Hyperimmune Rabbit Antisera Research Articles

Antibody deposition on vascular endothelial cells contributes to localized inflammation in salivary glands.

Oral and ocular dryness due to reduced saliva and tear production, exocrine gland inflammation, and autoantibodies to multiple cellular proteins are the cardinal features of Sjögren's Disease. Among the autoantibody specificities, anti-Ro52 is linked with higher disease severity. We have previously reported that mice immunized with recombinant Ro52 developed IgG deposits in salivary and lacrimal glands and showed reduced saliva and tear production. Furthermore, passive transfer of sera from Ro52-immunized mice rapidly induced glandular dysfunction without immune cell infiltration in recipient mice. To identify mechanisms driving antibody-mediated salivary gland dysfunction, hyperimmune rabbit antiserum to mouse Ro52 was passively transferred into NZM2758 female mice, pretreated with alum adjuvant. Alum-pretreated mice given hyperimmune rabbit antiserum to maltose-binding protein served as controls. Antibody deposition and its distribution in the salivary glands were studied by immunofluorescence staining for rabbit IgG, nerve fibers, and endothelial cells. The nCounter inflammation panel was used to determine differentially expressed genes in the salivary gland. Rabbit IgG deposits were detected in salivary glands of anti-Ro52 immune sera recipients. The rabbit IgG was present on the endothelial cells in small blood vessels, and it did not co-localize with nerve fibers. Ingenuity pathway analysis of the gene expression dataset predicted the canonical vascular endothelial growth factor (VEGF) pathway as the most activated and Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) as the most inhibited pathway in the salivary glands of anti-Ro52 sera recipients. Our study suggests that autoantibody deposition on salivary gland endothelial cells might play a critical role in the pathogenesis of Sjögren's Disease.

Antigenic cross-reactivity among human, swine, rabbit and avian hepatitis E virus capsid proteins

Hepatitis E virus (HEV), a zoonotic virus, infects many animal species, including humans. Capsid proteins of human, swine, rabbit and avian HEVs share 48 %–100 % amino acid identity. In the present study, antigenic cross-reactivity among human, swine, rabbit and avian HEV capsid proteins were analyzed in detail using indirect and blocking enzyme-linked immunosorbent assays (ELISAs). The C-terminal 268 amino acids of genotype 1 human, genotype 4 swine, genotype 3 rabbit and genotype B3 avian HEV capsid proteins served as coating antigens for ELISA. Hyperimmune rabbit antisera (against four HEV capsid proteins) and human, pig, rabbit and chicken clinical sera were as primary antibodies. Closely correlated and statistically indistinguishable results were obtained for detection of anti-HEV antibodies in human and pig sera using human, swine and rabbit HEV capsid proteins as coating antigens. Moderately correlated differences in detection of anti-HEV antibodies in rabbit sera were obtained using the three capsid proteins. Statistically significant differences with no correlations were obtained for anti-HEV antibodies detection in chicken sera between avian HEV capsid protein and human, swine and rabbit ones. Blocking ELISA results demonstrated that two common epitopes among the four species HEVs were immunodominant in avian HEV, but were non-immunodominant in human, swine and rabbit HEVs. Nevertheless, three epitopes common to human, swine and rabbit HEVs were all immunodominant epitopes for the three species HEVs. Collectively, these results demonstrate that anti-HEV antibodies in human and pig sera can be detected using human, swine and rabbit HEV capsid proteins. By contrast, for optimal detection of anti-HEV antibodies in rabbit and chicken sera, the respective rabbit and avian HEV capsid proteins should be used. These results provide insights to guide future development of serological assays for diagnosing HEV infections in various animal species.

Mm19, a Mycoplasma meleagridis Major Surface Nuclease that Is Related to the RE_AlwI Superfamily of Endonucleases.

