Rabbit Antibodies Research Articles

The Effect of Protein FAM172A on Proliferation in HepG2 Cells and Investigation of the Possible Molecular Mechanism.

Background In our previous study, we found that the FAM172A recombinant protein could promote proliferation of L02 cells. However, the underlying mechanisms are still unknown. The present study was aimed at investigating the effect of FAM172A on proliferation of HepG2 cells and exploring the possible molecular mechanisms and its role in hepatocellular carcinoma (HCC). Methods Cell proliferation was measured by MTT assay. Western blot test was carried out to investigate the mechanism. Rabbit antibodies against FAM172A and membrane proteins isolated from lysate of HepG2 cell were coprecipitated and the resultant precipitates were analyzed by mass spectrum. Results The MTT assay showed that recombinant protein FAM172A isoform 1 (FAM172A-1) could induce HepG2 cell proliferation at the concentration of 10-100 ng/mL, while protein FAM172A isoform 3 (FAM172A-3) was at the concentration of 80-100 ng/mL. Western blot demonstrated that both FAM172A-1 and FAM172A-3 could activate the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) pathway and the phosphatidylinositol 3-kinase/threonine-protein kinase (PI3K/Akt) pathway. Mass spectrum analysis suggested that there were some membrane proteins interacting with FAM172A. Several candidate interacting proteins might mediate proliferation signals induced by FAM172A recombinant protein, including seven membrane proteins. Conclusion In conclusion, FAM172A recombinant protein could induce proliferation of HepG2 cells, in which the MAPK/ERK and PI3K/Akt signaling pathways might be involved. The role of FAM172A in HepG2 cell proliferation also indicated its possible involvement in HCC. The receptor of FAM172A on cells still needs to be exploited.

Immunogenic Properties of Recombinant Enzymes from Bothrops Ammodytoides Towards the Generation of Neutralizing Antibodies against Its Own Venom.

Bothropic venoms contain enzymes such as metalloproteases, serine-proteases, and phospholipases, which acting by themselves, or in synergism, are the cause of the envenomation symptoms and death. Here, two mRNA transcripts, one that codes for a metalloprotease and another for a serine-protease, were isolated from a Bothrops ammodytoides venom gland. The metalloprotease and serine-protease transcripts were cloned on a pCR®2.1-TOPO vector and consequently expressed in a recombinant way in E. coli (strains Origami and M15, respectively), using pQE30 vectors. The recombinant proteins were named rBamSP_1 and rBamMP_1, and they were formed by an N-terminal fusion protein of 16 amino acid residues, followed by the sequence of the mature proteins. After bacterial expression, each recombinant enzyme was recovered from inclusion bodies and treated with chaotropic agents. The experimental molecular masses for rBamSP_1 and rBamMP_1 agreed with their expected theoretical ones, and their secondary structure spectra obtained by circular dichroism were comparable to that of similar proteins. Additionally, equivalent mixtures of rBamSP_1, rBamMP_1 together with a previous reported recombinant phospholipase, rBamPLA2_1, were used to immunize rabbits to produce serum antibodies, which in turn recognized serine-proteases, metalloproteases and PLA2s from B. ammodytoides and other regional viper venoms. Finally, rabbit antibodies neutralized the 3LD50 of B. ammodytoides venom.

CD80 expression and infiltrating regulatory T cells in idiopathic nephrotic syndrome of childhood.

CD80 (also known as B7-1) is a co-stimulatory molecule that is expressed in biopsies and also excreted in urine in patients with minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). CD80 is inhibited by the cytotoxic T-lymphocyte-associated-antigen 4 (CTLA4), which is mainly expressed on regulatory T cells (Tregs). Ineffective circulating Treg response is involved in the pathogenesis of nephrotic syndrome. In this study, we evaluated CD80 expression and infiltrating Tregs in children with MCD and FSGS. Evaluation of CD80 expression and semi-quantitative evaluation of Tregs (FOXP3-positive CD4 T cells) were carried out in 31 kidney biopsies (12 MCD, 19 FSGS) with immunofluorescence and immunohistochemistry staining. All MCD sections were stained negative; whereas six out of 19 FSGS sections (allfrom steroid-resistant (SR) patients), including one from a Wilms' tumor1(WT1) mutation-positive FSGS patient, stained positive for anti-CD80 goat antibody, and negative for anti-CD80 rabbit antibody. FSGS biopsy specimens had significantly higher FOXP3-positive cells/mm2 compared with MCD and control samples (P<0.001). Biopsy samples from SR-FSGS patients (n=12) with positive CD80 staining (n=6) had significantly less Tregs (FOXP3-positive CD4 T cells) compared with CD80 (-) biopsies (n=6; P=0.004). CD80 expression was not detected in the majority of the archival biopsy sections and the results were not consistent across the different antibodies. In the SR-FSGS sections, however, CD80-positive biopsies had decreased FOXP3-positive CD4 T cells, suggesting that a decreased anti-inflammatory milieu may be the cause of increased CD80 expression.

