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luxS mutants of Borrelia burgdorferi strain 297 naturally colonized their arthropod (Ixodes scapularis) vector, were maintained in ticks throughout the molting process (larvae to nymphs), were tick transmitted to uninfected mice, and elicited histopathology in mice indistinguishable from that induced by wild-type B. burgdorferi.
Whole-cell lysates and proteinase K-extracted lipopolysaccharide (LPS) of 19 strains of the group eugonic fermenter-4 (EF-4) were analyzed by electrophoresis and protein immunoblotting. These strains were isolated from dog- and cat-bite abscesses in human beings, ferret and human gastric lesions, and cat-lung infections. These strains represent 2 biovar groupings; EF-4a biovars ferment glucose and possess arginine dihydrolase activity, whereas EF-4b biovars do not. Electrophoresis of whole-cell lysates could distinguish between these biovars groups. Electrophoresis of LPS extracts revealed that all strains of EF-4 possess smooth chemotypes. Two strains of EF-4a reacted weekly in protein immunoblots and revealed distinct LPS profiles. These studies suggests that subgroups of EF-4 biovars may exist.
Reductive carboxylation is critical for the proliferation of cancer cells and the differentiation of T cells. However, the role of this reaction in cancer cell-mediated tumor immunity remains unclear. Analysis of TCGA database showed a negative correlation between IDH2 expression and the presence of CD8+ T cells in lung and breast cancers, whereas IDH1 expression didn't show such a correlation. Further GSEA analysis revealed a significant enrichment of immune-related genes within IDH2-associated genes, specifically those in the type Ⅰ interferon pathway. In lung cancer cells, the depletion of IDH2 expression indeed could induce the activation of the immune-related and specially type Ⅰ interferon pathway. Targeting IDH2 with shRNA or its inhibitor AGI-6780 caused an increase in intracellular α-ketoglutarate concentration and a decrease in ATP and SAM levels, leading to a reduction in the methylation of STING promoter and elevated levels expression of STING. The increase of STING expression underlies the activation of type I interferon pathway observed in IDH2 compromised tumor cells and increased defense responses in the tumors in mice. These results identify IDH2 as a potential target to enhance cancer immune therapy.
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We report the use of recombinant scorpion toxin in the form of fusion protein as antigen for mice immunisation. The aim is to produce protective antisera against lethal activity of the venom from Tunisian scorpion Buthus occitanus tunetanus, responsible for several annually reported human cases of scorpion stings. The gene encoding Bot III (the most toxic alpha toxin of Buthus occitanus tunetanus) was fused to the sequence encoding synthetic ZZ domains of staphylococcal protein A. The construct ZZ-Bot III was expressed in the periplasm of E. coli as a fusion protein and purified by affinity chromatography. The recombinant fusion protein was characterized and used as antigen to generate antibodies in mice. The antibodies against the recombinant protein neutralize the toxic venom (10 LD50/ml) and also confer protection for immunized mice against antigenically related mammal toxins.
Animal innate immunity evolved from ancient pathways in bacterial anti-phage defense. How bacterial immune components were first acquired and adapted within eukaryotic cells remains poorly understood. Here we identify a complete cGLR-STING signaling axis in choanoflagellates, the closest living relatives of animals, that exhibits a mosaic of features from both bacterial and animal immunity. Comparative genomics reveals choanoflagellate cGLR and STING genes organized in operon-like arrangements reminiscent of bacterial defense loci. Reconstitution of choanoflagellate cGLR-STING signaling in vitro demonstrates that activation occurs through the conserved nucleotide immune signal 2'3'-cGAMP. Structural analysis of a choanoflagellate STING-2'3'-cGAMP complex explains how retention of bacterial-like features in early eukaryotic proteins shapes ligand specificity and receptor activation. We analyze cGLR and STING evolution in unicellular eukaryotes and identify further STING homologs in choanoflagellates and fungi that support additional independent acquisition events. Our results reveal molecular fossils that bridge bacterial and animal immunity and illuminate early eukaryotic immune system evolution.
