Bivalent non-typhoidal Salmonella outer membrane vesicles immunized mice sera confer passive protection against gastroenteritis in a suckling mice model

Invasive non-typhoidal Salmonella (iNTS) serovars, especially Salmonella Typhimurium (ST) and Salmonella Enteritidis (SE), cause gastroenteritis worldwide. Due to the emergence of multi-drug resistance in iNTS, a broad-spectrum vaccine is urgently needed for the prevention of iNTS infection. Currently, there is no effective licensed vaccine against iNTS available in the market. We have formulated an outer membrane vesicles (OMVs) based bivalent immunogen as a vaccine candidate to generate broad-spectrum protective immunity against both recently circulating prevalent ST and SE. We have isolated OMVs from ST and SE and formulated the immunogen by mixing both OMVs (1:1 ratio). Three doses of bivalent immunogen significantly induced humoral immune responses against lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) as well as a cell-mediated immune response in adult mice. We also observed that proteins of OMVs act as an adjuvant for generation of high levels of anti-LPS antibodies through T cell activation. We then characterized the one-day old suckling mice model for both ST and SE mediated gastroenteritis and used the model for a passive protection study. In the passive protection study, we found the passive transfer of bivalent OMVs immunized sera significantly reduced ST and SE mediated colonization and gastroenteritis symptoms in the colon of suckling mice compared to non-immunized sera recipients. The overall study demonstrated that OMVs based bivalent vaccine could generate broad-spectrum immunity against prevalent iNTS mediated gastroenteritis. This study also established the suckling mice model as a suitable animal model for vaccine study against iNTS mediated gastroenteritis.     

Aberrant antibody affinity selection in SHIP‐deficient B cells

The strength of the Ag receptor signal influences development and negative selection of B cells, and it might also affect B‐cell survival and selection in the GC. Here, we have used mice with B‐cell‐specific deletion of the 5′‐inositol phosphatase SHIP as a model to study affinity selection in cells that are hyperresponsive to Ag and cytokine receptor stimulation. In the absence of SHIP, B cells have lower thresholds for Ag‐ and interferon (IFN)‐induced activation, resulting in augmented negative selection in the BM and enhanced B‐cell maturation in the periphery. Despite a tendency to spontaneously downregulate surface IgM expression, SHIP deficiency does not alter anergy induction in response to soluble hen‐egg lysozyme Ag in the MDA4 transgenic model. SHIP‐deficient B cells spontaneously produce isotype‐switched antibodies; however, they are poor responders in immunization and infection models. While SHIP‐deficient B cells form GCs and undergo mutation, they are not properly selected for high‐affinity antibodies. These results illustrate the importance of negative regulation of B‐cell responses, as lower thresholds for B‐cell activation promote survival of low affinity and deleterious receptors to the detriment of optimal Ab affinity maturation.     

High Tregs and systemic IL-10 expressions linked to the absence of sheath antibodies in lymphatic filariasis: implications on the persistence of residual infection

The persistence of residual infection is one of the major factors in failure of the Global Programme to Eliminate Lymphatic Filariasis (GPELF). The present study aims to explore the status of sheath antibody and regulatory T cells (Tregs) known to play key roles in clearance of parasite and patent filarial infection, in individuals with residual infection after MDA. A total of 61 microfilaremic (Mf) individuals were followed up after at least 6 rounds of MDA. Infection status of subjects was assessed through the detection of Mf and circulating filarial antigen (CFA). Antibodies to Mf sheath were determined by immuno-peroxidase assay (IPA). The expression of Tregs was measured by a flow cytometer. IL-10 and IFN-γ were evaluated using the commercially available ELISA kit. The sheath antibody was present in subjects who have cleared both Mf and CFA and absent in individuals who were found to be Mf /CFA positive. Further individuals carrying infection have significantly high levels of Tregs and IL-10. A positive correlation was observed between Tregs, IL-10, and CFA in infected individuals. In contrast, a negative correlation was observed between IFN-γ and IL-10 in both infected and uninfected subjects. Our study reveals that the absence of a sheath antibody and a high level of Tregs and IL-10 are the hallmarks of the persistence of residual filarial infection.