Mycoplasma meleagridis infection is widespread in turkeys, causing poor growth and feathering, airsacculitis, osteodystrophy, and reduction in hatchability. Like most mycoplasma species, M. meleagridis is characterized by its inability to synthesize purine and pyrimidine nucleotides de novo. Consistent with this intrinsic deficiency, we here report the cloning, expression, and characterization of a M. meleagridis gene sequence encoding a major surface nuclease, referred to as Mm19. Mm19 consists of a 1941- bp ORF encoding a 646-amino-acid polypeptide with a predicted molecular mass of 74,825 kDa. BLASTP analysis revealed a significant match with the catalytic/dimerization domain of type II restriction enzymes of the RE_AlwI superfamily. This finding is consistent with the genomic location of Mm19 sequence, which dispalys characteristics of a typical type II restriction-modification locus. Like intact M. meleagridis cells, the E. coli-expressed Mm19 fusion product was found to exhibit a nuclease activity against plasmid DNA, double-stranded DNA, single-stranded DNA, and RNA. The Mm19-associated nuclease activity was consistently enhanced with Mg2+ divalent cations, a hallmark of type II restriction enzymes. A rabbit hyperimmune antiserum raised against the bacterially expressed Mm19 strongly reacted with M. meleagridis intact cells and fully neutralized the surface-bound nuclease activity. Collectively, the results show that M. meleagridis expresses a strong surface-bound nuclease activity, which is the product of a single gene sequence that is related to the RE_AlwI superfamily of endonucleases.

Using specific synthetic peptide (p176) derived AgB 8/1-kDa accompanied by modified patient’s sera: A novel hypothesis to follow-up of Cystic echinococcosis after surgery

Cystic echinococcosis (CE) is caused by the larval stage of the tapeworm Echinococcus granulosus. Until now, CE does not have an effective follow-up after surgery. To date, CE follow-up is conducted based on either antibody or antigen detection assays by double antibody sandwich ELISA. Unlike antigen detection, antibodies to imply exposure to an Echinococcus infection while their titration could remain for a longer period (1–10years) after surgery. Likewise, antibody respond may be related to the location of a mature hydatid cyst. Antigen detection shows presence of CE infection, it is extremely important and necessary in follow-up of CE after surgery. The circulating antigen (CAg) titration decrease faster than circulating antibody (during 1–3weeks) after operation. Location of hydatid cyst in detecting antigen is affected less than antibody also. Regarding this subject, antigen detection has several limitations that lead to be used less in CE follow-up. Although, AgB 8/1-kDa subunit is considered as a principle and immunogenic CAg but sensitivity of CAg detection compared to with antibody has variable range, between 33% and 85% which owing to formation of circulating immune complexes (CICs) in result of antigen – antibody complex. The another problem is non using specific CAg (AgB 8/1-kDa subunit) for production of specific paratopes (rabbit hyper immune antiserum) against AgB 8/1-kDa which is used as capture (primary) antibody in double antibody sandwich ELISA assay. The designation of synthetic peptides from conserved regions of AgB 8/1-kDa can help to this problem. These regions (motifs) should be selected for allelic, dominant, immunogenic and conserved without any genetic variation. The first part of this hypothesis suggests which patient’s sera should be treated with acidic buffers such as boric acid, acetic acid, glycine/HCl, polyethylene glycol (PEG) or combination of each of them accompanied by boiling patient’s sera which causes breaking Ag-Ab complexes and in result of releasing AgB 8/1. These modifications are effective to releasing CAg from CIC. To date, the synthetic peptides have been widely used in CE serodiagnosis based on circulating antibody detection only. In second part of this hypothesis suggests the using synthetic peptide of p176 derived AgB 8/1-kDa subunit containing conserved specific epitopes for preparation of specific paratopes (rabbit hyper immune antiserum) based on CAg detection. So, there is no need any native antigens for preparation of non-specific rabbit hyper immune antiserum. These novel improvements can help to decrease the cross-reactivity with other parasitic diseases (specificity). Increasing antigen detection could make a chance in sensitivity of patient’s sera and in result of the best and suitable tool for CE follow-up.

