Prevalence of multi-antimicrobial resistant non-typhoidal Salmonella isolated from filth flies at wet markets in Klang, Malaysia, and their survival in the simulated gastric fluid.

Filth flies at wet markets can be a vector harbouring multiple antimicrobial-resistant (MAR) nontyphoidal Salmonella (NTS), and such strains are a significant threat to public health as they may cause severe infections in humans. This study aims to investigate the prevalence of antimicrobial-resistant NTS, especially Salmonella Enteritidis and S. Typhimurium harboured by filth flies at wet markets, and investigate their survival in the simulated gastric fluid (SGF). Filth flies (n = 90) were captured from wet markets in Klang, Malaysia, and processed to isolate Salmonella spp. The isolates (n = 16) were identified using the multiplex-touchdown PCR and assessed their antimicrobial susceptibility against 11 antimicrobial agents. Finally, three isolates with the highest MAR index were subjected to SGF survival tests. It was observed that 17.8 % of flies (n = 16/90) harbouring Salmonella, out of which 10 % (n = 9/90) was S. Enteritidis, 2.2 % (n = 2/90) was S. Typhimurium, and 5.6 % was unidentified serotypes of Salmonella enterica subsp. I. 43.8 % (n = 7/16) were confirmed as MAR, and they were observed to be resistant against ampicillin, chloramphenicol, kanamycin, streptomycin, and nalidixic acid. Three strains, F35, F75, and F85 demonstrated the highest MAR index and were able to survive (>6-log10) in the SGF (180 min), indicating their potential virulence and invasiveness. This study provides significant insights into the prevalence and severity of MAR nontyphoidal Salmonella harboured by filth flies in wet markets, which may help inform strategies for controlling the spread and outbreak of foodborne disease.

Characterisation and immunosuppressive activity of human cartilage-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) exert potent immuno-regulatory activities on various immune cells and also differentiate into various mesodermal lineages besides retaining a distinct self-renewal ability. Such exclusive characteristics had enabled MSCs to be recognised as an ideal source for cell-based treatment in regenerative medicine and immunotherapy. Thus, considering MSCs for treating degenerative disease of organs with limited regenerative potential such as cartilage would serve as an ideal therapy. This study explored the feasibility of generating human cartilage-derived MSCs (hC-MSCs) from sports injured patients and characterised based on multipotent differentiation and immunosuppressive activities. Cartilage tissues harvested from a non-weight bearing region during an arthroscopy procedure were used to generate MSCs. Despite the classic morphology of fibroblast-like cells and a defined immunophenotyping, MSCs expressed early embryonic transcriptional markers (SOX2, REX1, OCT4 and NANOG) and differentiated into chondrocytes, adipocytes and osteocytes when induced accordingly. Upon co-culture with PHA-L activated T-cells, hC-MSCs suppressed the proliferation of the T-cells in a dose-dependent manner. Although, hC-MSCs did not alter the activation profile of T cells significantly, yet prevented the entering of activated T cells into S phase of the cell cycle by cell cycle arrest. The present study has strengthened the evidence of tissue-resident mesenchymal stem cells in human cartilage tissue. The endogenous MSCs could be an excellent tool in treating dysregulated immune response that associated with cartilage since hC-MSCs exerted both immunosuppressive and regenerative capabilities.

Preliminary study on overproduction of reactive oxygen species by neutrophils in diabetes mellitus.

AIM: To assess the amount and pattern of reactive oxygen species (ROS) production in diabetic patient-derived neutrophils. METHODS: Blood samples from type 2 diabetes mellitus (DM) patients and volunteers (controls) were subjected to neutrophil isolation and the assessment of neutrophil oxidative burst using chemiluminescence assay. Neutrophils were activated by using phorbol myristate acetate (PMA) and neutrophils without activation were kept as a negative control. The chemiluminescence readings were obtained by transferring cell suspension into a 1.5 mL Eppendorf tube, with PMA and luminol. Reaction mixtures were gently vortexed and placed inside luminometer for a duration of 5 min. RESULTS: Our results showed that in the resting condition, the secretion of ROS in normal non-diabetic individuals was relatively low compared to diabetic patients. However, the time scale observation revealed that the secreted ROS declined accordingly with time in non-diabetic individuals, yet such a reduction was not detected in diabetic patients where at all the time points, the secretion of ROS was maintained at similar magnitudes. This preliminary study demonstrated that ROS production was significantly higher in patients with DM compared to non-diabetic subjects in both resting and activated conditions. CONCLUSION: The respiratory burst activity of neutrophils could be affected by DM and the elevation of ROS production might be an aggravating factor in diabetic-related complications.

