Assessment of Clinicopathological Status and Outcome of Children with Tuberculous Meningitis at a Tertiary Care Hospital

Objective: The aim of this study is to report the clinic-pathological profile of children with TBM and their treatment outcome. Methodology: A retrospective observational study was conducted in the Pediatrics Department over 6 months period. Medical records of children admitted with TBM from November 2017 to May 2018 were reviewed for data collection. Data regarding clinical presentation, laboratory investigations were recorded. Patients were treated with a standard ATT regimen, and their outcome was noted. The study was approved by hospital ethics committee. Data was entered in SPSS for statistical analysis. Results: Females were predominant 39 (55.7%) and age ranged from 4 months to 13 years in this study. Only 28 (42.0%) children were fully vaccinated and had BCG scar presence. Most TBM cases were of stage II 24 (42.8%) or stage III 29 (42.8%). The frequent symptoms were fever 61 (87.1%), rigidity/irritability 35 (50.0%), and seizures 26 (37.1%). WBCs count in CSF was found below 500 in 64 (91.4%) children. There were 55 (78.5%) children with lymphocytosis and 14 (20.0%) with polymorph nuclear cells. A CT scan was suggestive of TBM in 51 (72.8%) children. Only 21 (30.0%) cases had a complete recovery whereas 17 (24.2%) recovered with sequelae and 10 (14.2%) deaths were noted. Conclusion: TBM presents with a poor clinical and pathological state in the advanced stage of the disease, and the therapy outcome is also non-satisfactory with high mortality and sequelae posing constant challenges

Human mesenchymal stem cells protect neutrophil from serum deprived cell death.

We have previously shown that human MSC (mesenchymal stem cells) inhibit the proliferation of most of the immune cells. However, there are innate immune cells such as neutrophils and other PMN (polymorphonuclear) cells that do not require an extensive proliferation prior to their effector function. In this study, the effect of MSC on neutrophils in the presence of complete and serum-deprived culture media was investigated. In the presence of MSC, the viability of neutrophils increase as measured in 24 h of incubation at various supplementation of serum concentration. We have utilized Annexin V and PI (propidium iodide) staining to confirm whether the enhancement of neutrophil's viability is due to a reduction in PCD (programmed cell death). MSC significantly rescue neutrophils from apoptosis at 1, 5 and 10% of FBS (fetal bovine serum) supplementation. The fractions of viable and dead cells were increased and decreased respectively in the presence of MSC. Our results indicate MSC rescue neutrophils from nutrient- or serum-deprived cell death. However, whether this effect is exerted through a specific signalling pathway or confining neutrophils in resting state by MSC requires further investigation

(CDRGI)-Cancer detection through relevant genes identification

Cancer is a genetic disease that is categorized among the most lethal and belligerent diseases. An early staging of the disease can reduce the high mortality rate associated with cancer. The advancement in high throughput sequencing technology and the implementation of several Machine Learning algorithms have led to significant progress in Oncogenomics over the past few decades. Oncogenomics uses RNA sequencing and gene expression profiling for the identification of cancer-related genes. The high dimensionality of RNA sequencing data makes it a complex and large-scale optimization problem. CDRGI presents a Discrete Filtering technique based on a Binary Artificial Bee Colony coupling Support Vector Machine and a two-stage cascading classifier to identify relevant genes and detect cancer using RNA seq data. The proposed approach has been tested for seven different cancers, including Breast Cancer, Stomach Cancer (STAD), Colon Cancer (COAD), Liver Cancer, Lung Cancer (LUSC), Kidney Cancer (KIRC), and Skin Cancer. The results revealed that the CDRGI performs better for feature reduction while achieving better classification accuracy for STAD, COAD, LUSC and KIRC cancer types

Human mesenchymal stem cells impair the proliferation of monocytes through cell cycle interference

