomal miR-23b levels showed no difference among CHD groups.ConclusionsmiR-23a may serve as a non-invasive marker in predicting stable angina and AMI. The level of miR-23a was implicated in CREA-dependent CHD-renal damage.

Background: Female pattern hair loss (FPHL) is one of the most common types of hair loss with complex genetic predisposition. A frontal pattern hair loss with ponytail hairstyle is pervasively seen among young Chinese women. The purpose of this study is to investigate the association between the severity of FPHL and behavioral factors which include dietary, and sleep habits, and to test the hypothesis on whether ponytail hairstyle is an independent factor that increases the risks of being more severe on the FPHL scale. Methods: A cross-sectional survey was performed with a structured questionnaire in this study. The severity of FPHL was graded according to basic and specific (BASP) classifications. Ordinal logistic regression analysis was performed to investigate the factors related to the severity of FPHL. Results: 1,825 participants with different severities of FPHL completed the questionnaire. Ordinal logistic regression analysis revealed that the age group between thirty and forty years (OR:2.03, 95% CI: 1.56,2. 65), insufficient time with poor quality (OR:1.30, 95% CI: 1.05,1.62), presence of alcohol consumption (OR:2.15, 95% CI: 1.14,4.42), ponytail hairstyles (OR:2.03, 95% CI: 1.40,2.96), and oily scalps (OR:2.00, 95% CI: 1.65,2.43) were risk factors which increased the odds of being in the more severe type of FPHL, compared to the age group that ranged from eighteen to thirty years, sufficient sleep with good quality, without alcohol consumption, ponytail hairstyles, and oily scalps. Conclusion: Avoiding alcohol consumption and ponytail hairstyles, in combination with proper control of scalp oil, improve sleep quality with sufficient time may help prevent FPHL from deteriorating to the more severe type.

Neurite plasticity is a critical aspect of brain functional recovery after stroke. Emerging data suggest that Ras-related C3 botulinum toxin substrate 1 (Rac1) plays a central role in axonal regeneration in the injured brain, specifically by stimulating neuronal intrinsic growth and counteracting the growth inhibitory signaling that leads to growth cone collapse. Therefore, we investigated the functional role of Rac1 in axonal regeneration after stroke. Delayed treatment with a specific Rac1 inhibitor, NSC 23766, worsened functional recovery, which was assessed by the pellet reaching test from day 14 to day 28 after stroke. It additionally reduced axonal density in the peri-infarct zone, assessed 28 days after stroke, with no effect on brain cavity size or on the number of newly formed cells. Accordingly, Rac1 overexpression using lentivirus promoted axonal regeneration and functional recovery after stroke from day 14 to day 28. Rac1 inhibition led to inactivation of pro-regenerative molecules, including mitogen-activated protein kinase kinase (p-MEK)1/2, LIM domain kinase (LIMK)1, and extracellular signal-regulated kinase (p-ERK)1/2 at 14 days after stroke. Inhibition of Rac1 reduced axonal length and number in cultured primary mouse cortical neurons using microfluidic chambers after oxygen-glucose deprivation (OGD) without affecting cell viability. In contrast, inhibition of Rac1 increased levels of glial fibrillary acidic protein, an extrinsic inhibitory signal for axonal growth, after stroke in vivo and in primary astrocytes after OGD. In conclusion, Rac1 signaling enhances axonal regeneration and improve post-stroke functional recovery in experimental models of stroke.

