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Sodium glucose cotransporter-2 inhibitors (SGLT2-I) are the most recently approved drugs for type 2 diabetes (T2D). Recent clinical trials of these compounds reported beneficial cardiovascular (CV) and renal outcomes. A major cause of vascular dysfunction and CV disease in diabetes is hyperglycemia associated with inflammation and oxidative stress. Pre-clinical studies demonstrated that SGLT2-I reduce glucotoxicity and promote anti-inflammatory effects by lowering oxidative stress.To investigate the effects of SGLT2-I on markers of oxidative stress, inflammation, liver steatosis, and fibrosis in patients of T2D with non-alcoholic fatty liver disease (NAFLD).We referred fifty-two consecutive outpatients treated with metformin monotherapy and exhibiting poor glycemic control to our centre. We introduced the outpatients to an SGLT2-I (dapagliflozin, empagliflozin, or canagliflozin;Add-on therapy resulted in improved glycemic control and reduced fasting blood glucose in both groups. Of note, following treatment for six months, a reduction of FLI and APRI, as well as of the FibroScan result, was reported in patients treated with SGLT2-I, but not in the OTHER group; furthermore, in the SGLT2-I group, we reported lower circulating levels of interleukin (IL)-1β, IL-6, tumor necrosis factor, vascular endothelial growth factor, and monocyte chemoattractant protein-1, and higher levels of IL-4 and IL-10. We did not observe any modification in circulating interleukins in the OTHER group. Finally, serum HNE- and MDA-protein adducts decreased significantly in SGLT2-I rather than OTHER patients and correlated with liver steatosis and fibrosis scores.The present data indicate that treatment with SGLT2-I in patients with T2D and NAFLD is associated with improvement of liver steatosis and fibrosis markers and circulating pro-inflammatory and redox status, more than optimizing glycemic control.

BACKGROUND: Elevated levels of the enzymes gamma-glutamyltransferase (GGT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and C-reactive protein (CRP) have been shown to be associated with increased risk of cardiovascular disease (CVD). Objective: To assess cross-sectional relationships between biomarkers GGT, ALT, AST, ALP and CVD in adult Canadian population. METHODS: The Canadian Health Measures Surveys (CHMSs) are a series of cross-sectional national surveys and collect information on indicators of general health and wellness of Canadians. The CHMS has four components. We used data from the first three components (for Study participants ≥ 20 years) from CHMS cycles 1 through 5. RESULTS: Multivariable logistic regression revealed: immigration status [Odds ratio (OR)(95% Confidence Interval (95% CI)) = 0.67 (0.53–0.85), reference category (RC)-no-immigrant] education [1.38(1.10–1.75), RC- > secondary education]; smoking status [ex-smokers: 1.16(0.89–1.51); current smokers: 1.41(0.98–2.05), RC-non-smoker]; and income [middle income: 0.69(0.43–1.10); high income: 0.49(0.29–0.83); RC-lower income] were significantly associated with CVD prevalence. CONCLUSION: The relationship of GGT with CVD prevalence changed among age groups and body mass index categories; was different for males and females; and diabetes was an effect modifier in the relationship between AST and CVD prevalence. Socio-economic factors were significantly associated with CVD prevalence.

Balanced gut microbial composition of the host plays a crucial role in maintaining harmony among various physiological processes to maintain physiological homeostasis. Immunity and metabolism are the two physiologies mainly controlled by the gut microbiota. Reports suggested that gut microbial composition and diversity alteration are the leading causes of the host's healthy homeostasis alteration or a diseased state. The extent of gut perturbation depends on the perturbing agents' strength, chemical nature, and mode of action. In the current report, we have studied the effects of different perturbing agents on gut microbial dysbiosis and its impact on host immunity and metabolism.We studied the perturbation of gut microbial composition and diversity using next-generation sequencing and further investigated the changes in host immune and metabolic responses.Enrichment or abolition of a particular phylum or genus depended on the perturbing agents. In the current study, treatment with neomycin yielded an increase in the Bacteroidetes phylum. Vancomycin treatment caused a significant rise in Verrucomicrobia and Proteobacteria phyla. The treatment with AVNM and DSS caused a substantial increase in the Proteobacteria phylum. The gut microbial diversity was also lowest in AVNM treated group. The altered gut microbial composition ultimately altered the immune responses at localized and systemic levels of the host. Gut dysbiosis also changed the systemic level of SCFAs.This study will help us understand how the enrichment of a particular phylum and genus maintains the host's immune responses and metabolism.

