Recombinant Brugia malayi pepsin inhibitor (rBm33) exploits host signaling events to regulate inflammatory responses associated with lymphatic filarial infections.

Prolonged existence of filarial parasites and their molecules within the host modulate the host immune system to instigate their survival and induce inflammatory responses that contribute to disease progression. Recombinant Brugia malayi pepsin inhibitor (rBm33) modulates the host immune responses by skewing towards Th1 responses characterized by secretion of inflammatory molecules such as TNF-α, IL-6, nitric oxide (NO). Here we also specified the molecular signaling events triggered by rBm33 in peripheral blood mononuclear cells (PBMCs) of filarial endemic normals (EN). rBm33 predominantly enhanced the levels of nitric oxide in cultured PBMCs but did not result in oxidative stress to the host cells. Further, rBm33 treatment of human PBMCs resulted in higher GSH/GSSG levels. MYD88 dependent activation was found to be associated with rBm33 specific inflammatory cytokine production. rBm33 triggered intracellular signaling events also involved JNK activation in host PBMCs. In addition, c-Fos and not NF-κB was identified as the transcription factor regulating the expression of inflammatory cytokines in rBm33 stimulated PBMCs. rBm33 marked its role in filarial pathology by altered levels of growth factors but did not have a significant impact on matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs) activity of host PBMCs. Thus, the study outlines the signaling network of rBm33 induced inflammatory responses within the host immune cells.

Expression profile of key immune-related genes in Penaeus monodon juveniles after oral administration of recombinant envelope protein VP28 of white spot syndrome virus.

White spot syndrome virus (WSSV) is the most catastrophic pathogen the shrimp industry has ever encountered. VP28, the abundant envelope protein of WSSV was expressed in bacteria, the purified protein administered orally to Penaeus monodon juveniles and its immune modulatory effects examined. The results indicated significant up-regulation of caspase, penaeidin, crustin, astakine, syntenin, PmRACK, Rab7, STAT and C-type lectin in animals orally administered with this antigen. This revealed the immune modulations in shrimps followed by oral administration of rVP28P which resulted in the reduced transcription of viral gene vp28 and delay in mortality after WSSV challenge. The study suggests the potential of rVP28P to elicit a non-specific immune stimulation in shrimps.

Immunogenicity and protective efficacy of a major White Spot Syndrome Virus (WSSV) envelope protein VP24 expressed in Escherichia coli against WSSV.

The study reports cloning, expression and characterization of immunogenic activity of VP24, a major envelope protein of White Spot Syndrome Virus (WSSV). His-tagged VP24 was expressed as truncated protein and purified from inclusion bodies by metal affinity chromatography under denaturing conditions. The ability to confer protection from WSSV by oral administration of recombinant viral protein (rVP24) was examined in black tiger shrimp Penaeus monodon (P. monodon) juveniles (advanced post larvae). Animals were fed with rVP24 for 10 days, orally challenged with WSSV and assayed for expression of viral genes and shrimp immune genes on the 2nd, 5th and 8th days of challenge. The survival of juvenile shrimps in the vaccinated and challenged group was significantly higher compared to the unvaccinated and challenged group with lesser viral gene expression (DNA polymerase, latency 1 and vp28). Analysis of immune gene expression showed upregulation of syntenin and down regulation of STAT, Rab 7 and caspase during the experimental period. This study points to the feasibility of using rVP24 as candidate vaccine in P. monodon against WSSV.

Recombinant Brugia malayi pepsin inhibitor (rBm33) induced monocyte function and absence of apoptotic cell death: an in vitro study.

The effect of recombinant Brugia malayi pepsin inhibitor (rBm33) on human monocytes/macrophages has been examined using THP-1 cells. THP-1 cells stimulated with rBm33 showed enhanced levels of expression of pro-inflammatory cytokines (IL-1ß, TNF-α, IL-6) and diminished levels of IL-12, iNOS and anti-inflammatory cytokine (IL-10) expression suggesting the predominant features of Th1 response. Phorbol-12-myristate-13-acetate (PMA) treated THP-1 cells stimulated with rBm33 and subsequent incubation with GFP expressing Escherichia coli (E. coli) for 2 h enhanced the uptake of E. coli. Nitric oxide (NO) levels measured in the supernatants of these cultures did not show significant changes. Apoptotic studies with Peripheral Blood Mononuclear Cells (PBMCs) from normal individuals stimulated with rBm33 did not induce apoptosis of monocytes or lymphocytes. These observations suggest that rBm33 stimulates macrophages to induce Th1 response and does not promote apoptosis.

Autophagy protects monocytes from Wolbachia heat shock protein 60-induced apoptosis and senescence.