BACKGROUND: One of the most defining features of gastric cancer (GC) is harboring deficiency in DNA repair that subsequently contributes to carcinogenesis. The X-ray repair cross complementing 1 (XRCC1) protein is a key molecular scaffold required for efficient repair of DNA single-strand breaks (SSBs) to maintain genomic stability. However, further investigation is needed to uncover the role of XRCC1 in innate immune signaling and inflammation in GC. METHODS: We evaluated how loss of XRCC1 leads to accumulation of cytosolic DNA using immunofluorescence localization assay and measuring DNA from cytosolic extract. We applied ON-TARGETplus™ SMARTpool siRNAs to knockdown XRCC1 in gastric cell lines and examined the innate immune siganling and inflammation with and without ATM inhibitor treatment. Further, we examined Type I interferon gene expression in various gastric cancer cell lines and assessed its role in cGAS-STING signaling using RT-qPCR, RNA-Seq, and immunoblot analysis. In addition, we generated conditional knockout XRCC1 mice and characterized the innate immune signaling from stomach tissue extract using RT-qPCR, western blot. Further, the DNA damage and histological analysis was done by immunohistochemistry. RESULTS: In this work, we examined the role of XRCC1 in modulating the innate immune signaling axis via cGAS/STING pathway. We find that XRCC1 deficient gastric cancer cell lines and mouse stomach tissue shows activation of cGAS/STING signaling. Further, ATM inhibition enhances robust cGAS/STING mediate innate immune signaling and PD-L1 expression in XRCC1 deficient gastric cancer cells. CONCLUSIONS: Results from this work demonstrate that XRCC1 is essential to maintain innate immune homeostasis. Further, this work suggest that ATM inhibitors may provide a potential therapeutic strategy to enhance the PD-L1 expression that could increase the efficacy of an immune checkpoint blockade (ICB) in XRCC1 deficient or low expressing GC.
Animal venom can induce systemic alterations similar to those observed in acute-phase inflammatory response. In the present study, we report the systemic (circulatory) and local (peritoneal cavity) effects induced by Tityus serrulatus venom and its major toxin TsTX-I (Ts1) in mice over various time periods. Both the venom and TsTX-I elicited quite similar responses in most assays. Responses included reduction of albumin, increased C-reactive protein, IL-6, IL-1alpha and TNF-alpha. Local and systemic leucocytosis, with a predominance of polymorphonuclear cells, was also observed. These effects show that a systemic inflammation-like syndrome is triggered during the severe envenomation caused by the T. serrulatus sting. The initial increases of albumin and total protein were probably consequences of the dehydration that occurs at the beginning of envenomation. Time-course analysis of these effects shows that responses are most pronounced on the first day after poisoning. However, leucocytosis and changes in acute-phase protein concentrations can be observed up to 7 days after envenomation.
BACKGROUND: Mosquito bites cause immediate wheals and delayed bite papules in sensitized subjects having saliva-specific IgE and IgG4 antibodies. At present, mosquito saliva antigens are not well characterized. METHODS: To identify immunogenic proteins in mosquito saliva and study their cross-reactivity we immunized mice with Aedes communis, Aedes aegypti and Anopheles stephensi bites. Immune sera were used in immunoblotting and immunoblot inhibition experiments. RESULTS: The main A. communis saliva antigens were 22-, 30-, and 36-kD, A. aegypti saliva antigens 31-, 36-, 46- and 64- to 66-kD, and A. stephensi saliva antigen 46-kD proteins. Most of the saliva antigens appeared to be species-specific and only weak cross-reactivity was observed with heterologous immune sera. Distinct cross-reactivity was observed only between saliva proteins of A. communis and Aedes punctor, two taxonomically closely related species. Human IgE and IgG4 antibodies from mosquito-bite-sensitive children bound to the same saliva proteins as antibodies from the immunized animals. CONCLUSIONS: This study disclosed several immunogenic proteins in Aedes and Anopheles mosquito saliva and suggests that these proteins can also be allergenic in man.
