Biomedical subjects

E Jacobs

Publications and source records attributed to E Jacobs.

At least 127 records · Page 7Linked to original sources

PubMed1989

Comparison of vitamin E levels in plasma, bronchoalveolar lavage, and lung tissues of adult pulmonary patients.

Plasma, bronchoalveolar lavage fluids (BAL), and lung parenchyma were analyzed for vitamin E and polyunsaturated fatty acid (PUFA) concentrations in three groups of patients routinely receiving oxygen therapy--two with adult respiratory distress syndrome (ARDS and SARDS), a third with pneumonia (PNEU), as well as a fourth group of patients receiving little or no oxygen therapy (OTHER). Only plasma alpha- and gamma-tocopherols were significantly lower in patients receiving oxygen therapy compared to those not requiring oxygen. Among diagnosis groups, PNEU patients exhibited highest levels of alpha-tocopherol in BAL, though all groups on oxygen had greater amounts of alpha-tocopheryl quinone in BAL as compared to those of the OTHER group. No significant differences in BAL measures were observed between oxygen and non-oxygen groups, however. No statistical tests on lung measures could be performed between these groups because of insufficient sampling for the OTHER group. A highly significant relationship was observed (r = +0.73, p less than 0.004) between plasma vitamin E and lung vitamin E when expressed in terms of PUFA, whereas no significant relationship was observed if plasma vitamin E and lung vitamin E levels were compared directly. No relationship was obtained for BAL alpha-tocopherol (expressed per number of cells) and lung alpha-tocopherol. These findings support previous reports that in an appropriate setting plasma vitamin E:PUFA ratios along with smoking status may be used to evaluate lung vitamin E levels when also expressed in terms of PUFA.

Age Factors↗

PubMed1989

Binding sites of attachment-inhibiting monoclonal antibodies and antibodies from patients on peptide fragments of the Mycoplasma pneumoniae adhesin.

The adherence protein (P1 protein) of Mycoplasma pneumoniae was purified by electroelution and cleaved with cyanogen bromide. The resulting peptides were separated by two-dimensional electrophoresis. Spots reacting in Western immunoblots with two attachment-inhibiting monoclonal antibodies were isolated, and the amino-terminal ends of these peptides were microsequenced. The two monoclonal antibodies had different binding sites. One was associated with the amino-terminal region of the whole P1 protein beginning at amino acid position 237, and the other was associated with amino acid position 702, which was localized approximately in the middle of the P1 amino acid sequence. Serum samples from three M. pneumoniae-infected patients were tested by Western blotting against the cyanogen bromide peptide pattern. All three serum samples reacted with peptide fragments beginning at amino acid position 702, but the serum of only one patient also had antibodies against the oligopeptides beginning at amino acid position 237. These results indicate that the corresponding epitopes of the P1 protein are also immunogenic if they are presented at the surface of the infecting organism.

Adhesins, Bacterial↗

PubMed1989

The role of intestinal endotoxin in experimental peritonitis.

Escape of endotoxin from the intraintestinal site was investigated in experimental models of intestinal ischemia and during intraabdominal infection in rats. Following the instillation of Salmonella abortus equi endotoxin (S-form) into the proximal large bowel, we recorded the presence of the lipopolysaccharide molecule in the bowel wall, the intestinal lymph nodes, the peritoneal cavity, and in the liver sinusoids by immunohistochemical methods. At 3, 6, 12, 24, and 48 hr after the operative procedure, peritoneal fluid, blood, and tissue samples were taken. Survival rates were similar between the two test-groups (occlusion of the superior mesenteric artery [SMA] and cecal ligation and puncture [CLP]) and were not influenced by the amount of the injected endotoxin. There was no detectable morbidity in the sham-operated control animals with endotoxin doses up to 20 mg. Endotoxin could only be detected at 24 and 48 hr in the SMA group in the liver as well as in the peritoneal sediment and in intestinal lymph nodes. CLP and control samples remained negative throughout the observation period. Bacteria were found intraperitoneally within 12 to 24 hr in the SMA group and within 3 to 12 hr in the CLP group.