Monocyte dysfunction by filarial antigens has been a major mechanism underlying immune evasion following hyporesponsiveness during patent lymphatic filariasis. Recent studies have initiated a paradigm shift to comprehend the immunological interactions of Wolbachia and its antigens in inflammation, apoptosis, lymphocyte anergy, etc. Here we showed that recombinant Wolbachia heat shock protein 60 (rWmhsp60) interacts with TLR-4 and induces apoptosis in monocytes of endemic normal but not in chronic patients. Higher levels of reactive oxygen species (ROS) induced after TLR-4 stimulation resulted in loss of mitochondrial membrane potential and caspase cascade activation, which are the plausible reason for apoptosis. Furthermore, release in ROS owing to TLR-4 signaling resulted in the activation of NF-κB p65 nuclear translocation which leads to inflammation and apoptosis via TNF receptor pathway following the increase in IL-6 and TNF-α level. Here for the first time, we report that in addition to apoptosis, rWmhsp60 antigen in filarial pathogenesis also induces molecular senescence in monocytes. Targeting TLR-4, therefore, presents a promising candidate for treating rWmhsp60-induced apoptosis and senescence. Strikingly, induction of autophagy by rapamycin detains TLR-4 in late endosomes and subverts TLR-4-rWmhsp60 interaction, thus protecting TLR-4-mediated apoptosis and senescence. Furthermore, rapamycin-induced monocytes were unresponsive to rWmhsp60, and activated lymphocytes following PHA stimulation. This study demonstrates that autophagy mediates the degradation of TLR-4 signaling and protects monocytes from rWmhsp60 induced apoptosis and senescence.

A structural biology approach to understand human lymphatic filarial infection.

The presence of aspartic protease inhibitor in filarial parasite Brugia malayi (Bm-Aspin) makes it interesting to study because of the fact that the filarial parasite never encounters the host digestive system. Here, the aspartic protease inhibition kinetics of Bm-Aspin and its NMR structural characteristics have been investigated. The overall aim of this study is to explain the inhibition and binding properties of Bm-Aspin from its structural point of view. UV-spectroscopy and multi-dimensional NMR are the experiments that have been performed to understand the kinetic and structural properties of Bm-Aspin respectively. The human aspartic proteases that are considered for this study are pepsin, renin, cathepsin-E and cathepsin-D. The results of this analysis performed with the specific substrate [Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu (4-pyridylmethyl) ester] against aspartic proteases suggest that Bm-Aspin inhibits the activities of all four human aspartic proteases. The kinetics studies indicate that Bm-Aspin follows a competitive mode of inhibition for pepsin and cathepsin-E, non-competitive for renin and mixed mode for cathepsin-D. The triple resonance NMR experiments on Bm-Aspin suggested the feasibility of carrying out NMR studies to obtain its solution structure. The NMR titration studies on the interactions of Bm-Aspin with the proteases indicate that it undergoes fast-exchange phenomena among themselves. In addition to this, the chemical shift perturbations for some of the residues of Bm-Aspin observed from (15)N-HSQC spectra upon the addition of saturated amounts of aspartic proteases suggest the binding between Bm-Aspin and human aspartic proteases. They also provide information on the variations in the intensities and mode of binding between the proteases duly corroborating with the results from the protease inhibition assay method.

Functional Characterization of a c-type Lysozyme from Indian Shrimp Fenneropenaeus indicus.

Lysozyme gene from Fenneropenaeus indicus was cloned, expressed in Escherichia coli and characterized. The cDNA consists of 477 base pairs and encodes amino acid sequence of 159 residues. F. indicus lysozyme had high identity (98%) with Fenneropenaeus merguiensis and Fenneropenaeus chinensis and exhibits low to moderate identities with lysozymes of other invertebrates and vertebrates. This lysozyme is presumed to be chicken types as it possesses two catalytic and eight cysteine residues that are conserved across c-type lysozymes and a c-terminal extension, which is a characteristic of lysozymes from marine invertebrates. Further, the antimicrobial properties of the recombinant lysozyme from F. indicus were determined in comparison with recombinant hen egg white lysozyme. This exhibited high activity against a Gram-negative pathogenic bacterium Salmonella typhimurium and two fungal strains Pichia pastoris and Saccharomyces cerevisiae in turbidimetric assay. Distribution of lysozyme gene and protein in tissues of shrimps infected with white spot syndrome virus revealed that the high levels of lysozyme are correlated with low and high viral load in abdominal muscle and tail, respectively. In conclusion, lysozyme from F. indicus has a broad spectrum of antimicrobial properties, which once again emphasizes its role in shrimp innate immune response.

DNA vaccination with VP2 gene fragment confers protection against Infectious Bursal Disease Virus in chickens.

Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens by destruction of antibody producing B cells in the Bursa of Fabricius and poses a potential threat to the poultry industry. We have examined the protective efficacy of a subunit DNA vaccine against IBDV infection in chickens in this study. An immunodominant VP2 gene fragment (VP252-417) was cloned into CMV promoter based DNA vaccine vector pVAX1 and in vitro expression of the DNA encoded antigens was confirmed by transfection of CHO cells with vaccine constructs followed by RT-PCR and western blot analysis using IBDV-antiserum. Two weeks old chickens were immunized intramuscularly with pVAXVP252-417 and the in vivo transcription of the plasmid DNA was confirmed by RT-PCR analysis of DNA injected muscle tissue at different intervals of post immunization. Tissue distribution analysis revealed that the plasmid DNA was extensively distributed in muscle, spleen, kidney, liver, and bursa tissues. Chickens immunized with pVAXVP252-417 developed high titer (1:12,000) of anti-VP252-417 antibodies. Further, chicken splenocytes from pVAXVP252-417 immunized group showed a significantly high proliferation to the whole viral and recombinant antigen (P<0.01) compared to control groups, which implies that pVAXVP252-417 codes for immunogenic fragment which has epitopes capable of eliciting both B and T cell responses. This is evident by the fact that, pVAXVP252-417 immunized chicken conferred 75% protection against virulent IBDV (vIBDV) challenge compared to the control group. Thus, the present study confirms that the immunodominant VP2 fragment can be used as a potential DNA vaccine against IBDV infection in chickens.

Wolbachia heat shock protein 60 induces pro-inflammatory cytokines and apoptosis in monocytes in vitro.

Recombinant Wolbachia heat shock protein 60 (rWmhsp60) induces gene expression of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α in human monocytic cell line THP-1. In addition, it inhibits the phagocytic activity and does not alter the nitric oxide production by differentiated THP-1 macrophages, which corroborates with no significant change in inducible nitric oxide synthase gene expression in rWmhsp60 treated THP-1 monocytes. Further, 24 h stimulation of peripheral blood mononuclear cells from normal individuals by rWmhsp60 reveals that monocytes enter the late apoptotic stage, while lymphocytes do not show apoptosis. Thus these findings suggest that rWmhsp60 may contribute to inflammation mediated monocyte dysfunction in filarial pathogenesis.

Protective immune responses of recombinant VP2 subunit antigen of infectious bursal disease virus in chickens.

Infectious bursal disease virus (IBDV) is the causative agent of Gumboro disease and poses a huge threat to poultry industry. The risks associated with conventional attenuated viral vaccines make it indispensable to probe into the development of novel and rationally designed subunit vaccines which are safer as well as effective. VP2 is the major host-protective antigen found in IBDV capsid. It encompasses different independent epitopes responsible for the induction of neutralizing antibody. Here, we report the efficacy of the immunodominant fragment of VP2 which induces both humoral and cellular immunity against infectious bursal disease. A 366 bp fragment (52-417 bp) of the VP2 gene from an IBDV field isolate was amplified and expressed in Escherichia coli as a 21 kDa recombinant protein. The efficacy of rVP2(52-417) antigen was compared with two commercial IBDV whole virus vaccine strains. The rVP2(52-417) induced significantly high antibody titres in chicken compared to commercial vaccines and the anti-rVP2(52-417) sera showed reactivity with viral antigens from both commercial strains (P<0.0001) and field isolates. Also, the chicken splenocytes from rVP2(52-417) immunized group showed a significantly high proliferation (P<0.01) compared to other groups, which implies that the rVP2(52-417) fragment contains immunogenic epitopes capable of eliciting both B and T cell responses. Further, rVP2(52-417) conferred 100% protection against vIBDV challenge in the immunized chickens which was significantly higher (P<0.001) compared to 55-60% protection by commercial vaccine strains. Hence, the study confirms the efficacy of the immunodominant VP2 fragment that could be used as a potent vaccine against IBDV infection in chicken.

Recombinant Wolbachia heat shock protein 60 (HSP60) mediated immune responses in patients with lymphatic filariasis.

Wolbachia, an endosymbiont present in filarial nematodes, have been implicated in a variety of roles, including the worm development and survival. Elucidation of the role of Wolbachia in filarial nematode biology and pathogenesis has become the focus of many studies and its contribution to parasite survival or immune response is still unclear. Recombinant Wolbachia HSP60 decreases T cell activation and lymphoproliferation in filarial infected people compared to endemic controls as observed by the assessment of T cell activation markers and cytokine responses in the peripheral blood mononuclear cells. Reduced T cell activation may be linked to T regulatory cell activity since it is associated with increased expression of CTLA4 and CD25 on CD4(+) T cells in filarial infected group upon stimulation with recombinant Wolbachia HSP60. In addition, elevated interleukin-10 and TGF-ß cytokines corroborate the reduced CD4(+) T cell activation and interferon-γ observed upon recombinant Wolbachia HSP60 stimulation in filarial patients. Hence, these findings indicate that Wolbachia HSP60 may also contribute to the immune modulation seen in filarial patients.

