Polysialylation controls immune function of myeloid cells in murine model of pneumococcal pneumonia.

Polysialic acid (polySia) is a post-translational modification of a select group of cell-surface proteins that guides cellular interactions. As the overall impact of changes in expression of this glycan on leukocytes during infection is not known, we evaluate the immune response of polySia-deficient ST8SiaIV-/- mice infected with Streptococcus pneumoniae (Spn). Compared with wild-type (WT) mice, ST8SiaIV-/- mice are less susceptible to infection and clear Spn from airways faster, with alveolar macrophages demonstrating greater viability and phagocytic activity. Leukocyte pulmonary recruitment, paradoxically, is diminished in infected ST8SiaIV-/- mice, corroborated by adoptive cell transfer, microfluidic migration experiments, and intravital microscopy, and possibly explained by dysregulated ERK1/2 signaling. PolySia is progressively lost from neutrophils and monocytes migrating from bone marrow to alveoli in Spn-infected WT mice, consistent with changing cellular functions. These data highlight multidimensional effects of polySia on leukocytes during an immune response and suggest therapeutic interventions for optimizing immunity.

Immunohistochemical Study of p16INK4A, MIB-1 and CK17 in Pre-neoplastic and Neoplastic Epithelial Lesions of Cervix.

BACKGROUND: Cervical intraepithelial neoplasia (CIN) II and CIN III have a high progression rate to invasive squamous cell carcinoma (SCC). Histopathological assessment is known to have intra and inter-observer diagnostic discrepancies even among two panels of pathologist. Subsequently, to improve on the accuracy of histopathological examination, various IHC biomarkers have been evaluated in the biopsy of cervix. AIM: The present study was undertaken to evaluate the immunoexpression and interrelationship of p16INK4A, MIB-1 and CK17 in histopathologically diagnosed cases of CIN and invasive cervical carcinoma (ICC) which could aid in differentiating CIN and ICC from benign cervical lesions. MATERIALS AND METHODS: This study included 120 cases of cervical lesions; out of which 20 cases were each of negative for malignancy/dysplasia (NED), CIN I and CIN III, 10 cases of CIN II and 50 cases of ICC. A technique of manual tissue microarray was employed for the study of immunohistochemical markers such as p16INK4A, CK17 and MIB-1 in all cases. Results were subjected to statistical analysis. RESULTS: The difference in p16 immunoexpression between NED (0/20, 0%) and CIN+ICC (97/100, 97%) cases was statistically highly significant. (p<0.01) The sensitivity, specificity, positive and negative predictive value and diagnostic accuracy of p16 immunoexpression in comparison to histopathological diagnosis was 97%, 100%, 100%, 86.96% and 97.5% respectively. The overall agreement of p16 staining with histopathological diagnosis was 97.5% (?=0.9151 i.e. very good) The difference in MIB-1 immunoexpression between CIN-I (6/20, 30%) and CIN II+III (30/30, 100%), CIN (36/50,72%) and ICC (50/50, 100%) cases was statistically highly significant. (p<0.01) The difference in MIB-1 immunoexpression between NED (0/20, 0%) and CIN+IC (86/100, 86%) cases was statistically highly significant. (p<0.01) The sensitivity, specificity, positive and negative predictive value and diagnostic accuracy of MIB-1 immunoexpression in comparison to histopathological diagnosis was 86%, 100%, 100%, 58.82% and 88.33% respectively. The overall agreement of MIB-1 staining with H&E diagnosis was 88.33%. (?=0.6719 i.e. good) The difference in CK17 immunoexpression between CIN-I (11/20, 55%) and CIN-II+III (26/30, 86.67%) cases was statistically significant. (p=0.030) The difference in CK17 immunoexpression between CIN (37/50, 74%) and ICC (46/50, 92%) cases was statistically significant. (p=0.033) The difference in CK17 immunoexpression between NED (0/20, 0%) and CIN+ICC (83/100, 83%) cases was statistically highly significant. (p<0.01) The sensitivity, specificity, positive and negative predictive value and diagnostic accuracy of CK 17 immunoexpression in comparison to histopathological diagnosis was 82%, 100%, 100%, 52.63% and 85% respectively. The overall agreement of CK 17 staining with histopathological diagnosis was 85% (?=0.6029 i.e. moderate) The agreement between p16 and MIB-1 immunostaining was 89.16%. (?= 0.7 i.e., good) The agreement between CK17 and MIB-1 immunostaining was 86.6%. (?= 0.683 i.e., good) The agreement between p16 and CK17 immunostaining was 84.16%. (?= 0.5908 i.e., moderate) Conclusion: The findings of the present study indicate that the IHC report of p16, MIB-1 and CK-17 in CIN and ICC cases if included in each histopathology report could aid in accurate diagnosis which could facilitate in better patient management.