The relationship between liver enzymes and Metabolic Syndrome (MetS) in different populations, including Canadians, is not consistent and well understood. We used the Canadian Health Measures Survey data (Cycles 3 and 4) to examine the cross-sectional relationships between select liver biomarkers and MetS in the adult Canadian population. The biomarkers selected were gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST), and alkaline phosphatase (ALKP).Fasting blood samples (FBS) were collected from adults above the age of 20 years for Cycle 3 and Cycle 4 (MetS was prevalent in 32.3% of subjects. BMI was an effect modifier in the relationship between GGT and MetS prevalence, while sex was an effect modifier in the relationship between ALKP and MetS prevalence; and age was an effect modifier in the relationship between AST and MetS prevalence.Since the mechanisms to underpin the associations between the liver enzymes activity and MetS are unknown, further epidemiologic investigations using longitudinal designs are necessary to understand these associations.

The consequence of treatment with antibiotics on the gut microbiota can be destructive. The antibiotics, however, can be utilized to understand the role of gut microbiota on the host physiology. Earlier, we reported the efficacy of vancomycin in gut microbiota perturbation. We continued to understand the effect of restoration kinetics of perturbed gut microbiota on the immunity and behavior of Th1 (C57BL/6)- and Th2 (BALB/c)-biased mice. We studied restoration kinetics of the gut microbiota for two months following the withdrawal of vancomycin treatment in both mice strains. We analyzed cecal microbiome composition, different behavioral assays, and expression of select genes associated with stress and barrier function in gut and brain. Metagenomic analysis of gut microbiota revealed that the treatment with vancomycin caused a significant decrease in the relative abundance of Firmicutes and Bacteroidetes phyla with a time-dependent increase in Proteobacteria and Verrucomicrobia phyla. Maximum restoration (> 70%) of gut microbiota happened by the 15th day of withdrawal of vancomycin. BALB/c mice showed a more efficient restoration of gut microbiota compared to C57BL/6 mice. We established the correlation patterns of gut microbiota alteration and its effect on (a) the behavior of mice, (b) expression of key brain molecules, and (c) immunity-related genes. The results revealed that the gut microbiome profiling, behavior, and immune responses varied significantly between Th1- and Th2-biased mice. By withdrawing the treatment with vancomycin of major gut microbes, important physiological and behavioral changes of both mice strains returned to the normal (untreated control) level.

During the early postnatal life, gut microbiota development experiences dynamic changes in their structural and functional composition. The postnatal period is the critical window to develop a host defense mechanism. The maturation of intestinal mucosal barrier integrity is one of the essential defense mechanisms to prevent the entry of pathogens. However, the co-development of intestinal microbial colonization, formation of barrier integrity, and intestinal epithelial cell layer is not entirely understood.We studied the gut microbial composition and diversity using 16S rRNA marker gene-based sequencing in mice to understand postnatal age-dependent association kinetics between gut microbial and intestinal development. Next, we assessed the intestinal development by in vivo gut permeability assay, mRNA gene expression of different tight junction proteins and intestinal epithelial cell markers, goblet cells population, villus length, and cecal IgA quantification.Our results showed a significant shift in gut microbial structural and functional composition from postnatal day 14 onwards with early life Proteobacteria abundance. Relative abundance of Verrucomicrobia was maximum at postnatal day 14 and showed a gradual decrease over time. We also observed an age-dependent biphasic pattern in barrier integrity improvement and differentiation of intestinal epithelial cells (IECs). A significant improvement in barrier integrity between days 1 and 7 showed the host factor contribution, while that beyond day 14 revealed an association with changes in microbiota composition. Our temporal correlation analysis associated Bacteroidetes phylum with the mucosal barrier formation during postnatal development.The present study revealed the importance and interplay of host factors and the microbiome in gut development and intestinal mucosal homeostasis.

The health and economic burden of colitis is increasing globally. Understanding the role of host genetics and metagenomics is essential to establish the molecular basis of colitis pathogenesis. In the present study, we have used a common composite dose of DSS to compare the differential disease severity response in C57BL/6 (Th1 biased) and BALB/c (Th2 biased) mice with zero mortality rates. We employed multi-omics approaches and developed a newer vector analysis approach to understand the molecular basis of the disease pathogenesis. In the current report, comparative transcriptomics, metabonomics, and metagenomics analyses revealed that the Th1 background of C57BL/6 induced intense inflammatory responses throughout the treatment period. On the contrary, the Th2 background of BALB/c resisted severe inflammatory responses by modulating the host’s inflammatory, metabolic, and gut microbial profile. The multi-omics approach also helped us discover some unique metabolic and microbial markers associated with the disease severity. These biomarkers could be used in diagnostics.