Diagnosis and immunotherapy with venom extracts of patients sensitive to Hymenoptera stings has led to the problem of improving the standardization of Hymenoptera venom products. Current methods of standardization use the Lowry protein determination and a radial-diffusion assay for hyaluronidase activity. This study demonstrates that the results of these analyses do not always correlate with the actual quantity of allergenic protein present in the extracts. A method of standardization is examined herein that uses polyacrylamide gel electrophoresis to quantitate phospholipase A, antigen 5, and hyaluronidase, the proteins that together comprise most allergenic protein present in the venoms. Also discussed in this study is the biochemical variability of phospholipase A and antigen 5 in the venoms of the different vespid species examined.
Urtica dioica agglutinin (UDA) has previously been found in roots and rhizomes of stinging nettles as a mixture of UDA-isolectins. Protein and cDNA sequencing have shown that mature UDA is composed of two hevein domains and is processed from a precursor protein. The precursor contains a signal peptide, two in-tandem hevein domains, a hinge region and a carboxyl-terminal chitinase domain. Genomic fragments encoding precursors for UDA-isolectins have been amplified by five independent polymerase chain reactions on genomic DNA from stinging nettle ecotype Weerselo. One amplified gene was completely sequenced. As compared to the published cDNA sequence, the genomic sequence contains, besides two basepair substitutions, two introns located at the same positions as in other plant chitinases. By partial sequence analysis of 40 amplified genes, 16 different genes were identified which encode seven putative UDA-isolectins. The deduced amino acid sequences share 78.9-98.9% identity. In extracts of roots and rhizomes of stinging nettle ecotype Weerselo six out of these seven isolectins were detected by mass spectrometry. One of them is an acidic form, which has not been identified before. Our results demonstrate that UDA is encoded by a large gene family.
Nematocysts isolated from the stinging tentacles of the Atlantic Portuguese Man-of-War (Physalia physalis) possess a potent venom composed of several proteins. A hemolytic protein lethal to mice has been isolated from this nematocyst venom. This protein, physalitoxin, appears to be responsible for both the venom's hemolytic and lethal activities. The hemolysin has a molecular weight of approx. 240 000, a sedimentation coefficient of 7.8 S, and is rod-like in shape with a calculated axial ratio of about 1 : 10. It appears to be composed of three subunits of unequal size, each of which is glycosylated. Two of these subunits seem to have pKi values near 8.2 and the third near 5.5. Physalitoxin comprises about 28% of the total nematocyst venom protein. It is 10.6% carbohydrate by weight and represents the major glycoprotein of the venom. Physalitoxin is inactivated by concanavalin A and this inactivation can be blocked with alpha-methyl-mannoside. The inactivation by concanavalin A is temperature-dependent about 12 degrees C and the hemolytic activity of untreated venom is temperature-dependent below 12 degrees C. Physalitoxin is the first hemolytic toxin from a cnidarian to be purified directly from isolated nematocysts.
We studied the inhibition of tissue kallikrein by protein C inhibitor (PCI), a relatively unspecific heparin-dependent serine protease inhibitor present in plasma and urine. PCI inhibited the amidolytic activity (cleavage of H-D-valyl-L-leucyl-arginine-p-nitroaniline) of urinary kallikrein with an apparent second order rate constant of 2.3 x 10(4) M-1 s-1 and formed stable complexes (85 kDa) with urinary kallikrein as judged from silver-stained sodium dodecyl sulfate-polyacrylamide gels. Complex formation was time-dependent and was paralleled by a decrease in the intensity of the main PCI protein band (Mr = 57,000) and an increase in the intensity of the lower Mr (54,000) PCI form (cleaved inhibitor). Heparin interfered with the inhibition of tissue kallikrein by PCI and with the formation of tissue kallikrein-PCI complexes in a dose-dependent fashion and completely abolished PCI-tissue kallikrein interaction at 300 micrograms/ml. This is in contrast to findings on the interaction of PCI with all other target proteases studied so far (i.e. stimulation of inhibition by heparin) but is similar to the reaction pattern of 125I-labeled tissue kallikrein with so called kallikrein binding protein described in serum and other systems. To study a possible relationship between PCI and this kallikrein binding protein we incubated 125I-labeled urinary kallikrein in serum and in PCI-immunodepleted serum in the absence and presence of heparin and analyzed complex formation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In normal serum, formed complexes co-migrated with complexes of purified PCI and 125I-kallikrein and were less intense in the presence of heparin. No complex formation at all was seen in PCI-depleted serum. Our data indicate that PCI may be a physiologically important endogenous inhibitor of tissue kallikrein and provide evidence that PCI may be identical to the previously described kallikrein binding protein.