Animals↗

PubMed1988

Retrovirus-like vectors for Saccharomyces cerevisiae: integration of foreign genes controlled by efficient promoters into yeast chromosomal DNA.

Using modified Saccharomyces cerevisiae Ty1 elements located on a 2 mu plasmid, reverse-transcriptase-mediated transposition into yeast chromosomes of expression cassettes containing a foreign gene can be induced. These expression cassettes consist of the yeast ARG3 and CUP1 promoter sequences fused to the Escherichia coli galK structural gene. Expression cassettes as large as 2 kb can be inserted into Ty elements and transposed efficiently to various sites in the yeast genome. A third yeast promoter (from the yeast CAR1 gene) seems to be unsuitable for use in the expression cassette. This may be because it does not allow the transcription run-through necessary for Ty1 transposition. Ways of improving this vector system are discussed, as are its advantages over episomal vector systems.

Base Sequence↗

PubMed1988

Host reactions to Mycoplasma pneumoniae infections in guinea-pigs preimmunized systemically with the adhesin of this pathogen.

Guinea-pigs developed systemic and local humoral responses after intraperitoneal immunization with the isolated adhesin (168 kDa protein) of Mycoplasma pneumoniae cells. Hilar lymphocytes of these animals showed proliferation after in vitro stimulation with the 168 kDa protein or sonicated M. pneumoniae whole cell antigen. Animals preimmunized and subsequently infected with M. pneumoniae showed increased M. pneumoniae-specific IgG, IgA and adherence inhibiting antibody activities. Nevertheless these animals developed severe lung lesions of lympho-histiocyte infiltrations. Furthermore hilar lymph nodes were depleted of immunocompetent lymphocytes, suggesting a cell transfer of specific stimulable lymphocytes to the inflammation sites.

Adhesins, Bacterial↗

PubMed1988

Antibody response of patients against a 120 kDa surface protein of Campylobacter pylori.

Campylobacter pylori strains were isolated and serum samples were obtained from 63 patients. Immunoblots of 52 patients sera using their own isolates as antigen showed a 120 kDa band, which was missing in the other 11 isolates and the respective sera. This band was not detected in other Campylobacter species. Effects of trypsin treatment of bacteria and absorption of sera by live organisms suggest a C. pylori-specific surface protein.

Antibodies, Bacterial↗

PubMed1988

Immunological reaction of guinea-pigs following intranasal Mycoplasma pneumoniae infection and immunization with the 168 kDa adherence protein.

Humoral responses to Mycoplasma pneumoniae proteins, especially the 168 kDa protein, were demonstrated by Western blotting in sera and bronchial washings of all groups of infected or immunized guinea-pigs. However, infection was not prevented by these local and systemic antibodies. Hilar lymphocytes of infected and immunized guinea-pigs were stimulated in vitro by sonicated M. pneumoniae antigen and by the 168 kDa protein. Stimulation was significantly lower in animals which had been infected twice or had been preimmunized and challenged by infection. Histologically the most severe lesions were seen in the twice-infected group followed by the preimmunized group which was subsequently infected.

Animals↗

PubMed1988

Isolation of the adherence protein of Mycoplasma pneumoniae by fractionated solubilization and size exclusion chromatography.

The 168-kDa adherence protein of M. pneumoniae was solubilized and purified to homogeneity. Optimal yield was obtained by pretreatment of whole M. pneumoniae cells with buffer containing 1% Chaps and subsequent extraction with octylglucosid at a detergent to protein ratio of 5 and at octylglycoside concentrations between 1.5 and 2%. Contaminating membrane proteins with high molecular masses were removed by pretreatment with 1% Chaps and proteins of low molecular masses by size exclusion chromatography.

Adhesins, Bacterial↗

PubMed1988

Immunoprecipitation method for CK-MB analysis re-evaluated: influence of CK-BB and macro-CK on blank activities.

