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Depression is more frequent in women, affecting the early stages of child development. This study aimed to determine the association between maternal depression and self-regulation of emotions and behaviors in Peruvian children under five years. A cross-sectional analytical study of data collected by the 2019 Demographic and Family Health Survey (ENDES) was conducted. The outcome variable was emotion and behavior regulation in children aged 24 to 59 months, and exposure was the presence of depression in women aged 15 to 49 years during the 14 days prior to the survey using the Patient Health Questionnaire (PHQ-9). A generalized linear model of the binomial family was used for reporting crude prevalence ratios and adjusted. The overall prevalence of children who did not self-regulate their emotions and behaviors was 68.8%, while 3.8% of the mothers had moderate depressive symptoms and 2.2% severe symptoms. Regarding the association of interest, moderate and severe depressive symptoms of mothers decreased the probability of children regulating emotions and behaviors in the first model, whereas in the second model, an association was only found with severe depressive symptoms. In conclusion, children of mothers with moderate and severe depressive symptoms had a lower probability of self-regulating their emotions and behaviors. Therefore, it is necessary to develop maternal education, nutritional and social support programs and mental health strategies from the first level of care aimed at reducing social, economic and child factors to reduce the risk of depression in mothers and low early childhood development, which could reduce the risk of developing mental health disorders in adolescence and adulthood., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Background: Magnetic resonance imaging (MRI) is a recognized method to assess the morphological characteristics of subcutaneous adipose tissue (SAT). This study aimed to assess objectively different characteristics of SAT of women with different ages and body mass index (BMI) using MRI., Materials and Methods: Retrospective, observational study. Pelvic-MRI exams of 133 women aged between 15 and 80 years, with BMI 16-30 kg/m 2 were assessed., Results: There was a moderate positive correlation between the total fat thickness and the BMI (r = 0.64; p < 0.0001) considering the entire sample. Within the different age groups, strong positive correlation was observed for age groups 15 to 30 years (r = 0.76; p < 0.0001) and 46 to 60 years (r = 0.75; p < 0.0001), and moderate positive correlation in the others (r = 0.61; p < 0.0001 [31-45 years] and r = 0.53; p < 0.0001 [>61 years-old]). There was no correlation between age and fat layer thickness. Correlation between deep fat layer thickness and BMI showed similar pattern to that of the total fat layer. Within the different BMI groups, SAT thickness tends to decrease with aging in patients with low BMI., Conclusion: Inside each age group, the average thickness of the total fat layer increases according to BMI. The results suggest that BMI influences the thickness of SAT throughout life and how SAT changes with age in patients with different BMI. In patients with moderate BMI, the thickness of SAT remains stable., (© 2022 Wiley Periodicals LLC.)

Background: Invadopodia, actin-rich structures that release metallo-proteases at the interface with extra-cellular matrix, in a punctate manner are thought to be important drivers of tumour invasion. Invadopodia formation has been observed in-vitro and in-vivo in numerous metastatic cell lines derived from multiple tumour types. However, prostate cancer cell lines have not been routinely reported to generate invadopodia and the few instances have always required external stimulation., Methods: In this study, the invasive potential of primary prostate adenocarcinoma cell lines, which have never been fully characterised before, was investigated both in-vitro invadopodia assays and in-vivo zebrafish dissemination assay. Subsequently, circulating tumour cells from prostate cancer patients were isolated and tested in the invadopodia assay., Results: Retention of E-cadherin and N-cadherin expression indicated a transitional state of EMT progression, consistent with the idea of partial EMT that has been frequently observed in aggressive prostate cancer. All cell lines tested were capable of spontaneous invadopodia formation and possess a significant degradative ability in-vitro under basal conditions. These cell lines were invasive in-vivo and produced visible metastasis in the zebrafish dissemination assay. Importantly we have proceeded to demonstrate that circulating tumour cells isolated from prostate cancer patients exhibit invadopodia-like structures and degrade matrix with visible puncta. This work supports a role for invadopodia activity as one of the mechanisms of dissemination employed by prostate cancer cells., Conclusion: The combination of studies presented here provide clear evidence that invadopodia activity can play a role in prostate cancer progression., (© 2022. The Author(s).)

Human B cells and their expressed antibodies are crucial in conferring immune protection. Identifying pathogen-specific antibodies following infection is possible due to enhanced humoral immunity against well-described molecules on the pathogen surface. However, screening for cancer-reactive antibodies remains challenging since target antigens are often not identified a priori and the frequency of circulating B cells recognizing cancer cells is likely very low. We investigated whether combined ex vivo culture of human B cells with three innate stimuli, interleukin-17 (IL-17), B-cell activation factor (BAFF), and the toll-like receptor 9 (TLR-9) agonist DNA motif CpG ODN 2006 (CpG), each known to activate B cells through different signalling pathways, promote cell activation, proliferation, and antibody production. Combined IL-17+BAFF+CpG prolonged B-cell survival and increased proliferation compared with single stimuli. IL-17+BAFF+CpG triggered higher IgG secretion, likely by activating differentiated, memory and class-switched CD19+CD20+CD27+IgD- B cells. Regardless of anti-FOLR antibody seropositive status, IL-17+BAFF+CpG combined with a monovalent tumour-associated antigen (folate receptor alpha [FOLR]) led to secreted antibodies recognizing the antigen and the antigen-expressing IGROV1 cancer cells. In a seropositive individual, FOLR stimulation favoured class-switched memory B-cell precursors (CD27-CD38-IgD-), class-switched memory B cells and anti-FOLR antibody production, while IL-17+BAFF+CpG combined with FOLR, promoted class-switched memory B-cell precursors and antibody-secreting (CD138+IgD-) plasma cells. Furthermore, IL-17+BAFF+CpG stimulation of peripheral blood B cells from patients with melanoma revealed tumour cell-reactive antibodies in culture supernatants. These findings suggest that innate signals stimulate B-cell survival and antibody production and may help identify low-frequency antigen-reactive humoral responses., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Immunology.)

