Increased levels of pentraxins protein and cytokines bear good association in patients with severe dengue infection.

Dengue is an arboviral infection with high rates of morbidity and mortality throughout the tropics and sub-tropics. This work studied the status of pentraxin (CRP/SAP) protein, ferritin, TNF-α and IL-1ß levels in Dengue patients of different pathophysiological manifestations. Accordingly, clinically confirmed Dengue cases (n = 97) were enrolled and subsequently blood parameters were studied by Haematology cell counter and Biochemistry Autoanalyser. CRP, SAP, ferritin, TNF-α and IL-1ß ELISA were done in all the samples by using standard ELISA kits. Statistical Analysis was done in all the experiments. The levels of CRP (p < 0.0001), SAP (p < 0.0001), ferritin (p < 0.0001), TNF-α (p < 0.0001) and IL-1ß (p < 0.0001) were high in patients with Severe Dengue as compared to Dengue without warning signs. High levels of SGOT, SGPT and decreased platelet counts were found in severe patients as compared to Healthy donor. CRP/SAP as well as TNF-α/IL-1ß were independently associated with both dengue severity and overall disease manifestation. Statistically significant increased CRP, SAP, ferritin, TNF-α and IL-1ß titres were correlated in patients with severe clinical manifestations as compared to mild disease forms of dengue. Elevated levels of pentraxin, TNF-α/IL-1ß in blood during dengue infection could act as an early predictor in Severe Dengue infection.

Clinical Proteomics Profiling for Biomarker Identification Among Patients Suffering With Indian Post Kala Azar Dermal Leishmaniasis.

Background: Post Kala Azar Dermal Leishmaniasis (PKDL) is a non-fatal dermal sequel of Visceral Leishmaniasis (VL), affecting individuals worldwide. Available diagnostic tools lack sensitivity and specificity toward identifying macular (MAC) PKDL patients, due to low parasite load in patients' sample. Confirmatory test like punch biopsy are invasive and painful. Considering the rural nature of this disease and the prevailing situation of diagnostic scenario, PKDL patients mostly remains unattended from receiving proper medical care. They in turn act as "mobile parasite reservoir," responsible for VL transmission among healthy individuals (HI). This study aims to identify PKDL disease specific glycated protein biomarkers, utilizing the powerful LC-MS/MS technology, which is the tool of choice to efficiently identify and quantify disease specific protein biomarkers. These identified PKDL disease specific novel glycoproteins could be developed in future as immunochromatographic based assay for efficient case detection. Methodology: Previously our lab had identified importance of glycated (Circulating Immune Complexes) CICs, among PKDL patients. This study aims to further characterize disease specific glycated protein biomarkers, among MAC PKDL patients for both diagnostic and prognostic evaluation of the disease. LC-MS/MS based comparative spectral count analysis of MAC PKDL to polymorphic (POLY) PKDL, HI, and Cured (CR) individuals were performed. Proteins level alterations among all study groups were confirmed by Western blot and enzyme-linked immunosorbant Assay (ELISA). Results: Among MAC PKDL patients 43, 60, 90 proteins were altered compared to POLY PKDL, HI, and CR groups, respectively. Filtering for the most significant proteins, Plasminogen (PLG) and Vitronectin (VTN) were identified which promisingly identified MAC PKDL cases. Active surveillance results from endemic districts of West Bengal revealed drastic rise of MAC PKDL cases, alarming the urgency for field adaptive efficient biomarker. Conclusion: This current study aims to establish PLG and VTN as novel diagnostic and prognostic protein biomarker for MAC and POLY PKDL cases management.

Study of serum VEGF levels in patients with severe dengue infection admitted in a tertiary care hospital in Kolkata.

Dengue is the most common arboviral infection globally, but its pathogenesis is poorly explored. Vascular endothelial growth factor (VEGF) has an essential role in the host defense against viral infection. However, not much information is available regarding its status in dengue patients from the eastern zone of India. In the present investigation, the level of VEGF was investigated for its possible utility as a dengue severity marker. Accordingly, confirmed dengue cases were enrolled during 2016-2018. Serum from all the study subjects was subjected to the standard enzyme-linked immunosorbent assay test for VEGF analysis. In addition, we assessed the association of VEGF to dengue severity. The study revealed that VEGF titers (P < .0001) were significantly increased in severe dengue (SD) patients in contrast to those with a milder form of dengue. An association was obtained between VEGF and increased SGOT (r = 0.517 with P < .0001) while VEGF had a negative correlation with platelets in SD patients (r = -0.331 with P = .001). Enhanced VEGF titers along with decreased platelets had a good association with SD. The investigation revealed that high VEGF titers are novel indicators of dengue severity. However, our results must be verified in a study evaluating a larger number of dengue patients.