Human Plasma Significantly Reduces Bacteriophage Infectivity Against Staphylococcus aureus Clinical Isolates.

Bacteriophage therapy has been regaining interest as a potential therapeutic in treating a wide range of infections. However, there is a paucity of knowledge regarding numerous aspects of bacteriophage therapy, thereby hindering the development of proper treatment protocols and effective clinical trials. In this report, the activities of three bacteriophages are evaluated against clinical bacterial isolates in the presence and absence of human plasma (HP). The bacteriophages used in this experiment were residual therapeutic doses from the United States Food and Drug Administration (FDA) approved compassionate use cases to treat recalcitrant prosthetic joint infections (PJIs). Herein we demonstrate that in the presence of HP, the infectivity of these Staphylococcal bacteriophages was significantly reduced compared to the infectivity in the absence of HP. Inhibition of infectivity ranged from 48% to 81% for two methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates independently infected with the same bacteriophage and 98% for a third MRSA clinical isolate infected with a different bacteriophage. In contrast, bacteriophage infectivity of an Enterococcus faecalis clinical isolate was not affected by the presence of HP. We hypothesize that the inhibition is correlated with plasma proteins binding to Staphylococcal surface proteins masking the receptors associated with bacteriophage attachment, thereby reducing infectivity. This has clinical ramifications for bacteriophage therapy use in treating Staphylococcal bacteremia and periprosthetic joint infections.

Curcumin restores the engraftment capacity of aged hematopoietic stem cells and also reduces PD-1 expression on cytotoxic T cells.

Aging affects the functionality of hematopoietic stem cells (HSCs), and therefore, aged individuals are not preferred as donors in HSC transplantation. Such elimination leads to the restriction of donor cohort. Several efforts are being done to rejuvenate aged HSCs. Here, we show that treatment of aged mice with curcumin rejuvenates their HSCs by restoring the expression of autophagy-inducing messenger RNAs in them, and improves their engraftment capacity. Importantly, we show that curcumin is effective in rejuvenation of HSCs when administered via both, intraperitoneal as well as oral routes. Aging also affects the immune system. While elderly individuals are not immuno-deficient, they do not respond optimally to immunizations, and hence, a strategy needs to be developed to make them immunologically responsive. Programmed cell death 1 (PD-1), one of the inhibitory coreceptors, plays an important role in the regulation of autoimmunity, infectious immunity, and cancer immunity. Its expression on T cells is indicative of their exhaustion. Here, we show that curcumin reduces the frequency of PD1+ cytotoxic T cells in the spleens of aged mice. Curcumin has a proven safety profile, and hence, can be used to treat aged donors to boost the functionality of their HSCs and also to improve the immunological profile of aged individuals. These data could have implications in various other regenerative medicine protocols as well.

Autologous Hematopoietic Stem Cells Are a Preferred Source to Generate Dendritic Cells for Immunotherapy in Multiple Myeloma Patients.

