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Biomedical subjects

R A Cox

Publications and source records attributed to R A Cox.

At least 109 records · Page 6Linked to original sources
PubMed1988

Induction and expression of cell-mediated immune responses in inbred mice infected with Coccidioides immitis.

Comparisons of the course of coccidioidomycosis in two strains of inbred mice established that BALB/c mice are significantly more susceptible to pulmonary infection with Coccidioides immitis than are DBA/2 mice. The susceptibility of BALB/c mice does not reside in their inability to mount a delayed-type hypersensitivity response to C. immitis antigen. That is, BALB/c mice manifested footpad hypersensitivity to coccidioidin early during the course of disease, to a level comparable to that of DBA/2 mice. In contrast to the more resistant DBA/2 mouse strain, however, BALB/c mice developed anergy by day 15 postinfection. Suppression of the delayed-type hypersensitivity response was not specific for C. immitis antigen, as evidenced by the finding that BALB/c mice immunized with mycobacterial purified protein derivative prior to infection with C. immitis were suppressed in their footpad response to mycobacterial antigen at day 15 postinfection. Taken together, these results establish that genetically determined susceptibility to this fungus is associated with an acquired suppression of cell-mediated immune reactivity.

Animals↗
PubMed1988

Suppression of T-lymphocyte response by Coccidioides immitis antigen.

Intravenous injection of BALB/c mice with coccidioidin or an alkali-soluble cell wall extract of Coccidioides immitis mycelia resulted in the induction of a splenic cell population(s) that suppressed delayed-type hypersensitivity response to coccidioidal antigen. To determine whether the levels of C. immitis antigen produced during the course of active coccidioidal disease might also cause suppression of T-lymphocyte response, BALB/c mice were infected by intranasal instillation of arthroconidia, and 2 weeks later, their sera were evaluated for suppression of T-lymphocyte response in syngeneic recipients. Intravenous transfer of sera, which were shown to contain high levels of coccidioidal antigen by an enzyme-linked immunoadsorbent assay, suppressed the delayed-type hypersensitivity response of recipients to immunization with coccidioidin. Solid-phase immunoadsorption of the sera with goat antibodies to C. immitis antigens removed the suppressive component(s). To determine whether the suppressive effect of circulating coccidioidal antigen(s) was associated with the activation of a splenic suppressor cell(s), as was observed in mice injected intravenously with coccidioidal antigen, spleen cell lysates were prepared from infected donors, and after filtration to remove viable fungi, the lysates were transferred to syngeneic mice. Recipients of lysates from infected but not noninfected donors were suppressed in their response to immunization with coccidioidin. Collectively, these results provide evidence that depressed T-cell responses observed in coccidioidomycosis are associated with, and may be attributable to, the activation of a suppressor cell or factor by circulating C. immitis antigens.

Animals↗
PubMed1988

Induction of antigen-specific suppression by circulating Cryptococcus neoformans antigen.

Immunoaffinity chromatography of sera from mice infected with Cryptococcus neoformans (Inf-MS) on a column with rabbit anti-cryptococcal antibody as the ligand resulted in the adsorption of the component(s) that induce suppression of the cryptococcal delayed-type hypersensitivity (DTH) response. In contrast, immunoaffinity chromatography of Inf-MS on columns coupled with cryptococcal antigen or goat anti-mouse IgM, IgG, and IgA did not adsorb the suppressive component(s). Quantification of cryptococcal antigen and anti-cryptococcal antibody in Inf-MS and column fractions established a direct correlation between cryptococcal antigen levels and suppressive activity; no correlation was observed between anti-cryptococcal antibody levels and suppressive activity. The suppression induced by Inf-MS was shown to be specific in that suppressive sera did not affect the induction of DTH responses to Listeria monocytogenes or dinitrofluorobenzene. These collective results provide evidence that cryptococcal antigen is the component in Inf-MS that induces antigen-specific suppression of the cell-mediated immune response to C. neoformans.

Animals↗
PubMed1987

Biosynthesis and activity of DNA polymerase throughout the mitotic cycle of Physarum polycephalum.

