Use of the recombinant 38-kDa antigen of Mycobacterium tuberculosis as an immunogen for specific antisera production

The Mycobacterium tuberculosis 38-kDa protein antigen is one of the secreted immunodominant antigens showing high immunogenicity at B-cell and T-cell levels. Although monoclonal antibodies to this antigen have been produced, specific polyclonal antisera is required for standardization of specific immunodiagnostic assays. This protein has been overexpressed and purified from recombinant Escherichia coli using an inducible vector system. During each stage of expression and purification, the recombinant protein was used to immunize mice and rabbits by several methods: 1) as overexpressed protein present as inclusion bodies in recombinant E. coli; 2) embedded in a polyacrylamide gel; 3) fixed to a solid-phase nitrocellulose membrane and 4) emulsified with an adjuvant. All strategies yielded specific antisera as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. The results obtained, both quantitative (ELISA) and qualitative (immunoblot) demonstrate that the purified recombinant antigen retains its antigenicity and immunogenicity throughout the various steps in the process of expression and purification and serves as a potent antigen for production of specific antisera to be used in immunoassays.     

Synergistic cooperation between phorbol ester and IFN-gamma for induction of nitric oxide synthesis in murine peritoneal macrophages

The role of protein kinase C (PKC) in the induction of nitric oxide (NO) synthesis in murine peritoneal macrophages was examined. Phorbol ester, a PKC activator, had no effect on NO synthesis by itself, whereas IFN-gamma alone had modest activity. When phorbol ester was used in combination with IFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. This increase in NO synthesis was reflected as increased amount of inducible NO synthase (iNOS) mRNA, as determined by Northern blotting. The optimal effect of phorbol ester was shown at 6 h after treatment with IFN-gamma. Phorbol ester also induced the release of NO to the incubation medium by bacillus Calmette-Guerin-infected peritoneal macrophages. Prolonged incubation of cells with phorbol ester, which down-regulates PKC activity, abolished the synergistic cooperative effect on NO production with IFN-gamma. In addition, such PKC inhibitors as staurosporin or polymyxin B reduced NO production induced by IFN-gamma plus phorbol ester. When the cells were treated with both actinomycin D and phorbol ester after IFN-gamma stimulation, more NO was produced and more iNOS mRNA was expressed than in the cells treated with actinomycin D alone. On the basis of these observations, we conclude that PKC might not be directly involved in the expression of NO synthase, but, instead, might be involved in the stabilization of the iNOS mRNA already expressed by the treatment of IFN-gamma.     

Cloning and characterization of the NAD-linked glycerol-3-phosphate dehydrogenases of Trypanosoma brucei brucei and Leishmania mexicana mexicana and expression of the trypanosome enzyme in Escherichia coli

A polyclonal antiserum raised against the purified glycosomal glycerol-3-phosphate dehydrogenase of Trypanosoma brucei brucei has been used to identify the corresponding cDNA clone in a T.b. brucei expression library. This cDNA was subsequently used to obtain genomic clones containing glycerol-3-phosphate dehydrogenase genes. Two tandemly arranged genes were detected in these clones. Characterization of one of the genes showed that it codes for a polypeptide of 353 amino acids, with a molecular mass of 37,651 Da and a calculated net charge of +8. Using the T.b. brucei gene as a probe, a corresponding glycerol-3-phosphate dehydrogenase gene was also identified in a genomic library of Leishmania mexicana mexicana. The L.m. mexicana gene codes for a polypeptide of 365 amino acids, with a molecular mass of 39,140 Da and a calculated net charge of +8. The amino-acid sequences of both polypeptides are 63% identical and carry a type-1 peroxisomal targeting signal (PTS1) SKM and -SKL at their respective C-termini. Moreover, the L.m. mexicana polypeptide also carries a short N-terminal extension reminiscent of a mitochondrial transit sequence. Subcellular localisation analysis showed that in L.m. mexicana the glycerol-3-phosphate dehydrogenase activity co-fractionated both with mitochondria and with glycosomes. This is not the case in T. brucei, where the enzyme is predominantly glycosomal. The two trypanosomatid sequences resemble their prokaryotic homologues (32-36%) more than their eukaryotic counterparts (25-31%) and carry typical prokaryotic signatures. The possible reason for this prokaryotic nature of a trypanosomatid glycerol-3-phosphate dehydrogenase is discussed.     