First isolation of human Leptospira strains, Azores, Portugal

The aim of this study was the first identification of Leptospira isolates from Azorean inpatients. Whole blood samples from 68 inpatients attending the São Miguel Hospital between 2006 and 2008, with a clinical and epidemiological suspicion of leptospirosis, were inoculated in a transport medium broth at the patient's bedside and further processed using a serial dilution technique prior to culture. At admission, 62 (91%) patients were also analyzed for the presence of leptospiral DNA by a nested PCR and 40 (59%) for specific agglutinins by microscopic agglutination test (MAT). The isolates obtained were first assigned at the serogroup level by both MAT reactivity with hyperimmune rabbit antisera and a PCR-based assay with the single primer iRep1. The species identification was performed by DNA sequencing. The use of monoclonal antibodies allowed intraspecific discrimination at the serovar level. Of the 10 (14.7%) human Leptospira isolates, seven were identified as Leptospira interrogans serovar Copenhageni and three as Leptospira borgpetersenii serovar Arborea, which is in agreement with previous data from the Azorean rodent population. This study represents a great step towards the definitive identification of the pathogenic leptospires in Azorean patients and confirms the bacteriological human-rodent connection for the first time.

Protein and antigen profiles of Leptospira interrogans serovar Hardjo

The protein profile of the outer membrane of Leptospira interrogans serovar Hardjo subtype hardjoprajitno associated with the bovine natural immune response was investigated. The outer membrane proteins were extracted utilizing Triton X114 and precipitated with acetone. The protein sample was then resolved by SDS-PAGE and reacted in western blot against sera from a hyperimmune rabbit and from naturally infected bovines. In silver stained gels, 14 protein bands were observed, among which four proteins, with 22, 29, 47 and 63kDa, appeared as major constituents. Western blot tests with hyperimmune rabbit antiserum detected bands corresponding to proteins with 35; 27; 24; 21; 17 and 14kDa, while 32kDa and 45kDa proteins were the most immunoreactive with sera from naturally infected bovines.

Experimental Infection of Goats with an Unusual Strain of Mycoplasma mycoides subsp. capri Isolated in Jordan

Goats were infected experimentally with a mycoplasma (the "Irbid" strain) isolated previously from a goat with contagious agalactia in northern Jordan. The strain was unusual in that, although it had been identified by molecular methods as Mycoplasma mycoides subsp. mycoides LC/Mycoplasma mycoides subsp. capri, it showed no inhibition of growth by any of the hyperimmune rabbit antisera conventionally used to speciate members of the Mycoplasma mycoides cluster. Animals were infected either intratracheally or by aerosol and placed "in-contact" with other goats. After 2 weeks, those infected intratracheally became febrile, showing a nasal discharge and slight conjunctivitis, followed a week later by respiratory distress and polyarthritis; lesions seen at necropsy included coagulative necrotic pneumonia, fibrinous pleurisy with pleural exudate, and inflammatory exudates, necrosis and fibrosis in the joints. Animals infected by aerosol showed much milder clinical signs, including nasal discharge and occasional swollen joints. In the "in-contact" goats, seroconversion was first seen after 7 weeks, accompanied by coughing and laboured respiration; lesions in this group consisted of fibrinous pneumonia with focal areas of necrosis and abundant pleural exudate.

Surface modification of polystyrene microspheres with synthetic antigenic determinants of human immunodeficiency virus

The chemisorption of synthetic antigenic determinants of the human immunodeficiency virus on the surface of polystyrene microspheres with surface layers containing polystyrene-poly(vinylpyrrolidone) graft copolymers and carboxyl groups has been studied. The electrosurface properties of carrier particles before and after modification with peptides and agglutination of peptide-modified latexes with hyperimmune rabbit antisera have been investigated. It has been demonstrated that the stabilization of the structure of the hydrophilic surface layer via lengthening of polystyrene chains in the graft copolymers leads to an increase in the efficiency of exposure of antigenic determinants at the interface and improves the sensitivity of latex immunoreagents produced on their basis.