BackgroundUnlike its remarkable success in treating hematological malignancies, adoptive T cell therapy (ACT), such as CAR T therapy, targeting limited tumor-associated antigens (TAAs) is far less effective in patients with heterogeneous solid tumors. Oncolytic viruses, including oncolytic adenovirus Delta-24-RGDOX from our group [1, 2], are emerging as promising immunotherapy drugs. To take advantage of instant antitumor activity of T cells and oncolytic adenovirus mediated in situ autovaccination against heterogeneous cancer cells, we hypothesize that intratumorally injected Delta-24-RGDOX complements with ACT to overcome antigen escape, leading to more sustainable anti-cancer effect.MethodsWe used B16-OVA-C57BL/6 subcutaneous (s.c.)/s.c. melanoma model [2] to assess the systemic therapeutic effect in disseminated tumors. OVA (or gp100)-specific CD8+ T cells were injected into the first tumor, followed by three injections of Delta-24-RGDOX into the same tumor. T cells from the mice were profiled for surface markers with flow cytometry. Activity of splenocytes against tumor cells and specific TAAs was measured with ELISA. Tumor growth was monitored through measuring tumor size three times a week. The animal survival curves were plotted according to the Kaplan–Meier method.ResultsTAA-specific T cells injected into the first s.c. tumor showed tropism for disseminated s.c. and intracranial tumors, tumor draining lymph nodes, compared to normal tissue, spleen and peripheral blood. Delta-24-RGDOX increased total T cell presence within the tumors, and the activity of the T cells against the tumor cells and targeting other antigens than the one targeted by the transferred T cells. Consequentially, the combination of OVA-specific T cells and Delta-24-RGDOX was more potent to inhibit the injected first tumor and untreated disseminated second tumor growth and resulted in improved survival rate than either of the agent alone (p < 0.05). Interestingly, we observed relapse of the regressed tumors in the group treated by T cells alone. But the relapse was not observed in most of the mice in the combination group. The survivors from the combination therapy were protected from rechallenging with B16-OVA cells but not lung carcinoma cells, suggesting the development of immune memory.ConclusionsDelta-24-RGDOX collaborates with ACT to induce more potent systemic immunity against the tumors, leading to sustainable tumor regression and improved survival rate. Our study indicates the virus induces antigen spread, resulting in expansion of T cell repertoire to prevent cancer relapse in ACT.ReferencesJiang, H., et al., Localized Treatment with Oncolytic Adenovirus Delta-24-RGDOX Induces Systemic Immunity against Disseminated Subcutaneous and Intracranial Melanomas. Clin Cancer Res, 2019. 25(22): p. 6801–6814. https://cancerres.aacrjournals.org/content/77/14/3894.long.Jiang, H., et al., Oncolytic Adenovirus and Tumor-Targeting Immune Modulatory Therapy Improve Autologous Cancer Vaccination. Cancer Res, 2017. 77(14): p. 3894–3907.https://clincancerres.aacrjournals.org/content/25/22/6801.full-text.pdf

Diffuse Intrinsic Pontine Glioma (DIPG) is the leading cause of pediatric death by brain tumors with a median survival of only 9 months, in spite of the current standard of therapy which includes radiation. The purpose of our study is to explore the use of oncolytic adenoviruses as a new therapeutic modality. Our group is leading a phase I clinical trial in naive DIPGs treated with the oncolytic adenovirus Delta-24-RGD, also named DNX-2401 (NCT03178032), which results are still pending. Of interest, clinical trials using Delta-24-RGD in adult gliomas resulted in encouraging results with long-term survivors and the presence of a strong anti-tumor immunity. In this study, we aim to test the effect of armed Delta-24-RGD with immune costimulatory ligands such as GITRL (Delta-24-GREAT) and OX-40L (Delta-24-RGDOX or DNX-2440) for the treatment of DIPGs. We used a panel of murine DIPG cell lines (NP53 and XFMA) to test the effect of these armed-oncolytic adenoviruses. Infectivity assay using an Ad-RGD-GFP vector and flow cytometry showed the expression of the ectopic GFP in more than 80% of the NP53 cells (50 MOIs) and XFMA cells (30 MOIs). Consistent results were obtained using flow cytometry and western blot to detect the expression of the ectopic ligands GITRL and OX-40L in these cultures. These cells were semipermissive for viral replication as assessed by the detection of the viral structural protein hexon in western blot analysis. Preliminary in vivo data using immunocompetent mice models where DIPG cells have been implanted into the pons showed a dose-dependent effect and the generation of immune memory. Our results suggest that oncolytic adenoviruses expressing positive immunocheckpoints might prove be a new effective modality for treating DIPG and hence, increase survival in children suffering this dismal disease. Citation Format: Sumit Gupta, Virginia Laspidea, Juan Fueyo, Oren Becher, Teresa Nguyen, Dong Ho Shin, Hong Jiang, Sagar Sohoni, Xuejun Fan, Yanhua Yi, Joy Gumin, Frederick Lang, Candelaria Gomez-Manzano, Marta M. Alonso. Armed oncolytic virus for treatment of pediatric diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3053.