Diet maintains health by regulating host metabolism and immunity. The results revealed the consequence of starch-, unsaturated fat-, and saturated fat-rich diets on differentially immune-biased mice C57BL/6 and BALB/c. Time-course of various diets on differentially immune-biased mice revealed that starch-rich and unsaturated fat-rich diets reduced insulin resistance (IR) and visceral adiposity in BALB/c mice while a saturated fat-rich diet enhanced both parameters. In C57BL/6 mice, a fat-rich diet enhanced IR with time while visceral adiposity remained unchanged. Eight weeks' consumption of saturated fat-rich diet induced highest visceral adiposity in C57BL/6 mice, while the same diet resulted in the maximum IR in BALB/c mice. The current report presented a detailed metabolomic analysis of diets and evaluated differential index of each treatment for each mouse strain using a vector analysis of the multivariate linear discriminant data. The outcome identified metabolites that affected lipid and glucose metabolism to establish the inter-strain physiological differences.

Pathogenic infection is one of the major causes of death in newborns. Antibiotic based therapies are still the major mode of treatment for infection. Increased usage of antibiotics leads to selective evolution of microorganisms and causes diseases in adulthood. Attempts to develop alternatives to antibiotics did not yield much success. A recent viable trend is to identify novel probiotics that could alleviate problems associated with over usage of antibiotics. We screened three different Lactobacillus species to establish their efficacy in neonates in protecting against Salmonella challenge. The methodologies employed are metagenomics, metabonomics, transcriptional profiling, molecular assays and behavioral studies. Among the three probiotics used, only Lactobacillus rhamnosus GG (LGG) treatment of the neonates resulted in rescuing of 80% of the Salmonella-infected mice. We have shown that LGG (MTCC #1408) can prevent Salmonella mediated infection in neonates. In the current report, results from histopathology, gene expression, neutrophil infiltration, metabolite and metataxonomic profiling, and protein level data suggested that LGG treatment of the neonates enhanced anti-inflammatory cytokine expression and increased the gut barrier function. The current report establishes the potential use of LGG in clinical intervention of infectious diseases.

Higher organisms, especially mammals, harbor diverse microbiota in the gut that plays a major role in maintaining health and physiological homeostasis. Perturbation of gut flora helps identifying their roles. Antibiotics are potent perturbing agents of microbiome. Select antibiotics like vancomycin, neomycin, and AVNM (an antibiotic cocktail containing ampicillin, vancomycin, neomycin, and metronidazole) were used to perturb the gut microbiota of C57BL/6 male mice to understand their roles in host immunity and metabolism. The current study revealed that the resulting gut microbial composition was different, and diversity (at the phylum and genus level) was reduced differentially following each antibiotic treatment. Vancomycin treatment caused a significant increase in Verrucomicrobia and Proteobacteria phyla. The treatment with neomycin yielded an increase in the Bacteroidetes phylum, while the treatment with AVNM led to an increase in Proteobacteria phylum with lowest diversity of microbiome in the gut. The current results also revealed that the different antibiotics treatment caused variation in the cecal index, expression of immune genes (TNF-α, IL-10, IFN-γ) in the colon, and short-chain fatty acids (SCFA) level in the blood of mice. A strong correlation was observed for antibiotic-induced differential dysbiosis patterns of gut microbiota and the altered immune and SCFA profile of the host. The outcome of the present study could be clinically important.

A novel disease, COVID-19, is sweeping the world since end of 2019. While in many countries, the first wave is over, but the pandemic is going through its next phase with a significantly higher infectability. COVID-19 is caused by the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that seems to be more infectious than any other previous human coronaviruses. To understand any unique traits of the virus that facilitate its entry into the host, we compared the published structures of the viral spike protein of SARS-CoV-2 with other known coronaviruses to determine the possible evolutionary pathway leading to the higher infectivity. The current report presents unique information regarding the amino acid residues that were a) conserved to maintain the binding with ACE2 (Angiotensin-converting enzyme 2), and b) substituted to confer an enhanced binding affinity and conformational flexibility to the SARS-CoV-2 spike protein. The present study provides novel insights into the evolutionary nature and molecular basis of higher infectability and perhaps the virulence of SARS-CoV-2.

The gut is the largest reservoir of the resident microbiota. The microbiota can affect the host behavior and immunity. While the consequence of treatment with antibiotics on the gut microbiota can be destructive but can be utilized as a tool to understand the host immunity and behavior. The magnitude of perturbation and time needed for the restoration of gut microbiota can depend on the immune bias of the host. In the current study, we therefore, observed the perturbation and restoration kinetics of gut microbiota following treatment with vancomycin and its effect on the host physiology in both Th1-(C57BL/6) and Th2-(BALB/c) biased mice. A comparative metagenomic analysis revealed that the treatment with vancomycin caused a significant decrease in the abundance of Firmicutes and Bacteroidetes phyla and an initial increase in Proteobacteria. Increase in Proteobacteria decreased with continued treatment with vancomycin to result into a significant rise in Verrucomicrobia phylum. We established the patterns of gut microbiota alteration and its effect on a) the behavior of mice, b) expression of key brain molecules and b) immunity related genes. We followed the gut microbiome restoration for a period of two months following withdrawal of treatment with vancomycin. Maximum restoration (>70%) of gut microbiota happened by the 15th day of withdrawal. BALB/c mice showed a more efficient restoration of gut microbiota compared to C57BL/6 mice. The results, in general, revealed that along with the restoration of major gut microbes, important physiological and behavioral changes of both mice strains returned to the normal level.