Multiple antibodies targeting tumor-specific mutations redirect immune cells to inhibit tumor growth and increase survival in experimental animal models.

T cell therapy for cancer involves genetic introduction of a target-binding feature into autologous T cells, ex vivo expansion and single large bolus administration back to the patient. These reprogrammed T cells can be highly effective in killing cells, but tumor heterogeneity results in regrowth of cells that do not sufficiently express the single antigen being targeted. We describe a cell-based therapy that simultaneously targets multiple tumor-specific antigens. High-affinity polyclonal rabbit antibodies were generated against nine different surface-related tumor-specific mutations on B16F10 cells. Unsorted splenic effector cells from syngeneic mice were incubated with a cocktail of the nine anti-B16F10 antibodies. These 'armed' effector cells were used to treat mice previously inoculated with B16F10 melanoma cells. The cocktail of nine antibodies resulted in dense homogeneous binding to histological sections of B16F10 cells. Five treatments with the armed effector cells and PD1 inhibition inhibited tumor growth and improved survival. Shortening the interval of the five treatments from every three days to every day increased survival. Arming effector cells with the four antibodies showing best binding to B16F10 cells even further increased survival. This study demonstrates that ex vivo arming a mixed population of immune effector cells with antibodies targeting multiple tumor-specific mutated proteins in conjunction with PD1 inhibition delayed tumor growth and prolonged survival in mice inoculated with an aggressive melanoma. A remarkably low total antibody dose of less than 5µg was sufficient to accomplish tumor inhibition. Scaling up to clinical level may be feasible.

Comparative Analysis of the Efficiency of Chicken and Rabbit Antibodies in Competitive Enzyme Linked Immunoassay for the Detection of Bovine Beta-Casomorphin 7

Rabbit and chicken (IgY) polyclonal antibodies were compared with respect to their performance in competitive enzyme linked immunoassays (ELISA) for the determination of opioid peptide β-casomorphin 7 (BCM-7) released from variant A1 of bovine β-casein. To obtain antibodies, the animals (four rabbits and four hens) were immunized (with similar regimes) with BCM-7 conjugated to bovine serum albumin. Comparison of the binding curves of biotinylated BCM-7 obtained with affinity-purified mammalian and avian antibodies immobilized on the surface of polystyrene microtiter immunoplates via passive adsorption (in optimal conditions) showed that rabbit antibodies captured biotinylated antigen 100 times more efficiently than hen antibodies within the given antibody panel. The most efficient rabbit antibodies were used to construct a highly sensitive, competitive ELISA for the detection of BCM-7 (the minimal detection limit was 0.2 ng/mL). The chicken antibodies proved unsuitable for such use due to their low affinity. These results indicate that it is necessary to make comparisons with methods based on mammalian antibodies in the construction of quantitative ELISA based on chicken polyclonal antibodies.

Bisphenol A modified DNA: A possible immunogenic stimulus for anti-DNA autoantibodies in systemic lupus erythematosus

Anti-DNA antibodies are now considered as a universal diagnostic feature for the patients with systemic lupus erythematosus (SLE) but the mechanism(s) involved in the generation of these autoantibodies remains to be investigated. Bisphenol A (BPA) is a synthetic phenol extensively used in the manufacturing of polycarbonated plastics. Upon mixing in the diet, it causes several health hazards. This study was undertaken to investigate the contribution of BPA induced DNA damage in SLE patients. Human DNA was modified by BPA in-vitro and the binding characteristics of SLE circulating immunoglobulin Gs (SLE-IgGs) with BPA damaged DNA (BPA-DNA) were screened and compared with the IgGs from normal healthy humans (NH-IgGs). Immunogenicity of BPA-DNA was determined by immunisation in rabbits. DNA from SLE patients (SLE-DNA) or healthy humans (NH-DNA) were isolated and their binding specificity with rabbit anti-BPA-DNA-IgGs was studied. Treatment of human DNA with BPA caused extensive damaged. Circulating SLE-IgGs showed strong recognition of BPA-DNA. BPA-DNA induced high titre antibodies in rabbits. Rabbit anti-BPA-DNA-IgGs showed strong cross reaction with isolated DNA from SLE patients. In short, we concluded that the structural alterations in DNA by BPA, generate neo-epitopes that may be a factor responsible for the induction of anti-DNA autoantibodies in SLE.