New Findings: What is the central question of this study? Is lymphocyte DNA methylation differentially modulated by resistance training and aerobic exercise in older women? What is the main finding and its importance? The practice of resistance training led to an increased global DNA methylation in lymphocytes. The exercise-induced increase of inflammatory genes methylation may be associated with immune function impairment during ageing., Abstract: Ageing-induced increase in inflammatory gene expression through a reduction in DNA methylation might contribute to chronic diseases. Regular physical exercise practices, in turn, are associated with a decrease in the incidence of inflammatory diseases. We herein evaluated the effects of three exercise modalities on lymphocyte global and gene-specific (interferon γ (IFN-γ) and interleukin 17A (IL-17A) DNA methylation in aged women (68 ± 7.5 years). This cross-sectional study included 86 women, divided into four groups according to the physical exercise practice: 20 were practicing resistance training (RT); 24 were practicing water aerobics exercise (W); 22 were practicing water aerobics and resistance exercise (RWT), and 20 did not practice any physical exercise (CON). We evaluated volunteer functional capability using the Timed Up and Go (TUG) test, global lymphocyte DNA methylation by enzyme-linked immunosorbent assay, IFN-γ and IL-17A methylation by qPCR and CD4 + IFN-γ + and CD4 + IL-17 + cell percentage by flow cytometry. The three physically exercised groups performed functional capability tests in a shorter period and showed a higher global lymphocyte DNA methylation and methylated CpGs of IL-17A and IFN-γ promoter regions than the control group. The practice of resistance training (RT and RWT groups) lead to high global DNA methylation. The combination of resistance training and aerobic exercise led to the increase of lymphocyte IL-17A and IFN-γ gene methylation induced by each separately. However, the percentage of IFN-γ + and IL-17 + cells was lower only in the RT group. The exercise-induced increase of inflammatory-gene methylation may be associated with gene expression changes and immune function impairment during ageing., (© 2021 The Authors. Experimental Physiology © 2021 The Physiological Society.)

Histidine-containing dipeptides (HCDs) are abundantly expressed in striated muscles. Although important properties have been ascribed to HCDs, including H + buffering, regulation of Ca 2+ transients and protection against oxidative stress, it remains unknown whether they play relevant functions in vivo. To investigate the in vivo roles of HCDs, we developed the first carnosine synthase knockout (CARNS1 -/- ) rat strain to investigate the impact of an absence of HCDs on skeletal and cardiac muscle function. Male wild-type (WT) and knockout rats (4 months-old) were used. Skeletal muscle function was assessed by an exercise tolerance test, contractile function in situ and muscle buffering capacity in vitro. Cardiac function was assessed in vivo by echocardiography and cardiac electrical activity by electrocardiography. Cardiomyocyte contractile function was assessed in isolated cardiomyocytes by measuring sarcomere contractility, along with the determination of Ca 2+ transient. Markers of oxidative stress, mitochondrial function and expression of proteins were also evaluated in cardiac muscle. Animals were supplemented with carnosine (1.8% in drinking water for 12 weeks) in an attempt to rescue tissue HCDs levels and function. CARNS1 -/- resulted in the complete absence of carnosine and anserine, but it did not affect exercise capacity, skeletal muscle force production, fatigability or buffering capacity in vitro, indicating that these are not essential for pH regulation and function in skeletal muscle. In cardiac muscle, however, CARNS1 -/- resulted in a significant impairment of contractile function, which was confirmed both in vivo and ex vivo in isolated sarcomeres. Impaired systolic and diastolic dysfunction were accompanied by reduced intracellular Ca 2+ peaks and slowed Ca 2+ removal, but not by increased markers of oxidative stress or impaired mitochondrial respiration. No relevant increases in muscle carnosine content were observed after carnosine supplementation. Results show that a primary function of HCDs in cardiac muscle is the regulation of Ca 2+ handling and excitation-contraction coupling., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

We have outlined the approach of visualizing autophagy specifically in the epithelial follicle stem cells of the Drosophila ovary using the LysoTracker dye. The advantage of using this protocol is that it details several techniques, including ovary dissection, immunofluorescence, and western blotting, that positively identify autophagy changes in a very small population of cells. One of the limitations of this protocol is that it needs to be combined with other genetic manipulations and positive markers of the autophagy pathway. For complete details on the use and execution of this protocol, please refer to Singh et al., (2018)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Metagenomics is helping to expand the known diversity of viruses, especially of those with poorly studied hosts in remote areas. The Neotropical region harbors a considerable diversity of avian species that may play a role as both host and short-distance vectors of unknown viruses. Viral metagenomics of cloacal swabs from 50 Neotropical birds collected in French Guiana revealed the presence of four complete astrovirus genomes. They constitute an early diverging novel monophyletic clade within the Avastrovirus phylogeny, representing a putative new astrovirus species (provisionally designated as Avastrovirus 5) according to the International Committee on Taxonomy of Viruses (ICTV) classification criteria. Their genomic organization shares some characteristics with Avastrovirus but also with Mamastrovirus. The pan-astrovirus RT-PCR analysis of the cloacal samples of 406 wild Neotropical birds showed a community-level prevalence of 4.9% (5.1% in passerines, the highest described so far in this order of birds). By screening birds of a remote region, we expanded the known host range of astroviruses to the avian families Cardinalidae, Conopophagidae, Furnariidae, Thamnophilidae, Turdidae and Tyrannidae. Our results provide important first insights into the unexplored viral communities, the ecology, epidemiology and features of host-pathogen interactions that shape the evolution of avastroviruses in a remote Neotropical rainforest.

