Isolation of a functional antigen-Ia complex.

The helper T-cell recognition of globular protein antigens requires that the antigen be processed and presented by an I-region associated (Ia)-expressing antigen-presenting cell (APC). Processing involves the uptake of antigen into an intracellular, proteolytic, acidic compartment; release of peptide fragments containing the T-cell antigenic determinant; association of these peptides with Ia; and presentation of these complexes on the cell surface for recognition by the specific T cells. The molecular mechanisms by which processed antigenic peptides associate with Ia within the APC are poorly understood. To date, functional antigen-Ia complexes have not been isolated from cells that have processed native antigens, although the resolution of the structure of a major histocompatibility complex (MHC) class I protein indicates that peptide is bound in a groove between two alpha-helical regions of the molecule and synthetic peptides have been demonstrated to bind purified MHC both in detergent solution and incorporated into planar membranes, where the MHC-peptide complexes function to activate specific T cells. Here we demonstrate that Ia purified from APCs that have processed the native globular protein antigen cytochrome c, when incorporated into lipid membranes, stimulates cytochrome c-specific T cells in the absence of exogenous antigenic peptide. The T-cell response to Ia purified from cytochrome c-pulsed APCs shows the same MHC restriction and antigen fine specificity as the response to antigen-pulsed APCs. Indeed, T-cell recognition of pigeon cytochrome c (Pc) shows a well documented high-affinity heteroclitic cross-reaction to insect cytochromes c-namely, those of Drosophila melanogaster (DMc) and tobacco hornworm moth (THMc). The same heteroclitic response is observed when purified Ia from unpulsed cells, incorporated into lipid membranes, is used to present antigenic peptides of Pc and of THMc. Significantly, Ia purified from APCs that have processed DMc is approximately 50-fold more active in stimulating specific T cells compared to Ia purified from APCs that have processed Pc. The peptide-Ia complex isolated here may provide the necessary material for analysis of the physiochemical properties of the processed form of the antigen that is produced by the APC and associates with Ia.     

From the Cover: PACAP is a pathogen-inducible resident antimicrobial neuropeptide affording rapid and contextual molecular host defense of the brain

Defense of the central nervous system (CNS) against infection must be accomplished without generation of potentially injurious immune cell-mediated or off-target inflammation which could impair key functions. As the CNS is an immune-privileged compartment, inducible innate defense mechanisms endogenous to the CNS likely play an essential role in this regard. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide known to regulate neurodevelopment, emotion, and certain stress responses. While PACAP is known to interact with the immune system, its significance in direct defense of brain or other tissues is not established. Here, we show that our machine-learning classifier can screen for immune activity in neuropeptides, and correctly identified PACAP as an antimicrobial neuropeptide in agreement with previous experimental work. Furthermore, synchrotron X-ray scattering, antimicrobial assays, and mechanistic fingerprinting provided precise insights into how PACAP exerts antimicrobial activities vs. pathogens via multiple and synergistic mechanisms, including dysregulation of membrane integrity and energetics and activation of cell death pathways. Importantly, resident PACAP is selectively induced up to 50-fold in the brain in mouse models of Staphylococcus aureus or Candida albicans infection in vivo, without inducing immune cell infiltration. We show differential PACAP induction even in various tissues outside the CNS, and how these observed patterns of induction are consistent with the antimicrobial efficacy of PACAP measured in conditions simulating specific physiologic contexts of those tissues. Phylogenetic analysis of PACAP revealed close conservation of predicted antimicrobial properties spanning primitive invertebrates to modern mammals. Together, these findings substantiate our hypothesis that PACAP is an ancient neuro-endocrine-immune effector that defends the CNS against infection while minimizing potentially injurious neuroinflammation.