Evaluation of metabolic and immunological changes in streptozotocin-nicotinamide induced diabetic rats.

Type 2 diabetes is a chronic disease with growing public health concern globally. Finding remedies to assist this health issue requires recruiting appropriate animal model for experimental studies. This study was designated to evaluate metabolic and immunologic changes in streptozotocin-nicotinamide induced diabetic rats as a model of type 2 diabetes. Male rats were induced diabetes using nicotinamide (110 mg/kg) and streptozotocin (65 mg/kg). Following 42 days, biochemical and immunological tests showed that diabetic rats had higher levels of blood glucose, WBC, certain abnormalities in lipid profile and insufficient mitogenic responses of lymphocytes (p<0.05). However, the status of the total antioxidant, inflammatory biomarkers and other parameters of full blood count (except HCT) were not significantly altered. Phenotyping assay indicated insignificant lymphocyte subtype imbalances excluding a significant rise in the level of CD4+CD25+ marker (p<0.05). This model of diabetic animals may represent some but not all symptoms of human type 2 diabetes.

Mesenchymal stem cells of human placenta and umbilical cord suppress T-cell proliferation at G0 phase of cell cycle.

Mesenchymal stem cells (MSC) generated from human umbilical cord (UC-MSC) and placenta (PLC-MSC) were assessed and compared for their immunomodulatory function on T cells proliferation by analysis of the cell cycle. Mitogen stimulated or resting T cells were co-cultured in the presence or absence of MSC. T-cell proliferation was assessed by tritiated thymidine ((3) H-TdR) assay and the mechanism of inhibition was examined bycell cycle and apoptosis assay. Both UC-MSC and PLC-MSC profoundly inhibited the proliferation of T-cell, mainly via cell-to-cell contact. MSC-mediated anti-proliferation does not lead to apoptosis,but prevented T cells from entering S phase and they therefore accumulated in the G(0) /G(1) phases. The anti-proliferative activity of MSC was related to this cell cycle arrest of T-cell. UC-MSC produced a greater inhibition than PLC-MSC in all measured parameters.

Isolation and characterisation of mesenchymal stem cells derived from human placenta tissue.

AIM: To explore the feasibility of placenta tissue as a reliable and efficient source for generating mesenchymal stem cells (MSC). METHODS: MSC were generated from human placenta tissue by enzymatic digestion and mechanical dissociation. The placenta MSC (PLC-MSC) were characterized for expression of cell surface markers, embryonic stem cell (ECS) gene expression and their differentiation ability into adipocytes and osteocytes. The immunosuppressive properties of PLC-MSC on resting and phytohemagglutinin (PHA) stimulated allogenic T cells were assessed by means of cell proliferation via incorporation of tritium thymidine ((3)H-TdR). RESULTS: The generated PLC-MSC appeared as spindle-shaped cells, expressed common MSC surface markers and ESC transcriptional factors. They also differentiated into adipogenic and osteogenic lineages when induced. However, continuous cultivation up to passage 15 caused changes in morphological appearance and cellular senescence, although the stem cell nature of their protein expression was unchanged. In terms of their immunosuppressive properties, PLC-MSC were unable to stimulate resting T cell proliferation; they inhibited the PHA stimulated T cells in a dose dependent manner through cell to cell contact. In our study, MSC generated from human placenta exhibited similar mesenchymal cell surface markers; MSC-like gene expression pattern and MSC-like differentiation potential were comparable to other sources of MSC. CONCLUSION: We suggest that placenta tissues can serve as an alternative source of MSC for future experimental and clinical studies.

Immunomodulatory activity of polyphenols derived from Cassia auriculata flowers in aged rats.

The immunomodulatory activity of Cassia auriculata (CA)-derived polyphenols was tested on aged rats. Rats (24-26 months old) were given CA polyphenols supplementation at doses of 25, 50, and 100 mg/kg for 28 days. Flow cytometry analysis of CA polyphenols-treated aged rats showed increased T and B cells percentage along with enhanced proliferation of splenocytes in both resting and LPS-stimulated cells. Increased percentage of pan T cells is further supported by an elevation of CD4+, CD8+, and CD4+CD25+ regulatory cells. In terms of innate immune cell activity, CA polyphenol supplementation reduced the oxidative burst activity of neutrophils in response to PMA and Escherichia coli activation. Our results collectively show that polyphenols derived from CA boost T cell immunity by increasing the number of T cells and its sensitivity towards stimulants and decreasing ROS production by neutrophils that could potentially harm multiple biological systems in aged individuals.