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Monoclonal antibodies find broad application as therapy for various types of cancer by employing multiple mechanisms of action against tumors. Manipulating the Fc-mediated functions of antibodies that engage immune effector cells, such as NK cells, represents a strategy to influence effector cell activation and to enhance antibody potency and potentially efficacy. We developed a novel approach to generate and ascertain the functional attributes of Fc mutant monoclonal antibodies. This entailed coupling single expression vector (pVitro1) antibody cloning, using polymerase incomplete primer extension (PIPE) polymerase chain reaction, together with simultaneous Fc region point mutagenesis and high yield transient expression in human mammalian cells. Employing this, we engineered wild type, low (N297Q, NQ), and high (S239D/I332E, DE) FcR-binding Fc mutant monoclonal antibody panels recognizing two cancer antigens, HER2/ neu and chondroitin sulfate proteoglycan 4. Antibodies were generated with universal mutagenic primers applicable to any IgG1 pVitro1 constructs, with high mutagenesis and transfection efficiency, in small culture volumes, at high yields and within 12 days from design to purified material. Antibody variants conserved their Fab-mediated recognition of target antigens and their direct anti-proliferative effects against cancer cells. Fc mutations had a significant impact on antibody interactions with Fc receptors (FcRs) on human NK cells, and consequently on the potency of NK cell activation, quantified by immune complex-mediated calcium mobilization and by antibody-dependent cellular cytotoxicity (ADCC) of tumor cells. This strategy for manipulation and testing of Fc region engagement with cognate FcRs can facilitate the design of antibodies with defined effector functions and potentially enhanced efficacy against tumor cells.

Regulatory T cells (Tregs) play a pivotal role in maintaining immunological tolerance, but they can also play a detrimental role by preventing antitumor responses. Here, we characterized T helper (Th)-like Treg subsets to further delineate their biological function and tissue distribution, focusing on their possible contribution to disease states. RNA sequencing and functional assays revealed that Th2-like Tregs displayed higher viability and autocrine interleukin-2 (IL-2)-mediated activation than other subsets. Th2-like Tregs were preferentially found in tissues rather than circulation and exhibited the highest migratory capacity toward chemokines enriched at tumor sites. These cellular responses led us to hypothesize that this subset could play a role in maintaining a tumorigenic environment. Concurrently, Th2-like Tregs were enriched specifically in malignant tissues from patients with melanoma and colorectal cancer compared to healthy tissue. Overall, our results suggest that Th2-like Tregs may contribute to a tumorigenic environment due to their increased cell survival, higher migratory capacity, and selective T-effector suppressive ability., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

IgE antibodies are key mediators of antiparasitic immune responses, but their potential for cancer treatment via antibody-dependent cell-mediated cytotoxicity (ADCC) has been little studied. Recently, tumor antigen-specific IgEs were reported to restrict cancer cell growth by engaging high-affinity Fc receptors on monocytes and macrophages; however, the underlying therapeutic mechanisms were undefined and in vivo proof of concept was limited. Here, an immunocompetent rat model was designed to recapitulate the human IgE-Fcε receptor system for cancer studies. We also generated rat IgE and IgG mAbs specific for the folate receptor (FRα), which is expressed widely on human ovarian tumors, along with a syngeneic rat tumor model expressing human FRα. Compared with IgG, anti-FRα IgE reduced lung metastases. This effect was associated with increased intratumoral infiltration by TNFα + and CD80 + macrophages plus elevated TNFα and the macrophage chemoattractant MCP-1 in lung bronchoalveolar lavage fluid. Increased levels of TNFα and MCP-1 correlated with IgE-mediated tumor cytotoxicity by human monocytes and with longer patient survival in clinical specimens of ovarian cancer. Monocytes responded to IgE but not IgG exposure by upregulating TNFα, which in turn induced MCP-1 production by monocytes and tumor cells to promote a monocyte chemotactic response. Conversely, blocking TNFα receptor signaling abrogated induction of MCP-1, implicating it in the antitumor effects of IgE. Overall, these findings show how antitumor IgE reprograms monocytes and macrophages in the tumor microenvironment, encouraging the clinical use of IgE antibody technology to attack cancer beyond the present exclusive reliance on IgG. Cancer Res; 77(5); 1127-41. ©2017 AACR ., (©2017 American Association for Cancer Research.)

B cells participate in immune surveillance in human circulation and tissues, including tumors such as melanoma. By contrast, the role of humoral responses in cutaneous immunity is underappreciated. We report circulating skin-homing CD22+CLA+B cells in healthy volunteers and melanoma patients (n = 73) and CD22+ cells in melanoma and normal skin samples (n = 189). Normal and malignant skin featured mature IgG and CD22 mRNA, alongside mRNA for the transiently-expressed enzyme Activation-induced cytidine Deaminase (AID). Gene expression analyses of publically-available data (n = 234 GEO, n = 384 TCGA) confirmed heightened humoral responses (CD20, CD22, AID) in melanoma. Analyses of 51 melanoma-associated and 29 normal skin-derived IgG sequence repertoires revealed lower IgG1/IgGtotal representation compared with antibodies from circulating B cells. Consistent with AID, comparable somatic hypermutation frequencies and class-switching indicated affinity-matured antibodies in normal and malignant skin. A melanoma-associated antibody subset featured shorter complementarity-determining (CDR3) regions relative to those from circulating B cells. Clonal amplification in melanoma-associated antibodies and homology modeling indicated differential potential antigen recognition profiles between normal skin and melanoma sequences, suggesting distinct antibody repertoires. Evidence for IgG-expressing B cells, class switching and antibody maturation in normal and malignant skin and clonally-expanded antibodies in melanoma, support the involvement of mature B cells in cutaneous immunity.

IgG4 is the least abundant subclass of IgG in normal human serum, but elevated IgG4 levels are triggered in response to a chronic antigenic stimulus and inflammation. Since the immune system is exposed to tumor-associated antigens over a relatively long period of time, and tumors notoriously promote inflammation, it is unsurprising that IgG4 has been implicated in certain tumor types. Despite differing from other IgG subclasses by only a few amino acids, IgG4 possesses unique structural characteristics that may be responsible for its poor effector function potency and immunomodulatory properties. We describe the unique attributes of IgG4 that may be responsible for these regulatory functions, particularly in the cancer context. We discuss the inflammatory conditions in tumors that support IgG4, the emerging and proposed mechanisms by which IgG4 may contribute to tumor-associated escape from immune surveillance and implications for cancer immunotherapy.