Colitis is a complex and multifactorial disease with unknown etiology. To understand colitis, a number of models were developed in mouse. Colitis, in these models, was induced using either chemicals or select bacteria. Dextran sulfate sodium (DSS) induced colitis model was used widely, over other models, because of the simpler properties of DSS and similarities of the model with colitis in humans. The available models of DSS induced colitis require either longer time to understand the chronic stages of the disease or a shorter time period to study the acute phase. Currently, no composite models exist that can be used to study the acute, chronic and recovery stages of colitis within a reasonably shorter period of time. In the current study, we have developed a newer model system in two differently immune biased (Th-1 and Th-2) mice strains using DSS. We have developed the DSS model to compare the response in C57BL/6 (Th1 biased) and BALB/c (Th2 biased) mice models. We have standardized the dosage for both C57BL/6 and BALB/c mice with zero mortality rates. Using the model developed we studied the acute, chronic and recovery phases of colitis. The differential responses of C57BL/6 and BALB/c revealed that the disease was more severe in C57Bl/6. BALB/c, on the contrary, recovered from the diseased condition more quickly. The current report will further help to unravel the disease etiology based on immunological background of the host.

Aims The aim was to understand the time-dependent antibiotic-induced perturbation pattern of gut microbiota and its effect on the innate immune and metabolic profile of the host. Methods and results Vancomycin was administered at 50 mg kg-1 of body weight twice daily for six consecutive days to perturb the gut microbiota of C57BL/6 (Th1-biased) and BALB/c (Th2-biased) mice. Following treatment with vancomycin, we observed a reduction in the abundance of phyla Firmicutes and Bacteroides and an increase in Proteobacteria in the gut for both strains of mice following treatment with vancomycin till day 4. Abundance of Akkermansia muciniphila of Verrucomicrobia phylum also increased, from day 5 onwards following vancomycin treatment. The time-dependent variation of gut microbiota was associated with increased (i) expression of toll-like receptors and inflammatory genes such as TNF-α, IL-6, and IL-17, (ii) gut barrier permeability and (iii) blood glucose level of the host. The results also showed that (i) transplantation of cecal microbiota from vancomycin-treated day 6 mice to day 3 vancomycin-treated mice helped in restoring blood glucose level in C57BL/6 mice and (ii) short-chain fatty acids like acetate, butyrate and propionate changed with the alteration of gut microbiota to induce differential regulation of host immune response. Conclusions The current results revealed that an increase in A. muciniphila led to decreased inflammation and increased rate of glucose tolerance in the host. The treatment, with vancomycin till day 4, increased expression of inflammatory genes. The continuation of vancomycin for two more days reversed the effects. The effects were significantly more in C57BL/6 than BALB/c mice. Significance and impact of the study The current study established that the treatment with vancomycin till day 4 increased pathogenic bacteria but day 5 onwards provided significant health-related benefits to the host by increasing A. muciniphila more in C57BL/6 than BALB/c mice.

The composition, overtly abundance, and diversity of gut microbiota, play a significant role in maintaining physiological homeostasis with age. Reports revealed that the gut microbial profile might be correlated with immunity and metabolism. It is, therefore, tantamount to know if an older individual can achieve the immunity and metabolic profile of a younger individual by receiving the gut microbiome of a younger individual. In the current report, we have studied the effects of cecal microbiota transplantation (CMT) from younger to older mice.In this study, older BALB/c mice (23 weeks) received CMT from younger BALB/c mice (3 weeks).CMT recipient mice showed altered expressions of immune and tight junction protein genes in the colon of mice, while the non-CMT recipient mice did not. Older mice were treated with AVNM to make them compatible with CMT. Further data from metabolite studies revealed that AVNM treatment mainly affected the aromatic amino acid biosynthesis pathway while CMT mostly affected the metabolism of different carbohydrates. We repeated the analysis in C57BL/6 mice without any significant effects of CMT.Results revealed that mice who received CMT showed more efficient restoration of gut microbiota than non-CMT recipient mice. CMT caused the alleviation of Salmonella infection and efficient recovery of the cecal index in the mice following antibiotics treatment.