Oncolytic virotherapy and adoptive T cell therapy (ACT) are promising strategies for cancer treatment, but both are underdeveloped into routine clinical interventions for solid tumors. We reported previously that localized treatment with oncolytic adenovirus Delta-24-RGDOX (DNX-2440), which expresses immune co-stimulator OX40 ligand, stimulated tumor-specific T cells proliferation and migration to untreated disseminated tumors. We hypothesize that intratumorally injected Delta-24-RGDOX complements with ACT to overcome antigen escape, leading to more sustainable anti-cancer effect. To test the hypothesis, we used B16-OVA-C57BL/6 subcutaneous (s.c.)/s.c. melanoma model to assess the effect of the combination therapy. Briefly, 10 days after the first tumor implantation, we injected OVA-specific CD8+ T cells from OT-I mice into the first tumor, followed by three injections of Delta-24-RGDOX into the same tumor. We found the combination was more potent to inhibit the injected 1st tumor and untreated disseminated 2nd tumor growth and resulted in improved survival rate than either of the agent alone (p less than 0.05). Interestingly, we observed relapse of the regressed tumors in the group treated by T cells alone, which recapitulated the effect of CAR T therapy in cancer patients. But the relapse was not observed in most of the mice from the combination group, indicating the combination induces more sustainable tumor regression than T cells alone. The survivors from the combination therapy were protected from rechallenging with B16-OVA cells but not lung carcinoma cells, suggesting the development of immune memory against the same tumor cells. Furthermore, profiling the tumor-infiltrating lymphocytes with flow cytometry revealed that the combination promotes the T cell presence within the tumor and increase the population of the T cells targeting other antigens in addition to the original ACT targeted antigen. Collectively, the results demonstrate that Delta-24-RGDOX collaborates with ACT to induce more potent systemic immunity against the tumors, leading to more sustainable tumor regression and improved survival rate. Our study suggests the virus induces antigen spread, resulting in T cell repertoire to target more tumor-associated antigens to overcome cancer relapse due to cancer heterogeneity and limited targets in ACT and CAR T therapy. Citation Format: Hong Jiang, Teresa T. Nguyen, Dong Ho Shin, Sagar S. Sohoni, Sumit Gupta, Yanhua Yi, Xuejun Fan, Candelaria Gomez-Manzano, Juan Fueyo. Oncolytic adenovirus Delta-24-RGDOX collaborates with adoptive cell therapy to sustain anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1579.

Metastatic breast cancer is the second leading cause of cancer related death among women. Although, conventional therapies such as surgery, adjuvant and neoadjuvant therapies have shown promising outcomes in preclinical and clinical settings, long-term survival rates remain unacceptably low for patients with recurrent disease or disseminated metastases. Breast primary and metastasic niches are characterized by an immunosuppressive microenvironment. Hence, novel therapeutic regimens that modulate this challenging phenotype are desperately needed. Our group has developed and tested in preclinical models and in clinical trials for patients with recurrent glioblastoma the anti-tumor effect of oncolytic adenovirus Delta-24-RGD, also named DNX-2401, resulting with a 20% of long-term survivors, and the awaking of a antitumoral immunity after the administration of the pathogen. This paradigm shift suggests that viroimmunotherapy can be used for the treatment of metastasic cancer. Thus, here we hypothesize that viroimmunotherapy is capable of exerting abscopal effects to eradicate primary breast tumors and their metastatic niches. To test the hypothesis, 4T1 tumor bearing mice were subjected to the local administration of Delta-24-RGD and its armed derivatives, expressing expressing immune co-stimulatory ligands OX40L (Delta-24-RGDOX or DNX-2440) and GITRL (Delta-24-GREAT). We found that, the tumors treated with oncolytic adenoviruses exhibited increase of both CD8+ T cell infiltration and CD8+/CD4+ ratio. Additionally, T-cell activity measured by IFN-γ secretion of splenocytes when in co-culture with breast cancer cells was found to be increased in mice that underwent viroimmunotherapy (P Citation Format: Sagar Shashikant Sohoni, Teresa Nguyen, Dong Ho Shin, Xuejun Fan, Jiang Hong, Sumit Gupta, Ashley Ossimetha, Yanhua Yi, Chandra Bartholomeusz, Marta Alonso, Juan Fueyo, Candelaria Gomez-Manzano. Targeting the immunosuppressive microenvironment of metastatic breast cancer with viroimmunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1786.