We evaluated the analytical performance of the immunoprecipitation technique for quantification of creatine kinase (CK; EC 2.7.3.2) isoenzyme MB, focusing on specimens with increased blank activities. The samples we studied had blank values that were equal to or greater than either 15 U/L or the uncorrected CK-MB value. Of 134 specimens selected, 16 and 21 contained macro CK type 1 and CK-BB, respectively. All samples containing macro CK type 1 gave negative CK-MB values, even though four contained significant quantities of CK-MB. Correcting the blank value for the fluid displacement of the second antibody suspension eliminated all but three of the negative results. However, 45 specimens, negative for CK-MB by electrophoresis, gave CK-MB values ranging from 1 to 21 U/L. Furthermore, 11 of the specimens containing macro-CK type 1 were in this group. Thus, the presence of macro-CK type 1 definitely interferes with the immunoprecipitation technique. Our results indicate the need to re-evaluate the diagnostic accuracy of the immunoprecipitation technique, especially when the un-adjusted blank activities are increased.

Creatine Kinase↗

PubMed1987

Mapping of monoclonal antibody-defined epitopes associated with carcinoembryonic antigen, CEA.

Six immunoglobulin G monoclonal antibodies reactive with carcinoembryonic antigen (CEA) were evaluated with respect to parameters implicated in their potential diagnostic application and use as tumor targeting agents for cytotoxic drugs or plant or bacterial toxins. Antibody reactivity with surface antigens of the MKN-45 gastric tumor cell line was demonstrated by flow cytofluorimetry. In a subcellular membrane binding assay, each antibody reacted preferentially with membranes isolated from colorectal tumor tissue in comparison with their reaction with membranes from adjacent, apparently normal colonic mucosa. Three of the antibodies (NCRC-23, C228, and 11.285.14) reacted specifically with CEA with little or no reaction with the cross-reacting antigen, NCA. The remaining three antibodies (C24, C161, and C198) were reactive with both CEA and NCA. Analysis of the epitopes defined by these antibodies was performed by competitive binding inhibition assays evaluating the capacity of unlabeled antibodies to compete with 125I-labeled antibodies in their binding to CEA. In addition, double determinant or 'sandwich' radioimmunoassays were employed to examine the coexpression of epitopes on CEA molecules. These studies permitted an epitope map to be constructed which describes the coincidence, overlapping, or independent expression of both CEA specific epitopes and epitopes shared between CEA and NCA. The map may be employed for the selection of antibodies for diagnostic and therapeutic use.

Antibodies, Monoclonal↗

PubMed1987

Antibodies in the sera of Mycoplasma pneumoniae-infected patients against proteins of Mycoplasma genitalium and other mycoplasmas of man.

Frequency and pattern of anti-Mycoplasma genitalium antibodies in sera of 50 patients with Mycoplasma pneumoniae-infection were examined by the Western immunoblot method. The sera reacted with several proteins of M. genitalium. However, during the course of infection there was only a moderate increase of antibodies mainly against the bands of 135 and 105 kd in contrast to the more intense increase of numerous bands of M. pneumoniae. If antibodies against the 168 kd-adhesin of M. pneumoniae were isolated by affinity chromatography, only the IgG-, but not the IgM-fraction reacted with the 135 kd protein of M. genitalium. Results with rabbit antisera supported these findings, additionally indicating a lack of substantial cross reactions between M. pneumoniae and other species except M. genitalium. The development of anti M. genitalium antibodies at early age and their relatively constant pattern suggest an early immunization by so far unknown cross reacting microbial antigens. Antigenic cross reactions between proteins of M. pneumoniae and M. genitalium apparently do not play a substantial role in the diagnostic serology of M. pneumoniae disease.

Adult↗

PubMed1987

Amino acid sequence and antigenicity of the amino-terminus of the 168 kDa adherence protein of Mycoplasma pneumoniae.