     

Inhibition of the MRP1-mediated transport of the menadione-glutathione conjugate (thiodione) in HeLa cells as studied by SECM

Oxidative stress induced in live HeLa cells by menadione (2-methyl-1,4-napthaquinone) was studied in real time by scanning electrochemical microscopy (SECM). The hydrophobic molecule menadione diffuses through a living cell membrane where it is toxic to the cell. However, in the cell it is conjugated with glutathione to form thiodione. Thiodione is then recognized and transported across the cell membrane via the ATP-driven MRP1 pump. In the extracellular environment, thiodione was detected by the SECM tip at levels of 140, 70, and 35 µM upon exposure of the cells to menadione concentrations of 500, 250, and 125 µM, respectively. With the aid of finite element modeling, the kinetics of thiodione transport was determined to be 1.6 × 10^-7 m/s, about 10 times faster than menadione uptake. Selective inhibition of these MRP1 pumps inside live HeLa cells by MK571 produced a lower thiodione concentration of 50 µM in presence of 500 µM menadione and 50 µM MK571. A similar reduced (50% drop) thiodione efflux was observed in the presence of monoclonal antibody QCRL-4, a selective blocking agent of the MRP1 pumps. The reduced thiodione flux confirmed that thiodione was transported by MRP1, and that glutathione is an essential substrate for MRP1-mediated transport. This finding demonstrates the usefulness of SECM in quantitative studies of MRP1 inhibitors and suggests that monoclonal antibodies can be a useful tool in inhibiting the transport of these MDR pumps, and thereby aiding in overcoming multidrug resistance.Multidrug resistance (MDR) pumps play a critical role in the detoxification pathway and cell survival under the oxidative stress caused by quinone or quinone-based chemotherapeutic drugs. Among the MDR pumps, the multidrug resistance protein (MRP1) pump is known to pump a broad variety of organic anions out of cells (1). According to the accepted model, MRP1 pumps out glutathione-S-conjugates (GS-conjugates), oxidized glutathione (GSSH), and reduced glutathione (GSH) as well as the unmodified drugs in the presence of physiological concentration of GSH; for example vincristine or daunorubicin are transported out of the cells by MRP1 in unmodified form in the presence of GSH (2). The cytotoxicity of a particular drug also depends on the types of MDR pumps and whether they are overexpressed in a cell under oxidative stress. For example, MRP pumps are known to be highly expressed in colon, breast and ovarian cancer cells whereas P-glycoprotein (Pgp) pumps are widely expressed in colon, renal and liver cancer cells but poorly expressed in breast, lung, and ovarian tumors (3). Hence, there are differences between the oxidative stress response of one type of cell to another and this is significant when comparing the effects of xenobiotics being added to different cells. In rat platelets, 85% intracellular GSH was reported to deplete as menadione-GSH conjugate, whereas in hepatocytes, 75% of intracellular GSH was depleted by menadione due to formation of GSSG (4).Depending on their modifications, quinones induce cytotoxicity in living cells by different pathways (4). A recycler such as 2,3-dimethoxy-1,4-napthaquinone exhibits oxidative stress purely by redox cycling, forming semiquinones, superoxide and hydroxyl radicals; thus depleting the reduced glutathione or GSH pool present inside the cell by forming oxidized glutathione or GSSH. A second type of quinone, an arylator such as 1,4-benzoquinone, exhibits cytotoxicity through arylation, forming GS-conjugates and thus depleting the intracellular GSH. Quinone-based oxidative stress in living cells differs from oxidative stress based on extracellularly administered hydrogen peroxide. The later agent is capable of inducing lipid peroxidation and subsequently rupturing the cell membrane before even entering the cell. Other types of quinone such as menadione (2-methoxy-1,4-napthaquinone) can act as both a redox cycler and arylator. Because of its hydrophobicity, menadione can pass through an intact cell membrane and induce oxidative stress by producing superoxide and hydroxyl radical. As part of the cells defense against such oxidative stress, GSH present inside the cell subsequently undergoes sacrificial nucleophilic addition or arylation with menadione in presence of the GS-transferase enzyme, forming menadione-S-glutathione (thiodione). However, the conjugate retains the ability to carry out redox recycling to form superoxide and hydroxyl radical, and this is not, by itself, an effective detoxification pathway unless the thiodione has been recognized by GS-X or MDR pumps as a substrate and pumped out of the cell by an ATP-driven process (Fig. 1) (5–10).Open in a separate windowFig. 