Epidemiological studies implicate stress as an important factor contributing to the increasing prevalence of metabolic disorders. Studies have correlated visceral obesity and atherosclerosis with hyper-cortisolemia, a sequela of chronic psychological stress in humans and animals. Although several hormonal markers of stress have been associated with various metabolic disorders, the mechanism by which these hormones alter metabolic functions have not been established. We used an in vitro model system, culturing 3T3-L1 pre-adipocytes and RAW 264.7 macrophages in the presence or absence of cortisol, to analyze cell signaling pathways mediating changes in metabolic functions. Our analysis revealed that cortisol up-regulated the expression and function of two serotonin (S) receptors, HTR2c and HTR5a. HTR2c and HTR5a were also directly involved in mediating cortisol enhanced adipogenesis when pre-adipocytes were cultured alone or in the presence of macrophages. Finally, cortisol treatment of pre-adipocytes co-cultured with macrophages enhanced adipogenesis in both macrophages and pre-adipocytes.

Gut microbiota play important role in maintaining health. Probiotics are believed to augment it further. We aimed at comparing effects of probiotics, Lactobacillus acidophilus (LA) and Bacillus clausii (BC) (a) on the gut microbiota abundance and diversity and (b) their contributions to control intestinal dysbiosis and inflammation in Th1- and Th2-biased mice following Salmonella infection. We report how could gut microbiota and the differential immune bias (Th1 or Th2) of the host regulate host responses when challenged with Salmonella typhimurium in the presence and absence of either of the probiotics. LA was found to be effective in ameliorating the microbial dysbiosis and inflammation caused by Salmonella infection, in Th1 (C57BL/6) and Th2 (BALB/c)-biased mouse. BC was able to ameliorate Salmonella-induced dysbiosis and inflammation in Th2 but not in Th1-biased mouse. These results may support probiotics LA as a treatment option in the case of Salmonella infection.

Metabolic disorders are a prime global threat, primarily aided by consuming excess carbohydrate or fat-rich diet. Being related to a chronic inflammatory state, metabolic disorders may have a difference in pathophysiology; like adiposity and insulin resistance (IR), based on the individual immune status. Thus, we aim to explore the effects of different diets on the development of adiposity and IR under different immune backgrounds, using immune-biased mouse strains, BALB/c and C57BL/6. Mouse from each strain was randomly divided based on the diets like a) chow-fed control, b) non-resistant starch-rich, c) unsaturated fat-rich or d) saturated fat-rich groups, and observed for eight weeks, to evaluate the extent of various metabolic disease-related pathophysiology. Results revealed that the consumption of a) non-resistant starch-rich diet enhanced lipidemia, b) both the fat-rich diets enhanced visceral adiposity, and c) saturated fat-rich diet increased meta-inflammation. The extent of lipidemia and visceral adiposity were higher in C57BL/6 while the meta-inflammatory responses were higher in BALB/c. IR was severer in all the treatment diet groups of BALB/c mice compared to the control and corresponding C57BL/6 groups. Maximum IR was observed in the saturated fat-rich group which may have related to the intrinsic immune biases of the mice.

Vaginal microbiota contributes in maintaining and protecting the urogenital niche from infections and their sequelae. Despite extensive research, microbiome studies have often ignored asymptomatic bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC). The present study aimed to explore the cultivable vaginal bacterial and mycological communities in women asymptomatic for BV and VVC using multiplex PCR and species-specific PCR. Vaginal swabs collected from 199 participants asymptomatic for urogenital infections, scored by Nugent criteria indicated 73.9% had normal microbiota, 11.6% intermediate and 14.5% BV. The most frequent Lactobacillus species in normal women were L. iners (69.4%), L. crispatus (24.5%), L. reuteri (20.4%). Women with BV colonized L. iners (62.1%); L. rhamnosus (41.4%); L. salivarius (13.7%) and L. reuteri (7.2%). Furthermore, L. crispatus was associated with normal microbiota, whereas L. iners was a frequent member of normal and dysbiotic microbiota. Lactobacillus abundance and species richness reduced in asymptomatic BV. Also L. crispatus, L. fermentum, L. acidophilus and L. delbruckii were absent in these women. L. iners significantly co-existed with other Lactobacillus species, indicating its failure in independently maintaining the healthy vaginal niche. Of 30.4% women detected with Candida, 72.1% constituted non-albicans Candida. Predominance of C. albicans increased from 18.4% in healthy to 60% in women with asymptomatic BV; whereas distribution of BV related bacteria did not vary across the groups. Heterogeneous population of lactobacilli in 80.8% of normal women calls attention towards cumulative effects of these species in safeguarding the vaginal microenvironment. Since the microbiota of asymptomatic BV was different from healthy, screening and management could be encouraged to avoid further complications of infections.

