Do we really need to differentiate mesenchymal stem cells into insulin-producing cells for attenuation of the autoimmune responses in type 1 diabetes: immunoprophylactic effects of precursors to insulin-producing cells.

BACKGROUND: Type 1 diabetes (T1D) is a multifactorial autoimmune disorder where pancreatic beta cells are lost before the clinical manifestations of the disease. Administration of mesenchymal stem cells (MSCs) or MSCs differentiated into insulin-producing cells (IPCs) have yielded limited success when used therapeutically. We have evaluated the immunoprophylactic potentials of precursors to insulin-producing cells (pIPCs) and IPCs in nonobese diabetic (NOD) mice to ask a basic question: do we need to differentiate MSCs into IPCs or will pIPCs suffice to attenuate autoimmune responses in T1D? METHODS: Bone marrow-derived MSCs from Balb/c mice were characterized following the International Society for Cellular Therapy (ISCT) guidelines. MSCs cultured in high-glucose media for 11 to 13 passages were characterized for the expression of pancreatic lineage genes using real-time polymerase chain reaction. Expression of the PDX1 gene in pIPCs was assessed using Western blot and fluorescence-activated cell sorting (FACS). Triple-positive MSCs were differentiated into IPCs using a three-step protocol after sorting them for cell surface markers, i.e. CD29, CD44, and SCA-1. Nonobese diabetic mice were administered pIPCs, IPCs, or phosphate-buffered saline (PBS) into the tail vein at weeks 9 or 10 and followed-up for 29-30 weeks for fasting blood glucose levels. Two consecutive blood sugar levels of more than 250 mg/dl were considered diabetic. RESULTS: MSCs grown in high-glucose media for 11 to 13 passages expressed genes of the pancreatic lineage such as PDX1, beta2, neurogenin, PAX4, Insulin, and glucagon. Furthermore, Western blot and FACS analysis for PDX-1, a transcription factor necessary for beta cell maturation, confirmed that these cells were precursors of insulin-producing cells (pIPCs). NOD mice administered with pIPCs were better protected from developing diabetes with a protective efficacy of 78.4% (p < 0.009); however, administration of IPCs gave protective efficacy of 55% at the end of 28-30 weeks. CONCLUSIONS: Precursors to insulin-producing cells seem to have better potential to arrest autoimmune response in type 1 diabetes when administered before the onset of the disease in NOD mice. When translated to humans, autologous mesenchymal stem cells grown in high-glucose media for 10 to 13 passages may have beneficial effects in individuals at high risk of developing type 1 diabetes.

Classical autophagy proteins LC3B and ATG4B facilitate melanosome movement on cytoskeletal tracks.

Macroautophagy/autophagy is a dynamic and inducible catabolic process that responds to a variety of hormonal and environmental cues. Recent studies highlight the interplay of this central pathway in a variety of pathophysiological diseases. Although defective autophagy is implicated in melanocyte proliferation and pigmentary disorders, the mechanistic relationship between the 2 pathways has not been elucidated. In this study, we show that autophagic proteins LC3B and ATG4B mediate melanosome trafficking on cytoskeletal tracks. While studying melanogenesis, we observed spatial segregation of LC3B-labeled melanosomes with preferential absence at the dendritic ends of melanocytes. This LC3B labeling of melanosomes did not impact the steady-state levels of these organelles but instead facilitated their intracellular positioning. Melanosomes primarily traverse on microtubule and actin cytoskeletal tracks and our studies reveal that LC3B enables the assembly of microtubule translocon complex. At the microtubule-actin crossover junction, ATG4B detaches LC3B from melanosomal membranes by enzymatic delipidation. Further, by live-imaging we show that melanosomes transferred to keratinocytes lack melanocyte-specific LC3B. Our study thus elucidates a new role for autophagy proteins in directing melanosome movement and reveal the unconventional use of these proteins in cellular trafficking pathways. Such crosstalk between the central cellular function and housekeeping pathway may be a crucial mechanism to balance melanocyte bioenergetics and homeostasis.

