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Pyoverdine, a fluorescent siderophore that have high- affinity for iron is produced by Pseudomonas putida and Pseudomonas aeruginosa that synthesizes it under iron-deficient growth conditions. Pseudomonas species are often encountered in diverse ecological habitats along side being censurable for nosocomial infections spread round the world. Due to these characteristics, there's a growing interest during this microbe for a spread of uses. One such is the production of Pyoverdine, it influences the uptake of iron, along with eliminating the limited iron availability condition. Pyoverdine is important for Pseudomonas putida and Pseudomonas aeruginosa to cause acute infections. Intense research and study led to the invention of Pyoverdines being a crucial source of chelating Iron. This study is predicated on Pyoverdine extracted from two different species of Pseudomonas which will act as an antimicrobial agent for various species including Escherichia coli and Staphylococcus aureus. The organism, i.e., Pseudomonas putida and Pseudomonas aeruginosa, were isolated from soil sample using medias like: Cetrimide media, King’s B media. It had been confirmed using primary biochemical tests, along side species level identification (MALDI - TOF). Isolation was followed by studying the antimicrobial activity of Pyoverdine on different organisms using antibiotics as standard for the same. Results for the tests were obtained, colonies were observed on specific media and zone of inhibition was observed on Muller Hinton plate. Comparative studies were carried out to find which organism used up Pyoverdine or Pyoverdine - Fe complex without using FeCl3 as sole standard source. Thus, these compounds can synergize with conventional antimicrobials, forming a simpler treatment with serving as a useful gizmo

Arthritis is a musculoskeletal system disorder which involves destabilization of normal mechanisms. Due to changing habits of living life, the number of arthritic people will increase rapidly. Currently, the existing anti-rheumatic drugs mostly show side effects like acne, blurred vision, high blood pressure and more effective to reduce pain and improve disease conditions but still, they do not treat the disease completely. Literature available indicates that most of the people seek complementary alternative treatments (CAM). Ginger (Zingiber officinale) has empirically explored its use as an anti-inflammatory agent. Amongst others, the Topical route of drug delivery has built up popularity because it avoids first-pass effects, metabolic breakdown associated with oral administration and gastrointestinal irritation. Also, they are less greasy and can be easily washed off from the skin surface. The current study aimed to formulate antiarthritic gel containing ginger extract and to evaluate its drug release activity. The topical ginger gels were prepared using Carbopol 934 as a gelling agent with varying concentrations, i.e., 0.5 %, 1 %, and 1.5 % w/w. The gel was assayed to determine percent purity and cumulative drug release. Results indicated that the 1.5 % w/w concentration of carbopol in ginger gel exhibited an adequate drug release. In conclusion, an antiarthritic gel containing 1.5 % w/w of carbopol had a good consistency, acceptable spreadability, and showed a good drug release profile. The topical prepared herbal ginger gel is a simple, easily formulated, convenient and economical alternative that needs to be weighed in the treatment of RA.

Combinatorial drug treatment strategies perturb biological networks synergistically to achieve therapeutic effects and represent major opportunities to develop advanced treatments across a variety of human disease areas. However, the discovery of new combinatorial treatments is challenged by the sheer scale of combinatorial chemical space. Here we report a high-throughput system for nanoliter-scale phenotypic screening that stabilizes a chemical library in nanoliter droplet emulsions and automates the formulation of chemical combinationsen massusing parallel droplet processing. We apply this system to predict synergy between more than 4,000 investigational and approved drugs and a panel of 10 antibiotics againstE. coli, a model Gram-negative pathogen. We found a range of drugs not previously indicated for infectious disease that synergize with antibiotics. Our validated hits include drugs that synergize with the antibiotics vancomycin, erythromycin, and novobiocin, which are used against Gram-positive bacteria but are not effective by themselves to resolve Gram-negative infections.

Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of “randomly paired” scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.