Emerging evidence suggests pathological and immunoregulatory functions for IgG4 antibodies and IgG4 + B cells in inflammatory diseases and malignancies. We previously reported that IgG4 antibodies restrict activation of immune effector cell functions and impair humoral responses in melanoma. Here, we investigate IgG4 as a predictor of risk for disease progression in a study of human sera (n = 271: 167 melanoma patients; 104 healthy volunteers) and peripheral blood B cells (n = 71: 47 melanoma patients; 24 healthy volunteers). IgG4 (IgG4/IgG total ) serum levels were elevated in melanoma. High relative IgG4 levels negatively correlated with progression-free survival (PFS) and overall survival. In early stage (I-II) disease, serum IgG4 was independently negatively prognostic for progression-free survival, as was elevation of IgG4 + circulating B cells (CD45 + CD22 + CD19 + CD3 - CD14 - ). In human tissues (n = 256; 108 cutaneous melanomas; 56 involved lymph nodes; 60 distant metastases; 32 normal skin samples) IgG4 + cell infiltrates were found in 42.6% of melanomas, 21.4% of involved lymph nodes and 30% of metastases, suggesting inflammatory conditions that favor IgG4 at the peripheral and local levels. Consistent with emerging evidence for an immunosuppressive role for IgG4, these findings indicate association of elevated IgG4 with disease progression and less favorable clinical outcomes. Characterizing immunoglobulin and other humoral immune profiles in melanoma might identify valuable prognostic tools for patient stratification and in the future lead to more effective treatments less prone to tumor-induced blockade mechanisms.

Background: Intellectual disability (ID) is a highly heterogeneous condition affecting 2% of the population worldwide. In a field study conducted in a highly inbred area of Northeastern Brazil, we investigated a consanguineous family in which seven adults presented syndromic ID., Methods: Genome-Wide Human SNP Array 6.0 (Affymetrix) microarray was used to determine regions of homozygosity-by-descent and whole exome sequencing (WES) was performed in one affected individual using Extended Nextera Rapid-Capture Exome and Illumina HiSeq2500., Results: We found two regions with an logarithm of the odds (LOD) score of 3.234: a region spanning 4.0 Mb in 19q13.32-q13.33 and a pericentromeric 20 Mb area in chromosome 2 (2p12-q11.2). WES disclosed in the critical region of chromosome 19 a homozygous variant (c.418C>T, p.Arg140Trp) in Mediator complex subunit 25 (MED25), predicted as deleterious by PolyPhen-2, Provean, Mutation Taster and Sorting Intolerant From Tolerant (SIFT). MED25 is a component of the Mediator complex, involved in regulation of transcription of nearly all RNA polymerase II-dependent genes. Deleterious mutations in MED12, MED17 and MED23 have already been associated with ID., Conclusions: These findings demonstrate that the combination of field investigation of families in highly inbred regions with modern technologies is an effective way for identifying new genes associated with ID., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Malignant melanoma is associated with poor clinical prognosis; however, novel molecular and immune therapies are now improving patient outcomes. Almost 50% of melanomas harbor targetable activating mutations of BRAF that promote RAS-RAF-MEK-ERK pathway activation and melanoma proliferation. Recent evidence also indicates that melanomas bearing mutant BRAF may also have altered immune responses, suggesting additional avenues for treatment of this patient group. The small molecule inhibitors selective for mutant BRAF induce significant but short-lived clinical responses in a proportion of patients, but also lead to immune stimulatory bystander events, which then subside with the emergence of resistance to inhibition. Simultaneous BRAF and MEK inhibition, and especially combination of BRAF inhibitors with new immunotherapies such as checkpoint blockade antibodies, may further enhance immune activation, or counteract immunosuppressive signals. Preclinical evaluation and ongoing clinical trials should provide novel insights into the role of immunity in the therapy of BRAF-mutant melanoma., (©2014 American Association for Cancer Research.)

Host-induced antibodies and their contributions to cancer inflammation are largely unexplored. IgG4 subclass antibodies are present in IL-10-driven Th2 immune responses in some inflammatory conditions. Since Th2-biased inflammation is a hallmark of tumor microenvironments, we investigated the presence and functional implications of IgG4 in malignant melanoma. Consistent with Th2 inflammation, CD22+ B cells and IgG4(+)-infiltrating cells accumulated in tumors, and IL-10, IL-4, and tumor-reactive IgG4 were expressed in situ. When compared with B cells from patient lymph nodes and blood, tumor-associated B cells were polarized to produce IgG4. Secreted B cells increased VEGF and IgG4, and tumor cells enhanced IL-10 secretion in cocultures. Unlike IgG1, an engineered tumor antigen-specific IgG4 was ineffective in triggering effector cell-mediated tumor killing in vitro. Antigen-specific and nonspecific IgG4 inhibited IgG1-mediated tumoricidal functions. IgG4 blockade was mediated through reduction of FcγRI activation. Additionally, IgG4 significantly impaired the potency of tumoricidal IgG1 in a human melanoma xenograft mouse model. Furthermore, serum IgG4 was inversely correlated with patient survival. These findings suggest that IgG4 promoted by tumor-induced Th2-biased inflammation may restrict effector cell functions against tumors, providing a previously unexplored aspect of tumor-induced immune escape and a basis for biomarker development and patient-specific therapeutic approaches.

Dipeptidyl peptidase-4 (DPP-4) inhibition is a well- characterized treatment for type 2 diabetes mellitus (T2DM). The objective of this model-based meta-analysis was to describe the time course of HbA1c response after dosing with alogliptin (ALOG), saxagliptin (SAXA), sitagliptin (SITA), or vildagliptin (VILD). Publicly available data involving late-stage or marketed DPP-4 inhibitors were leveraged for the analysis. Nonlinear mixed-effects modeling was performed to describe the relationship between DPP-4 inhibition and mean response over time. Plots of the relationship between metrics of DPP-4 inhibition (ie, weighted average inhibition [WAI], time above 80% inhibition, and trough inhibition) and response after 12 weeks of daily dosing were evaluated. The WAI was most closely related to outcome, although other metrics performed well. A model was constructed that included fixed effects for placebo and drug and random effects for intertrial variability and residual error. The relationship between WAI and outcome was nonlinear, with an increasing response up to 98% WAI. Response to DPP-4 inhibitors could be described with a single drug effect. The WAI appears to be a useful index of DPP-4 inhibition related to HbA1c. Biomarker to response relationships informed by model-based meta-analysis can be leveraged to support study designs including optimization of dose, duration of therapy, and patient population.