1.Schematic diagram of cellular response to menadione in the presence or absence of MRP1 blocker MK571.MRP1 transports both endogenous substrates such as glutathione, steroids, LTC₄, LTD₄, LTE₄ as well as substrates like doxorubicin, daunorubicin, GS-conjugates, and vinblastine. However, LTC₄ has the highest affinity for MRP1 (2, 6, 9, 11–15). The inhibition of these MRP1 pumps increases the accumulation of intracellular xenobiotics or their conjugates; which therefore increases the cytotoxicity of the drugs towards the cell. MK571 (5-(3-(2-(7-chloroquinolin-2-yl) ethenyl) phenyl)-8-dimethylcarbamyl-4,6-dithiaoctanoic acid), an LTD₄ receptor antagonist, has been reported to act as competitive inhibitor for MRP1-mediated transport, both for GS-conjugate transport (such as thiodione) as well as for the transport of unconjugated GSH-mediated xenobiotics, such as daunorubicin (15–26).To understand mechanistically the function of this MRP1 pump in physiological condition, several immunoblot, immunoprecipitate and immunofluorescence based studies (27–35) have been made with MRP1-specific antibodies such as QCRL-1, QCRL-2, QCRL-3, QCRL-4, and QCRL-6. These IgG class antibodies have been developed to recognize a specific sequence of amino acids in the MRP proteins. For example, QCRL-1,-2,-3 recognize 918–924, 617–858, 617–932 amino acid sequences respectively; whereas QCRL-4 and QCRL-6 bind overlapping sequences of 1294–1531 amino acids, -COOH proximal nucleotide binding site (NBD2). Hipfner and coworkers (27–30) have used these antibodies to map the topology of this entire transmembrane protein. An inhibitory effect of this antibody has also been reported with the endogenous substrate, LTC₄, whereas QCRL-3 has been reported (30, 35) to inhibit the photolabeling of MRP1 by LTC₄, proving that the 617–932 sequence is the major substrate binding site. Thus different kinds of antibodies can be used to understand the functionally important domain of the MRP1 pump, especially in terms of binding sites of different xenobiotic substrates and pumping out by an ATP-driven process.Although there have been numerous studies on oxidative stress with different arrays of drugs and xenobiotics on diverse mammalian cell lines, most of them have been done with assays developed on lyzed cells after they were exposed to xenobiotics. Very few quantitative studies have been performed using live intact cells and their response in presence of xenobiotics, and fewer, particularly in terms of determining how transmembrane flux is affected by various antibodies that recognize different epitopes. Most of the immunoblot-based studies with antibodies and MDR pumps used antibodies to detect the pumps qualitatively, but very few studies have been done to demonstrate the blocking of a MRP1 pump efflux with an antibody in the dynamic environment of a live intact cell.In previous studies (36, 37), we studied this process in yeast and heptablastoma cells with the SECM. In this paper we show that HeLa cells exposed to menadione form the GS-conjugate, which is then pumped into the extracellular environment by ATP-driven MDR pumps. The quantitative estimation of thiodione flux out of the living cells was measured by SECM on a real time basis. The selective blocking by MK571 of these MRP1 pumps present in a live HeLa cell was also demonstrated; thus confirming that thiodione is indeed a substrate for MRP1 pumps and plays an important role in the cellular defense mechanism against quinone-based oxidative stress. In addition, a monoclonal antibody such as QCRL-4 was able to inhibit the thiodione flux under oxidative stress, again demonstrating the relevant function of MRP1.     