Hyaluronic Acid-Antibody Fragment Bioconjugates for Extended Ocular Pharmacokinetics.

Treatment of ocular diseases associated with neovascularization currently requires frequent intravitreal injections of antivascular endothelial growth factor (anti-VEGF) therapies. Reducing the required frequency of anti-VEGF injections and associated clinical visits may improve patient adherence to the prescribed treatment regimen and improve outcomes. Herein, we explore conjugation of rabbit and fragment antibodies (Fab) to the biopolymer hyaluronic acid (HA) as a half-life modifying strategy, and assess the impact on Fab biophysical properties and vitreal pharmacokinetics. HA-Fab conjugates of three distinct molecular weights and hydrodynamic radii (RH) were assessed for in vivo pharmacokinetic performance relative to unconjugated Fab after intravitreal injection in rabbits. Covalent conjugation to HA did not significantly alter the thermal stability or secondary or tertiary structure, or diminish the potency of the Fab, thereby preserving its pharmacological properties. Conjugation to HA did significantly slow the in vivo clearance of Fab from the rabbit vitreous in an RH-dependent manner. Compared to free Fab (observed vitreal half-life of 2.8 days), HA-Fab conjugates cleared with observed half-lives of 7.6, 10.2, and 18.3 days for 40 kDa, 200 kDa, and 600 kDa HA conjugates, respectively. This work elucidates a possible strategy for long-acting delivery of proteins intended for the treatment of chronic posterior ocular diseases.

In Vivo Phosphorylation: Development of Specific Antibodies to Detect the Phosphorylated PEPC Isoform for the C4 Photosynthesis in Zea mays.

Phosphoenolpyruvate carboxylases (PEPCs), mostly known as the enzymes responsible for the initial CO2 fixation during C4 photosynthesis, are regulated by reversible phosphorylation in vascular plants. The phosphorylation site on a PEPC molecule is conserved not only among isoforms but also across plant species. An anti-phosphopeptide antibody is a common and powerful tool for detecting phosphorylated target proteins with high specificity. We generated two antibodies, one against a peptide containing a phosphoserine (phosphopeptide) and the other against a peptide containing a phosphoserine mimetic, (S)-2-amino-4-phosphonobutyric acid (phosphonopeptide). The amino acid sequence of the peptide was taken from the site around the phosphorylation site near the N-terminal region of the maize C4-isoform of PEPC. The former antibodies detected almost specifically the phosphorylated C4-isoform of PEPC, whereas the latter antibodies had a broader specificity for the phosphorylated PEPC in various plant species. The following procedures are described herein: (1) preparation of the phosphopeptide and phosphonopeptide; (2) preparation and purification of rabbit antibodies; (3) preparation of cell extracts from leaves for analyses of PEPC phosphorylation with antibodies; and (4) characterization of the obtained antibodies. Finally, (5) two cases involving the application of these antibodies are presented.

The first report of molecular characterized BRD4-NUT carcinoma in Brazil: a case report

BackgroundNUT midline carcinoma is a rare and aggressive subset of squamous cell carcinoma, which is characterized by the translocation of nuclear protein in testis gene that is mostly fused with bromodomain and extraterminal family proteins. We describe here the first Brazilian case of NUT midline carcinoma with BRD4-NUT fusion detected in a next-generation sequencing panel and we present the clinical evolution of this patient.Case presentationA 42-year-old Caucasian man was diagnosed with poorly differentiated squamous cell carcinoma of the left maxillary sinus, with negative in situ hybridization for Epstein–Barr encoding region and human papillomavirus genotyping. He received induction therapy, chemoradiotherapy with weekly systemic chemotherapy, and, concurrently, weekly intra-arterial chemotherapy. New imaging evaluation, 1 month after the end of the last treatment, revealed a good partial response in the primary lesion. However, positron emission tomography-computed tomography showed multiple suspicious lesions in his bones and lungs, which were histologically confirmed. He died exactly 2 months after metastatic disease was diagnosed.ConclusionsNUT midline carcinoma is usually very aggressive. Currently, there is no standard of care for treatment of NUT midline carcinoma. The definitive diagnosis must be by demonstration of NUTM1 rearrangement. Immunohistochemical staining of greater than 50% of tumor nuclei on formalin-fixed paraffin-embedded tissue using the monoclonal rabbit antibody to NUT (clone C52B1), has a specificity of 100%, and sensitivity of 87% for the diagnosis of NUT midline carcinoma. Our case is the first Brazilian case of NUT midline carcinoma with BRD4-NUT fusion.