The Secreted Protein Rv1860 of Mycobacterium tuberculosis Stimulates Human Polyfunctional CD8+ T Cells.

We previously reported that Rv1860 protein from Mycobacterium tuberculosis stimulated CD4(+)and CD8(+)T cells secreting gamma interferon (IFN-γ) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulent M. tuberculosis We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latently M. tuberculosis-infected individuals dominated by CD8(+) T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8(+) PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studied M. tuberculosis antigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4(+) T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8(+) T-cell-stimulating antigens has the potential to prevent progression of latent M. tuberculosis infection to TB disease.

IFN-γ signaling maintains skin pigmentation homeostasis through regulation of melanosome maturation.

Cellular homeostasis is an outcome of complex interacting processes with nonlinear feedbacks that can span distinct spatial and temporal dimensions. Skin tanning is one such dynamic response that maintains genome integrity of epidermal cells. Although pathways underlying hyperpigmentation cascade are recognized, negative feedback regulatory loops that can dampen the activated melanogenesis process are not completely understood. In this study, we delineate a regulatory role of IFN-γ in skin pigmentation biology. We show that IFN-γ signaling impedes maturation of the key organelle melanosome by concerted regulation of several pigmentation genes. Withdrawal of IFN-γ signal spontaneously restores normal cellular programming. This effect in melanocytes is mediated by IFN regulatory factor-1 and is not dependent on the central regulator microphthalmia-associated transcription factor. Chronic IFN-γ signaling shows a clear hypopigmentation phenotype in both mouse and human skin. Interestingly, IFN-γ KO mice display a delayed recovery response to restore basal state of epidermal pigmentation after UV-induced tanning. Together, our studies delineate a new spatiotemporal role of the IFN-γ signaling network in skin pigmentation homeostasis, which could have implications in various cutaneous depigmentary and malignant disorders.

Lsr2 of Mycobacterium leprae and its synthetic peptides elicit restitution of T cell responses in erythema nodosum leprosum and reversal reactions in patients with lepromatous leprosy.

The Lsr2 protein of Mycobacterium leprae and its synthetic peptides have been shown to elicit lymphoproliferation and gamma interferon (IFN-γ) release by peripheral blood mononuclear cells (PBMCs) of patients with lepromatous leprosy (M. Chaduvula, A. Murtaza, N. Misra, N. P. Narayan, V. Ramesh, H. K. Prasad, R. Rani, R. K. Chinnadurai, I. Nath, Infect. Immun. 80:742-752, 2012). PBMCs from 16 patients with lepromatous leprosy who were undergoing erythema nodosum leprosum (ENL) (type 2) and 5 patients with reversal reactions (RR) (type 1) were stimulated with M. leprae, recombinant Lsr2, and six end-to-end synthetic peptides (A through F) spanning the Lsr2 sequence. During the reaction all patients with ENL showed lymphoproliferation (stimulation index, >2) in response to peptides A and F, with other peptides eliciting responses in 75 to 88% of the subjects. In PBMC cultures, both lymphoproliferation and IFN-γ release for peptide E were significantly higher than for peptides B and C and recombinant Lsr2 (P < 0.05, Wilcoxon signed-rank test). Five patients with RR also showed enhanced lymphoproliferative responses and IFN-γ release in response to Lsr2, M. leprae, and peptide E. Six months postreaction, 14 patients with ENL continued to exhibit responses to Lsr2 and its peptides, with the highest responses being elicited by peptide E. However, 5 subjects showed no lymphoproliferation and had reduced IFN-γ release in response to Lsr2 peptides (P < 0.001, Kruskal-Wallis test) but responded to recombinant Lsr2. Six patients with ENL had HLA-A*68.01, which the STFPEITHI program showed to have high peptide-binding scores of 20 to 21 for peptides E, B, and C. Eleven patients had HLA-DRB1*1501 and HLA-DRB1*1502, which had high binding scores for peptides C and E. Thus, Lsr2 and its peptides are recognized in leprosy reactions during and well after the subsidence of clinical signs.