Cross-reactivity among sapovirus recombinant capsid proteins

Sapovirus (SaV), a member of the genus Sapovirus in the family Caliciviridae, is an agent of human and porcine gastroenteritis. SaV strains are divided into five genogroups (GI-GV) based on their capsid (VP1) sequences. Human SaV strains are noncultivable, but expression of the recombinant capsid protein (rVP1) in a baculovirus expression system results in the self-assembly of virus-like particles (VLPs) that are morphologically similar to native SaV. In this study, rVP1 constructs of SaV GI, GII, and GV strains were expressed in a baculovirus expression system. The structures of the GI, GII, and GV VLPs, with diameters of 41-48 nm, were morphologically similar to those of native SaV. However a fraction of GV VLPs were smaller, with diameters of 26-31 nm and spikes on the outline. This is the first report of GII and GV VLP formation and the first identification of small VLPs. To examine the cross-reactivities among GI, GII, and GV rVP1, hyperimmune rabbit antisera were raised against Escherichia coli-expressed GI, GII, and GV N- and C-terminal VP1. Western blotting showed the GI antisera cross-reacted with GV rVP1 but not GII rVP1; GII antisera cross-reacted weakly with GI rVP1 but did not cross-react with GV rVP1; and GV antisera reacted only with GV rVP1. Also, hyperimmune rabbit and guinea pig antisera raised against purified GI VLPs were used to examine the cross-reactivities among GI, GII, and GV VLPs by an antigen enzyme-linked immunosorbent assay (ELISA). The ELISA showed that the GI VLPs were antigenically distinct from GII and GV VLPs.

A set of recombinant antigens from Echinococcus granulosus with potential for use in the immunodiagnosis of human cystic hydatid disease.

Several recombinant clones expressing antigens from Echinococcus granulosus were isolated previously from a parasite cDNA library using cystic hydatid disease (CHD) patients' sera or rabbit hyperimmune antiserum against a lipoproteic fraction from bovine cyst fluid. Six of these antigens were expressed in Escherichia coli and the purified recombinant proteins were tested in enzyme-linked immunosorbent assay (ELISA) for specific IgG with a panel of sera from patients with surgically confirmed (n = 58) or immunologically diagnosed (n = 71) CHD. Sera from clinically normal individuals (n = 203) and sera from individuals with other helminthic infections (n = 65) were assayed for the assessment of specificity. A cut-off value was determined by receiver-operating-characteristic plots for each antigen. A recombinant antigen B subunit (AgB8/2) presented the highest sensitivity (93.1%), considering the group of sera from patients with CHD surgically confirmed, and specificity (99.5%) and is proposed as the basis for an immunodiagnostic test. The other recombinant antigens tested presented sensitivities between 58.6% and 89.7%, and three of them were considered of complementary value. In subclass-specific ELISA, different IgG isotypes showed dominance in the response for each of the recombinant antigens. There was a clear predominance of IgG4 response for all antigens tested, indicating that this would be the subclass of choice to be assessed for these recombinant proteins.

Complement-mediated enhancement of antibody function for neutralization of pseudotype virus containing hepatitis C virus E2 chimeric glycoprotein.

We previously reported a number of features of hepatitis C virus (HCV) chimeric glycoproteins related to pseudotype virus entry into mammalian cells. In this study, pseudotype virus was neutralized by HCV E2 glycoprotein-specific antibodies and infected human sera. Neutralization (50% reduction of pseudotype virus plaque formation) was observed with two human immunoglobulin G1 monoclonal antibodies (MAbs) at concentrations of between 2.5 and 10 microg/ml. A hyperimmune rabbit antiserum to an E2 hypervariable region 1 (HVR1) mimotope also exhibited an HCV E2 pseudotype virus neutralization titer of approximately 1/50. An E1 pseudotype virus used as a negative control was not neutralized to a significant level (<1/10) by these MAbs or rabbit antiserum to E2 HVR1. Since HCV probably has a lipid envelope, the role of complement in antibody-mediated virus neutralization was examined. Significant increases in the neutralization titers of the human MAbs (approximately 60- to 160-fold higher) and rabbit antiserum to HVR1 mimotopes (approximately 10-fold higher) were observed upon addition of guinea pig complement. Further, these studies suggested that complement activation occurred primarily by the classical pathway, since a deficiency in the C4 component led to a significant decrease in the level of virus neutralization. This same decrease was not observed with factor B-deficient complement. We also determined that 9 of 56 HCV-infected patient sera (16%) had detectable pseudotype virus neutralization activity at serum dilutions of between 1/20 and 1/50 and that complement addition enhanced the neutralization activity of some of the HCV-infected human sera. Taken together, these results suggest that during infection, HCV E2 glycoprotein induces a weak neutralizing antibody response, that those antibodies can be measured in vitro by the surrogate pseudotype virus plaque reduction assay, and that neutralization function can be augmented by complement.