Introduction: Information is accumulating on exceptional cases of oily orange anal leakage, probably caused by certain foods and their form of consumption. The leakage is usually inadvertent and is worrisome for patients., Method: Clinical data was gathered on a case series presenting with massive and inadvertent orange oily anal leakage, without sphincter sensation, causing the patients to feel alarmed. This condition usually occurs after eating fish, especially Japanese cuisine., Results: Eleven patients were included (four women and seven men). The mean age was 47.3 years. Colonoscopy had previously been performed in four patients, with normal results. The symptoms, described as keriorrhea, were probably produced by accumulation of indigestible "wax esters", es Mero present in some oily fish, recently incorporated in raw fish dishes (sashimi) or as Japanese Mero sea bass. These oils, which cannot be absorbed or digested, accumulate in the rectum and may also filter spontaneously and induce toxin-related sphincter dysfunction. The same symptoms are sometimes produced after consumption of cooked versions of these species. The diagnosis is given by clinical data, prior ingestion of these types of fish and the self-limiting nature of the symptoms, without the need for further investigations., Conclusion: We describe a case series of a recently described entity associated with dietary changes. This entity is benign and self-limiting. Clinicians should be familiar with the symptoms to inform patients correctly and avoid unnecessary laboratory studies., (Copyright © 2011 Elsevier España, S.L. All rights reserved.)

The nuclear protein PLU-1/JARID1B/KDM5 is widely expressed in breast cancers while showing highly restricted expression in normal adult tissues. To investigate whether JARID1B is a potential target antigen for immunotherapy of breast cancer, we have analyzed the responses of CD8(+) T cells to JARID1B HLA-A*0201 peptides in vitro and used peptide multimers to detect the presence of JARID1B reactive T cells in the circulation of breast cancer patients. Peptides were selected using two web-based algorithms: criteria for inclusion being a high score in both prediction algorithms, and nonhomology with retinoblastoma binding protein-2 (RBP2/JARID1A/KDM5A). A 65-peptide panel was selected and assayed for binding strength by competition assay to obtain the IC(50). The immunogenicity in vitro of these peptides was assessed by T cell stimulation experiments, using autologous dendritic cells as APCs in the first rounds followed by autologous lymphoblasts. Fourteen of the peptides assayed produced cultures having >2% of the CD8(+) cells being IFN-γ(+) after 3-6 rounds of stimulation. An HLA-A*0201 cell line could activate the specific T cells if pulsed with peptide, but endogenous peptide levels were insufficient for activation. Nevertheless, multimer staining of circulating T cells from breast cancer patients showed a significantly higher percentage of multimer positive CD8(+) T cells, as compared to healthy adults for two of three JARID1B epitopes tested. One of these, peptide 73 (QLYALPCVL), was analyzed for memory phenotype, and found to have a significantly higher proportion of central memory T cells than the control group, demonstrating a previous exposure to the peptide., (Copyright © 2011 UICC.)

Loss of tolerance toward commensal bacteria has been invoked as a mechanism for Crohn's disease. IL-10 is a key anti-inflammatory cytokine that plays a role in induction and maintenance of tolerance. The aim of this study is to determine IL-10 production in response to bacterial components in Crohn's disease patients, who were classified according to their phenotypes as stricturing, penetrating, or inflammatory. Peripheral blood was obtained from Crohn's disease patients and healthy controls. Cytokine production was measured in whole blood cultures, isolated CD4(+) cells, and monocyte-derived dendritic cells (MDDCs). Under unstimulated conditions, IL-10, but not IL-12, was down-regulated significantly in blood cultures of patients with severe phenotypes, compared with inflammatory, nonpenetrating, nonstricturing Crohn's disease patients. In response to LPS, IL-10 was up-regulated more significantly in patients with no fistulae or fibrosis. Study of IL-10 production by isolated cell subsets showed that DCs, but not CD4(+) T cells, from penetrating Crohn's disease produced significantly less IL-10 in response to LPS. Differences were not associated with the 1082A/G polymorphism in the IL-10 gene promoter. We show a defect in IL-10 production in whole blood cell cultures and MDDCs in patients with severe forms of Crohn's disease. This defect in IL-10 production by a group of Crohn's disease patients may represent a mechanism mediating more severe manifestations of the disease. We propose that treatment with IL-10 or IL-10-inducing therapies could be of particular benefit to these group of patients.

The epithelial mucin MUC1 is one of the few tumour-associated antigens identified for breast cancer. Several MUC1-derived peptides binding HLA-A*0201 molecules have been identified that correspond to sequences outside the tandem repeat. Immunisation with some of these peptides induces protective antitumour immunity in mice. Another HLA-A*0201-binding peptide has been identified in a human system. We have evaluated the CD8(+) T-cell responses to all these peptides using peripheral blood lymphocytes from breast cancer patients and normal donors. Specific CD8(+) T-cell responses could be generated in vitro against some of these peptides but only after several rounds of in vitro restimulation, and they did not recognise human cells endogenously expressing the antigen. Nevertheless, T cells recognised by HLA-A*0201 tetramers carrying a peptide from the signal sequence (LLLLTVLTV) could be detected in the peripheral blood of some HLA-A*0201(+) breast cancer patients but not in healthy adults. This peptide is the only one of those tested which was identified in the human system, and the results emphasize the potential problems involved in translation of data from laboratory animal models to the human system., ((c) 2005 Wiley-Liss, Inc.)