Neuropeptides enable interneuronal communication and signaling (1), mediating diverse functions ranging from endocrine stimulation and homeostatic regulation to immune signaling, pain modulation, and circadian rhythm maintenance. At present, over 100 neuropeptides are known in mammals (2). These peptides originate from neurons in the central, enteric, or peripheral nervous systems and within immune organs (3). Canonically, neuropeptides exert their biological function by binding to a cognate receptor (usually a G-coupled protein receptor [GPCR]), triggering a signal transduction pathway that leads to a functional change in the target cell (1). Neuropeptides are typically considered neurotransmitters or neurohormones, but recent work has illuminated their potential roles in modulating immune responses and neuroinflammation (4–8).Human innate and adaptive immunity have evolved via two parallel and complementary paradigms in host defense against microbial invasion: molecular and cellular. Molecular defense mediators are secreted or activated rapidly and locally to directly inhibit pathogens. Prototypic examples include host-defense peptides (HDPs), the acute-phase reactants, and the complement cascade. Cellular defense involves infiltration of professional immune phagocytes (neutrophils and macrophages) and lymphocytes into infected tissues. Cellular infiltration into the central nervous system (CNS) is a double-edged sword, given its anatomically confined space and physiologically delicate context. On one hand cellular defense may be necessary to control or clear certain pathogens. On the other hand, neutrophils and other phagocytes can cause counterproductive damage to tissue parenchyma due to production and release of reactive oxygen species and other cytotoxic constituents from phagolysosomes. Thus, molecular defenses that are rapidly deployable in immediate settings of infection to obviate the need for infiltration of potentially harmful immune cells would be of special relevance in context of the CNS.To explore putative molecular host-defense mediators within the CNS that may have both neuro- and immunomodulatory properties, we used a support vector machine (SVM) trained on HDPs (9, 10) to identify neuropeptides with potential host defense capabilities. Among the human neuropeptides identified as potential HDPs for molecular host defense of the CNS is pituitary adenylate cyclase-activating polypeptide (PACAP). PACAP is a member of the vasoactive intestinal peptide (VIP)/PACAP/secretin family (11) that regulates neurodevelopment (12), metabolism, emotion, mood, and stress responses via GPCRs (13). PACAP is known to interact with the immune system (14, 15) and modulate T helper type 1 (TH1)/TH2 cytokine production (3). Important previous work on structure activity relationships (SAR) of PACAP have also shown that it possess antimicrobial activity in vitro against a range of organisms (16–18), as well as anti-cancer activity against tumor cell lines. (Interestingly, our use of an SVM classifier that can scan different fragments of the same peptide allows us to identify antimicrobial activity in previously identified metabolites of PACAP as well^* (19)). However, host defense functions, contextual bioactivity, or pathogen-specific inducibility of PACAP or other neuropeptides regarding antimicrobial activity in vivo are not known. More specifically, the role of PACAP in the larger context of innate immunity and its in vivo relevance to antimicrobial defense of the CNS and in other tissues remains unclear, given that antimicrobial activity is strongly dependent on biochemical and physiological context (20, 21, 22). Here, we examine PACAP inducibility in response to infection in the CNS and other tissues, and whether PACAP exerts antimicrobial activity against relevant organisms in the specific biochemical context relevant to those tissues. Bioinformatic and structural analyses showed PACAP to possess almost identical structural similarity to human cathelicidin LL-37, despite having overall low sequence similarity to other known HDPs. Synchrotron X-ray scattering revealed that PACAP can induce negative Gaussian curvature (NGC) in microbial membranes, a general requirement for membrane-permeating antimicrobial processes such as pore formation, blebbing, and other membrane-perturbing events (23–25). Moreover, extending from prior work (18), antimicrobial assays and mechanistic fingerprinting analyses showed that PACAP exerts potent antimicrobial mechanisms against drug-resistant bacteria and fungi via multiple synergistic pathways, including permeabilization, disruption of cellular energetics, and activation of regulated cell death pathways. In mouse models of bacterial or fungal infection, we demonstrated that PACAP is strongly induced up to 50-fold in brain, spleen, or kidney. Further, in media simulating these tissue contexts, PACAP exerted robust microbiostatic and microbicidal efficacy. Taken together, these findings imply that PACAP is an infection-inducible, tissue-specific host-defense effector that affords rapid and contextual antimicrobial host defense in the CNS and periphery. Beyond immediate contributions to better understanding of antimicrobial defense, the present discoveries reveal specific intersections of neurological and immunological systems and establish insights into antiinfective strategies that preserve critical functions of the CNS.     

