The impact of frost-damage on the quality and quantity of the secreted antigen-specific IgG repertoire

Freezing of alum-based vaccines drastically alters their colloidal composition and leads to irreversible cluster formation. The loss of stability is well described, but the impact of frost damage on the functionality of the induced and secreted antibody repertoire has not been studied in detail. We therefore applied our single-cell measurement platform to extract the frequencies of Immunoglobulin G-secreting cells in combination with individual secretion rates and affinities. We showed that, frost-damaged or not, the tested vaccine was able to generate similar frequencies of total and antigen-affine IgG-secreting cells. Additionally, the frost-damaged vaccine stimulated a similar T-cell cytokine secretion pattern when compared to the regularly stored vaccine. However, frost-damaged vaccines induced no efficient affinity maturation and a complete collapse of the affinity distribution was observed. This study unveiled the impact of frost-damage to alum-based vaccines on the induced secreted antibody repertoire, and illustrated the power of functional single-antibody analysis.     

Novel HLA-A2-restricted human metapneumovirus epitopes reduce viral titers in mice and are recognized by human T cells

Human metapneumovirus (HMPV) is a major cause of morbidity and mortality from acute lower respiratory tract illness, with most individuals seropositive by age five. Despite the presence of neutralizing antibodies, secondary infections are common and can be severe in young, elderly, and immunocompromised persons. Preclinical vaccine studies for HMPV have suggested a need for a balanced antibody and T cell immune response to enhance protection and avoid lung immunopathology. We infected transgenic mice expressing human HLA-A*0201 with HMPV and used ELISPOT to screen overlapping and predicted epitope peptides. We identified six novel HLA-A2 restricted CD8⁺ T cell (T_CD8) epitopes, with M_39–47 (M39) immunodominant. Tetramer staining detected M39-specific T_CD8 in lungs and spleen of HMPV-immune mice. Immunization with adjuvant-formulated M39 peptide reduced lung virus titers upon challenge. Finally, we show that T_CD8 from HLA-A*0201 positive humans recognize M39 by IFNγ ELISPOT and tetramer staining. These results will facilitate HMPV vaccine development and human studies.     

AAV-CRISPR Gene Editing Is Negated by Pre-existing Immunity to Cas9

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Suppression of PRDX4 inhibits cell proliferation and invasion of ectopic endometrial stromal cells in endometriosis

Abstract

Oxidative stress (OS) has been proposed to play a role in the development of EMs. Peroxiredoxins are a family of antioxidant proteins that exhibit peroxidase activity in a thioredoxin-dependent manner, protecting cells against OS. The Western blotting results showed that the relative expression of PRDX4 was significantly increased in ectopic endometria compared with the normal endometria of EMs-free (p?<?.05). The H₂O₂ concentration was also significantly higher in the ectopic endometrium. PRDX4 siRNA was transfected into primary ectopic endometrial stromal cells (EESCs). The viability of the transfected EESCs was measured by CCK-8 assay, and the results showed significantly decreased cell viability. Furthermore, the apoptosis rate and ROS generation in flow cytometry assays were significantly increased after the knockdown of PRDX4 expression (p?<?.05). Scratch assays and transwell assays revealed that decreased expression of PRDX4 mediated by siRNA inhibited EESC migration and invasion. In conclusion, these findings indicate the potential role of PRDX4 in the development of EMs and PRDX4 as a possible therapeutic target for EMs treatment.     

Cost-effective differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules

Significant efforts have been invested into the differentiation of stem cells into functional hepatocyte-like cells that can be used for cell therapy, disease modeling and drug screening. Most of these efforts have been concentrated on the use of growth factors to recapitulate developmental signals under in vitro conditions. Using small molecules instead of growth factors would provide an attractive alternative since small molecules are cell-permeable and cheaper than growth factors. We have developed a protocol for the differentiation of human embryonic stem cells into hepatocyte-like cells using a predominantly small molecule–based approach (SM-Hep). This 3 step differentiation strategy involves the use of optimized concentrations of LY294002 and bromo-indirubin-3’-oxime (BIO) for the generation of definitive endoderm; sodium butyrate and dimethyl sulfoxide (DMSO) for the generation of hepatoblasts and SB431542 for differentiation into hepatocyte-like cells. Activin A is the only growth factor required in this protocol. Our results showed that SM-Hep were morphologically and functionally similar or better compared to the hepatocytes derived from the growth-factor induced differentiation (GF-Hep) in terms of expression of hepatic markers, urea and albumin production and cytochrome P450 (CYP1A2 and CYP3A4) activities. Cell viability assays following treatment with paradigm hepatotoxicants Acetaminophen, Chlorpromazine, Diclofenac, Digoxin, Quinidine and Troglitazone showed that their sensitivity to these drugs was similar to human primary hepatocytes (PHHs). Using SM-Hep would result in 67% and 81% cost reduction compared to GF-Hep and PHHs respectively. Therefore, SM-Hep can serve as a robust and cost effective replacement for PHHs for drug screening and development.     