Introduction: Monocytes are essential phagocytic cells of the innate immune system as they are required for the maintenance of tissue homeostasis. However, accumulation of monocytes is implicated in various chronic inflammatory diseases like coronary heart disease, atherosclerosis and in autoimmune disorders. Therefore, the number of monocytes must be carefully regulated to avoid monocyte induced inflammatory disorders. Mesenchymal stem cells (MSCs) have shown to be effective against various inflammatory diseases due to their immunosuppressive properties. The present study was designed to evaluate the less understood immunomodulatory effect of MSCs on monocyte proliferation and survival. Method: Primary monocytes were isolated from peripheral human blood using CD14+ monocyte isolation kit. The in house produced umbilical cord MSCs were co-cultured with monocytes at different ratio and time; assessed for the monocyte viability, proliferation and cell cycle. Results: Mesenchymal stem cells suppressed monocyte proliferation in a dose-dependent manner. The antiproliferative effect of MSCs was mediated by cell cycle arrest, whereby monocytes were arrested in the G0/G1 phase of the cell cycle by preventing them from progress into S and G2/M phases. Although cell cycle arrest could potentially lead to apoptosis; however, MSCs significantly enhanced the monocytes survival and inhibited apoptosis. Conclusion: Human MSCs inhibit the stimulated monocyte proliferation without inducing cellular apoptosis at in vitro. These results reveal that MSCs can be utilised to control monocytes’ quantity during an unwanted immune response to maintain homeostasis

Characterization and expression of senescence marker in prolonged passages of rat bone marrow-derived mesenchymal stem cells

The present study is aimed at optimizing the in vitro culture protocol for generation of rat bone marrow- (BM-) derived mesenchymal stem cells (MSCs) and characterizing the culture-mediated cellular senescence. The initial phase of generation and characterization was conducted using the adherent cells from Sprague Dawley (SD) rat’s BM via morphological analysis, growth kinetics, colony forming unit capacity, immunophenotyping, and mesodermal lineage differentiation. Mesenchymal stem cells were successfully generated and characterized as delineated by the expressions of CD90.1, CD44H, CD29, and CD71 and lack of CD11b/c and CD45 markers. Upon induction, rBM-MSCs differentiated into osteocytes and adipocytes and expressed osteocytes and adipocytes genes. However, a decline in cell growth was observed at passage 4 onwards and it was further deciphered through apoptosis, cell cycle, and senescence assays. Despite the enhanced cell viability at later passages (P4-5), the expression of senescence marker, β-galactosidase, was significantly increased at passage 5. Furthermore, the cell cycle analysis has confirmed the in vitro culture-mediated cellular senescence where cells were arrested at the G0/G1 phase of cell cycle. Although the currently optimized protocols had successfully yielded rBM-MSCs, the culture-mediated cellular senescence limits the growth of rBM-MSCs and its potential use in rat-based MSC research

Optimisation of laboratory procedures for isolating human peripheral blood derived neutrophils.

Functional analysis of neutrophils requires isolation of these cells in the laboratory. Current isolation procedures are time consuming and can potentially activate the resting neutrophils. Thus, in this present study, we have optimised an existing laboratory protocol for human neutrophil isolation from peripheral blood. Twenty ml of blood samples were subjected to optimised density gradient separation and dextran sedimentation to obtain a pure population of neutrophils. The efficacy of the optimised manual post isolation of neutrophils was compared with pre isolation count performed by an automated haematology analyzer. The recovery of neutrophils via our optimised methods was 65.5% in comparison with neutrophils counts at pre-isolation. The morphological analysis of isolated neutrophils indicated the purity level more than 95% using Leishman staining. Our optimised laboratory procedures for neutrophils isolation successfully harvested neutrophils with good viability, purity and post recovery yield. This procedure provides an ideal platform to separate neutrophils for in vitro studies

The effect of human Mesenchymal stem cell on neutrophil oxidative burst

Objective: Mesenchymal stem cells (MSC) are multipotent, non-haematopoietic stem cells that are capable of differentiating into different varieties of mature cell types such as osteoblasts, chondrocytes, adipocytes. and myoblasts. There is abundant evidence showing that MSC not only affect the differentiation of haematopoietic progenitors, but also the function of mature cells like lymphocytes and neutrophils. However the effect of MSC on neutrophil function and its responses is not well studied. Therefore, this study was conducted to assess the effect of MSC on neutrophil nitric oxide production. Method: Neutrophils from heparanised venous blood were isolated using Ficoll-Hypaque density gradient centrifugation followed by Dextran sedimentation and red blood cell (RBC) lysis. Isolated neutrophils were on average of 97% purity as determined by morphologic analysis. MSC were generated from human bone marrow and characterised by immunophenotyping (monoclonal antibodies CD1O5, CD73 and CD34) using a flowcytometer. In order to test the effects of MSC on neutrophil function, isolated neutrophils were co-cultured in the presence or absence of MSC at different ratios for 24 and 48 hours. The amount of nitric oxide released was used as an indication of oxidative burst and measured using the Griess assay. Result: The results indicate that MSC neither elevate the NO level when cocultured with resting neutrophils nor alone. However MSC profoundly inhibit the secretion of nitric oxide in PMA stimulated neutrophils after 241w of incubation. Conclusion: MSC exert an immunomodulatory effect on neutrophil by suppressing neutrophil oxidative burst in vitro