Plants produce a large number of cellulases that are either secreted or anchored in the plasma membrane where they likely function in various aspects of cellulose synthesis, modification and degradation during plant growth and development. Very few of these enzymes have been characterized in any detail, however. Here we attempted to produce two Arabidopsis modular cellulases, which contain a catalytic domain belonging to glycoside hydrolase family 9 (GH9) and a carbohydrate binding module (CBM), in the yeast Pichia pastoris. Neither of the intact modular enzymes was detectably produced, although the independently expressed GH9 catalytic domain of one enzyme was secreted when the protein was expressed at low temperature. Expression of intact and truncated cellulases at the standard temperature caused extensive cell lysis, with release of high concentrations of endogenous proteins into the culture medium. Cell lysis appeared to result from misfolding of cellulase proteins within the Pichia secretory pathway. The toxicity of these misfolded cellulases potentially could be exploited to derive host strains with enhanced capability to fold recombinant secretory proteins.

Objective: To evaluate different solvent extracts of selected Indian traditional medicinal plant against Mycobacterium tuberculosis, its antioxidant potential and cytotoxicity. Methods: Acacia catechu (L.) Willd (Root extract) and Ailanthus excelsa Roxb., leaf extracts of Aegle marmelos Corr., Andrographis paniculata Nees. and Datura metel L. were sequentially extracted in water, ethanol, chloroform and hexane and evaluated for their anti-tuberculosis (TB) activity against Mycobacterium tuberculosis using agar diffusion assay. The zone of inhibition ( at 20 and 40 mg/ ml) was measured and MIC were calculated. The results were compared with Rifampicin as a standard anti TB drug. The extracts were also evaluated for DPPH and OH radical scavenging activities to understand their antioxidant potential. MTT based cytotoxicity assay was used for evaluating cytotoxicity of the selected samples against Chang liver cells. Results: The selected botanicals were sequentially extracted in water, ethanol, chloroform and hexane and tested for growth inhibition of M. tuberculosi. The hexane extract of A. catechu root and ethanol extract of A. paniculata leaf showed promising activity against M. tuberculosis while remaining extracts showed moderate anti TB activity. The samples were found to possess considerable DPPH and OH radical scavenging activities with no demonstrable cytotoxicity against Chang liver cells. Conclusions: Five traditional medicinal plants were selected for the present study. The selection of medicinal plants was based on their traditional usage for the treatment of tuberculosis, asthma and chronic respiratory diseases. Herein we report for the first time, the anti TB activity of root extracts of Acacia catechu and Ailanthus excelsa while leaf extract of Andrographis paniculata, Aegle marmelos and Datura metel. The study holds importance in the midst of multi drug resistance (MDR) crisis in the TB management, since it unravels the scientific basis of use of these plant species for the management of TB and related disorders which will be useful for searching the lead compounds from natural products as potential antimycobacterial agents.

Breast cancer being the most fatal form of cancer for female population, justifies exploration of immunotherapy as an alternative treatment. Here, we have formulated and evaluated an oral microparticulate breast cancer vaccine to provide a new line of therapy. The whole cell lysate of 4T07 murine breast cancer cells was incorporated in an aqueous polymer matrix and spray dried to formulate an enteric protected vaccine microparticle. These particles were characterized in vitro and then administered orally to female Balb/c mice in successive boosters. Serum antibody titers during the study were analyzed using enzyme-linked immunosorbent assay. Postvaccination animals were challenged with live 4T07 cells, and tumor growth was monitored. Flow cytometry studies were performed to analyze the role of T cells. Results show that the vaccine microparticles were 1-4 µm in volume diameter and neutral in charge. The particles were protected enterically and had sustained-release profile. Serum antibody titers of vaccinated animals increased significantly after boosters compared with controls (p < 0.05). Tumor challenge studies revealed that vaccinated animals developed significantly smaller tumors (p < 0.05). Significantly higher numbers of CD4(+) cells occurred in vaccinated animals (p < 0.05). Thus, we conclude that the particulate oral breast cancer vaccine was effective in providing protective immune response in the murine model.