Detection of circulating antigens in Gabonese patients with Loa loa filariasis.

Circulating antigens were detected in sera of Gabonese patients with Loa loa filarial infection by co-electrosyneresis (Co-ES). The serum samples selected for this study were exposed to rabbit hyperimmune antifilarial antisera and human immune antisera: specific arcs were observed in 35/60 filarial patients, of whom 24/47 were microfilaraemic and 11/13 amicofilaraemic. The identity of rabbit hyperimmune antisera and loiasial immune sera was confirmed by the continuity of precipitation arcs. These preliminary studies were carried out to develop diagnostic tools for loiasis and to study the physiopathology of immune complexes.

Polyclonal B-Cell activation byNeisseria meningitidis capsular polysaccharides elicit antibodies protective againstTrypanosoma cruzi infection in vitro

A hyperimmune rabbit antiserum against group C Neisseria meningitidis agglutinated and lysed Trypanosoma cruzi metacyclic trypomastigotes in a complement-mediated reaction. Immunization of rabbits with the purified polysaccharide C from N. meningitidis and of human volunteers with the AC-polysaccharide vaccine against meningitis also resulted in antibody production cross-reactive with T. cruzi infective forms. The rabbit antibodies bound to parasites, lysed metacyclic forms, and recognized several components from lysates of cell-derived trypomastigotes. The sera from six human volunteers reacted with cell-cultured trypomastigotes in vitro, lysed these forms, and recognized glycoconjugates migrating diffusely on the top of immunoblots. One serum also reacted with the isolated mucin-like glycoconjugate carrying the Ssp-3 epitope from cell-derived trypomastigotes, but treatment with sialidase did not abolish this reactivity. The anti-AC human antiserum also protected against HeLa cell infection and markedly decreased the number of parasites liberated after cell burst. The polyclonal response that resulted from human immunization with N. meningitidis polysaccharides A and C comprised trypanolytic antibodies that recognized nonsialylated epitopes expressed on infective forms of the parasite. It is suggested that human AC vaccination could be potentially helpful as an adjuvant to a specific immunotherapy of Chagas disease, developed with native or recombinant antigens of the parasite.

Late bioprosthetic valve endocarditis caused byPhialemoniumaff.curvatumandStreptococcus sanguis: a case report

Dual fungal and Streptococcus sanguis endocarditis is reported in a 63-year-old woman 7 months after placement of a porcine aortic valve prosthesis. Both micro-organisms were isolated by blood cultures, and the patient succumbed after a full course of antibacterial chemotherapy without having received antifungal chemotherapy. The best possible designation of the fungus was Phialemonium aff. curvatum W. Gams & W. B. Cooke, as represented by CBS 331.93. At autopsy hyphae were revealed in the porcine valve tissue by conventional staining. A hyperimmune rabbit antiserum raised towards strain CBS 331.93 and extensively absorbed with heterologous fungal antigens reacted strongly with hyphae in the valve tissue by indirect immunofluorescence technique. We consider it most likely that the Phialemonium infection evolved insidiously from the time of open heart surgery and led to a haematogenous streptococcal infection of a more fulminant course.

Major membrane proteins and lipoproteins as highly variable immunogenic surface components and strain-specific antigenic markers of Mycoplasma arthritidis

