AmpliSeq screening of genes encoding the C-Type lectin receptors and their signaling components reveals a common variant in MASP1 associated with pulmonary tuberculosis in an Indian population

Tuberculosis (TB) is a multifactorial disease governed by bacterial, host and environmental factors. On the host side, growing evidence shows the crucial role that genetic variants play in the susceptibility to Mycobacterium tuberculosis (Mtb) infection. Such polymorphisms have been described in genes encoding for different cytokines and pattern recognition receptors (PRR), including numerous Toll-like receptors (TLRs). In recent years, several members of the C-type lectin receptors (CTLRs) have been identified as key PRRs in TB pathogenesis. Nevertheless, studies to date have only addressed particular genetic polymorphisms in these receptors or their related pathways in relation with TB. In the present study, we screened the main CTLR gene clusters as well as CTLR pathway-related genes for genetic variation associated with pulmonary tuberculosis (PTB). This case-control study comprised 144 newly diagnosed pulmonary TB patients and 181 healthy controls recruited at the Bhagwan Mahavir Medical Research Center (BMMRC), Hyderabad, India. A two-stage study was employed in which an explorative AmpliSeq-based screening was followed by a validation phase using iPLEX MassARRAY. Our results revealed one SNP (rs3774275) in MASP1 significantly associated with PTB in our population (joint analysis p = 0.0028). Furthermore, serum levels of MASP1 were significantly elevated in TB patients when compared to healthy controls. Moreover, in the present study we could observe an impact of increased MASP1 levels on the lectin pathway complement activity in vitro. In conclusion, our results demonstrate a significant association of MASP1 polymorphism rs3774275 and MASP1 serum levels with the development of pulmonary TB. The present work contributes to our understanding of host-Mtb interaction and reinforces the critical significance of mannose-binding lectin and the lectin-complement pathway in Mtb pathogenesis. Moreover, it proposes a MASP1 polymorphism as a potential genetic marker for TB resistance

Recognition of microbial viability via TLR8 drives TFH cell differentiation and vaccine responses

Live attenuated vaccines are generally highly efficacious and often superior to inactivated vaccines, yet the underlying mechanisms of this remain largely unclear. Here we identify recognition of microbial viability as a potent stimulus for follicular helper T cell (TFH cell) differentiation and vaccine responses. Antigen-presenting cells (APCs) distinguished viable bacteria from dead bacteria through Toll-like receptor 8 (TLR8)-dependent detection of bacterial RNA. In contrast to dead bacteria and other TLR ligands, live bacteria, bacterial RNA and synthetic TLR8 agonists induced a specific cytokine profile in human and porcine APCs, thereby promoting TFH cell differentiation. In domestic pigs, immunization with a live bacterial vaccine induced robust TFH cell and antibody responses, but immunization with its heat-killed counterpart did not. Finally, a hypermorphic TLR8 polymorphism was associated with protective immunity elicited by vaccination with bacillus Calmette-Guérin (BCG) in a human cohort. We have thus identified TLR8 as an important driver of TFH cell differentiation and a promising target for TFH cell–skewing vaccine adjuvants

Assessment of Biochemical Parameters, Kidney Function, and Long-term Outcome in Renal Transplant Recipients

Background and Aims: New-onset of diabetes after transplantation (NODAT) is the most significant complications arising post-renal transplantation and affecting the long-term graft outcome and recipient survival. Assessment of renal function in kidney transplant recipients might help in understanding the better outcome of the graft and also the factors associated with NODAT. The present study was aimed to estimate the biochemical parameters, electrolytes, and minerals in the serum among renal transplant recipients and healthy controls (HC) and to evaluate the graft function, graft outcome and patient survival. Materials and Methods: Biochemical parameters (creatinine, urea, and uric acid), electrolytes (sodium, potassium, and chloride), and minerals (calcium and phosphorus) were estimated in serum by enzymatic method using commercially available kits in 100 HC, 80 NODAT, and 80 Non-NODAT subjects. The graft outcome was assessed by comparing serum creatinine levels and urinary creatinine clearance at 0 month and 60 months. The survival rate was evaluated by Kaplan-Meier survival curve. Results: The mean age was significantly higher in NODAT versus non-NODAT at P < 0.0009. Significant gender difference was observed in NODAT and non-NODAT versus HC at P < 0.0001. The levels of creatinine, urea, and uric acid were significantly more in NODAT versus HC at P < 0.0001, P < 0.0001, and P < 0.006. The mean levels of sodium and phosphorus were significantly lower in NODAT versus HC at P < 0.008 and P < 0.029. In multinomial logistic regression analysis, age, male gender, creatinine, and urea significantly predicted the outcome and the Receiver Operating Characteristic analysis revealed creatinine to be better marker for assessing kidney function. The Kaplan-Meier survival curve analysis showed decreased survival rates in NODAT than non-NODAT. Conclusion: Older age (above 40), hyponatremia, and hypophosphatemia could be significant risk factors for NODAT development