Development of an enzyme-linked immunosorbent assay for detection of Chlorpyrifos-ethyl and its metabolites 3,5,6-Trichloro-2-Pyridinol and Diethylthiophosphate

ABSTRACT A competitive enzyme-linked immunosorbent assay (ELISA) test using polyclonal rabbit antibodies for detection of Chlorpyrifos-ethyl (CPE) and its two major metabolites of degradation [3,5,6-Trichlororpyridin-2-ol (TCP) and O,O’-diethylthiophosphoric acid (DETP)], has been developed. We have first synthetized haptens of these three molecules, with 4 and 5 carbons spacer arms having carboxyl function at their ends. Haptens with 5 carbons’ spacer arm have been coupled to Bovine Serum Albumin (BSA) then injected to rabbits for polyclonal antibodies production. Haptens with 4 carbons spacer arm have been coupled to Human Albumin (HAS) to be in well plates coating to perform the ELISA tests. After adjusting the ELISA parameters, we have plotted the B/B0 (%) values against the logarithm of analyst concentrations. The IC50 values and detection limits were obtained through fitting the curves. The IC50 value of CPE and TCP were 651 and 5.61 ppb, with detection limits about 2.3410−2 and 2.18 10−3 ppb, respectively. We report for the first time, ELISA system developed for DETP with IC50 of 76.5 and a detection limit of 8.81 10−3 ppb. To evaluate the potential interferences that may be encountered in environmental water samples, pH, ionic strength, humic acid and some heavy metals were tested upon IC50 of the developed ELISA systems. In addition, several pesticides as well as CPE metabolites were tested for cross reactivity study. In all cases, the interference of the CPE assay was negligible. TCP assay represented a high cross reactivity to CPE but no interference with the others compounds. For DETP, no cross reactivity with the TCP was detected, but it was higher with CPE and the others pesticides. The developed ELISA systems have been compared with HPLC technique. Both techniques gave values of the same order of CPE and TCP prepared concentrations with a better sensitivity for ELISA system.

Serodiagnostic potential of Brucella outer membrane and periplasmic proteins

The aim of this study was to evaluate the serological diagnostic potential of the Brucella recombinant outer membrane (rOMP25, rOMP31) and periplasmic proteins (rBP26, rSOD) in a comparative way using an indirect enzyme-linked immunosorbent assay (i-ELISA). Rabbit and/or mouse antibodies to Brucella whole cell and/or soluble protein preparations recognized all recombinant proteins used, which confirms the expression of target antigens in E. coli in active form. The recombinant proteins showed different antigenicity to antibodies of cattle kept on a brucellosis-affected (endemic) farm and/or a new focus of infection. Thus, the presence of anti-Brucella antibodies was confirmed by i-ELISA/rSOD in 79% of cows from endemic conditions with positive results by conventional serological tests (RBPT and/or CFT). However, antibodies specific to this protein were detected in only 14% of seropositive animals kept in the hotbed of a new brucellosis infection. Moreover, rSOD-specific antibodies were not detected in the sera of vaccinated cattle from a brucellosis-free farm, whereas antibodies to other recombinant proteins were found in 2%-8% of animals. Using recombinant proteins in immunoassays significantly reduced the number of cows positive for brucellosis. Furthermore, there was not a single protein among the rOMPs that would show the total positive results of all proteins used. Thus, the development of reliable ELISA tests for the diagnosis of brucellosis requires further comprehensive study of the recombinant proteins in order to design a multiprotein antigen that consists of a combination of several proteins with diagnostic potential.

Delineating Surface Epitopes of Lyme Disease Pathogen Targeted by Highly Protective Antibodies of New Zealand White Rabbits.