Presence of strong association of the major histocompatibility complex (MHC) class I allele HLA-A*26:01 with idiopathic hypoparathyroidism.

CONTEXT: The pathogenesis of isolated hypoparathyroidism, also referred to as idiopathic hypoparathyroidism (IH), is not clear. There is a paucity of information related to the immunogenetic basis of the disease due to its rarity. A recurrent theme of several autoimmune disorders is aberrant antigen presentation. OBJECTIVE: We investigated for the association of alleles of the human leukocyte antigen (HLA) class I and II loci with IH. PATIENTS AND CONTROLS: A total of 134 patients with IH and 902 healthy controls from the same ethnic background participated in the study. RESULTS: There was a significant increase of HLA class I alleles HLA-A*26:01 [P < 1.71 × 10(-34); odds ratio (OR) = 9.29; 95% confidence interval (CI) = 6.08-14.16] and HLA-B*08:01 (P < 8.19 × 10(-6); OR = 2.59; 95% CI = 1.63-4.04) in patients with IH compared to healthy controls. However, the association of A*26:01 was primary because B*08:01 was in linkage disequilibrium with A*26:01. Although the major histocompatibility complex (MHC) is very polymorphic, several alleles of HLA loci share key residues at anchor positions in the peptide binding pockets such that similar peptides may be presented by different MHC molecules encoded by the same locus. These allelic forms with similar anchoring amino acids have been clustered in supertypes. An analysis of HLA-A locus supertypes A01, A02, A03, and A04 revealed that supertype A01 was significantly increased (P < 9.18 × 10(-9); OR = 2.95) in IH compared to controls. However, this increase in the supertype A01 was contributed by A*26:01 because 68.7% of the A01 samples had A*26:01. Other alleles of the supertype did not show any significant differences. CONCLUSION: The strong association of HLA-A*26:01 suggests an important role of MHC class I-mediated presentation of autoantigenic peptides to CD8(+) cytotoxic T cells in the pathogenesis of IH. These data provide evidence for the autoimmune etiology of IH akin to other autoimmune disorders like type 1 diabetes and rheumatoid arthritis.

Lsr2 peptides of Mycobacterium leprae show hierarchical responses in lymphoproliferative assays, with selective recognition by patients with anergic lepromatous leprosy.

Lsr2 protein of Mycobacterium leprae was shown earlier to elicit B and T cell responses in leprosy patients (20, 28). Lymphoproliferation to M. leprae and Lsr2 antigens was observed in >70% of tuberculoid (T) patients and in 16 and 34% of lepromatous (L) patients, respectively. We focused on the M. leprae nonresponders in the lepromatous group using 22 synthetic Lsr2 peptides (end-to-end peptides A to F and overlapping peptides p1 to p16) in in vitro T cell responses. A total of 125 leprosy and 13 tuberculosis patients and 19 healthy controls from the area of endemicity (here, healthy controls, or HC) were investigated. The highest responses were observed (67 to 100%) in HC for all peptides except p1 to p3, and the lowest was observed in tuberculosis patients. Significant differences in lymphoproliferation were observed in T, L, and HC groups (analysis of variance [ANOVA], P = 0.000 to 0.015) for all end-to-end peptides except B and for p5 and p7 to p10. Hierarchical recognition between lepromatous and tuberculoid leprosy was noted for p8 (P < 0.05) and between the HC and L groups for p7 to p10, p15, and p16 (P < 0.005 to P < 0.02). Significant lymphoproliferation was observed to peptides A to F and p1 to p9, p11, p12, p15, p16 (P = 0.000 to 0.001) with 40% responding to peptides C and p16 in L patients. Lepromatous patients also showed significantly higher levels of a gamma interferon (IFN-γ) response to peptide C than to other peptides (P < 0.05). Major histocompatibility complex (MHC) class II bias for peptide recognition was not observed. These studies indicate that Lsr2 has multiple T cell epitopes that induce in vitro T cell responses in the highly infective lepromatous leprosy patients.