Assessment of renal function and acute rejection using Cystatin C and kidney injury Molecule-1 in renal transplant recipients

Purpose: Kidney injury molecule (KIM)-1, a transmembrane-tubular protein, is excreted in the urine within 12 h of the initial ischemic insult and before regeneration of the epithelium. Cystatin C (CysC) a low-molecular-weight nonglycosylated protein has been shown to be a good marker of kidney function. We aimed to evaluate the levels of KIM-1 and CysC immediately after transplantation as an early marker. Subjects and Methods: Urine and blood samples were collected from prospective renal transplant patients with chronic allograft dysfunction (CAD) at baseline and during the follow-up period at the interval of 12, 24 h, 7th postoperative day 15th and 6 m, and compared with healthy controls (HC) for KIM-1 CysC S.creatinine (SCr) and creatinine clearance. Results: Kidney transplant recipients showed significantly higher KIM-1and CysC values than the control group. Nonparametric receiver-operating characteristic (ROC) curve of the renal function with an area under the curve of 0.518 KIM-1, CysC was 0.841 and creatinine 0.74 indicating CysC at 12 h posttransplant (post-Tx) is a better biomarker among three. ROC of acute rejection (AR) of KIM-1 at 24 h post-Tx showed sensitivity of 0.938. ROC for distinguishing between graft survival and failure at 1 year showed a sensitivity of 0.763 for CysC, In CAD, both KIM-1and CysC were increased as compared to HC. Conclusion: Both KIM-1 and CysC are useful markers for predicting AR, renal function. Elevated urinary levels of KIM-1 independently predict AR. CysC is a valuable marker of predicting the long-term outcome

The N-terminal fragment of PPE17 (N-PPE17) shows better seroreactivity than full-length PPE17 in active TB patients (n = 143).

<p>(A) The full-length PPE17 or N-PPE17 protein was coated on EIA plate at various concentrations starting from 25 ng/well/50 μl to 1 μg/well/50 μl and antibody responses of TB patient sera were compared. The negative control (no antigen) displayed a background absorbance value of 0.056. Data shown in Fig 4A were re-plotted to compare the antibody levels of TB patients between PPE17 and N-PPE17 at 50 ng/well/50 μl (B1), 100 ng/well/50 μl (C1) and 250 ng/well/50 μl (D1) antigen coating concentrations. Data from Fig 4A were re-plotted to compare the PPE17 and N-PPE17-specific antibody levels in sera samples of smear positive (n = 58), smear negative (n = 38) and extrapulmonary TB (n = 47) patients at antigen concentration of 50 ng/well/50 μl (B2), 100 ng/well/50 μl (C2) and 250 ng/well/50 μl (D2).</p

Characteristics of study participants.

<p>Characteristics of study participants.</p

The N-terminal fragment of PPE17 (N-PPE17) is more sensitive to detect TB cases than full-length PPE17 protein.

<p>(A) The percentages of high-level responders (patients showing antibody levels greater than or equal to the cut-off values) shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179965#pone.0179965.g001" target="_blank">Fig 1A</a> were calculated using a cut-off value (mean OD₄₉₂ of healthy control sera plus 3 SD) for PPE17 and N-PPE17. (B) The percentage of high level responders was compared for pulmonary (smear-positive and smear-negative) and extrapulmonary TB cases.</p

N-PPE17-specific antibodies in sera obtained from TB patients did not significantly cross-react with N-terminal domains of other PPE proteins.

<p>Sera samples (n = 50) pre-incubated with recombinantly purified full-length PPE17 or PPE18 or PPE44 or PPE65 protein were added to EIA plates coated with recombinantly purified N-PPE17. The plates were further incubated with anti-human IgG-HRP and absorbance was read at 492 nm using the chromogenic substrate OPD.</p