Trends of lipoprotein variables from childhood to adulthood in offspring of parents with coronary heart disease: The Bogalusa heart study

Although dyslipidemia among offspring of parents with coronary heart disease (CHD) has been known, the development of this adverse relationship with respect to specific lipoprotein variables from childhood to young adulthood has not been elucidated. This aspect was examined in a young adult cohort with (n = 271) and without (n = 805) a parental history of CHD followed longitudinally since childhood by repeated surveys from 1973 to 1991. Trends in fasting lipoprotein variables by parental CHD status were assessed by Lowess smoothing curve and Generalized Estimating Equations (GEE). In multivariate analyses adjusted for race and sex, parental CHD associated positively with low-density lipoprotein cholesterol (LDL-C, P <.01) and triglycerides (P <.05) mainly at the young adulthood age, whereas a positive association was noted with very-low-density lipoprotein cholesterol (VLDL-C) during both childhood and young adulthood (P <.05). The positive association between parental CHD and LDL-C in young adulthood persisted independently of body mass index (BMI) and fasting insulin, but disappeared when fasting glucose was added to the model. With respect to triglycerides and VLDL-C, inclusion of BMI, insulin, and/or glucose eliminated the adverse association with parental CHD. These observations suggest that parental CHD is just one more explanatory variable that loses its partial contribution to lipoprotein profiles in their offspring when other strongly interrelated contributory variables such as age, body fatness, and measures of glucose homeostasis are taken into account. Information on these risk variables in conjunction with parental or family history of CHD may enhance the potential of CHD risk assessment in youth.     

Nutritional Factors Associated with Antenatal Depressive Symptoms in the Early Stage of Pregnancy Among Urban South Indian Women

Many women of reproductive age from developing countries have poor nutritional status, and the prevalence of depression during pregnancy is high. The objective of the present study was to assess the prevalence of antenatal depressive symptoms in early pregnancy, and to identify the demographic and nutritional factors associated with these symptoms in a sample of urban South Indian pregnant women. This cross-sectional study was the baseline assessment of a prospective randomized controlled trial of vitamin B₁₂ supplementation in urban pregnant south Indian women between the ages of 18 and 40 years (www.clinicaltrials.gov: NCT00641862). 365 women in their first trimester of pregnancy were screened for depressive symptoms at an urban clinic in Karnataka, South India, using the Kessler Psychological Distress Scale (K-10). Nutritional, clinical and biochemical factors were also assessed. Mean (SD) age of the cohort was 22.6 (3.7) years and mean (SD) BMI was 20.4 (3.3) kg/m². 121 (33 %) of the women in the 1st trimester had symptoms consistent with depression (K-10 score >6). In multivariate log binomial regression analysis, presence of antenatal depressive symptoms in the first trimester were positively associated with vomiting, prevalence ratio (PR) = 1.54 (95 % CI 1.10, 2.16) and negatively with anemia, PR = 0.67 (95 % CI 0.47, 0.96). Nutrient intakes, serum vitamin B₁₂, methylmalonic acid, homocysteine and red cell folate levels were not associated with measures of depression. Antenatal depressive symptoms in early pregnancy are highly prevalent in urban Indian women and are more common in women with vomiting and without anemia. In this cross-sectional data, blood concentrations of vitamin B₁₂ and folate were not associated with depressive symptoms. The relationship between nutritional status and depressive symptoms may require larger and longitudinal studies.     

Metachronous Multiple Primary Malignant Neoplasms of the Stomach and the Breast: Report of Two Cases With Review of Literature