AimsVancomycin, an antibiotic, is used to treat infection of multi-drug resistant strains ofClostridium difficileandStaphylococcus. Post-usage effects of vancomycin may lead to many unwanted effects including perturbation of gut microbiota. Perturbation of the gut microbiota, by Vancomycin, was used to understand the altered metabolic and innate immune profile of C57BL/6(Th1-biased) and BALB/c (Th2-biased) mice.Methods and ResultsFollowing treatment with vancomycin till day 4, we observed reduction in abundance of phyla Firmicutes and Bacteroides and increase in Proteobacteria in the gut for both strains of mice. Results further revealed a significant increase in the phylum Verrucomicrobia, from day 5 onwards following treatment with vancomycin led to decreased inflammation and increased rate of glucose tolerance in the host.ConclusionsContinued dosage of vancomycin was more beneficial in C57BL/6 than BALB/c miceSignificance and Impact of the studyThe current study established that initial doses of vancomycin increased pathogenic bacteria but the continued doses of vancomycin provided significant health-related benefits to the host by decreasing pathogenic load and by increasing beneficial microbes of Verrucomicrobia phylum (A. muciniphila) more in C57BL/6 (Th-1) than BALB/c (Th-2) mice.

Synthetic unmethylated cytidine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) is an effective immune stimulant in chicken. To be effective CpG dosage requirement is high. High dosage increases cost of treatment and introduces toxicity. A delivery system using multi-walled carbon nanotubes (MWCNT) is utilized in this study to aid in lowering the effective dose of the immune stimulant. CpG ODNs were attached non-covalently in different ways to multi-walled carbon nanotubes (MWCNT). We assessed and selected an appropriate linking method of CpG ODN with MWCNT followed by cellular uptake studies to establish that MWCNT-conjugated CpG ODNs were delivered better than free CpG ODNs into the cell. It was observed that MWCNT-conjugated CpG ODNs were equally effective in priming the cells in vitro at 1000-fold less concentration than free CpG ODN. In vivo studies revealed that a significantly lower dose of CpG ODN, when given subcutaneously, was enough to protect chickens from a lethal challenge of bacteria. The mechanism of immune stimulation was examined by in vivo cell recruitment and in vitro cytokine production studies. MWCNT-conjugated CpG ODNs are significantly more efficacious and less toxic than free CpG ODN to qualify as a potential immune stimulant.

Aims Secretory clusterin (sCLU) plays an important role in tumor development and cancer progression. However, the molecular mechanisms and physiological functions of sCLU in oral cancer is unclear. We examined the impact of sCLU-mediated autophagy in cell survival and apoptosis inhibition in oral cancer. Main methods Immunohistochemical analysis was performed to analyze protein expression in patient samples. Autophagy and mitophagy was studied by immunofluorescence microscopy and Western blot. The gain and loss of function was studied by overexpression of plasmid and siRNA approaches respectively. Cellular protection against nutrient starvation and therapeutic stress by sCLU was studied by cell viability, caspase assay and meta-analysis. Key findings The data from oral cancer patients showed that the expression levels of sCLU, ATG14, ULK1, and PARKIN increased in grade-wise manners. Interestingly, sCLU overexpression promoted autophagy through AMPK/Akt/mTOR signaling pathway leading to cell survival and protection from long exposure serum starvation induced-apoptosis. Additionally, sCLU was demonstrated to interact with ULK1 and inhibition of ULK1 activity by SBI206965 was found to abolish sCLU-induced autophagy indicating critical role of ULK1 in induction of autophagy. Furthermore, sCLU was observed to promote expression of mitophagy-associated proteins in serum starvation conditions to protect cells from nutrient deprivation. The meta-analysis elucidated that high CLU expression is associated with therapy resistance in cancer and we demonstrated that sCLU-mediated mitophagy was revealed to inhibit cell death by cisplatin. Significance The present investigation has highlighted the probable implications of the clusterin-induced autophagy in cell survival and inhibition of apoptosis in oral cancer.

Probiotic and potential probiotic bacterial strains are routinely prescribed and used as supplementary therapy for a variety infectious diseases, including enteric disorders among a wide range of individuals. While there are an increasing number of studies defining the possible mechanisms of probiotic activity, a great deal remains unknown regarding the diverse modes of action attributed to these therapeutic agents. More precise information is required to support the appropriate application of probiotics. To address this objective, we selected two probiotics strains, Lactobacillus acidophilus MTCC-10307 (LA) and Bacillus clausii MTCC-8326 (BC) that are frequently prescribed for the treatment of intestinal disorders and investigated their effects on the RAW 264.7 murine macrophage cell line. Our results reveal that LA and BC are potent activators of both metabolic activity and innate immune responses in these cells. We also observed that LA and BC possessed similar activity in preventing infection simulated in vitro in murine macrophages by Salmonella typhimurium serovar enterica.