Various approaches have been evaluated for generation of efficient immune response against tumor antigens. Our approach exploits usage of particulate delivery to generate immune response against prostate cancer antigens.The aim of this study was to evaluate the efficacy of prostate cancer vaccine derived from a murine prostate cancer cell line, TRAMP C2 in murine model via oral route using aleuria aurantia lectin as a targeting ligand for M-cells in the intestinal Peyer's patches.The whole cell lysate (WCL) was obtained from TRAMP C2 murine prostate cancer cell line and was formulated into particles using one step spray drying process. For in vivo studies, 4-6 week old C57BL/6 male mice were vaccinated orally biweekly for 10 weeks. Serum samples were analyzed at regular intervals to determine serum IgG levels. The mice were then challenged with live TRAMP C2 cells to determine efficacy of the vaccine.The serum IgG levels of vaccinated animals were higher compared to that of the controls. Moreover, the tumor growth was retarded significantly in the vaccinated mice compared to that of controls (p0.001).The above findings suggest that oral particulate WCL vaccine can trigger an immune response against prostate cancer antigens.

Summary Background Patients allergic to cashew nuts often report allergy to pistachio, which could be a result of cross-reactivity between the two as both are members of the Anacardiaceae family. Objective Because cashew 7S globulin (vicilin, Ana o 1) is a recognized major allergen, we cloned the pistachio homologue and assayed it for IgE reactivity and cross-reactivity with Ana o 1. Methods Degenerate primers for 7S globulin were used in PCR to amplify DNA from a pistachio cDNA library. An isolate was sequenced, cloned and expressed in Escherichia coli. Reactivity to the allergen was screened by dot blot using 19 pistachio and/or cashew-allergic patients’ sera. Cross-reactivity was investigated by inhibition dot- and Western immunoblot assays using pistachio/cashew-allergic patients’ sera, and monoclonal antibodies (MAbs) raised against recombinant Ana o 1 (rAna o 1). Results An isolate was found that coded for a 7S vicilin-like protein, designated Pis v 3. IgE reactivity to Pis v 3 was found in the serum of seven of the 19 (37%) patients with histories of allergy to both pistachio and cashew or who were allergic to cashew but had never eaten pistachio. The seven patients with IgE that recognized rPis v 3 also recognized rAna o 1. Six of nine anti-rAna o 1 MAbs also showed reactivity to rPis v 3 on dot blots. Conclusion Of the 37% of pistachio/cashew-allergic patients’ sera that recognized the pistachio allergen, rPis v 3, all showed complete cross-reactivity with rAna o 1. The data does not identify the primary sensitizing agent but suggests that IgE reactivity to rPis v 3 and rAna o 1 is focused on the most conserved regions of the proteins. Clinical histories suggest that in some cases, cashew was the sensitizing agent. rPis v 3 is a likely contributor to the observed co-sensitivity to pistachio and cashew in some patients.

Cashew apple juice has the potential to be a natural source of vitamin C and sugar in processed foods. The juice of the cashew apple is obtained by pressing the fleshy peduncle or receptacle, which forms a rounded apple that sits above the true fruit, the cashew nut. Cashew nut allergy is the second most commonly reported tree nut allergy in the United States. To determine if cashew apple juice contains cashew nut allergens, immunoblotting was performed using a cashew apple juice 6X concentrate that was extracted and further concentrated through dialysis, lyophilization, and resuspension. Serum IgE of individuals allergic to cashew nut bound proteins in the cashew apple juice concentrate extract. For some serum samples, IgE reactivity could be inhibited by preincubation of the serum with cashew nut extract, suggesting the presence of cashew nut-related allergens. Using monoclonal antibodies specific for cashew nut allergens, the concentrate was found to contain Ana o 1 (vicilin) and Ana o 2 (legumin). Neither IgE from cashew nut allergic sera nor the monoclonal antibodies bound any peptides in 5 kDa filtered cashew apple juice concentrate. The cashew apple juice concentrate used in these studies contains proteins with IgE-reactive epitopes, including cashew nut legumin and vicilin. No IgE-binding peptides remained after 5 kDa filtration of the concentrate.