Surface antigenic variation was investigated in Mycoplasma arthritidis, an agent that produces chronic arthritis in rats which shares several features with many mycoplasma-induced diseases and thus defines a well-characterized model system. Hyperimmune rabbit antisera (anti-ISR1, anti-PG6, anti-H606 and anti-158p10) to whole M. arthritidis organisms were used as immunological probes in Western immunoblots of four M. arthritidis prototype strains (ISR1, PG6, H606 and D263) and five rat-passaged substrains (ISR1p1, ISR1p7, ISR1p8, 158p10 and D263p1). Several prominent antigens were identified that varied in expression. By Triton X-114 phase fractionation and treatment of whole cells with trypsin and carboxypeptidase Y, these strain-variant antigens were shown to be integral membrane proteins with C-termini and portions of the polypeptide chains oriented outside the membrane. Western blot immunoscreening of a large number of randomly selected clonal isolates and well-established clonal lineages from stock cultures of M. arthritidis ISR1p7, 158p10, PG6 and H606 revealed an expanded repertoire of variant membrane proteins whose expression was subject to independent, reversible phase variation. Colony immunoblots of these clonal populations with a hyperimmune rabbit antiserum to a gel-purified variant membrane protein (P36) showed that this phase switching occurred at a high frequency (10(-4) to 10(-2) per generation). Detailed immunological and biochemical characterization of the phase-variant membrane proteins demonstrated that they are: (i) antigenically related or distinct; (ii) apparently specific to particular strain populations; (iii) proteins or lipoproteins; (iv) major immunogens of M. arthritidis, recognized by serum antibodies from convalescent rat; and (v) able to undergo variation in expression during in vivo passage. Thus, M. arthritidis possesses a complex system capable of creating large repertoires of cell surface phenotypes which may affect the multiple interactions of this organism with its host and dictate its potential as a successful infectious agent and pathogen.

Immunomagnetic separation and PCR for detection of Helicobacter pylori in water and stool specimens.

The detection of Helicobacter pylori in clinical and environmental samples by PCR sometimes requires removal of polymerase inhibitors. We have used a magnetic immunoseparation technique as pre-PCR treatment to facilitate direct detection of H. pylori in stool and water specimens. Rabbit hyperimmune antiserum was produced and magnetic beads were coated with purified immunoglobulin G, which reacted with and bound to both coccoid and rod-shaped forms of H. pylori. When PCR was applied for the detection of H. pylori from cultured samples, the number of organisms that was required for positive scores varied significantly. For a 3-day culture of H. pylori, samples containing 10(2) bacteria per ml are needed for a positive score; for a 6-day culture, samples containing 10(4) bacteria per ml are needed; and for a 10-day culture, samples containing 10(6) bacteria per ml are needed. These results indicate that the coccoid forms of H. pylori may have a different antigenicity and DNA content and are therefore more difficult to detect by immunomagnetic separation and PCR than the rod-shaped forms. Spiked samples with the addition of feces, spiked water samples, and a patient stool specimen were all scored positive with this technique.

Antibodies to capsular polysaccharides are not protective against experimental Staphylococcus aureus endocarditis.

The protective efficacy of antibodies to the Staphylococcus aureus capsular polysaccharide was examined in a rat model of catheter-induced endocarditis. Capsular antibodies were induced either by active immunization with killed S. aureus or by passive immunization with hyperimmune rabbit antiserum to S. aureus. Control rats were injected with phosphate-buffered saline or passively immunized with normal rabbit serum or rabbit antiserum to a nonencapsulated strain. Animals with indwelling catheters were challenged intravenously with 5 x 10(4) to 4 x 10(6) CFU of the homologous S. aureus strain (capsular serotype 5 strain Reynolds or serotype 1 strain SA1 mucoid). Both immunized and control rats developed S. aureus endocarditis. The numbers of S. aureus cells recovered from the blood and aortic valve vegetations of immunized rats were similar to those of control rats, indicating that capsule-specific antibodies were not protective. To determine whether the presence of an indwelling catheter interfered with antibody-mediated protection against S. aureus endocarditis, catheters were removed 2 h after insertion in additional groups of rats. An inoculum of 10(8) CFU of strain Reynolds was needed to provoke endocarditis in rats catheterized for 2 h, compared with 5 x 10(4) CFU for rats with indwelling catheters. Passively transferred capsular antibodies were not protective since both immunized and nonimmunized animals developed endocarditis, and quantitative cultures of blood and valvular vegetations revealed no differences between immunized and control animals. The findings of this study indicate that antibodies to the capsular polysaccharide are not protective in the rat model of experimental S. aureus endocarditis.