In multiple myeloma (MM), dendritic cells (DCs), and their precursors are prone to malignant cell-mediated regulation of function leading to low efficacy of DC vaccine. DCs taken directly from MM patient's body or derived from monocytes are fewer in numbers and are also dysfunctional. Here, we investigated the functionality of Hematopoietic stem cell-derived DCs (SC-DCs) from MM patients. Mature-MM-SC-DCs showed all essential functions like antigen uptake, allogenic T cells simulation and migration comparable to those derived from healthy donor (HD) samples. A comparison of Mo-DCs and SC-DCs obtained from the same MM patients' samples revealed that the expression of IL-6 was higher in the precursors of Mo-DCs leading to their impaired migration. In addition, expression of CCR7 which is responsible for DCs migration was found to be lower in MM-Mo-DCs. The chromatin permissiveness as observed by H3K4me3 histone modification at the Ccr7 promoter in MM-Mo-DCs was significantly lower than those in MM-SC-DCs. Levels of Zbtb46- a hall mark DC transcription factor mRNA was also found to be reduced in MM-Mo-DCs. Cytotoxic T cells generated from MM-SC-DCs from autologous naïve T cells exhibited reduced antitumor activity because the T cells were exhausted. Blocking of CTLA-4 on autologous T cells could partially restore T cell proliferation and activation. Thus, a combination of MM-SC-DC vaccine and anti-CTLA-4 antibody may serve as a better candidate for immunotherapy of MM. This study has implications in increasing the efficacy of cancer immunotherapy in MM.

A comparative study of safety and efficacy of ultrasound-guided infra-clavicular axillary vein cannulation versus ultrasound-guided internal jugular vein cannulation in adult cardiac surgical patients.

Background: Ultrasound (US)-guided internal jugular vein (IJV) cannulation is a widely accepted standard procedure. The axillary vein (AV) in comparison to the subclavian vein is easily visualized, but its cannulation is not extensively studied in cardiac patients. Aims: This study is an attempt to study the efficacy of real-time US-guided axillary venous cannulation as a safe alternative for the time-tested US-guided IJV cannulation. Design: This is a prospective randomized controlled study. Materials and Methods: A total of 100 adult patients scheduled for cardiac surgery were divided equally in Group A-US-guided IJV cannulation, and Group B-US-guided axillary venous cannulation. Under local anesthesia and real-time US guidance the IJV or AV was secured. The access time, guidewire time, and procedure time were noted. Furthermore, the number of needle attempts, malposition, change of site, and complications were noted. Results: The data were analyzed for 49 patients in Group A and 48 patients in the Group B due to exclusions. The access time and the guidewire time were comparable in both groups. The first attempt needle puncture was successful for the IJV group in 98% of patients in comparison to 95% of patients in Group B. Guidewire was passed in the first attempt in 94% in Group A and 89% in the Group B. Except for arterial puncture in one case in group A, the complications were insignificant in both groups. Conclusion: The study shows that the US-guided AV cannulation may serve as an effective alternative to the IJV cannulation in cardiac surgery.

Freezing of dendritic cells with trehalose as an additive in the conventional freezing medium results in improved recovery after cryopreservation.

BACKGROUND: Dendritic cell (DC) vaccination involves administration of multiple doses. Cryopreservation of tumor antigen-pulsed DCs can provide a ready to use vaccine source and eliminate the need of frequent withdrawal of the patient's blood for vaccine preparation. The aim of this study was to assess the effect of addition of trehalose in the freezing medium on the recovery of DCs after cryopreservation. STUDY DESIGN AND METHODS: DCs were generated from mononuclear cells from apheresis samples of healthy donors. For long-term storage of 6 months, cells were frozen with a rate-controlled programmable freezer and stored in liquid nitrogen. For short-term storage of 1 month, cells were frozen and stored at -80°C. DCs frozen with Iscove's Modified Dulbecco's Medium + 10% dimethyl sulfoxide + 20% fetal bovine serum served as the control group, while the test group was additionally supplemented with 50 µg/mL of trehalose. After revival of control and test DCs, they were assessed for viability, morphology, phenotype, and functions. RESULTS: The addition of trehalose to the conventional freezing medium helped to preserve the viability and functionality of DCs better than dimethyl sulfoxide alone in both long- and short-term cryopreservation. Trehalose also protected the mitochondrial membrane potential and cytoskeleton integrity of DCs, which are necessary for their functionality. Mediators of the intrinsic apoptotic pathway like Caspase-9 and Bim-1 were found to be low in the test. CONCLUSION: Supplementation of conventional freezing medium with trehalose results in better quality of DCs revived after cryopreservation. This finding could help improve DC vaccine preparation for cancer immunotherapy.