Background The identity of allergenic almond proteins is incomplete. Objective Our objective was to characterize patient IgE reactivity to a recombinant and corresponding native almond allergen. Methods An almond cDNA library was screened with sera from patients with allergy for IgE binding proteins. Two reactive clones were sequenced, and 1 was expressed. The expressed recombinant allergen and its native counterpart (purified from unprocessed almond flour) were assayed by 1-dimensional and 2-dimensional gel electrophoresis, dot blot, and ELISA, and screened for cross-reactivity with grass profilin. Results The 2 selected clones encoded profilin (designated Pru du 4) sequences that differed by 2 silent mutations. By dot-blot analyses, 6 of 18 patient sera (33%) reacted with the recombinant Pru du 4 protein, and 8 of 18 (44%) reacted with the native form. ELISA results were similar. Almond and ryegrass profilins were mutually inhibitable. Two-dimensional immunoblotting revealed the presence of more than 1 native almond profilin isoform. The strength of reactivity of some patients' serum IgE differed markedly between assays and between native and recombinant profilins. Conclusion Almond nut profilin is an IgE-binding food protein that is cross-reactive with grass pollen profilin and is susceptible to denaturation, resulting in variable reactivity between assay types and between patients. Clinical implications Serum IgE of nearly half of the tested patients with almond allergy reacts with almond nut profilin. Because most patients also had pollinosis, the well-known cross-reactivity between pollen and food profilins could account for this pattern of reactivity.

Background: The allergens responsible for cashew food allergy have not been well characterized. Objectives: We initiated a study to clone cDNAs encoding cashew food allergens. Methods: A cashew cDNA library was screened with human serum for IgE-reactive clones and rabbit IgG anti-cashew extract antisera. Reactive clones were sequenced and expressed, and linear epitopes were identified by means of solid-phase overlapping peptide analysis. Immunoblot inhibition was used to identify the native peptide in cashew extract. Results: Four closely related clones reactive with both human and rabbit antisera were sequenced. Sequence analysis showed that these encode members of the vicilin/sucrose-binding protein family of plant seed storage proteins. Screening of the recombinant protein with sera from 20 patients with cashew allergy and 8 cashew-tolerant patients with allergies to other tree nuts showed that 50% and 25% of sera from patients with cashew allergy and cashew-tolerant subjects, respectively, bound the recombinant protein. The corresponding native allergen protein, designated Ana o 1, was located at approximately 50 kd. Epitope mapping revealed 11 linear IgE-binding epitopes, of which 3 appear to be immunodominant. None of the epitopes were shared in common with those of the peanut vicilin allergen Ara h 1. Conclusion: Ana o 1, a vicilin-like protein, is a major food allergen in cashews. Cashew and peanut vicilins do not share linear epitopes. (J Allergy Clin Immunol 2002;110:160-6.)

Several major histocompatibility complex class II (MHC II) complexes with known minimal immunogenic peptides have now been solved by X-ray crystallography. Specificity pockets within the MHC II binding groove provide distinct peptide contacts that influence peptide conformation and define the binding register within different allelic MHC II molecules. Altering peptide ligands with respect to the residues that contact the T-cell receptor (TCR) can drastically change the nature of the ensuing immune response. Here, we provide an example of how MHC II (I-A) molecules may indirectly effect TCR contacts with a peptide and drive functionally distinct immune responses. We modeled the same immunogenic 12-amino acid peptide into the binding grooves of two allelic MHC II molecules linked to distinct cytokine responses against the peptide. Surprisingly, the favored conformation of the peptide in each molecule was distinct with respect to the exposure of the N- or C-terminus of the peptide above the MHC II binding groove. T-cell clones derived from each allelic MHC II genotype were found to be allele-restricted with respect to the recognition of these N- vs. C-terminal residues on the bound peptide. Taken together, these data suggest that MHC II alleles may influence T-cell functions by restricting TCR access to specific residues of the I-A-bound peptide. Thus, these data are of significance to diseases that display genetic linkage to specific MHC II alleles, e.g. type 1 diabetes and rheumatoid arthritis.