Endogenous retroviral envelope peptide expression is involved in a regulation of lymphocyte and hematopoietic precursor activity

A biological function of endogenously expressed MuLV p15E-related proteins for lymphocyte and hematopoietic precursor activity in mice was examined. A high level of endogenous p15E-related peptide expression in spleen cells of mice with hemolytic anemia rendered by phenylhydrazine (PHZ) treatment was observed, detected by hyperimmune rabbit antisera against amino acid sequence which compose the immunosuppressive domain (ISD) of exogenous viral transmembrane (TM) p15E protein. The conditioned medium of these cultured cells (PHZ/CM) was inhibitory for lymphocyte blastogenesis and granulocyte-macrophage (GM) precursor activity, but stimulatory for the erythroid colony growth. When added to PHZ/CM, anti-ISD/p15E antibodies were capable to abrogate these effects. These antibodies bound 14K and 48K structural peptides contented in PHZ/CM as presumably smaller components of env gene products. Given together, the results indicate that erythroid immature cells produce proteins appearing in cell culture medium which exert p15E-related properties. These peptides are suggested to exert a down regulation for both lymphocyte and GM precursor activities, and the colony-promoting effect towards erythroid compartment cells.

A 9.0-kilobase-pair circular plasmid of Borrelia burgdorferi encodes an exported protein: evidence for expression only during infection

In this study, we report the cloning, sequencing, and molecular analysis of a gene located on a 9.0-kbp circular plasmid of virulent Borrelia burgdorferi B31 designated eppA (exported plasmid protein A). This gene encodes a precursor protein of 174 amino acids including a signal peptide of 20 amino acids and a type I signal peptidase cleavage site. The mature EppA protein of 154 amino acids has a calculated molecular weight of 17,972. Several lines of evidence suggest that eppA is not expressed by B. burgdorferi B31 during in vitro cultivation. Immunoblot analysis using hyperimmune rabbit antiserum to recombinant EppA (rEppA) did not detect the presence of EppA in B. burgdorferi B31 cultivated in vitro. Northern blot analysis using total RNA isolated from in vitro-cultivated virulent B. burgdorferi B31 failed to detect an eppA transcript. EppA was not detected in culture supernatants of virulent B. burgdorferi B31 in a sensitive antigen-capture enzyme-linked immunosorbent assay. In contrast, evidence for expression of eppA during infection was based on the observation that patients with Lyme disease as well as rabbits experimentally infected with B. burgdorferi B31 produced antibodies that recognized rEppA. Because the cellular location of EppA in B. burgdorferi cannot be determined in vivo because of very small numbers of organisms present in vertebrate infection, we examined the cellular location of rEppA expressed in Escherichia coli. In E. coli, rEppA is targeted to the outer membrane. In addition, purified E. coli outer membranes containing rEppA treated with chaotrophic agents did not result in rEppA release. These findings are consistent with the idea that EppA is not peripherally associated with the outer membrane of E. coli but rather has an integral outer membrane association.

Identification and Characterization of a Mycoplasma synoviae 55,000-Molecular-Weight Antigen Associated with Hemagglutination

Serological studies have shown that some antigenic determinants are conserved among several pathogenic Mycoplasma species, including Mycoplasma pneumoniae, M. genitalium, and M. gallisepticum. M. synoviae, an avian pathogen that shares certain morphological and biological features with the above-mentioned mycoplasmas, was examined by the protein immunoblot procedure for its reactivity with hyperimmune rabbit antiserum specific for the major (190,000 molecular-weight [MW]) adhesion P1 protein of M. pneumoniae. A single polypeptide of M. synoviae of approximately 55,000 MW was recognized by the anti-P1 antiserum. The 55,000-MW antigen was electroeluted following electrophoretic separation of M. synoviae polypeptides, and the eluted protein was used for immunization of mice for the production of monoclonal antibodies (MAbs) and polyclonal antiserum. Immunoelectron microscopy with MAbs and gold-conjugated secondary antibodies showed that the 55,000-MW antigen was located at the cell surface and was more densely clustered around the bleb-like protuberance of the cell. Immuno-affinity-purified 55,000-MW antigen, as well as the antibodies produced against it, blocked the hemagglutination by M. synoviae.