Multiple primary malignant neoplasm is the occurrence of a second primary malignancy in the same patient within 6 months of the detection of first primary (synchronous), or 6 months or more after primary detection (metachronous). Multiple primary malignant neoplasms are not very frequently encountered in clinical practice. The relative risk for a second primary malignancy increases by 1.111-fold every month from the detection of the first primary malignancy in any individual. We present 2 patients treated for carcinoma of the breast who developed a metachronous primary malignancy in the stomach to highlight the rare occurrence of multiple primary malignant neoplasms. These tumors were histologically dissimilar, with distinct immunohistochemical parameters. The importance lies in carefully identifying the second primary malignancies, not dismissing them as metastases, and treating them accordingly.Key words: Breast neoplasms, Stomach neoplasms, Neoplasms, Second primaryBreast cancer is the most common malignancy among women worldwide. With proper screening, earlier detection, and improved treatment, survival has greatly increased, with the result that there is now a large population of women with a present or past history of breast cancer. This has led to an increased detection of second primary malignancies among these women. The relative risk for a second primary malignancy increases by 1.111-fold every month from the detection of the first primary malignancy in any individual.¹ Several authors have reported on a lesion in the stomach being labeled as a second primary malignancy and subsequently found to be metastasis. When the primary breast tumor is positive for estrogen and progesterone receptors (ER/PRs) and the stomach tumor is ER/PR negative, the diagnosis is established easily.² However, studies have shown that some primary gastric cancers can have ER/PR positivity. Further, if the primary breast lesion is ER/PR negative, the same cannot be used as a marker. Here, we present 2 breast cancer patients who developed second primary malignancies in the stomach and the final diagnosis was established based on histopathology and immunohistochemistry.     

The use of ABO-incompatible grafts in living donor liver transplantation—First report from India

ABO incompatibility is the commonest reason for rejection of donors in living donor liver transplantation (LDLT). The donor pool could be expanded by 25 % to 35 % if the ABO barrier is overcome. In the absence of pre-conditioning, transplantation across the blood groups is fraught with the almost universal risk of antibody-mediated rejection (AMR) that rapidly leads to graft loss. However, AMR can be prevented by removal of preformed antibodies and reducing their production by B cells. We describe our initial experience of three cases of ABO-incompatible (ABO-i) LDLT: a 42-year-old male, an 8-month-old male and a 28-month-old female, all of blood group O+ who received blood group B + right lobe, B + left lateral segment, and A + left lateral segment liver grafts, respectively. Pre-LDLT conditioning included administration of anti-CD20 antibody (Rituximab^®) to the adult 4 weeks prior, and four to seven sessions of double-filtration plasmapheresis to all, to remove preformed antibodies and achieve anti-donor blood group antibody (ADA) titers of ≤1:16 IgG and ≤1:8 IgM, respectively. In addition, cases 1 and 3 received mycophenolate mofetil for 7 days prior to LDLT. After LDLT, all three patients achieved normal graft function over 8–17 days with no evidence of AMR and without the need for further plasmapheresis. Postoperative complications included portal vein thrombosis (one successfully re-explored), CMV (one), Pseudomonas and Klebsiella sepsis (one each), and abdominal collection (one treated with percutaneous drainage). All are currently well with normal graft function and low ADA titers at 8, 16, and 19 months after ABO-i LDLT.     

Anti-HBs response to hepatitis B immunoglobulin prophylaxis in liver transplant recipients

Background

Hepatitis B virus (HBV) recurrence after a liver transplant (LT) is a global issue. Several strategies have been adopted to prevent this recurrence. Most strategies recommend a combination of hepatitis B immunoglobulin (HBIG) and or nucleos(t)ide analogue.

Aim of the Study

The aim of the study is to determine the anti-HBs response to HBIG among Indian patients who had undetectable pre-transplant HBV DNA.

Methods

Seven adult HBV-related LT recipients of Indian origin with low pre-transplant HBV titres who had a liver transplant between August 2009 and June 2012 were included in the study. The protocol followed for post-liver transplant HBIG dose was titrated to achieve an anti-HBs titre of at least 100 IU/L. All recipients were on entecavir. Anti-HBs titre, and HBsAg status was checked at regular intervals. A retrospective analysis of the anti-HBs response to a loading and maintenance dose of HBIG was done.

Results

Seven adult HBV-related LT recipients on post-transplant prophylaxis with HBIG and nucleoside analogue (entecavir) fulfilled the criteria for the study. The median anti-HBs response to the anhepatic and loading dose of HBIG was high at 555 IU/L. In two, the response was less than 100 IU/L. The median dose of HBIG reduced at end of 1 month to 800 IU, and the median titre was 223 IU/L. For the next 11 months, the median requirement of HBIG was 3,000 and 4,000 IU, and the titre was low at 53.8 and 60.9 IU/L at end of 6 and 12 months, respectively.

Conclusions

The anti-HBs response to HBIG was variable, and titres even below 100 IU/L did not result in HBV recurrence when HBIG was given in combination with entecavir.