A single-chain Fv reactive with the Goodpasture antigen

Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement membrane and characterized clinically by rapidly progressive glomerulonephritis and pulmonary hemorrhage. P1, a murine monoclonal antibody to the Goodpasture antigen (the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1), has been a valuable reagent in investigating the pathogenesis of this disorder. The purpose of this study was to generate and characterize a recombinant form of P1 as a single-chain Fv (scFv). First strand cDNA was made from RNA extracted from the P1 hybridoma cell line, and DNA encoding the antibody light and heavy chain variable domains was amplified by polymerase chain reaction, using universal oligonucleotides. The purified products were ligated sequentially into an expression plasmid separated by a sequence encoding a 15 amino acid flexible oligopeptide linker. The resulting scFv was expressed in E. coli. Functional scFv, designated HBR-3, was obtained by denaturing and refolding the expressed product. HBR-3 was shown by ELISA, immunoblotting, and immunohistologic techniques, to have the same specificity for alpha 3(IV)NC1 as P1 and autoantibodies from patients with Goodpasture's disease. HBR-3 and P1 were shown to have similar affinity for their mutual ligand. On sections of normal human kidney, the scFv bound only to glomerular basement membrane and distal tubular basement membrane. It did not bind to the glomerular basement membrane of patients with Alport's syndrome, in whom the Goodpasture antigen is often not expressed in an antigenic form. We have, therefore, generated a scFv which reproduces the specific binding properties of the parent monoclonal antibody, P1. The potential of HBR-3 as a diagnostic reagent in Alport's syndrome has been demonstrated. The development of this recombinant molecule should permit new approaches to the investigation of Goodpasture's disease.     

Conditions for the induction of long-term potentiation in layer II/III horizontal connections of the rat motor cortex

1. The present studies investigated conditions for the induction of long-term potentiation (LTP) in the local horizontal pathways of layers II/III in the primary motor cortex (MI) of the adult rat. Field potential and intracellular recordings demonstrated synaptic interactions across the superficial layers within MI that could be enhanced transiently by focal application of the gamma-aminobuturic acid-A receptor antagonist bicuculline methiodide (Bic) at the recording site. 2. Field potentials evoked in the superficial MI horizontal pathways increased in amplitude after tetanizing, theta burst stimulation (TBS), but only when Bic was applied transiently at the recording site immediately before TBS. In the absence of Bic, TBS failed to produce long-lasting increases in horizontally evoked field responses. By contrast, TBS delivery during focal Bic application increased field potential amplitudes by 25-35% when measured 25-30 min after stimulation. The amount of potentiation was greater when two converging horizontal inputs were stimulated together but was not increased with higher intensity stimulation. Persistent effects of Bic application alone were evident. However, these effects were small unless Bic application continued until evoked field potential amplitude increase exceeded 200% of baseline. 3. The synaptic nature of field potential increases were confirmed using intracellular recordings of layer II/III neurons located near field potential electrodes. 4. LTP also could be induced without Bic application by cotetanization of vertical pathways simultaneously with horizontal activation. Vertical conditioning alone at 2 Hz, which affects inhibitory efficacy, was shown to transiently relieve depression of successive responses that ordinarily occurs during a burst of three horizontal stimuli. These results suggest that LTP of horizontal pathways may be regulated by spatiotemporal interactions between horizontal and vertical pathways. 5. Horizontal LTP was blocked reversibly by bath application of the N-methyl-D-aspartate (NMDA) antagonist 2-amino-5-phosphonovaleric acid, thereby implicating NMDA-receptor activation in LTP induction for these pathways. 6. The results confirm and extend our previous finding that the potential for activity-dependent modification of synaptic connections exists within the intrinsic horizontal connections of the superficial cortical layers. Synaptic modification across horizontally connected neurons appears to be regulated both by the arrangement of intrinsic circuitry and by the availability of mechanisms for modification at individual synapses. The properties of horizontal connections indicate that they form a spatial substrate and provide an activity-dependent mechanism for plasticity of adult cortical representations.     

Cardio-pulmonary function during acute unilateral occlusion of the pulmonary artery in broilers fed diets containing normal or high levels of arginine-HCl

Cardio-pulmonary function was measured in male broilers reared on diets formulated to contain 1.5% arginine (NORMAL group) or 2.5% arginine (ARGININE group). A snare placed around the right pulmonary artery permitted acute shunting of the entire cardiac output (CO) through the left pulmonary artery, resulting in sustained increases in blood flow (BF) through the left lung in both groups. The unilateral increase in BF was accompanied by sustained increases in pulmonary arterial pressure (PAP) and pulmonary vascular resistance (PVR) in the NORMAL group. However, following initial transient increases in PAP and PVR in the ARGININE group, subsequent pulmonary vasodilation gradually reduced PVR, and thus PAP, in spite of the ongoing elevation of BF through the left lung. The capacity of the pulmonary vasculature in the ARGININE group to accommodate an increased BF at a normal PAP accounts for the previously reported lower incidence of pulmonary hypertension syndrome (PHS, ascites) in cold-stressed broilers fed supplemental dietary arginine. Hypoxemia and respiratory acidosis ensued rapidly in both groups after tightening the pulmonary artery snare, in spite of a compensatory increase in the respiratory rate. The gradual return of PVR and PAP to presnare levels in the ARGININE group did not eliminate the concurrent ventilation-perfusion mismatch caused by the increased rate of BF through the left lung. Tightening the pulmonary artery snare caused mean systemic arterial pressure (MAP) to drop from control levels of approximately 98 mm Hg to sustained hypotensive levels of approximately 65 mm Hg in both groups. This systemic hypotension was caused by decreases in CO and total peripheral resistance (TPR). The reduction in CO were caused by reduction in stroke volume (SV) rather than heart rate (HR), suggesting that acutely tightening the pulmonary artery snare increased PVR sufficiently to impede left ventricular filling. Accordingly, the maximum increment in PAP attainable by the right ventricle during acute increases in PVR apparently was inadequate to propel the entire CO through the pulmonary vasculature, setting the stage for the congestive right-sided pooling of blood routinely associated with PHS in broilers.     