The amino-terminal end of the 168 kDa adherence protein from the membrane of Mycoplasma pneumoniae was sequenced up to 12 amino acids. A synthetic peptide containing nine amino acids of this sequence was used to study the antigenicity of the amino-terminus of the 168 kDa protein and the involvement of the homologous sequence of the protein in the adherence process. The synthetic peptide when coupled to ovalbumin was immunogenic in rabbits. Antibodies against this peptide epitope could be demonstrated in sera taken during natural M. pneumoniae infection in humans. The structural domain of the 168 kDa protein homologous with the synthetic peptide did not appear to be involved in adherence, as the synthetic peptide or its homologous antibody failed to inhibit adherence of M. pneumoniae.

Amino Acid Sequence↗

PubMed1987

Comparative biodistribution of methotrexate and monoclonal antibody-methotrexate complexes in mice.

The biodistribution of radiolabelled methotrexate and immune complexes of methotrexate and a murine monoclonal anti-methotrexate antibody has been compared in mice. Complexes formed in-vitro with the antibody, but not with control immunoglobulin. The complexes were, characteristically, acid labile. In-vivo, blood levels, organ distribution and whole body catabolism of methotrexate in immune complexes were similar to those of free antibody, and markedly different from those of free drug. These findings suggest the feasibility of prolonging the survival of drugs and altering in-vivo distribution using complexes with monoclonal antibodies.

Animals↗

PubMed1987

Use of adherence protein of Mycoplasma pneumoniae as antigen for enzyme-linked immunosorbent assay (ELISA).

Antibodies against the adherence protein of Mycoplasma pneumoniae are regularly found in patients with M. pneumoniae infection. Therefore, this 168-kilodalton (kDa) protein was used as an antigen in a dot-ELISA for serological diagnosis of M. pneumoniae disease. M. pneumoniae proteins were separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), gels were stained with Coomassie Blue, and the 168-kDa protein band was cut out and eluted using a special electroelution device. Isolated proteins or sonicated whole-cell antigens, respectively, were immobilized on a 96-well filtration plate with a nitrocellulose bottom (dot-ELISA). The test procedure was performed as in conventional ELISA tests, using alkaline phosphatase-labeled antihuman IgM or IgG antibodies, respectively, to detect antigen-antibody complexes. All results were confirmed by immunoblotting. The dot-ELISA using the 168-kDa antigen proved to be sensitive and specific. The specificity was tested on 53 sera of M. pneumoniae infections and on 490 serum specimens of patients with other respiratory diseases due to other pathogens, or with clinical conditions such as pancreatitis, meningitis or endocarditis. With regard to IgM antibodies, no false-positive reactions were found in non-M. pneumoniae diseases against the 168-kDa antigen, but there were such reactions against other M. pneumoniae proteins in immunoblots.

Adhesins, Bacterial↗

PubMed1986

Electroelution of fixed and stained membrane proteins from preparative sodium dodecyl sulfate-polyacrylamide gels into a membrane trap.

The membrane trap is a new device for the electroelution of all kinds of charged macromolecules from gels. Instead of dialysis membranes, the membrane trap uses a new membrane. Retention of macromolecules in an electric field by dialysis membranes depends on the presence of sodium dodecyl sulfate (SDS) in the buffer. The new membrane retains all charged macromolecules larger than approximately 5000 Da without adsorbing them, independent of the use of SDS. Here we report the electroelution of five different lipophilic membrane proteins (33 to 193 kDa) of Mycoplasma pneumoniae from preparative SDS-polyacrylamide gels into a 300-microliter recovery volume. After an 8-h elution period, recovery ranged from 80 (193 kDa) to 97% (33 kDa). The "losses" were generally due to proteins still remaining in the gel slice. All of the eluted proteins tested in a dot-blot assay proved to be antigenically active. The advantages of the device described here are easy handling (insertion of membranes, open system), quantitative recovery, and high reproducibility of the elution results.

Antigens, Bacterial↗

PubMed1986

A 168-kilodalton protein of Mycoplasma pneumoniae used as antigen in a dot enzyme-linked immunosorbent assay.