Transcriptional upregulation of Nrf2-dependent phase II detoxification genes in the involved epidermis of vitiligo vulgaris.

Oxidative stress is widely believed to be a contributing factor in vitiligo pathogenesis. To explore mechanisms by which epidermis responds to mounting oxidative stress, we investigated the involvement of phase II detoxification genes in vitiligo. Phase II detoxification pathways have recently been identified as being important in the regulation of epidermal skin homeostasis. In this study we show that the key transcription factor nuclear factor E2-related factor 2 (Nrf2) and the downstream genes NAD(P)H:quinone oxidase-1 (NQO-1), γ-glutamyl cystine ligase catalytic subunit (GCLC), and γ-glutamyl cystine ligase modifying subunit (GCLM) are upregulated in the lesional epidermal skin of subjects with vitiligo vulgaris. The differences between lesional and nonlesional skin were further investigated by studying the induced expression of Nrf2-dependent transcripts in skin punch biopsies using curcumin and santalol. Surprisingly, nonlesional skin showed induction of all transcripts while a similar effect was not observed for the skin punches from the lesional skin. The use of curcumin and santalol on epidermal cells showed that keratinocytes were more susceptible to apoptosis, whereas melanocytes induced phase II genes under the same concentrations with negligible apoptosis. Our studies provide new insights into the role of phase II detoxification pathway in maintaining skin homeostasis and sustaining redox balance in vitiligo patients.

Evaluation of Japanese encephalitis virus DNA vaccine candidates in rhesus monkeys [Macaca mulatta].

We have previously described DNA vaccine candidates against Japanese encephalitis virus (JEV) that were immunogenic in mice. Present study was conducted to evaluate their immunogenicity in rhesus monkeys (Macaca mulatta) and compare it with the commercial mouse brain-derived, formalin-inactivated vaccine. Groups of four monkeys were immunized with either pMEa (expressing the anchored form of the envelope protein along with the pre-membrane protein of JEV) or pMEs (expressing the secretory form of the envelope protein along with pre-membrane protein of JEV) by intra-muscular (IM, using needle) or intra-dermal (ID, using gene gun) routes. Following primary immunization with 1mg plasmid DNA given IM, or 5 microg plasmid DNA given ID, the monkeys were boosted after 1 and 2 months with 0.5mg DNA given IM or 5 microg DNA given ID, and observed for a period of 6 months. After the second booster, most of the monkeys sero-converted and developed JEV neutralizing antibodies, albeit of low titer. Importantly however, following a sham challenge with the mouse brain-derived inactivated JEV vaccine given 6 months after immunization, the neutralizing antibody titers rose rapidly indicating a vigorous anamnestic response. Based on the JEV neutralizing antibody response following the vaccination and the extent of anamnestic response generated in the immunized monkeys, plasmid pMEa was superior to pMEs. This study indicates that the JEV candidate DNA vaccine is capable of generating protective levels of JEV neutralizing antibodies in rhesus monkeys and prime the immune system effectively against a subsequent exposure to JEV.

Phenotype frequencies of autosomal minor histocompatibility antigens display significant differences among populations.

Minor histocompatibility (H) antigens are allogeneic target molecules having significant roles in alloimmune responses after human leukocyte antigen-matched solid organ and stem cell transplantation (SCT). Minor H antigens are instrumental in the processes of transplant rejection, graft-versus-host disease, and in the curative graft-versus-tumor effect of SCT. The latter characteristic enabled the current application of selected minor H antigens in clinical immunotherapeutic SCT protocols. No information exists on the global phenotypic distribution of the currently identified minor H antigens. Therefore, an estimation of their overall impact in human leukocyte antigen-matched solid organ and SCT in the major ethnic populations is still lacking. For the first time, a worldwide phenotype frequency analysis of ten autosomal minor H antigens was executed by 31 laboratories and comprised 2,685 randomly selected individuals from six major ethnic populations. Significant differences in minor H antigen frequencies were observed between the ethnic populations, some of which appeared to be geographically correlated.