Lyme disease (LD), the most prevalent vector-borne illness in the United States and Europe, is caused by Borreliella burgdorferi No vaccine is available for humans. Dogmatically, B. burgdorferi can establish a persistent infection in the mammalian host (e.g., mice) due to a surface antigen, VlsE. This antigenically variable protein allows the spirochete to continually evade borreliacidal antibodies. However, our recent study has shown that the B. burgdorferi spirochete is effectively cleared by anti-B. burgdorferi antibodies of New Zealand White rabbits, despite the surface expression of VlsE. Besides homologous protection, the rabbit antibodies also cross-protect against heterologous B. burgdorferi spirochetes and significantly reduce the pathology of LD arthritis in persistently infected mice. Thus, this finding that NZW rabbits develop a unique repertoire of very potent antibodies targeting the protective surface epitopes, despite abundant VlsE, prompted us to identify the specificities of the protective rabbit antibodies and their respective targets. By applying subtractive reverse vaccinology, which involved the use of random peptide phage display libraries coupled with next-generation sequencing and our computational algorithms, repertoires of nonprotective (early) and protective (late) rabbit antibodies were identified and directly compared. Consequently, putative surface epitopes that are unique to the protective rabbit sera were mapped. Importantly, the relevance of newly identified protection-associated epitopes for their surface exposure has been strongly supported by prior empirical studies. This study is significant because it now allows us to systematically test the putative epitopes for their protective efficacy with an ultimate goal of selecting the most efficacious targets for development of a long-awaited LD vaccine.

Structural and functional peculiarities of the endothelium of heart vessels of mature rats according to immunistochemical studies

The relevance of the study is connected with the modern point of view that endothelial dysfunction may be the cause of such socially significant diseases as atherosclerosis, diabetes, varicose veins, etc. The aim of the work is to study the morphofunctional peculiarities of the endothelium of the cardiovascular vessels of the mature rat using Willebrand factor (vWF) immunohistochemistry.Material and methods. We used mature Wistar rats (n = 12). Polyclonal rabbit antibodies were used for the immunohistochemical detection of vWF. The reaction was carried out on paraffin sections, made through the whole heart.Results. It was shown that the immunopositive reaction to vWF (vWF+ ) in the endothelium of different regions of the rat heart is not uniform. A tendency was found to weaken the vWF+ reaction in the direction from the base of the heart to its apex. Most functionally active endothelial cells with signs of exocytosis were observed in the aortic root, the large coronary arteries of the epicardium, the fibrous ring, the valves and the subaortal cone. vWF+ was less pronounced in the endothelium lining the atrial and ventricular cavities and in the myocardial capillaries.Conclusions. Using immunohistochemical detection of the vWF, the endothelium features of different parts of the rat heart were identified. Structural features due to increased secretion of vascular endothelial cells of the heart were revealed. Further research in this direction may be important to explain the mechanisms and diagnosis of endothelial dysfunction.

Proteasomal degradation within endocytic organelles mediates antigen cross-presentation.

During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human β2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.

Immunohistochemical Analysis of Somatostatin Receptors in Endometriosis Tissue Samples: A Retrospective Study.

Three types of endometriosis are described: superficial peritoneal endometriosis (SPE), ovarian endometrioma (OMA), and deep infiltrating endometriosis (DIE). The expression of somatostatin receptors (SSTR1, 2, and 5) in human endometrial tissue and its ectopic form has been previously studied and may be different in each type of endometriosis. The aim of this study was to assess the immunohistochemical expression of SSTR1, 2, and 5 in tissue samples of SPE, OMA, and DIE. We performed a retrospective analysis in the pathology department database. Patients aged <50 yr and diagnosed with endometriosis have been identified and sorted into 3 groups according to their endometriosis type: SPE, OMA, and DIE. For each selected patient, formalin-fixed paraffin-embedded blocks were retrieved in order to make new sections to be incubated with polyclonal rabbit antibodies anti-SSTR1, 2, and 5. Receptor status was considered as positive on the sections when >50% of the cells showed immunostaining. Seventy-six patients were included in the analysis. SSTR1 and 5 were expressed in 95.4% and 77.2% of SPE, respectively, in 95.8% and 83.3% of OMA, respectively, and in 100% and 80% of DIE, respectively. There was no significant difference between SPE, OMA, and DIE with regard to the SSTR1 (P=0.5) and SSTR5 (P=0.9) expression. We observed a significant difference between SPE (9.0%), OMA (16.6%), and DIE (63.3%) with regard to SSTR2 expression (P<0.05). The present study identifies 2 different immunohistochemical patterns of endometriosis lesions with regard to their SSTR expression: SSTR1+/SSTR2-/SSTR5+ for SPE and OMA, and SSTR1+/SSTR2+/SSTR5+ for DIE.