The attachment protein of Mycoplasma pneumoniae (molecular weight 168 kd) was used as antigen in a special enzyme-linked immunosorbent assay (dot ELISA) and compared with a sonicate of the whole organism. In control sera the intensity of the 168-kd band on immunoblots correlated well with the ELISA IgG values derived from isolated protein. The diagnostic significance of the 168-kd antigen was tested on paired sera from 33 patients with Mycoplasma pneumoniae infection (24 children, 9 adults). The ELISA values with the isolated protein were slightly lower than with cell antigen, but the protein also showed a lower basic activity in controls. In first sera of specimens of children collected within the first week of infection the 168-kd IgM response was more distinct than that to the whole cell antigen. Similarly the IgG response to the purified protein antigen differed significantly from the controls already in the first serum. In sera of adult patients the increased levels of IgG antibody were more evident with the 168-kd protein antigen. Use of the protein 168 kd as antigen increased the sensitivity of the ELISA for detecting early stages of disease, especially in children.

Adolescent↗

PubMed1986

Stability of lactate dehydrogenase at different storage temperatures.

The effect of storing human serum, cord blood serum or heparinized plasma at 25 degrees C, 4 degrees C & -20 degrees C on the activity and isoenzyme distribution of lactate dehydrogenase (LD) was studied. Cellulose acetate and agarose electrophoresis, as well as an immunochemical inhibition technique, were used for isoenzyme quantification. In contrast to previous reports, cryo-instability was found only in specimens stored at 4 degrees C. Serum specimens stored at 25 degrees C and -20 degrees C retained 74% and 87% of total activity after 45 days of storage. LD-1 was stable at all three temperatures, with a maximum loss of 10%. LD-2, LD-3, LD-4, & LD-5 were most labile at 4 degrees C. Specimens that are to be analyzed for total LD or LD isoenzymes should be stored frozen or, if necessary, at room temperature, but not in a refrigerator. Thus, separate storage of specimens for cardiac isoenzymes (LD & creatine kinase) is not necessary. This may eliminate a possible source of falsely elevated LD-1/LD-2 ratios, as well as reducing the labor factor and the corresponding cost of cardiac isoenzyme determinations.

Binding Sites, Antibody↗

PubMed1986

Reaction pattern of human anti-Mycoplasma pneumoniae antibodies in enzyme-linked immunosorbent assays and immunoblotting.

In serological diagnosis of Mycoplasma pneumoniae disease the frequently used complement fixation test is based on a cross-reacting glycolipid. Recently enzyme-linked immunoassays have been developed to overcome this lack of specificity. To study the involvement of the various proteins and the influence of age on the level of antiprotein antibodies present, we investigated by enzyme-linked immunoassays (immunoglobulin M [IgM] and IgG) and immunoblotting the sera of healthy persons of different age groups as well as sera of patients (including paired sera) with M. pneumoniae infection. In sera of children with nonrespiratory diseases and in healthy blood donors the IgM antibodies rose during the first 2 years of life to a relatively constant background level (optical density at 405 nm of 0.15 to 0.21). In contrast IgG remained low up to the seventh year and then increased to moderate levels (optical density of 0.15). The blotting patterns showed few IgM bands in the age group of 20 to 30 years. IgG blots revealed, up to 7 years, only very few reactions with a 168-kilodalton protein, but in higher age groups a considerable number of reactions with proteins of 193, 168, 84, 69, and 56 kilodaltons were detected. In the sera of patients, positive IgM blots were most numerous in the third week, whereas the number of IgG blots increased up to the fourth and the fifth week. At this time all sera contained antibodies against the 168-kilodalton protein, which is identical with the adhesin of M. pneumoniae. In a patient with acute infection, who had a high preinfection IgG level, no IgM response developed. The data indicate a relatively high background of antibodies against M. pneumoniae proteins in older age groups, suggesting a requirement for paired sera. Furthermore, reinfections of adults may occur without a concomitant IgM response.

Adult↗