Oncolytic viruses are a new form of cancer immunotherapy that harnesses the combined abilities of the virus to directly lyse cancer cells and to induce immunogenic environments to activate anti-tumor immunity. Our oncolytic adenovirus Delta-24-RGD has demonstrated significant clinical benefits for a subset of patients with recurrent malignant gliomas in a recent phase 1 clinical trial. However, the backbone of Delta-24-RGD is derived from adenovirus serotype 5 to which 40 percent of the human population has prior exposure and harbor neutralizing antibodies that can block virus infection and replication. We hypothesized that replacing the major targets of antibodies in the oncolytic virus will protect it from neutralizing antibodies, resulting in enhanced efficacy of the cancer virotherapy. Because hexon hypervariable regions are the dominant targets of antibodies, we replaced these regions with those from a lesser prevalent serotype 43 to create a hybrid virus called Delta-24-RGD-H43m. Transmission electron microscopy imaging of the viruses revealed the assembly of viable virions after infection with either Delta-24-RGD or Delta-24-RGD-H43m in human and mouse cancer cell lines, signifying successful virus replication, that was corroborated by quantification of viral replication using hexon-staining dilution method. Accordingly, the inherent anti-tumor effect of the virus was not perturbed after the genetic modification, as Delta-24-RGD and Delta-24-RGD-H43m showed comparable efficacies both in vitro (P= 0.568) and in vivo using mice without prior immunity to adenovirus (P= 0.228). Importantly, the oncolytic ability of Delta-24-RGD-H43m was not compromised by neutralizing antibodies generated against adenovirus serotype 5 that efficiently neutralized Delta-24-RGD (P< 0.0001) as assessed by measuring cell viability after virus infection. In immunized mice with prior adenovirus exposure, Delta-24-RGD-H43m treatment yielded higher occurrences of complete tumor remission compared to Delta-24-RGD (89% versus 67%). Despite our growing understanding of viruses, the immune system, and cancer, the specific interaction between oncolytic viruses and the host immune system is still very limited. Strategies to protect the therapeutic viruses from virus-specific immune responses should prolong their persistence in tumors and result in better therapeutic outcomes. Further research in this field will aid the development of enhanced cancer virotherapies. Citation Format: Dong Ho Shin, Teresa T. Nguyen, Hong Jiang, Xuejun Fan, Yanhua Yi, Sagar S. Sohoni, Sumit Gupta, Ashley Ossimetha, Candelaria Gomez-Manzano, Juan Fueyo. Evasion of neutralizing antibodies and enhanced anti-tumor effects of hexon-modified Delta-24-RGD oncolytic adenovirus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1912.

Metformin, an inexpensive and well-tolerated oral agent commonly used in the first-line treatment of type 2 diabetes, has become the focus of intense research as a candidate anticancer agent. Here, we discuss the potential of metformin in cancer therapeutics, particularly its functions in multiple signaling pathways, including AMP-activated protein kinase, mammalian target of rapamycin, insulin-like growth factor, c-Jun N-terminal kinase/mitogen-activated protein kinase (p38 MAPK), human epidermal growth factor receptor-2, and nuclear factor kappaB pathways. In addition, cutting-edge targeting of cancer stem cells by metformin is summarized.

Background The use of medial sural vessels (MSVs) as recipient vessels to repair wounds in the lower limbs has been previously reported. However, there is controversy regarding the methods used to expose MSVs and the repairable area of the wound. Methods Eight fresh cadavers were used to determine the shape, outer diameter, and length of the MSVs arising from the popliteal vessels. Lower extremity wounds were repaired in 7 patients using free latissimus dorsi flaps with the MSVs as recipient vessels. The flap area ranged from 18 × 11 cm to 24 × 17 cm, and the postoperative follow-up time ranged from 7 to 60 months. Results The outer diameter of the medial sural artery from the starting point of the popliteal artery was 2.48 ± 0.24 mm. After descending 29.98 ± 4.62 mm, the vessels entered the muscle; the outer diameter of the blood vessel upon entry was 2.24 ± 0.17 mm. Notably, the outer diameter of the vessel was 1.88 ± 0.09 mm only 2 cm after entering the muscle. Seven cases were treated clinically. In 6 cases, we used an approach between the medial head of the gastrocnemius muscle and the tibia to expose and anastomose the MSVs. In 1 case, the vascular diameter was mismatched. After surgery, the flap survived completely in 6 cases. The remaining case experienced epidermal necrosis in the distal part of the flap, which resolved spontaneously. There were no obvious complications. Conclusions An approach between the medial head of the gastrocnemius muscle and the tibia can easily expose and anastomose the MSVs in a lateral position. Latissimus dorsi flaps were useful in repairing limb wounds as far as the talocrural region with the MSVs as the recipient vessels.