Using a C57Bl/6 mouse model system, where intramuscular (i.m.) injection of full length (FL) MUC1 cDNA protects against subsequent challenge with MUC1-expressing syngeneic tumour cells, we have investigated the importance of the tandem repeat (TR) domain in the induction of T cell-dependent tumour rejection. A MUC1 construct engineered to remove the TR domain (MUC1 0TR) was found to be as effective as the full length MUC1 cDNA in inhibiting the growth of RMA MUC1 cells in C57Bl/6 mice. Protection by i.m. injection of either the FL-MUC1 cDNA or the MUC1 0TR construct depended on the presence of functional CD4+ and CD8+ T cells. Specific CD8+ T cell responses, however, could not be detected in vitro using mouse spleen cells taken after only cDNA injection, but only after challenge in vivo with MUC1-expressing tumour cells. To attempt to enhance the responses of CD4+ T cells, a cDNA construct was developed, where the extracellular domain of MUC1 was fused to the transmembrane and cytoplasmic domain of Lamp1 (MUC1/Lamp1). This construct was equally effective in inducing tumour rejection but did not induce MUC1-specific CTL in mice before challenge with MUC1-expressing tumour cells. Our results indicate that, in this model, T cell responses necessary for protection against MUC1-expressing tumours that are induced by IM injection of MUC1 cDNA are independent of the tandem repeat domain as well as the transmembrane and cytoplasmic domains. A low level of protection was seen with all constructs in BALB/c mice, which show a defect in Th1 responses. C57Bl/6xBALB/c hybrids were, however, well protected against both H2(d) and H2(b) expressing tumour challenge, emphasizing the importance of the host background., (Copyright 2004 Wiley-Liss, Inc.)

MUC1 is a transmembrane mucin that is expressed on ductal epithelial cells and epithelial malignancies and has been proposed as a target antigen for immunotherapy. The expression of MUC1 has recently been reported on T and B cells. In this study we demonstrate that following activation in vivo or activation by different stimuli in vitro, human T cells expressed MUC1 at the cell surface. However, the level of expression in activated human T cells was significantly lower than that seen on normal epithelial cells or on breast cancer cells. In contrast, resting T cells did not bind MUC1-specific monoclonal antibodies (mAbs), nor was MUC1 mRNA detectable by reverse transcription-polymerase chain reaction (RT-PCR) or Northern blot analysis in these cells. The profile of activated T-cell reactivity with different MUC1-specific antibodies suggested that the glycoform of MUC1 expressed by the activated T cells carried core 2-based O-glycans, as opposed to the core 1 structures that dominate in the cancer-associated mucin. Confocal microscopy revealed that MUC1 was uniformly distributed on the surface of activated T cells. However, when the cells were polarized in response to a migratory chemokine, MUC1 was found on the leading edge rather than on the uropod, where other large mucin-like molecules on T cells are trafficked. The concentration of MUC1 at the leading edge of polarized activated human T cells suggests that MUC1 could be involved in early interactions between T cells and endothelial cells at inflammatory sites.

Background: p53 has a remarkable number of biological activities, including a central role in cell cycle checkpoints, apoptosis, senescence, and maintenance of genomic integrity. Its expression is modified by estradiol in some epithelial cancer-derived cell lines from the reproductive tract. The aim of this study was to evaluate the effect of low and high doses of estradiol in p53 gene expression in epithelial cancer-derived cell lines from the reproductive tract., Methods: p53 gene expression was assessed by Northern and Western blot methods in three human epithelial cancer-derived cell lines after estradiol treatment., Results: These indicated that no changes in p53 mRNA content occurred after estradiol treatment at both low (10 nM) and high (1 micro M) doses of estradiol in HeLa, CaLo, and C-33 cell lines. p53 protein content was nearly constant in HeLa and C-33 cell lines at administration of 10 nM of estradiol. However, when estradiol was administered at a higher dose (1 micro M), an increase in p53 protein was observed over time in HeLa and CaLo cell lines. In contrast, estradiol was without variations in C-33., Conclusions: Overall results indicate that estradiol induces variations of p53 protein levels in epithelial cancer-derived cell lines from the reproductive tract in vitro and that this effect may be related with status p53 and/or presence of E6/E7 from human papillomavirus.

The membrane epithelial mucin MUC1 is expressed at the luminal surface of most simple epithelial cells, but expression is greatly increased at lactation and in most breast carcinomas. The increase in level of expression of MUC1 in breast cancer is accompanied by changes in the profile of glycosyl transferases involved in the synthesis of the O-glycans attached to the MUC1 core protein. The cancer-associated mucin is therefore structurally different from the normal mucin, and expresses novel B cell epitopes. MUC1 antibodies are used for in vivo targeting of breast and ovarian tumors, and there is considerable interest in MUC1 as a possible target antigen for the immunotherapy of breast cancer. The different glycoforms can affect cell interactions differently, depending on whether specific interactions with lectins occur. In the absence of such lectin interactions, the long sialylated and negatively charged molecule can inhibit intercellular interactions between other cell surface molecules. The potential role of the different components of the immune system in MUC1 responses are discussed within the framework of how to develop logical strategies for designing clinical studies.

During the past decade there has been renewed interest in the use of vaccine immunotherapy for the treatment of cancer. This review focuses on HER2/neu, a tumour-associated antigen that is overexpressed in 10-40% of breast cancers and other carcinomata. Several immunogenic HER2/neu peptides recognized by T lymphocytes have been identified to be included in cancer vaccines. Some of these peptides have been assessed in clinical trials of patients with breast and ovarian cancer. Although it has been possible to detect immunological responses against the peptides in the immunized patients, no clinical responses have so far been described. Immunological tolerance to self-antigens like HER2/neu may limit the functional immune responses against them. It will be of interest to determine whether immune responses against HER2/neu epitopes can be of relevance to cancer treatment.