Background/Aims NAFLD has today emerged as the leading cause of liver disorder. There is scanty data on risk factors associated with NAFLD emanating from India. The present study was conducted to identify the risk factors associated with NAFLD. Methods 464 consecutive NAFLD patients and 181 control patients were subjected to detailed questionnaire regarding their lifestyle and dietary risk factors. Anthropometric measurements were obtained and biochemical assays were done. Comparison of different variables was made between NAFLD patients and controls using principal component analysis (PCA). Results NAFLD patients had higher BMI [26.25 ± 3.80 vs 21.46 ± 3.08 kg/m2, P = 0.000], waist-hip ratio [0.96 ± 0.12 vs 0.90 ± 0.08, P = 0.000] and waist-height ratio [0.57 ± 0.09 vs 0.50 ± 0.06, P = 0.000] compared to controls. Fasting blood sugar [101.88 ± 31.57 vs 90.87 ± 10.74 mg/dl] and triglyceride levels [196.16 ± 102.66 vs 133.20 ± 58.37 mg/dl] were significantly higher in NAFLD group. HOMA-IR was also higher in NAFLD group [2.53 ± 2.57 vs 1.16 ± 0.58, P = 0.000]. Majority (90.2%) of NAFLD patients were sedentary. Family history of metabolic syndrome (MS) was positively correlated with NAFLD. Dietary risk factors associated with NAFLD were non-vegetarian diet [35% vs 23%, P = 0.002], fried food [35% vs 9%, P = 0.000], spicy foods [51% vs 15%, P = 0.001] and tea [55% vs 39%, P = 0.001]. Diabetes, hypertension, snoring and sleep apnoea syndrome were common factors in NAFLD. On multivariate PCA, waist/height ratio and BMI were significantly higher in the NAFLD patients. Conclusion The risk factors associated with NAFLD are sedentary lifestyle, obesity family history of MS, consumption of meat/fish, spicy foods, fried foods and tea. Other risk factors associated with NAFLD included snoring and MS.

Regulation of adipogenesis, the root cause for obesity, is very poorly understood. However, studies have presented evidence of immuno-metabolic regulation of adipose tissue during periods of chronic psychological stress, leading to adverse conditions related to stress manifestation, including visceral obesity and atherosclerosis. Despite pronounced association of hormonal markers of stress with dys-regulated metabolic states, the contributing signalling events are yet to be established. It is apparent that to understand contributing signalling events we need a model. Although an in vivo model is preferred, it is difficult to establish. The current report, therefore, presents an in vitro model system for the simulation of adipose tissue in a chronic stress micro-environment by growing pre-adipocytes with macrophages in the presence and absence of stress hormones. In this report, effects of cortisol and serotonin on the kinetics of immune and metabolic changes in adipocytes and macrophage (alone and co-cultured) was studied through whole genome transcriptome profiling. A transition from pro- to anti-inflammatory response in the immune profile of pre-adipocytes, with increasing time in co-culture with macrophages, was observed. This transition was reversed by stress hormones cortisol and/or serotonin., Summary: Stress-induced obesity is poorly understood in vivo at the molecular level. The current report established a novel molecular basis of adipogenesis in vitro.

Additional file 1: Table S1. List of primers used to validate microarray through q-RTPCR.

Understanding of probiotic-induced regulatory gene expression and networking is critical to further explore their roles in controlling infection. Transcriptional profile of selected innate immune genes in primary bovine intestinal epithelial cells was assessed over a time course of incubation with the probiotic Lactobacillus plantarum 299v. Based on gene expression results, a time point was chosen to prime epithelial cells with the probiotic prior to infection with rotavirus. Plaque assays and genomic analysis provided the basis for establishing the efficacy of probiotics in preventing a rotaviral infection. Plaque assays revealed that the probiotic is capable of decreasing (at least by 100-fold) the levels of live virus when the cells were primed with the probiotic. Results from gene expression studies (a) suggested that homeostasis in the gut is maintained in probiotic-primed cells despite infection with rotavirus and (b) revealed preliminary mechanisms for understanding the pathway of pathogen protection by using probiotics.