The efficiency of cell sorting and harvesting methods for in vitro expansion of human umbilical cord blood derived CD34+ hematopoietic stem cells

Introduction: During the last three decades hematopoietic stem cell transplantation (HSCT) has become a well-established treatment for many hematologic malignancies. The most important limitation for HSC transplantation is the low number of hematopoietic stem cells (HSC) that can lead to delayed engraftment or graft failures. Numerous attempts have been made to improve in vitro HSC expansion via optimization of various methods such as isolation techniques, supplementing with growth factors, utilizing stromal cells as feeder layer and other culture conditions. Objective: This project is aimed to decipher the efficiency of an isolation technique and retrieval of culture expanded HSC from feeder layer using two different harvesting methods. Materials and Methods: Hematopoietic stem cells from human umbilical cord blood were isolated via MACS mediated CD34+ double sorting. Then, the cells were cultured onto MSC feeder layer for 3 and 5 days. Culture expanded cells were harvested using two different harvesting method namely cell aspiration and trypsinization methods. Hematopoietic stem cell expansion index were calculated based on harvesting methods for each time point. Results: The numbers of HSC isolated from human umbilical cord blood were 1.64 x 106 and 1.20 x106 cells at single and double sortings respectively. Although the number of sorted cells diminished at the second sorting yet the yield of CD34+ purity has increased from 43.73% at single sorting to 81.40% at double sorting. Employing the trypsinization method, the HSC harvested from feeder layer showed a significant increase in expansion index (EI) as compared to the cell aspiration harvesting method (p≤ 0.05). However, the purity of CD34+ HSC was found higher when the cells were harvested using aspiration method (82.43%) as compared to the trypsinization method (74.13%). Conclusion: A pure population of CD34+ HSC can be retrieved when the cells were double sorted using MACS and expanded in culture after being harvested using cell aspiration method

The Prevalence of Hypomagnesemia in Critically Ill Patients Admitted in Medically Intensive Care Unit

Background Magnesium is the fourth most abundant cation in the human body. Hypomagnesemia can result from decreased intake, redistribution of magnesium from the extracellular to the intracellular space, or increased renal or gastrointestinal loss. Hypomagnesemia can cause severe outcomes in ill patients. So, we conducted this study to determine the frequency of hypomagnesemia in critically ill medical patients. Methods This is a Descriptive cross-sectional study involving 120 patients admitted in the medical intensive care unit (MICU) of the Holy Family Hospital, Rawalpindi, Pakistan. The study was conducted from July 2020 to September 2021. About 1 ml sample of blood was taken from each patient included and sent to the hospital laboratory for evaluation of serum magnesium levels. All the collected data was entered and analyzed on SPSS v. 23. A p-value of ≤ 0.05 was taken significant. Results In our study, the mean age of the patients was 42.76±12.77 years, and the male-to-female ratio of the patients was 1:1. The mean value of the APACHE II score of the patients was 29.68±2.571. Hypomagnesemia was found in 28 (23.33%) patients. Conclusion According to our study, the frequency of hypomagnesemia in critically ill medical patients was 23.33% (28 patients)

Generation and characterisation of human umbilical cord derived mesenchymal stem cells by explant method

Mesenchymal stem cells (MSCs) derived from human umbilical cord (UC) have been considered as an important tool for treating various malignancies, tissue repair and organ regeneration. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are better alternative to MSCs that derived from bone marrow (BM-MSCs) as they are regarded as medical waste with little ethical concern for research and easily culture-expanded. In this present study, the foetal distal end of human UC was utilised to generate MSC by explant method. Upon in vitro culture, adherent cells with fibroblastic morphology were generated with rapid growth kinetics. Under the respective inductive conditions, these cells were capable of differentiating into adipocytes and osteocytes; express an array of standard MSC’s surface markers CD29, CD73, CD90, CD106 and MHC-class I. Further assessment of immunosuppression activity revealed that MSCs generated from UC had profoundly inhibited the proliferation of mitogen-activated T lymphocytes in a dosedependent manner. The current laboratory findings have reinforced the application of explant method to generate UCMSCs thus, exploring an ideal platform to fulfil the increasing demand of MSCs for research and potential clinical use