Ovarian cancer is the fifth most leading cause of cancer related deaths in women in the US. Customized immunotherapeutic strategies may serve as an alternative method to control the recurrence or progression of ovarian cancer and to avoid severe adverse effects of chemotherapy. In this study, a microparticulate vaccine using whole cell lysate of a murine ovarian cancer cell line, ID8 was prepared with the use of a spray dryer. These particles were designed for oral delivery using enteric polymers such as methacrylic copolymer, Eudragit(®) FS30D and hydroxyl propyl methyl cellulose acetate succinate. These particles were targeted for uptake via microfold cell (M-cell) in Peyer's patches of small intestine using M-cell targeting ligand, Aleuria aurantia lectin. The interleukins (ILs) such as IL-2 and IL-12 were added to the vaccine formulation to further enhance the immune response. The particles obtained were of 1.58±0.62 μm size with a charge of 12.48±2.32 mV. The vaccine efficacy was evaluated by administering the particles via oral route to C57BL/6 female mice. At the end of vaccination, mice were challenged with live tumor cells. Vaccinated mice showed significant (around six-fold) retardation of tumor volume in comparison to non-vaccinated animals for 3 weeks after the tumor challenge (p

gp140, 0.44% (0.35–0.52%) for the gp120, and 0.35% (0.13–0.56%) for the D368R gp140 immunized animals. However, no significant correlations can be drawn between the titer of Env specific antibodies and frequency of Env specific B cells in any group. The wt-gp140 was a more effective immunogen than m-gp120 in eliciting weak cross-NAbs. Interestingly, the wt-gp140 was also more effective in eliciting cross-NAbs than the D368Rgp140; potentially suggesting that the development of cross-NAbs by wt-gp140 depends on the structural integrity of the CD4-binding site. Conclusion Protein boosting is required for the development of high frequencies of anti-HIV Env antibodies, and ability of trimeric gp140 to elicit cross-NAbs will depend on the structural integrity of the CD4-binding site.

Two mechanisms that repair DNA double-strand breaks in mammalian cells are homologous recombination and non-homologous DNA end-joining (NHEJ). Previous studies showed that a critical component of the NHEJ pathway, the DNA-activated protein kinase (DNA-PK), was poorly expressed in non-lactating (resting) breast tissue. Therefore, we proposed to identify the mechanisms responsible for regulating levels of non-homologous end-joining DNA repair components in human breast tissue and to measure the DNA double-strand break repair capacity of breast epithelial cells. We reexamined the expression of DNA-PK in human breast tissues by immuno-histochemistry and extended these studies to two other components of the NHEJ repair pathway, XRCC4 and DNA ligase IV, as well as other DNA repair components including NBSl and MRE11. In contrast to the original report, 90% of the epithelial cells in normal resting breast tissues from 10 different patients expressed both components of DNA-PK, DNAPKcs and Ku. In contrast, stromal cells failed to express NHEJ proteins, but a cell line derived from breast stromal tissue did. No polymorphisms were detected in the Ku7O gene of 14 breast cancer patients, but a high frequency fragment length polymorhism was identified in the promoter region of the Ku8O gene from breast cancer patients. We also showed that 11.3% of breast cancer patients amplified the gene for the Wip1 phosphatase that regulates p53 activity. Furthermore, mouse cells lacking the Wip1 gene were resistant to cell transformation, and mice lacking the Wip1 gene were resistant to tumor formation, suggesting that Wip1 is a potential target for anti-cancer drugs.