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The antibody responses of subjects who presented with a definite Ixodes scapularis (Say) tick bite were measured to determine the utility of the antibody response against recombinant tick calreticulin (rTC) as a biologic marker of tick exposure. Subjects bitten by I. scapularis evidenced an increase in anti-rTC antibody levels between visit 1 and visit 2 from 24.3 to 27.1 ng/microl serum (n = 88, p = 0.003), and levels remained elevated at visit 3 (p = 0.005). These anti-rTC antibody levels during visits 2 and 3 were significantly higher than those in four non-exposed controls. Tick engorgement indices, measured on the biting ticks, were found to be correlated with anti-rTC antibody levels (e.g., for visit 3: Pearson's r = 0.357, p = 0.001). Tick engorgement index (TEI), ratio of body length to scutal width, was identified to be the only independent predictor of anti-rTC antibody levels in linear regression models. Logistic regression revealed that a bite from an I. scapularis tick that became engorged (TEI >3.4) was a risk factor for anti-rTC antibody seropositivity (adjusted odds ratio for age and bite location = 7.4 (95% confidence interval 2.1-26.4)). The anti-rTC antibody test had a sensitivity of 0.50 and a specificity of 0.86 for a bite from I. scapularis that became engorged. Immunoblotting revealed that subjects made a specific anti-rTC antibody response.
Pemphigus foliaceus (PF) and the endemic form Fogo Selvagem (FS) are mediated by pathogenic antibodies to the EC1-2 domains of desmoglein-1. There is a preclinical phase with antibodies to only EC5. Based on geographic clustering of cases, FS is thought to have an, as yet unidentified, environmental trigger. In this study we have searched for anti-desmoglein-1 antibodies in sera from parasitic (leishmaniasis, Chagas, and onchocerciasis), and infectious diseases (leprosy and South American (SA) blastomycosis), which are prevalent in the same geographic regions of Brazil as FS. A specific and sensitive desmoglein-1 ELISA detected antibodies in 34 of 41 onchocerciasis (83%), 38 of 88 leishmaniasis (43%), 18 of 31 Chagas disease (58%), 7 of 28 SA blastomycosis (25%), and 14 of 83 leprosy sera (17%). These sera recognized epitopes restricted to the EC5 domain. These findings identify several etiological factors for FS. It is hypothesized that a component of insect vector saliva, rather than the parasite itself may trigger an antibody response to EC-5. In persons with the known HLA susceptibility alleles and living in endemic areas, a response to the EC1-2 domains may subsequently develop by epitope spreading with associated clinical signs of FS.
The intimate contact between mosquitoes and the immune system of their hosts is generally not considered important because of the transient nature of mosquito feeding. However, when hosts are exposed to many feeding mosquitoes, they develop immune responses against a range of salivary antigens. Understanding the importance of these responses will provide new tools for monitoring vector populations and identifying individuals at risk of mosquito-borne diseases, and allow the development of novel methods for monitoring control and mosquito-release programmes. Antibodies targeting the mosquito midgut are also important in the development of mosquito vaccines. The feasibility of this approach has been demonstrated and future research opportunities are considered in this review. The potential impact of mosquito vaccines is also discussed. Our understanding of the interplay between mosquitoes and the immune system of their hosts is still in its infancy, but it is clear that there is great potential for exploiting this interplay in the control of mosquito-borne diseases.
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