Compromised functionality of monocyte-derived dendritic cells in multiple myeloma patients may limit their use in cancer immunotherapy.

Dendritic cells (DCs) have the potential to elicit long-lasting anti-tumour immune responses. Most of the clinical trials of anti-cancer DC vaccines are based on monocyte-derived DCs (Mo-DCs). However, their outcomes have shown limited promise especially in multiple myeloma (MM) patients. Here, we investigated whether in vitro generated Mo-DCs from MM patients (MM-DCs) possess impaired functionality, thus contributing to the limited success of DC vaccines. We generated MM-DCs and compared them with DCs from healthy donors (HD-DCs). The yield of DCs in MM was 3.5 fold lower than in HD sets. However morphology, phenotype, antigen uptake and allo-T cell stimulation were comparable. Migration and secretion of IL12p70 and IFN-γ (in DC-T cell co-cultures) were significantly reduced in MM-DCs. Thus, MM-DCs were compromised in functionality. This impairment could be attributed to autocrine secretion of IL6 by MM-monocytes and activation of their P38 MAPK pathway. This indicates a need to look for alternative sources of DCs.

Derivation of human iPSC line NCCSi002-A from umbilical cord blood (UCB) CD34+cells of donor from Indian ethnicity.

We discuss the reprogramming of CD34+ cells isolated from UCB of a healthy female child of Indian ethnicity. The CD34+cells were nucleofected using episomal vectors expressing Oct4, Sox2, L-Myc, Klf4, Lin28 and p53DD (negative mutation in p53). The colonies were stained for alkaline phosphatase and evaluated for pluripotency marker expression by PCR, immunofluorescence and flow-cytometry. The safety of cells was confirmed by absence of plasmid in subsequent passages by PCR. G-banded karyotype demonstrated a stable genome. The ability of tri-lineage differentiation was confirmed by specific marker expression by immunofluorescence invitro and teratoma formation invivo.

Establishment of human iPSC line NCCSi003-A from CD34+cells of peripheral blood collected during apheresis of healthy donor from Indian ethnicity.

We present generation of iPSCs from CD34+ cells isolated from peripheral blood, collected during apheresis of a healthy female individual. We nucleofected the CD34+cells by episomal vectors containing Oct4, Sox2, L-Myc, Lin28, Klf4 and p53DD (dominant negative mutation in p53). The resultant colonies showed cobble-stone appearance and stained positive for alkaline phosphatase. The colonies demonstrated presence of pluripotency markers by immunofluorescence, flow-cytometry and PCR. The plasmids were lost from cells subsequently during passages as assessed by PCR. Karyotype analysis demonstrated a stable genome. The cells had capability to differentiate to cells from all three-germ lineages in vitro.

The brain-dead donor: An anaesthesiologist's perspective.

The brain-dead organ donation programme is slowly gathering momentum in India. There is a long way to go before our cadaver donor numbers, currently at 0.35 per million reaches 35 per million as is the case in countries like Spain. Each donor, therefore, has to be managed immaculately. The anaesthesiologists will be well served by familiarising themselves with the challenges during the crucial period preceding and during the actual harvest of organs in a brain-dead donor. There are significant opportunities for anaesthesiologists to make great contributions in this cause due to their unique skill sets and perspective. A robust brain-dead cadaver programme will go a long way in saving numerous lives as well as reduce the requirements of the living donor programme. A well-managed harvest will ensure good quality organs and an overall superior outcome in the recipients.