“Diabetes care at doorsteps”: A customised mobile van for the prevention,screening, detection and management of diabetes in the urban underprivileged populations of Delhi

Diabetes is on the rise in India and recently shown to be increasing in the urban underprivileged. Lack of awareness of the disease, its complications, combined with lack of financial resources among the underprivileged, often results in late detection and more complications in them. To combat this, healthcare delivered at the doorstep through the use of a customised mobile medical van is a potentially attractive option.We used a customized mobile van (included trained personnel, glucose meters, fundus evaluation camera, apparatus for detection of neuropathy and foot circulation and net enabled Skype calling for remote consultation) for educating general population regarding healthy lifestyle and screening, management and intervention in patients with diabetes.The project covered 10 underprivileged areas (n, 2,31,000 people) in Delhi. Total of 24,072 individuals (10.9% of total population) attended 352 awareness sessions. A total 3,12,347 visits (included repeat visits) were carried out for screening, education and management for obesity and diabetes. During screening (n, 16,834), 2933 subjects (18.7%) had high random blood glucose levels (>200 mg/dL) and had a blood pressure averaging 127.1 ± 23.6/81.3 ± 16.6 mm of mercury (n, 16,339). A pre-post intensive lifestyle counselling for 6 months in a subset of 352 diabetic patients (of which 77.8% i.e. n, 274 were overweight/obese) showed a significant lowering in weight (p < 0.001). In addition, 292 frontline workers and 256 paramedical workers were given training regarding lifestyle and diabetes, over 20 sessions.Based on achievements of this project of spreading awareness, screening, and management of diabetes and obesity in the large number of individuals in urban underprivileged colony, we believe this project could be extended to other cities and rural areas of India, and to other developing countries as well.     

Functional beta-cells derived from umbilical cord blood mesenchymal stem cells for curing rats with streptozotocin-induced diabetes mellitus

INTRODUCTIONThis study aimed to investigate the therapeutic response to injected human umbilical cord blood mesenchymal stem cells (UCBMSCs) among albino rats with streptozotocin (STZ)-induced diabetes mellitus.METHODSControl group (GI; n = 25) rats were fed with standard rat diet. Rats with STZ-induced diabetes mellitus without (GII; n = 25) and with (GIII; n = 25) differentiated human UCBMSCs implantation were the test groups. Rats were sacrificed in Week 11 following implantation. Liver biopsies were sectioned and stained in order to highlight both the presence and function of impregnated cells in the liver tissue.RESULTSHaematoxylin and eosin-stained sections in GI and GII rats showed normal liver architecture while GIII rats showed presence of cell clusters inside the liver tissue and around the central veins. Cell clusters with blue cytoplasm were present in sections in GIII rats but absent in GI and GII rats, indicating the presence of injected differentiated human UCBMSCs. The anti-human insulin immunostaining of GIII rats showed clusters of cells within the liver parenchyma and around central veins, indicating that these cells were active and secreting insulin.CONCLUSIONUCBMSCs are proficient in differentiating into insulin-producing cells in vivo under specific conditions and, when transplanted into the liver of albino rats with STZ-induced diabetes mellitus, were able to secrete insulin and partially control the status of diabetes mellitus in rats.     

Damage-associated molecular pattern (DAMP) activation in melanoma: investigation of the immunogenic activity of 15-deoxy, Δ12,14 prostamide J2

Metastatic melanoma is the most deadly skin neoplasm in the United States. Outcomes for this lethal disease have improved dramatically due to the use of both targeted and immunostimulatory drugs. Immunogenic cell death (ICD) has emerged as another approach for initiating antitumor immunity. ICD is triggered by tumor cells that display damage-associated molecular patterns (DAMPs). These DAMP molecules recruit and activate dendritic cells (DCs) that present tumor-specific antigens to T cells which eliminate neoplastic cells. Interestingly, the expression of DAMP molecules occurs in an endoplasmic reticulum (ER) stress-dependent manner. We have previously shown that ER stress was required for the cytotoxic activity of the endocannabinoid metabolite, 15-deoxy, Δ^12,14 prostamide J₂ (15dPMJ₂). As such, the current study investigates whether 15dPMJ₂ induces DAMP signaling in melanoma. In B16F10 cells, 15dPMJ₂ caused a significant increase in the cell surface expression of calreticulin (CRT), the release of ATP and the secretion of high-mobility group box 1 (HMGB1), three molecules that serve as surrogate markers of ICD. 15dPMJ₂ also stimulated the cell surface expression of the DAMP molecules, heat shock protein 70 (Hsp70) and Hsp90. In addition, the display of CRT and ATP was increased by 15dPMJ₂ to a greater extent in tumorigenic compared to non-tumorigenic melanocytes. Consistent with this finding, the activation of bone marrow-derived DCs was upregulated in co-cultures with 15dPMJ₂-treated tumor compared to non-tumor melanocytes. Moreover, 15dPMJ₂-mediated DAMP exposure and DC activation required the electrophilic cyclopentenone double bond within the structure of 15dPMJ₂ and the ER stress pathway. These results demonstrate that 15dPMJ₂ is a tumor-selective inducer of DAMP signaling in melanoma.