Palok Aich, Andrew A Potter, Philip J GriebelVaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, CanadaAbstract: Studies in animals and humans link both physical and psychological stress with an increased incidence and severity of respiratory infections. For this manuscript we define stress as the physiological responses an individual undergoes while adjusting to a continually changing environment. It is known that stressors of various types (psychological/physical) can alter the physiological levels of certain hormones, chemokines and cytokines. These alterations send information to the central nervous system to take necessary action which then sends messages to appropriate organs/tissues/cells to respond. These messages can either activate or suppress the immune system as needed and failure to compensate for this by the body can lead to serious health-related problems. Little is known how stress affects disease susceptibility, yet understanding this mechanism is important for developing effective treatments, and for improving health and food quality. The current review focuses on (a) the effects of psychological stressors in humans and animals, (b) various methodologies employed to understand stress responses and their outcomes, and (c) the current status of the attempts to correlate stress and disease with respiratory disease as model system. The methodologies included in this review span traditional epidemiological, behavioral and immunological studies to current high throughput genomic, proteomic, metabolomic/metabonomic approaches. With the advent of various newer omics and bioinformatics methodologies we postulate that it will become feasible to understand the mechanisms through which stress can influence disease onset. Although the literature in this area is limited because of the infancy of this research area, the objective of this review is to illustrate the power of new approaches to address complex biological questions. These new approaches will also aid in our understanding how these processes are related to the dynamics and kinetics of changes in expression of multiple genes at various levels.Keywords: stress, disease, biomarker discovery, systems biology, omics, respiratory disease

Host defense peptides (historically called antimicrobial peptides, AMPs) are key components in the mammalian innate immune system, and are responsible for both direct killing and immunomodulatory effects in host defense against pathogenic organisms. In order to identify novel host defense peptides by sequence analysis, we constructed the AMPer resource (http://www.cnbi2.com/cgi-bin/amp.pl) that utilizes hidden Markov models to recognize sequences of antimicrobial peptides. In the current work, we utilized the AMPer resource to search bovine expressed sequence tags from the NCBI dbEST project and the bovine genome sequence for novel host defense peptides. Of the 34 known bovine AMPs, 27 were identified with high confidence in the AMPs predicted from ESTs. A further potential 68 AMPs predicted from the EST data were found that appear to be novel giving a total estimate of 102 AMPs present in the genome. Two of these were cathelicidins and selected for experimental verification in RNA derived from bovine tissue. One predicted AMP, most similar to rabbit '15 kDa protein' AMP, was confirmed to be present in infected bovine intestinal tissue using PCR. These findings demonstrated the practical applicability of the developed bioinformatics approach and laid a foundation for future discoveries of gene-coded AMPs. No members of the alpha-defensin family were found in the bovine sequences. Since we could find no technical reasons these would be missed and no references to bovine alpha-defensins in the literature, this suggests that cattle lack this important family of host defense peptides.

Bovine rotavirus (BRV) and bovine coronavirus (BCV) are important causes of diarrhoea and death in newborn calves. Although these viruses belong to distinct viral classes, they both infect intestinal epithelial cells and induce similar clinical symptoms. Rotavirus usually causes an acute infection, but coronavirus infection can persist and reoccur in adults. Differences in viral structure and clinical outcome prompted us to postulate that innate mucosal immune responses would be markedly different following rotavirus and coronavirus infections. To address this hypothesis, gene expression following BRV and BCV infection was analysed in surgically prepared intestinal loops from 1-day-old colostrum-deprived calves. Gene expression was profiled at 18 h post-infection using bovine cDNA microarrays; the majority of differentially expressed significant genes were associated with the cell cycle and innate immune responses. A select group of these genes was validated by quantitative real-time PCR (qRT-PCR). The expression of genes associated with interferons (IFNs), cytokines and Toll-like receptors, which were not present on the microarray, was analysed further by qRT-PCR. Strong activation of TLR3, IL-6 and p65 was observed in BRV-infected host tissues, but not in tissues infected with BCV. Both viruses also downregulated IFN- and pro-inflammatory cytokine-associated pathways.In vitrostudies confirmed that IFN inhibited viral replication. All of these results together suggested either that very early events of host responses at 18 h post-infection were being observed, or that both viruses have unique effective strategies to evade host immune responses.

Antibiotic resistance is concern of today's world. Search for alternative molecules, for treatment and immune stimulation, remains at the forefront. One such group of biomolecules with promise, along the line of immune stimulation or therapy, is host defense peptide (HDP). These molecules, however, are required at a higher dose to be effective which leads to high cost. To alleviate such problems, an aid can be used to achieve similar efficacy but at a smaller effective dose of the immune stimulant. We hypothesised that by conjugating HDPs with carbon nanotubes and/or gold nanoparticles, it would be possible to stimulate a protective immune response in host system at a lower dosage of HDP. In this report, we characterized, using biophysical methodologies, conjugation of Indolicidin, as a representative of HDP. We further established efficacy of peptide-nanomaterial conjugates in activating innate immunity and protecting against pathogen infection in vitro at a significantly small dose.