Measurements of DNA polymerase protein levels and polymerase activity through the naturally synchronous mitotic cycle of Physarum polycephalum show that active DNA polymerase-alpha is synthesized throughout the G2 phase, in step with the profile of general protein biosynthesis. Three main components of P. poly-cephalum DNA polymerase of 200, 112 and 70 kDa were found to be immunologically related.

DNA Polymerase II↗
PubMed1987

Differential expression of an alpha-tubulin gene during the development of Physarum polycephalum.

The expression of an alpha-tubulin gene (altB1 (N alpha Tu) [(1987) J. Mol. Biol. 193, 427-438]) of Physarum polycephalum (strain CLdAXE) was found to be governed by a developmental switch since mRNA transcripts were detected, by S1 nuclease analysis, in the plasmodial but not the amoebal phase of the life-cycle. The conclusion that the altB1 (N alpha Tu) allele codes for a plasmodial specific alpha-tubulin isotype is supported by recent amino acid sequence data.

Actins↗
PubMed1987

Primary structure of an alpha-tubulin gene of Physarum polycephalum.

An alpha-tubulin gene of Physarum was isolated as a phage-lambda NM1149 recombinant (designated phage-lambda N alpha Tu). Phage-lambda N alpha Tu contained a 4700 base-pair HindIII nuclear DNA fragment of an allele of the altB locus of Physarum (one of four unlinked alpha-tubulin gene loci). Subfragments of the 4700 base-pair insert of phage-lambda N alpha Tu were cloned into phage M13 and the nucleotide sequence was determined by the dideoxy chain termination method. The start point of transcription was identified by primer extension and a putative polyadenylation site was located by S1 nuclease analysis. The 4650 base-pair HindIII insert into phage-lambda N alpha Tu spans the complete gene; sequences upstream from the 5' end contain the RNA transcription promoter elements (the TATA and CCAAT boxes). The nucleotide sequence encoding alpha-tubulin contains seven intervening sequences, ranging from 63 to 222 nucleotides in size. The exons have a sequence that is identical with a Physarum alpha-tubulin cDNA clone, except for three base changes, one leading to a Val codon in place of a Met codon, another leading to a Glu codon in place of an Asp codon, and the third change is silent. The genomic clone provides the nucleotide sequence coding for the last 26 amino acid residues missing from the cDNA clone. The new sequence data indicate that the alpha-tubulin gene has a C-terminal methionine codon and not a tyrosine codon, which has been found in all alpha-tubulin genes sequenced to date.

Amino Acid Sequence↗
PubMed1987

Isolation of Coccidioides immitis F antigen by immunoaffinity chromatography with monospecific antiserum.

Detection of antibody to Coccidioides immitis F antigen is of proved value in the diagnosis of coccidioidomycosis. This antibody is demonstrable by use of an immunodiffusion assay with reference coccidioidin antigen and antiserum to C. immitis. Using a combination of lectin affinity and immunoaffinity chromatography, we isolated the F antigen from coccidioidin and prepared monospecific antibody to the purified antigen. The availability of these reagents will enable the development of a sensitive and specific assay for detecting serologic reactivity to this antigen.

Antibodies, Bacterial↗
PubMed1987

Antigenic identity of biologically active antigens in coccidioidin and spherulin.

We recently reported the isolation of three clinically relevant antigens from coccidioidin; viz., the antigen that is reactive in the immunodiffusion (ID) assay for detecting tube precipitin (TP) antibody (designated IDTP); the antigen that is reactive in the ID assay for detecting complement-fixing antibody (designated IDCF); and the heat-stable (HS) antigen which, when demonstrated in soluble extracts of fungal cultures by using the IDHS assay, establishes the mycologic identification of Coccidioides immitis. This investigation was undertaken to determine whether the IDTP, IDCF, and IDHS antigens isolated from coccidioidin are of antigenic identity with components in spherulin. By employing the purified coccidioidin antigens in line-immunoelectrophoresis with spherulin and by assaying coccidioidin and spherulin by tandem two-dimensional immunoelectrophoresis against antisera produced to the purified antigens, we report that these three coccidioidin-derived antigens are antigenically identical to precipitinogens in spherulin.