Bio-nanoparticle assembly: a potent on-site biolarvicidal agent against mosquito vectors

Background: Vector-borne diseases such as malaria, dengue, yellow fever, encephalitis and filariasis are considered serious human health concerns in the field of medical entomology. Controlling the population of mosquito vectors is one of the best strategies for combating such vector-borne diseases. However, the use of synthetic insecticides for longer periods of time increases mosquito resistance to the insecticides. Recently, the search for new environmentally friendly and efficient insecticides has attracted major attention globally. With the evolution of material sciences, researchers have reported the effective control of such diseases using various sustainable resources. The present investigation demonstrates a potent on-site biolarvicidal agent against different mosquito vectors such as Aedes albopictus, Anopheles stephensi and Culex quinquefasciatus. Methods: Stable and photo-induced colloidal silver nanoparticles were generated via the surface functionalization of the root extract of Cyprus rotundas. Characterizations of the nanoparticles were performed using assorted techniques, such as UV-visible spectroscopy, FTIR spectroscopy, DLS and HRTEM. The bioefficacy of the synthesized nanoparticles was investigated against different species of mosquito larvae through the evaluation of their life history trait studies, fecundity and hatchability rate of the treated larvae. Histopathological and polymerase chain reaction-random amplified polymorphic DNA (RAPD) analyses of the treated larvae were also examined to establish the cellular damage. Results: The synthesized nanoparticles showed remarkable larvicidal activity against mosquito larvae in a very low concentration range (0.001–1.00) mg L⁻¹. The histopathological study confirmed that the present nanoparticles could easily enter the cuticle membrane of mosquito larvae and subsequently obliterate their complete intestinal system. Furthermore, RAPD analysis of the treated larvae could assess the damage of the DNA banding pattern. Conclusion: The present work demonstrates a potent biolarvicidal agent using sustainable bioresources of the aqueous Cyprus rotundas root extract. The results showed that the synthesized nanoparticles were stable under different physiological conditions such as temperature and photo-induced oxidation. The effectiveness of these materials against mosquito larvae was quantified at very low dose concentrations. The present biolarvicidal agent can be considered as an environmentally benign material to control the mosquito vectors with an immense potential for on-site field applications.

The present work demonstrates a potent and stable biolarvicidal agent using sustainable bioresources. The synthesized nanomaterials can control the mosquito vectors at a very low concentration range (0.01–1.00 mg L⁻¹) for on-site field applications.     

Telomerase inhibition by siRNA causes senescence and apoptosis in Barrett's adenocarcinoma cells: mechanism and therapeutic potential

Background  

In cancer cells, telomerase induction helps maintain telomere length and thereby bypasses senescence and provides enhanced replicative potential. Chemical inhibitors of telomerase have been shown to reactivate telomere shortening and cause replicative senescence and apoptotic cell death of tumor cells while having little or no effect on normal diploid cells.     

Severe acute respiratory syndrome: a preliminary review

Severe acute respiratory syndrome (SARS) is a new disease of unknown aetiology and probably a new strain of coronavirus is thought to be responsible for the disease. After originating from south-east China it has spread to several countries across the world. Patients of SARS suffer from fever with cough and dyspnoea. The virus spreads by droplet to nearby contacts and has high tendency to spread to the healthcare providers. Since the aetiology is not yet clear exact treatment is not yet defined and hence prevention is of utmost importance.     

Diclofenac-induced biochemical and histopathological changes in white leghorn birds (Gallus domesticus)

Objective:

Objective: To evaluate diclofenac-induced biochemical and histopathological changes in White Leghorn birds.

Materials and Methods:

Six-week-old birds were equally divided into three groups of six birds each. Group I served as control and received vehicle orally. The birds of Group II and III were orally administered with a single low (2 mg/kg) and high dose (20 mg/kg) of diclofenac sodium, respectively, and were observed for 7 days. The acute toxicity was assessed by observing the clinical signs and symptoms, mortality, alterations in blood biochemistry, and necropsy findings.

Results:

The birds of Group II showed only mild symptoms of diarrhea. In Group III, 50% of birds died in between 24 and 36 h post-treatment showing the symptoms of segregatory behavior, lethargy, terminal anorexia, and severe bloody diarrhea. The birds of Group II and the surviving birds of Group III showed a significantly (P<0.05) increased plasma uric acid, creatinine and plasma glutamic pyruvic transaminase (PGPT), and decreased total protein and albumin at 12 and 24 h post-treatment which returned to the normal levels at 36 h post-treatment. The dead birds of the high-dose group also showed similar pattern of biochemical changes at 12 and 24 h post-treatment and revealed extensive visceral gout with characteristic histopathological lesions in liver, kidney, heart, spleen, and intestine on post-mortem.

Conclusion:

The results indicate that diclofenac sodium has hepatotoxic, nephrotoxic, and visceral gout inducing potentials in White Leghorn birds, especially at higher dose.