Glycosylation of erythrocyte spectrin and its modification in visceral leishmaniasis.

Using a lectin, Achatinin-H, having preferential specificity for glycoproteins with terminal 9-O-acetyl sialic acid derivatives linked in α2-6 linkages to subterminal N-acetylgalactosamine, eight distinct disease-associated 9-O-acetylated sialoglycoproteins was purified from erythrocytes of visceral leishmaniaisis (VL) patients (RBC(VL)). Analyses of tryptic fragments by mass spectrometry led to the identification of two high-molecular weight 9-O-acetylated sialoglycoproteins as human erythrocytic α- and ß-spectrin. Total spectrin purified from erythrocytes of VL patients (spectrin(VL)) was reactive with Achatinin-H. Interestingly, along with two high molecular weight bands corresponding to α- and ß-spectrin another low molecular weight 60 kDa band was observed. Total spectrin was also purified from normal human erythrocytes (spectrin(N)) and insignificant binding with Achatinin-H was demonstrated. Additionally, this 60 kDa fragment was totally absent in spectrin(N). Although the presence of both N- and O-glycosylations was found both in spectrin(N) and spectrin(VL), enhanced sialylation was predominantly induced in spectrin(VL). Sialic acids accounted for approximately 1.25 kDa mass of the 60 kDa polypeptide. The demonstration of a few identified sialylated tryptic fragments of α- and ß-spectrin(VL) confirmed the presence of terminal sialic acids. Molecular modelling studies of spectrin suggest that a sugar moiety can fit into the potential glycosylation sites. Interestingly, highly sialylated spectrin(VL) showed decreased binding with spectrin-depleted inside-out membrane vesicles of normal erythrocytes compared to spectrin(N) suggesting functional abnormality. Taken together this is the first report of glycosylated eythrocytic spectrin in normal erythrocytes and its enhanced sialylation in RBC(VL). The enhanced sialylation of this cytoskeleton protein is possibly related to the fragmentation of spectrin(VL) as evidenced by the presence of an additional 60 kDa fragment, absent in spectrin(N) which possibly affects the biology of RBC(VL) linked to both severe distortion of erythrocyte development and impairment of erythrocyte membrane integrity and may provide an explanation for their sensitivity to hemolysis and anemia in VL patients.

9-O-acetylated sialoglycoproteins are important immunomodulators in Indian visceral leishmaniasis.

Overexpression of disease-associated 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) on peripheral blood mononuclear cells (PBMC) of visceral leishmaniasis (VL) patients (PBMC(VL)) compared to their levels of expression in healthy individuals has been demonstrated using a lectin, achatinin-H, with specificity toward 9-O-acetylated sialic acid derivatives alpha2-6 linkage with subterminal N-acetylgalactosamine (9-O-AcSAalpha2-6GalNAc). The decreased presence of disease-associated 9-O-AcSGPs on different immune cells of parasitologically cured individuals after successful treatment relative to the levels in patients with active VL prior to treatment was demonstrated. However, their contributory role as immunomodulatory determinants on PBMC(VL) remained unexplored. Accordingly, 9-O-AcSGPs on PBMC(VL) were sensitized with achatinin-H, leading to their enhanced proliferation compared to that observed with different known mitogens or parasite antigen. This lymphoproliferative response was characterized by evaluation of the TH1/TH2 response by intracellular staining and enzyme-linked immunosorbent assay for secreted cytokines, and the results were corroborated by their genetic expression. Sensitized PBMC(VL) evidenced a mixed TH1/TH2 cellular response with a predominance of the TH1 response, indicating the ability of 9-O-AcSGPs to modulate the host cell toward a favorable response. Interestingly, the humoral and cellular responses showed a good correlation. Further, high levels of anti-9-O-AcSGP antibodies with an order of distribution of immunoglobulin M (IgM) > IgG1 = IgG3 > IgG4 > IgG2 > IgE could be explained by a mixed TH1/TH2 response. A good correlation of enhanced 9-O-AcSGPs with both the cell-mediated (r = 0.98) and humoral (r = 0.99) response was observed. In summary, it may be concluded that sensitization of 9-O-AcSGPs on PBMC(VL) may provide a basis for the modulation of the host's immune response by their controlled expression, leading to a beneficial immune response and influencing the disease pathology.