Antibodies, Fungal↗
PubMed1987

Serum-mediated suppression of lymphocyte transformation responses in coccidioidomycosis.

Lymphocyte transformation (LT) responses to coccidioidin (CDN) and spherulin were suppressed in 11 (73%) of 15 patients with active coccidioidomycosis when their mononuclear cells were assayed in autologous serum as compared to serum from healthy, CDN skin test-positive subjects. Suppressed LT responses were specific for Coccidioides immitis antigens in 7 (64%) of the 11 patients. Immunoaffinity chromatography of patient sera with Staphylococcus protein A adsorbed the suppressor component(s) and thereby established that suppression was attributed to immunoglobulin G, either alone or complexed with antigen. The possibility that suppression was mediated by immune complexes was examined by adding complexes formed in vivo or in vitro to mononuclear cell cultures of healthy CDN-reactive persons before LT assays. Although complexes prepared in this manner were reactive in an enzyme-linked immunosorbent assay designed to detect Coccidioides antigen-specific immune complexes, no suppression of LT responses was observed. We conclude that serum-mediated suppression of LT responses in coccidioidomycosis is attributed to monomeric and not immune-complexed immunoglobulin G antibody.

Antigen-Antibody Complex↗
PubMed1986

Correlation of urokinase activity from biopotency and high-performance liquid chromatographic assays.

A simpler, less expensive, and faster high-performance liquid chromatographic method was shown to be an alternative to urokinase potency determinations by the Ploug method. Post-elution recovery of the low-molecular-weight form was 104 +/- 2.4% as determined by the Ploug method. Two analysts reported relative standard deviations of 1.6% and 1.1% based on peak height determination of eight replicate injections of a single sample of low-molecular-weight material. Linearity at the same wavelength for low- and high-molecular-weight forms was 0.9999 and 0.9992, respectively, for peak height versus potency.

Chromatography, High Pressure Liquid↗
PubMed1986

Evidence for genetic divergence in ribosomal RNA genes in mycobacteria.

DNA was isolated from Mycobacterium phlei and from M. smegmatis. Each DNA sample was restricted with endonucleases, the fragments were separated by agarose gel electrophoresis and transferred to nitrocellulose film. Fragments of DNA containing rRNA sequences were identified by means of 125I-labelled rRNA of M. phlei or of M. smegmatis. The distributions of restriction endonuclease sites within the rRNA gene(s) and flanking sequences were found to be characteristic for each of the two species. Hybridizations with heterologous probes indicate that although M. phlei rRNA and M. smegmatis rRNA share regions of sequence homology, they are probably not identical in primary structure. The results suggest that the rRNA genes might prove to be useful taxonomic markers for mycobacteria.

Biological Evolution↗
PubMed1986

Immunoadsorption of Cryptococcus-specific suppressor T-cell factors.

In the murine cryptococcal suppressor cell circuit, two different T-cell suppressor factors, TsF1 and TsF2, have been identified which specifically suppress the delayed-type hypersensitivity (DTH) response to cryptococcal culture filtrate antigen (CneF). TsF1 is produced by a first-order T suppressor (Ts1) cell population and suppresses the afferent limb of the DTH response, whereas TsF2 is produced by a second-order T suppressor (Ts2) cell population and suppresses the efferent limb of the cryptococcal DTH response. The objective of this study was to ascertain whether TsF1 or TsF2 could bind to cryptococcal antigen. To assess this, adsorption of TsF1 and TsF2 was performed with heat-killed Cryptococcus neoformans cells and by solid-phase immunoadsorption (SPIA) on columns containing cryptococcal antigens, i.e., CneF covalently bound to Sepharose 4B. The suppressive effect of TsF1 was removed by adsorption with intact heat-killed cryptococci and by SPIA on CneF-Sepharose 4B. The binding of cryptococcal TsF1 to the cryptococcal SPIA column was shown to be specific since Sepharose 4B columns either coupled with Saccharomyces cerevisiae mannan or blocked with glycine did not adsorb the suppressor activity. In contrast, the suppressive component of TsF2 did not bind to heat-killed cryptococci, CneF-Sepharose 4B, S. cerevisiae mannan-Sepharose 4B, or glycine-Sepharose 4B columns. These results, together with the finding that cryptococcal antigen, anticryptococcal antibody, and C1q-binding immune complexes were not demonstrated in either TsF1 or TsF2, establish that TsF1 and TsF2 can be differentiated on the basis of their affinity for cryptococcal antigen.

