Metabolic and immunologic alterations of ginger rhizome among streptozotocin-nicotinamide induced diabetic rats

Introduction: This study was conducted to determine immunological and metabolic effects of different concentrations of ginger rhizome (Zingiber officinale Roscoe) in streptozotocin (STZ)-nicotinamide (NA) induced diabetic rats. Methods: Forty-eight fasted male Sprague-Dawley rats were induced diabetes using a single intraperitoneal injection of NA(110 mg/kg b.w.) and STZ (65 mg/kg b.w, 15 min after NA). Diabetic rats orally received either different concentrations (250, 500 and 750 mg/kg body weight) of ginger rhizome suspension or glibenclamide (10 mg/kg body weight) for 6 weeks. Two control diabetic and normal groups were gavaged with only distilled water as a vehicle. Results: The results indicated that the lower concentrations of ginger modulated body weight, fasting blood glucose, level of triglyceride and tumor necrosis factor-α (TNF-α) (p0.05). Conclusion: Ginger indicated better impact on metabolic and immunologic parameters in lower doses of supplementation compared with high doses of treatment

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

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

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

Generation and characterisation of mesenchymal stem cells derived from human cartilage

Mesenchymal stem cells (MSCs) were initially discovered as stromal cells that possess unique characteristics as compared to other counterparts of multipotent stem cells. Besides the capability of self-renewal and differentiating into a variety of mature cells, MSCs also exert potent immuno-regulatory activities on various immune cells. This exclusive characteristic has enabled MSCs to be recognised as an ideal cell based treatment in the field of regenerative medicine, gene therapy and immunotherapy. As regeneration of cartilage tissue in situ is hampered by limited intrinsic growth, this study explores the feasibility of generating human MSCs from sports injured patients‟ cartilages and investigates the possibility to differentiate them into cartilage tissues. For this, MSCs were generated from tissues that was harvested from a non-weight bearing region of cartilage during an arthroscopy procedure and characterised based on morphology, immunophenotype and immunomodulatory properties. Furthermore, the MSCs generated from their original physiognomy (cartilage) are believed to support the cartilage regeneration much greater. The cartilage tissues in laboratory were subject to enzymatic digestions and cultured in plastic culture ware. A series of experiments were designed using the cells from passage three onwards. Initially, the cells were cultured at 200 cells/cm2 and harvested at day 10 and 12 respectively to determine the cells‟ growth kinetics and population doubling time. Cells generated from these tissues showed spindleshaped fibroblast morphology with a population doubling time of approximately 27 hours. Next, the cells were then stained with respective antibodies with fluorescent conjugated markers and analysed in flow cytometer. When the right cells‟ populations were gated, a common surface markers that are related to mesenchymal origin however not haematopoietic were observed (CD29+, CD73+, CD90+, CD105+, HLA-ABC+, CD271-, CD14 , CD19-, CD45-, CD86-, CD80-, CD34- and HLA-DR-). Besides that, these cells were also subjected to the cell differentiation analysis. The cells were allowed to confluent before cultured with the respective differentiation media according to the manufacturer‟s instructions. Cells‟ cytostaining assay and PCR analysis on isolated total RNA showed the cells are capable of differentiating into mesodermal lineages (chondrocytes, adipocytes and osteocytes). In term of stemness, human cartilage derived cells expressed the early embryonic markers of SOX2, REX1, OCT4, NANOG; hence indicating their inherent pluripotency. Such results has confirmed cartilage tissues hold the aptitude to generate mesenchymal stem cells and these cells were termed as human cartilage derived mesenchymal stem cells (hC-MSCs). Further experiments reveal that the hC-MSCs are able to suppress proliferation of activated T-lymphocytes, demonstrating that their immunomodulatory effects are analogous to bone marrow derived MSCs. In the presence of hC-MSCs, the proliferation of the T cells was severely inhibited in dose dependent manner but their activation profile was well preserved. They further affirm the requirement for the cell-to-cell contact during their immuno-inhibitory activity. These outcomes were confirmed in the hC-MSCs: T cells co-culture assay and further analysis of CD25 expressions by activated T cells shows no variations when they were cultured either with or without the presence of hC-MSCs. Furthermore when the activated T cells were co-cultured with hC-MSCs, the immune cells were arrested in G0/G1 phase of the cell cycles and their commitments into S phase were not permissible. Based on the acquired laboratory data, it has been shown that human cartilage sample could serve as a good source to generate mesenchymal stem cells and the functional properties of human cartilage mesenchymal stem cells in term of differentiating into mature chondrocytes plus ability to prevent the expansion of activated T cells has endeavoured as a new paradigm to treat destructive autoimmune diseases of joints such as rheumatoid arthritis. Moreover, this study has further strengthened the fundamental findings on human cartilage mesenchymal stem cells biology, thus adding value to the existing clinical therapy