Pancreas Proapoptotic Proteases in Patients with Chronic Pancreatitis

Aim. To study features of localization of the DNA-ase I and endonuclease-G proapoptotic proteases in the pancreas in chronic pancreatitis (CP). Materials and methods. Histological pancreas preparations from 60 patients with various CP forms were studied: group I — 10 patients with obstructive CP; group II — 21 patients with calcific CP; group III — 13 patients with fibroparenchymal CP; group IV — 16 patients with CP complicated by pseudocyst. Pancreas biopsies were obtained during planned organ operations, as well as using a fine-needle biopsy under ultrasound control. Tissue material was fixed in Bowen medium. Microscopic tissue sections were prepared and subsequently stained with hematoxylin-eosin and by Mallory-Slinchenko. Immunohistochemical typing of proapoptotic proteases was performed according to the indirect avidin-streptavidin-peroxidase reaction (“Elite”, USA) using rabbit antibodies to DNA-I and endonuclease-G (“Chemicon”, USA, 1: 500 dilution — 1: 2000, incubation 12:00, + 4 °С).Results. Patients in all the groups demonstrated signs of chronic inflammation, with 31.7 % of cases showing signs of its exacerbation. Atrophic changes were found in most patients (96.7 %). No significant differences were observed with regard to the severity and frequency of fibrosis of various degrees in the groups: mild, moderate, severe and full fibrosis was detected in 6.7 %, 20.0 %, 16.7 % and 56.6 %, respectively. The study of the localization of proapoptotic nucleases in the structures of the pancreas showed proapoptotic nucleases of DNA-ase I to be exclusively located in the cytoplasm of pancreatic acinar cells. At all stages of CP fibrosis, single acinar cells with translocation of the nuclease from the cytoplasm into the cell nucleus were found in the lobes of the pancreas. Endonuclease-G was found in large numbers in the cytoplasm of pancreatic islets, with it lower number being detected in the cytoplasm of the epithelial cells of the ducts. Conclusions. In CP, proapoptotic proteases of DNase I and endonuclease G are expressed in the cytoplasm of cells located in different pancreas zones. Thus, DNase I is expressed in the cytoplasm of acinar cells, while endonuclease G is most typical for insular cells and those in the epithelium of the ducts. This proves the existence of various apoptosis mechanisms in the exo- and endocrine portions of the pancreas.

New Zealand White Rabbits Effectively Clear Borrelia burgdorferi B31 despite the Bacterium's Functional vlsE Antigenic Variation System.

Borrelia burgdorferi is a tick-borne bacterium responsible for approximately 300,000 annual cases of Lyme disease (LD) in the United States, with increasing incidences in other parts of the world. The debilitating nature of LD is mainly attributed to the ability of B. burgdorferi to persist in patients for many years despite strong anti-Borrelia antibody responses. Antimicrobial treatment of persistent infection is challenging. Similar to infection of humans, B. burgdorferi establishes long-term infection in various experimental animal models except for New Zealand White (NZW) rabbits, which clear the spirochete within 4 to 12 weeks. LD spirochetes have a highly evolved antigenic variation vls system, on the lp28-1 plasmid, where gene conversion results in surface expression of the antigenically variable VlsE protein. VlsE is required for B. burgdorferi to establish persistent infection by continually evading otherwise potent antibodies. Since the clearance of B. burgdorferi is mediated by humoral immunity in NZW rabbits, the previously reported results that LD spirochetes lose lp28-1 during rabbit infection could potentially explain the failure of B. burgdorferi to persist. However, the present study unequivocally disproves that previous finding by demonstrating that LD spirochetes retain the vls system. However, despite the vls system being fully functional, the spirochete fails to evade anti-Borrelia antibodies of NZW rabbits. In addition to being protective against homologous and heterologous challenges, the rabbit antibodies significantly ameliorate LD-induced arthritis in persistently infected mice. Overall, the current data indicate that NZW rabbits develop a protective antibody repertoire, whose specificities, once defined, will identify potential candidates for a much-anticipated LD vaccine.