Generation of the HLA-A0201 (A2) influenza Matrix 58-66 epitope contained within the full-length Matrix protein is impaired in cells lacking the proteasome subunits low molecular protein 2 (LMP2) and LMP7. This Ag presentation block can be relieved by transfecting the wild-type LMP7 cDNA into LMP7-deficient cells. A mutated form of LMP7, lacking the two threonines at the catalytic active site, was equally capable of relieving the block in presentation of the influenza Matrix A2 epitope. These observations were extended by analyzing whether modification of the influenza Matrix protein could overcome the block in presentation of the A2 Matrix epitope. Expression of either a rapidly degraded form of the full-length Matrix protein or shorter Matrix fragments led to an efficient presentation of the A2 influenza Matrix epitope by LMP7-negative cells. These findings demonstrate two main points: 1) LMP7 incorporation into the proteasome is of greater importance for the generation of the influenza A2 Matrix epitope than the presence of the LMP7's catalytic site; and 2) the interplay between cytosolic proteases and stability of target proteins is of importance in optimization of Ag presentation. These observations may have relevance to the immunodominance of tumor and viral epitopes and raise the possibility that generation of shorter protein fragments could be a mechanism to ensure optimal Ag presentation by cells expressing low levels of LMP7.

Natural killer cells in the mouse express class I MHC-specific inhibitory receptors of the Ly49 protein family. The receptors mediate inhibition of the lysis of tumor cells and normal cells, and mediate the specificity of bone-marrow graft rejection by NK cells in vivo. The function of these receptors may be to confer upon NK cells the capacity to distinguish normal self cells from cells that have down-regulated expression of some or all self-class I molecules. Ly49 receptors discriminate between different class I molecules, and are distributed in expression to overlapping subsets of NK cells. The receptors appear to interact with class I-MHC residues and associated N-glycans, with little or no discrimination of the class I-bound peptide. The Ly49 receptor repertoire may be initially generated by a stochastic process that distributes receptors randomly to different cells and treats the two alleles of a given Ly49 gene independently. However, class I-MHC-dependent "education" processes shape the functional repertoire. The education processes silence potentially auto-aggressive NK cells, probably by ensuring that each NK cell expresses at least one self-specific Ly49 receptor. In addition, NK cell clones that express multiple self-specific Ly49 receptors are disfavored by the education processes, perhaps to confer greater discrimination on to individual NK cells.

This review addresses the physiological role of class I-mediated inhibition of NK cell lysis. It is suggested that several distinct activating receptors can stimulate NK lysis, all of which can be inhibited by class I molecules on the target cell. Evidence that most or all peptides that bind a class I molecule can cause inhibition is discussed, supporting a model in which NK cells detect loss of class I molecules, rather than loss of specific peptide/class I complexes. Finally, the acquisition of self-tolerance among NK cells is addressed with respect to data suggesting that autoaggressive NK cells are not deleted but rather exhibit altered characteristics which may render them unable to lyse autologous cells.

The microbial transformation rates of propanil, a commonly used herbicide, were investigated using water from a pristine lake in northeast Georgia. Microbial degradation rates were measured using natural water microflora, the natural water microflora amended with five bacterial species (Aerobacter aerogenes, Aeromonas hydrophila, Acinetobacter calcoaceticus, Proteus mirabilis, and Aeromonas salmonicida) isolated from the same lake, and the five isolates individually. Transformation rate constants for propanil were compared for the mixed microbial assemblages and isolates at similar initial bacterial concentrations (approximately 5.0 x 10(-3) bacteria/mL). Degradation started within 60 hours and was completed by 160 hours in all experiments. The mean first-order rate constant for natural microflora was -(4.80 +/- 0.620) x 10(-3) h-1. Natural waters amended with the bacterial isolates yielded rate constants ranging from -(0.39 +/- 0.186) x 10(-3) h-1 to -(2.13 +/- 0.029) x 10(-3) h-1 with an overall mean of -(1.63 +/- 0.242) x 10(-3) h-1. After 660 hours following the first amendment of propanil, (i.e., 500 hours after propanil degradation was complete), each sample was again amended with propanil. Subsequent degradation rates ranged from -(21.3 +/- 0.186) x 10(-3) h-1 to -(64.2 +/- 0.786) x 10(-3) h-1 and the mean rate constant was -(37.5 +/- 0.922) x 10(-3) h-1. No significant differences were observed between first-order rate constants among isolates following the first or the second addition of propanil. After the second spike, however, the average of rate constants was approximately 20 times greater than that following the first spike. Rates for the individual isolates varied greatly from one isolate to another, ranging from virtually no degradation with A. calcoaceticus to -(21.6 +/- 0.332) x 10(-3) h-1 for the composite treatment of all isolates.

Several lines of evidence indicate that major histocompatibility complex class I molecules expressed by target cells can prevent natural killer cell (NK) lysis, possibly by engaging inhibitory receptors expressed by NK cells. On the other hand it is likely that NK cells must be activated to lysis by the recognition of unidentified NK target structures on target cells. To investigate the relationship between positive activation of NK cells by NK target structures versus inhibition by target cell class I molecules, we have examined various NK/target cell interactions for which the expression of inhibitory class I molecules by the target cells is known. The results suggests that specific properties of the target cell other than the absence of class I expression are necessary to activate NK-mediated lysis. Furthermore, different effector cell populations, i.e. freshly isolated versus interleukin-2 activated NK cells, differ in their capacity to kill class I-deficient lymphoblast target cells. In general, class I-deficient target cells that are resistant to direct lysis by a given NK population can be lysed by the NK cells when the reaction is mediated by antibody-dependent cellular cytotoxicity (ADCC). Most significantly, all types of NK-mediated lysis of lymphoblasts, of tumor cells and of almost any target by ADCC can be inhibited by appropriate class I gene expression in the target cell. These results suggest a model in which lysis by NK cells must be triggered by any one of a set of distinct target cell ligands, but that all of these signals can be overruled by class I-mediated inhibition.

We describe the use of random peptide sequences for the mapping of antigenic determinants. An oligonucleotide with a completely degenerate sequence of 17 or 23 nucleotides was inserted into a bacterial expression vector. This resulted in an expression library producing random hexa- or octapeptides attached to a beta-galactosidase hybrid protein. Mimotopes, or antigenic sequences that mimic an epitope, were selected by immunoscreening of colonies with monoclonal antibodies, which were specific for antigenic sites on the spike protein of the coronavirus transmissible gastroenteritis virus. We report one mimotope for antigenic site II, eight for site III and one for site IV. The site III and site IV mimotopes were closely similar to the corresponding linear epitopes, localized previously in the amino acid sequence of the S protein. An alignment of the site II mimotope and the sequence of the S protein around Trp97, which is substituted in escape mutants, suggests that this mimotope mimics a conformational epitope located around residues 97-103. Applications of mimotopes to epitope mapping, serodiagnosis and vaccine development are discussed.