Cinnamic acid, from the bark of Cinnamomum cassia, regulates glucose transport via activation of GLUT4 on L6 myotubes in a phosphatidylinositol 3-kinase-independent manner.

BACKGROUND: Cinnamomum cassia (Family: Lauraceae) is an Ayurvedic medicinal plant used traditionally for the treatment of a number of diseases, including diabetes. The hypoglycemic effect of this plant has been established in vivo. However, the effects of cinnamic acid, isolated from C. cassia, on the insulin signaling cascade in an in vitro model have not been elucidated. Hence, the aim of the present study was to evaluate the anti-diabetic effect of cinnamic acid on glucose transport by L6 myotubes. METHODS: The mechanism of action of cinnamic acid was determined using specific targets in the insulin signaling pathway, including protein tyrosine phosphatase (PTP) 1B, phosphatidylinositol 3-kinase (PI3-K) and the glucose transporter GLUT4. After differentiation of myoblast to myotubes, the cells were serum deprived for 5 h and then treated with 1 ng/mL cinnamic acid and 50 µmol/L rosiglitazone for 18 h and 100 nmol/L insulin for 20 min for gene expression studies. RESULTS: Expression of GLUT4 mRNA was increased following treatment of L6 myotubes with 1 ng/mL cinnamic acid. Furthermore, cinnamic acid inhibited PTP1B activity (by 96.5%), but had no significant effect on PI3-K activity. CONCLUSION: On the basis of the results of the present study, we postulate that cinnamic acid isolated from the hydro-alcoholic extract of Cinnamomum cassia activates glucose transport by a PI3-K-independent pathway. However, the detailed mechanism of action requires further analysis.

Impairment of tetanus-specific cellular and humoral responses following tetanus vaccination in human lymphatic filariasis.

To investigate the consequences of the impaired parasite-specific immune response in lymphatic filariasis, the effect of concurrent Wuchereria bancrofti infection on the immune response to tetanus toxoid (TT) following tetanus vaccination was studied in 20 asymptomatic microfilaremic (MF) patients, 20 patients with chronic lymphatic obstruction/elephantiasis (chronic pathology [CP]), and 10 endemic normal (EN) control individuals at baseline and at 3 and 6 months after TT vaccination. Peripheral blood mononuclear cell (PBMC) proliferative responses to TT before vaccination were not significantly different between the EN control and CP groups, but the MF group showed significantly lower baseline proliferative responses to TT compared with either the EN or CP group. Six months following vaccination, the change in proliferative response to TT was significantly greater in the EN and CP groups than in the MF group. This difference in proliferative response was reiterated in the gamma interferon (IFN-gamma) response in the EN group, in that they increased IFN-gamma production by 400% at 6 months, in contrast to that seen in the filaria-infected groups. In contrast to the IFN-gamma responses, PBMCs from the MF group produced significantly increased levels of TT-specific IL-10 compared with PBMCs from the EN group. Although there was significantly greater TT-specific immunoglobulin G (IgG) production at baseline between the EN and MF groups, postvaccination IgG (and IgG1 isotype) responses did not differ among the groups, whereas TT-specific IgG2, IgG3, and IgG4 were all increased in the EN group compared with the filaria-infected groups. These studies indicate that concurrent infection with W. bancrofti can diminish the immune response to an unrelated antigen by a mechanism that is likely to involve IL-10.

Development of antigen detection ELISA for the diagnosis of brugian and bancroftian filariasis using antibodies to recombinant filarial antigens Bm-SXP-1 and Wb-SXP-1.

Antibodies specific to recombinant filarial antigens Wb-SXP-1 and Bm-SXP-1 have been used to develop a sandwich ELISA for the detection of circulating filarial antigen (CFA) in sera from patients with lymphatic filariasis caused by Wuchereria bancrofti of Brugia malayi. In patients with W. bancrofti infections, a high proportion of microfilaria (mf) positive (MF) and low proportions of patients with chronic pathology (CP) and endemic normals (EN) showed the presence of CFA. Similarly in patients with brugian infections a high proportion of mf positive individuals contained CFA while none of the patients with chronic pathology or endemic normals showed the presence of CFA. Sera from patients with other parasitic infections (OPI) like O. volvulus, Loa loa, Ascaris lumbricoides and from individuals residing in areas non-endemic to filariasis did not exhibit any reactivity. This assay shows promise for the detection of microfilaremic infections in lymphatic filariasis and its usefulness as a diagnostic tool especially in B. malayi infections, needs to be further evaluated.