Determination of hyaluronidase activity in Tityus spp. Scorpion venoms and its inhibition by Brazilian antivenoms

Hyaluronidases (HYALs) are enzymes ubiquitously found in venoms from diverse animals and seem to be related to venom spreading. HYAL activity might be important to Tityus spp. envenoming, since anti-Tityus serrulatus HYAL (TsHYAL) rabbit antibodies neutralize T. serrulatus venom (TsV) lethality. The present work aimed to verify and compare HYAL activity of venoms from other Brazilian Tityus spp. (Tityus bahiensis, Tityus stigmurus and Tityus obscurus) and to test whether anti-TsHYAL antibodies and Brazilian horse therapeutic scorpion antivenom (produced by Fundação Ezequiel Dias (FUNED), Butantan and Vital Brazil Institutes) can recognize and inhibit HYAL activity from these venoms. In ELISA assays, anti-TsHYAL and scorpion antivenoms recognized T. serrulatus, T. bahiensis and T. stigmurus venoms, however, they demonstrated weaker reaction with T. obscurus, which was also observed in Western blotting assay. Epitope mapping by SPOT assay revealed different binding patterns for each antivenom. The assay showed a weaker binding of scorpion antivenom produced by FUNED to peptides recognized by anti-TsHYAL antibodies. Anti-TsHYAL antibodies and antivenoms produced by Butantan and Vital Brazil institutes inhibited HYAL activity of all tested venoms in vitro, whereas FUNED antivenom did not show the same property. These results call attention to the importance of hyaluronidase inhibition, that can aid the improvement of antivenom production.

A multivalent Kaposi sarcoma-associated herpesvirus-like particle vaccine capable of eliciting high titers of neutralizing antibodies in immunized rabbits

Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and the causative agent of multiple cancers in immunocompromised patients. To date, there is no licensed prophylactic KSHV vaccine. In this study, we generated a novel subunit vaccine that incorporates four key KSHV envelope glycoproteins required for viral entry in diverse cell types (gpK8.1, gB, and gH/gL) into a single multivalent KSHV-like particle (KSHV-LP). Purified KSHV-LPs were similar in size, shape, and morphology to KSHV virions. Vaccination of rabbits with adjuvanted KSHV-LPs generated strong glycoprotein-specific antibody responses, and purified immunoglobulins from KSHV-LP-immunized rabbits neutralized KSHV infection in epithelial, endothelial, fibroblast, and B cell lines (60–90% at the highest concentration tested). These findings suggest that KSHV-LPs may be an ideal platform for developing a safe and effective prophylactic KSHV vaccine. We envision performing future studies in animal models that are susceptible to KSHV infection, to determine correlates of immune protection in vivo.

Molecular docking explores heightened immunogenicity and structural dynamics of acetaldehyde human immunoglobulin G adduct.

Acetaldehyde is a metabolite of ethanol, an important constituent of tobacco pyrolysis and the aldehydic product of lipid peroxidation. Acetaldehyde induced toxicity is mainly due to its binding to cellular macromolecules resulting in the formation of stable adducts accompanied by oxidative stress. The aim of this study was to characterize structural and immunological alterations in human immunoglobulin G (IgG) modified with acetaldehyde in the presence of sodium borohydride, a reducing agent. The IgG modifications were studied by various physicochemical techniques such as fluorescence and CD spectroscopy, free amino group estimation, 2,2-azobis 2-amidinopropane (AAPH) induced red blood cell hemolysis as well as transmission electron microscopy. Molecular docking was also employed to predict the preferential binding of acetaldehyde to IgG. The immunogenicity of native and acetaldehyde-modified IgG was investigated by immunizing female New Zealand white rabbits using native and modified IgG as antigens. Binding specificity and cross reactivity of rabbit antibodies was screened by competitive inhibition ELISA and band shift assays. The modification of human IgG with acetaldehyde results in quenching of the fluorescence of tyrosine residues, decrease in free amino group content, a change in the antioxidant property as well as formation of cross-linked structures in human IgG. Molecular docking reveals strong binding of IgG to acetaldehyde. Moreover, acetaldehyde modified IgG induced high titer antibodies (>1:12800) in the experimental animals. The antibodies exhibited high specificity in competitive binding assay toward acetaldehyde modified human IgG. The results indicate that acetaldehyde induces alterations in secondary and tertiary structure of IgG molecule that leads to formation of neo-epitopes on IgG that enhances its immunogenicity.