Two separate field trials involving naturally infected cattle were carried out on two farms known to have a history of Fasciola hepatica infection. On the first farm, 15 animals per group were allocated as follows: G1, triclabendazole (TCBZ) four times a year; G2, TCBZ twice a year (May and September); G3, untreated control. All groups grazed together and after 3.5 years the animals were slaughtered and their livers examined by federal meat inspectors who condemned 100% of livers in G3 and 8.3% in G2 owing to the presence of lesions of fasciolosis. In G1 no livers were condemned. Significant differences in weight gains were not detected and fluke counts remained at low levels in the treated groups. Also, in the control group, egg counts started to decrease when animals were 2 years old. On the second farm, groups of 20 animals were treated as follows: G1, TCBZ three times a year (May, September and December); G2, TCBZ twice a year (May and September); G3, nitroxynil twice a year (May and September); G4, rafoxanide twice a year (May and September); G5, untreated controls. All animals were weighed and faecal samples examined at approximately 28-day intervals. During the period of the study, larger weight gains were detected in the TCBZ treated groups than in the others. TCBZ treatment kept F. hepatica egg counts at a lower level for longer periods than the other drugs and significant differences in weight gains were only obtained between the group receiving TCBZ three times a year and the control group.

The S glycoprotein of transmissible gastroenteritis virus (TGEV) has been shown to contain four major antigenic sites (A, B, C, and D). Site A is the main inducer of neutralizing antibodies and has been previously subdivided into the three subsites Aa, Ab, and Ac. The residues that contribute to these sites were localized by sequence analysis of 21 mutants that escaped neutralization or binding by TGEV-specific monoclonal antibodies (MAbs), and by epitope scanning (PEPSCAN). Site A contains the residues 538, 591, and 543, which are essential in the formation of subsites Aa, Ab, and Ac, respectively. In addition, mar mutant 1B.H6 with residue 586 changed had partially altered both subsite Aa and Ab, indicating that these subsites overlap in residue 586; i.e. this residue also is part of site A. The peptide 537-MKSGYGQPIA-547 represents, at least partially, subsite Ac which is highly conserved among coronaviruses. This site is relevant for diagnosis and could be of interest for protection. Other residues contribute to site B (residues 97 and 144), site C (residues 50 and 51), and site D (residue 385). The location of site D is in agreement with PEPSCAN results. Site C can be represented by the peptide 48-P-P/S-N-S-D/E-52 but is not exposed on the surface of native virus. Its accessibility can be modulated by treatment at pH greater than 11 (at 4 degrees) and temperatures greater than 45 degrees. Sites A and B are fully dependent on glycosylation for proper folding, while sites C and D are fully or partially independent of glycosylation, respectively. Once the S glycoprotein has been assembled into the virion, the carbohydrate moiety is not essential for the antigenic sites.

Transmissible gastroenteritis virus (TGEV) was neutralized more than 10(9)-fold with antibodies of a single specificity [monoclonal antibodies (MAbs)]. Most of the virus was neutralized in the first 2-3 min of a reversible reaction, which was followed by a second phase with a decreased neutralization rate and, in some cases, by a persistent fraction, which was a function of the MAb and of the antibody-to-virus ratio. Neutralization of TGEV is a specific event that requires the location of the epitope involved in the neutralization in the appropriate structural context, which is present in the wild-type virus but not in certain MAb escaping mutants. In neutralization of TGEV by binary combinations of MAbs specific for the same or for different antigenic sites, either no cooperation or a synergistic effect, respectively, was observed. Mechanisms of TGEV neutralization by MAbs were characterized at high, intermediate, and low antibody-to-virus ratios. Under these conditions, mainly three steps of the replication cycle were inhibited: binding of virus to the cell, internalization, and a step that takes place after internalization. In addition, virus aggregation could be responsible for the neutralization of 10 to 20% of virus infectivity.

The antigenic homology of 26 coronavirus isolates, of which 22 were antigenically related to transmissible gastroenteritis virus (TGEV), was determined with 42 monoclonal antibodies. Type, group, and interspecies specific epitopes were defined. Two group specific MAbs distinguished the enteric TGEV isolates from the respiratory variants. An antigenic subsite involved in neutralization was conserved in porcine, feline, and canine coronavirus. The classification of the human coronavirus 229E in a taxonomic cluster distinct from TGEV group is suggested.

The antigenic structure of transmissible gastroenteritis (TGE) virus E2 glycoprotein has been defined at three levels: antigenic sites, antigenic subsites and epitopes. Four antigenic sites (A, B, C and D) were defined by competitive radioimmunoassay (RIA) using monoclonal antibodies (MAbs) selected from 9 fusions. About 20% (197) of the hybridomas specific for TGE virus produced neutralizing MAbs specific for site A, which was one of the antigenically dominant determinants. Site A was differentiated in three antigenic subsites: a, b and c, by characterization of 11 MAb resistant (mar) mutants, that were defined by 8, 3, and 3 MAbs, respectively. These subsites were further subdivided in epitopes. A total of 11 epitopes were defined in E2 glycoprotein, eight of which were critical for virus neutralization. Neutralizing MAbs were obtained only when native virus was used to immunize mice, although to produce hybridomas mice immunizations were made with antigen in the native, denatured, or mixtures of native and denatured form. All neutralizing MAbs reacted to conformational epitopes. The antigenic structure of the E2-glycoprotein has been defined with murine MAbs, but the antigenic sites were relevant in the swine, the natural host of the virus, because porcine sera reacted against these sites. MAbs specific for TGE virus site C reacted to non-immune porcine sera. This reactivity was not directed against porcine immunoglobulins. These results indicated that TGE virus contains epitope(s) also present in some non-immunoglobulin component of porcine serum.