Activity of the plasma membrane H(+)-ATPase and optimal glycolytic flux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid

The weak acid sorbic acid transiently inhibited the growth of Saccharomyces cerevisiae in media at low pH. During a lag period, the length of which depended on the severity of this weak-acid stress, yeast cells appeared to adapt to this stress, eventually recovering and growing normally. This adaptation to weak-acid stress was not due to metabolism and removal of the sorbic acid. A pma1-205 mutant, with about half the normal membrane H+-ATPase activity, was shown to be more sensitive to sorbic acid than its parent. Sorbic acid appeared to stimulate plasma membrane H+-ATPase activity in both PMA1 and pma1-205. Consistent with this, cellular ATP levels showed drastic reductions, the extent of which depended on the severity of weak-acid stress. The weak acid did not appear to affect the synthesis of ATP because CO2 production and O2 consumption were not affected significantly in PMA1 and pma1-205 cells. However, a glycolytic mutant, with about one-third the normal pyruvate kinase and phosphofructokinase activity and hence a reduced capacity to generate ATP, was more sensitive to sorbic acid than its isogenic parent. These data are consistent with the idea that adaptation by yeast cells to sorbic acid is dependent on (i) the restoration of internal pH via the export of protons by the membrane H+-ATPase in an energy-demanding process and (ii) the generation of sufficient ATP to drive this process and still allow growth.     

Interleukin-1 beta increases leukemia inhibitory factor mRNA levels through transient stimulation of transcription rate

The aim of this study was to determine immunogenetic markers of susceptibility in Crohn's disease (CD), taking the different features of the clinical course of the disease into account. HLA class I, HLA class II and TAP transporter gene polymorphisms were studied using DNA typing methods. Gene and antigen frequencies were analysed and compared in a group of 102 CD patients and 200 unrelated healthy controls from the same area. Analysis of the whole CD patient population revealed no definite association with either HLA or TAP gene alleles, with the exception of an association with DRB1*1302 (Pc < 0.05). However, when clinical subgroups of patients were considered, specific associations with some genetic markers were found. The most definitive results involved a genetic association in the group of patients who did not respond to glucocorticoid therapy. This group was characterized by a high frequency of HLA-DRB1*04 (P < 0.05). Conversely, a positive association with the TAP2-A allele was found in cortico-responder patients (Pc < 0.03). Furthermore, analysis of the distribution of HLA class II alleles in relation to the presence of extra-intestinal manifestations revealed an association with the DQB1*0501 or *0503 suballele of DQ5 (P < 0.05). Finally, patients with lesions in the small bowel were more frequently HLA DRB1*07 (P < 0.05). The present study supports the concept of clinical heterogeneity in Crohn's disease associated with a background of genetic heterogeneity.     

IgD-receptor up-regulation on human peripheral blood T cells in response to IgD in vitro or antigen in vivo correlates with the antibody response to influenza vaccination

Aged individuals (more than 65 years) were classified as antibody (Ab) responders on the basis that they showed increases to more than or = 1:40 in serum Ab titers to all influenza virus strains present in the trivalent influenza vaccine within 4 weeks after immunization. The peripheral blood mononuclear cells (PBMC) from pre-immunization samples of blood taken from seven Ab-responders and seven Ab-nonresponders were examined for their ability to exhibit up-regulation of IgD-receptor (IgD-R) after exposure for 2 h to immobilized cross-linked IgD, as shown by rosetting with IgD-coated ox erythrocytes. The responsiveness to IgD was found to be predictive of the ability to produce Ab responses to viral protein Ag: the IgD-R up-regulation was greater than 5% in all Ab-responders and less than 4% in all the Ab-nonresponders. In addition, there was an excellent correlation between mean Ab titers (to the three viruses in sera collected 4 weeks after immunization) and the percentage of IgD-R+ cells obtained in response to IgD in PBMC from the same individual prior to immunization: p = 0.894. Injection of influenza vaccine itself also induced IgD-R on PBMC in vivo. The percentage of IgD-R+ cells peaked after 24 h, was still detectable above background by day 7 or 14, and returned to pre-injection levels by day 28 in young subjects and aged Ab-responders, but not in Ab-nonresponders. Similarly, purified peripheral blood T cells obtained from aged Ab-responders exhibited IgD-R upon immunization in vivo. These findings suggest that Ag injection causes rapid up-regulation of IgD-R by cross-linking IgD in humans as well as in mice as shown previously. In analogy with results in mice, the present data are consistent with a role for IgD-R+ T cells in the humoral response in man. Proliferative responses to influenza proteins in peripheral blood T cells from vaccinated individuals were found to peak on day 7 and were higher in Ab-responders than in Ab-nonresponders.