Background In Plasmodium falciparum-infected children, the relationships between blood cell histopathology, blood plasma components, development of immunocompetence and disease severity remain poorly understood. Blood from Nigerian children with uncomplicated malaria was analysed to gain insight into these relationships. This investigation presents evidence for circulating neutrophil extracellular traps (NETs) and antinuclear IgG antibodies (ANA). The presence of NETs and ANA to double-stranded DNA along with the cytokine profiles found suggests autoimmune mechanisms that could produce pathogenesis in children, but immunoprotection in adults. Methods Peripheral blood smear slides and blood samples obtained from 21 Nigerian children under six years of age, presenting with uncomplicated malaria before and seven days after initiation of sulphadoxine-pyrimethamine (SP) treatment were analysed. The slides were stained with Giemsa and with DAPI. Levels of the pro-inflammatory cytokines IFN-γ, IL-2, TNF, CRP, and IL-6, select anti-inflammatory cytokines TGF-β and IL-10, and ANA were determined by immunoassay. Results The children exhibited circulating NETs with adherent parasites and erythrocytes, elevated ANA levels, a Th2 dominated cytokine profile, and left-shifted leukocyte differential counts. Nonspecific ANA levels were significant in 86% of the children pretreatment and in 100% of the children seven days after SP treatment, but in only 33% of age-matched control samples collected during the season of low parasite transmission. Levels of ANA specific for dsDNA were significant in 81% of the children both pre-treatment and post treatment. Conclusion The results of this investigation suggest that NET formation and ANA to dsDNA may induce pathology in falciparum-infected children, but activate a protective mechanism against falciparum malaria in adults. The significance of in vivo circulating chromatin in NETs and dsDNA ANA as a causative factor in the hyporesponsiveness of CpG oligonucleotide-based malaria vaccines is discussed.

Rationale The aim of our study was to make hypoallergenic variants of Ana o 2, a major cashew allergen for possible specific immunotherapy. Methods Epitope mapping done using the SPOTs technology. The critical amino acid residues in the epitopes identified by alanine scanning. Using the QuikChange sup>tm site-directed mutagenesis we introduced point mutations into the IgE epitopes of Ana o 2 and generated seven mutants. Reactivity of mutant proteins to anti-Ana o 2 monoclonal antibodies, rabbit anti-cashew antibodies, and cashew allergic patients sera studied by ELISA and Immunoblotting. Results Epitope mapping studies of Ana o 2 with synthetic peptides identified several epitopes reactive with cashew allergic human sera. Mutational analysis using alanine scanning revealed that single amino acid changes at critical residues in the epitopes could reduce IgE binding. Critical amino acids were mutated in the Ana o 2 gene and seven mutants were expressed and characterized. We observed reduced immuno-reactivity to anti-Ana o 2 monoclonal antibodies, rabbit polyclonal anti-cashew antibodies by Inhibition ELISA (3mutants showed 80% inhibition activity), and to human cashew allergic sera by immunoblot suggesting that these may be hypo-allergenic mutants of Ana o 2. Conclusion Engineering of these hypoallergenic mutants of Ana o 2 could serve as agents for cashew immunotherapy.

Bovine serum albumin (BSA) was reacted with the bifunctional reagent p,p′ -difluoro- m,m′ -dinitrodiphenylsulfone (FNPS) under varying conditions. While intramolecular cross linkages involving the lysine and tyrosine residues were the major modifications, formation of intermolecular linkages (dimerization), favored by high protein concentrations, was observed under all conditions studied. The modified derivatives exhibited decreased precipitin reactions with the antiserum prepared against the native protein indicating that the protein was denatured to some extent during the modification. FNPS appears useful as a general reagent for conjugation of two proteins.