Animals↗
PubMed1986

Isolation and identification of an exoantigen specific for Coccidioides immitis.

Results of previous studies have established that mycelial-phase cells of Coccidioides immitis produce a heat-stable (HS) exoantigen that is specific for this fungus. In the present study, the HS exoantigen was isolated from a heterogeneous culture filtrate of C. immitis mycelia by using a combination of physicochemical procedures. Affinity chromatography of the culture filtrate on concanavalin A yielded two fractions: an effluent fraction that did not bind to the lectin and an eluate fraction that eluted in alpha-2-methylmannoside. Antigenic analyses by two-dimensional immunoelectrophoresis established that of the seven antigens detected in the unfractionated culture filtrate preparation, only two were present in the column effluent, and of these, only one was stable to heating at 56 degrees C for 30 min. Reactivity in the immunodiffusion assay for the HS exoantigen was demonstrable with the column effluent fraction and not the column eluate. The detection of only one precipitinogen in two-dimensional immunoelectrophoresis of the heat-treated concanavalin A effluent fraction, coupled with the reactivity of this fraction in the immunodiffusion assay for the HS antigen, provides strong, if not definitive, evidence that this antigen is the HS exoantigen. Purification of the HS antigen and the production of monospecific antiserum will provide the necessary reagents for the development of a sensitive and specific immunoassay for detecting the HS antigen in C. immitis cultures.

Antigens, Fungal↗
PubMed1986

Isolation of a coccidioidin component that reacts with immunoglobulin M precipitin antibody.

Detection of immunoglobulin M (IgM) precipitin antibody to coccidioidin, the autolysate of mycelial-phase cells of Coccidioides immitis, is an important serologic aid in establishing a diagnosis of primary coccidioidomycosis. In the present study, the component of coccidioidin that reacts with IgM precipitin antibody was isolated by a combination of immunoaffinity and anion-exchange chromatography. Antigenic analysis of the purified antigen in two-dimensional immunoelectrophoresis against goat anti-coccidioidin revealed a precipitinogen characterized by a complete cathodal leg and a partial anodal leg. The reactivity of this incomplete precipitating antigen with anti-C. immitis IgM was established by serologic assays and by the adsorption of reference IgM precipitin antibody on solid-phase immunosorbents containing the purified precipitinogen. The isolation of the coccidioidin component that reacts with IgM precipitin antibody and the production of monospecific antibody will provide the necessary reagents for the development of a sensitive immunoassay for detecting this serodiagnostic response.

Antigens, Fungal↗
PubMed1986

Step-wise isolation of RNA and DNA from mycobacteria.

Mycobacterium phlei and M. smegmatis were lysed at -15 degrees C in the presence of guanidinium salts and poly(A)+-RNA. Nonpolyadenylated RNA (mainly rRNA) and DNA were isolated in successive steps from the same lysate. DNA isolated by this procedure was sufficient to yield distinct bands on treatment with restriction endonucleases as shown by hybridization to 125I-labeled rRNA. The successive isolation of poly(A)+-RNA, nonpolyadenylated RNA, and high molecular weight DNA from the same lysate by this procedure, which is being reported for the first time, provides a very economical approach for isolation and purification of nucleic acids from mycobacteria and other organisms. This could be of special value for various genetic recombination studies, particularly in the case of mycobacteria which are available in very limited quantities, e.g., noncultivable or difficult-to-grow mycobacteria, especially M. leprae.

DNA, Bacterial↗