A Simple Characterisation of Violacein Compound Derived from Chromobacterium sp. strain Dyh27s2016 and its Antimicrobial Activity Against Pseudomonas aeruginosa

Chromobacterium sp. strain Dyh27s2016 was isolated from the lake at Manipal International University. Its purple pigmented violacein is hypothesised for a broad spectrum of intriguing biological properties, specifically as an antibacterial agent against Gram-negative bacteria. Hence, the current study aimed to isolate, characterise, and review the antimicrobial property of violacein from the Dyh27s2016 strain against P. aeruginosa. The bacterial strain was cultured in nutrient broth with L-tryptophan (0.1 mg/ml); after 24 h, the bacteria were lysed with methanol (3:1 ratio) and mixed with ethyl acetate (4:1 ratio). The top layer was then separated to obtain a concentrated purple pigment. The pH was changed at varying ranges and measured with UV/Vis to characterize this pigment. Then the antimicrobial test was performed against P. aeruginosa using the microdilution method and gauged its minimal inhibitory concentration (MIC). Violacein pigment derived from Chromobacterium violacein was used as a control in all experiments. For the result, crude violacein from Chromobacterium sp. strain Dyh27s2016 was obtained, and the purple pigment in different pH was observed in varying colours; the compound was particularly decolourised at a highly alkaline solution. The pigment was also able to significantly inhibit the growth of P. aeruginosa from 200 µg/mL - 800 µg/mL. Tests show that this pigment has a maximum absorption of light wavelength at approximately 600 nm and antibiotic test results show that the pigment can be considered a potential antimicrobial drug against P. aeruginosa

Generation and characterisation of human mesenchymal stem cells derived from umbilical cord and placenta

Mesenchymal stem cells (MSC) have emerged as a great therapeutic potential in regenerative medicine and tissue engineering, hence created a vast demand for its large clinical scale production. In current study, we have generated MSC from human umbilical cord and placenta tissues that are easily accessible and direct comparisons were made in opting for a better alternate source of MSC in replacement of bone marrow. MSC were successfully generated, assessed for the morphological changes; surface protein expression via immunophenotyping; early embryonic stem cell (ESC) transcriptional factor expression via RT-PCR and mesodermal differentiation ability. UC-MSC and PLC-MSC appeared fibroblastic-like cells and expressed the common mesenchymal surface markers. Both MSC expressed the ESC markers and were able to differentiate into adipocytes, osteocytes and chondrocytes upon induction. In comparison, UC-MSC showed a significantly rapid growth kinetic with higher cell yield and shorter doubling time as compared to PLC-MSC. In our findings, both UC-MSC and PLC-MSC shared similar mesenchymal markers and properties however; UC-MSC appears as a better source of MSC as they display superior differentiation potential and growth kinetics than PLC-MSC

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

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 (3H-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

Isolation and characterization of Chromobacterium sp. fromlake water at Manipal International University

Introduction: The violet-pigmented Chromobacterium spp. are vastly located in soil and surface water of subtropical regions. Majority of the species have been identified as highly potential in bio-industries; however, the bacterial pathogenicity is largely understudied. These bacteria are resistant to multiple-drugs and infections may cause sepsis and liver abscissions. Thus, this study aimed to characterize the violet-pigmented bacteria isolated from the lake in Manipal International University and further examine its antibiotic susceptibility. Methods: The isolated violet bacteria (Dyh27s2016) were subjected to the morphology, physiology, biochemical and antibiotic susceptibility tests. Also, the species were scrutinized via the 16S rRNA and phylogenetic analysis. In addition, the lake water physicochemical properties were examined to understand the bacterial adaptability in this region. Results: Dyh27s2016 strain was found to exhibit similar morphology and physiology characteristics to Chromobacterium spp. and be closely related to Chromobacterium amazonense (98% sequence-homology). However, the biochemical analysis indicated that this strain was capable of indole production; contrarily, Chromobacterium spp. were found mostly indole negative. On top of that, this strain also tested resistant to most β-lactam and aminoglycoside antibiotics. The adaptability of the Dyh27s2016 strain in this region might be supported by the satisfactory physiochemical properties of the lake water and mainly by the low dissolve oxygen concentration. Conclusion: The morphology, physiology, biochemical and molecular characterizations of Dyh27s2016 isolate show high similarity to Chromobacterium spp. and the multi-drug resistance of this strain can potentially harbour a threat to public health if contacted by humans or animals via food or water