Purpose: Intratumoral injection of oncolytic adenovirus Delta-24-RGDOX induces efficacious antiglioma immunity in syngeneic glioma mouse models. We hypothesized that localized treatment with the virus is effective against disseminated melanomas. Experimental Design: We tested the therapeutic effect of injecting Delta-24-RGDOX into primary subcutaneous (s.c.) B16-Red-FLuc tumors in s.c./s.c. and s.c./intracranial (i.c.) melanoma models in C57BL/6 mice. Tumor growth and in vivo luciferase-expressing ovalbumin-specific (OT-I/Luc) T cells were monitored with bioluminescence imaging. Cells were profiled for surface markers with flow cytometry. Results: In both s.c./s.c. and s.c./i.c. models, 3 injections of Delta-24-RGDOX significantly inhibited the growth of both the virus-injected s.c. tumor and untreated distant s.c. and i.c. tumors, thereby prolonging survival. The surviving mice were protected from rechallenging with the same tumor cells. The virus treatment increased the presence of T cells and the frequency of effector T cells in the virus-injected tumor and mediated the same changes in T cells from peripheral blood, spleen, and brain hemispheres with untreated tumor. Moreover, Delta-24-RGDOX decreased the numbers of exhausted T cells and regulatory T cells in the virus-injected and untreated tumors. Consequently, the virus promoted the in situ expansion of tumor-specific T cells and their migration to tumors expressing the target antigen. Conclusions: Localized intratumoral injection of Delta-24-RGDOX induces an in situ antovaccination of the treated melanoma, the effect of which changes the immune landscape of the treated mice, resulting in systemic immunity against disseminated s.c. and i.c. tumors.

Hydroxytyrosol (HT), a primary phenolic antioxidant in olive oil, can afford protection from oxidative stress (OS) in different cells, including skin cells. In particular, it regulates several inflammation-associated processes as well as in improving the antioxidant defense system. However, there is no information about HT used in the treatment of hair loss. This work aimed at exploring the potential protective actions of HT against OS in rat dermal papilla cells. After treatment, the related expression of protein and messenger RNA were detected using morphological and molecular analyses. The results showed that HT significantly reduced intracellular reactive oxygen species level, apoptotic markers and inflammation induced by OS and enhanced cell survival by regulating autophagy. Furthermore, HT enhanced the secretion of hair growth factors in the anti-inflammation process. These results suggest that HT has a significant protective ability against OS and encourage the use of this biological ingredient as a possible tool to prevent alopecia.

Platelet-rich plasma (PRP) has received growing attention as a valuable therapeutic tool in androgenetic alopecia (AGA). However, knowledge regarding specific effectiveness and satisfaction of PRP for different grades of AGA in male pattern hair loss (MPHL) and female pattern hair loss (FPHL) is missing. This study aims to ascertain and compare the efficacy and safety of PRP treatment for different grades of AGA in males and females over 6 months. In this study, 51 MPHL patients with Norwood-Hamilton stage II-V and 42 FPHL patients with Ludwig stage I to III were enrolled for 6 monthly sessions of PRP injections. A longitudinal analysis was used to compare the hair density, thickness, and hair pull test over 6 months for MPHL and FPHL through generalized estimating equation (GEE) models. Phototrichograms of scalp inflammation and oil secretion, global photographs and overall patient satisfaction were also assessed. Consequently, improvement of hair density, hair thickness, hair pull test, the level of scalp inflammation and oil secretion were observed with statistical significance in all stages for both MPHL and FPHL at 6 months. Noteworthy, lower level of alopecia (Grade II, III in MPHL and Grade I in FPHL) had better response to PRP, and also had a better tendency of increment of hair growth than that of high-grade patients with prolonged treatment. PRP injections, as an efficacious and reliable therapy, can be recommended for Grade II and Grade III in MPHL and Grade I in FPHL.

Background We present a case of spurious low WBC count in a liver transplant patient. The patient is a 56-year-old man with liver cancer. Methods His routine blood test revealed a decrease in WBC count: 0.03 x 109/L compared to 19.30 x 109/L before. The WBCs in the blood smear appeared higher than that reported by the XN without any aggregation. We diluted the blood sample to 1:7 with the DCL of the XN. Results The diluted result matches the blood smear. Conclusions Dilution mode may be a good choice when there is WNR and WDF discordance, and a smear must be reviewed.