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Biomedical subjects

H Karch

Publications and source records attributed to H Karch.

At least 145 records · Page 8Linked to original sources
PubMed1993

The spectrum of immune responses to Campylobacter jejuni and glycoconjugates in Guillain-Barré syndrome and in other neuroimmunological disorders.

An acute infectious illness frequently precedes the Guillain-Barré syndrome. Recently, Campylobacter jejuni was claimed to be a predominant precipitating agent that may also trigger a humoral immune response to glycoconjugates of peripheral myelin in Guillain-Barré syndrome. Because of conflicting reports, we determined the frequency of a recent infection with C. jejuni in 38 patients with Guillain-Barré syndrome using a highly sensitive and specific immunoblot technique, and of the presence of circulating antibodies to gangliosides. We detected IgM and/or IgG C. jejuni directed antibodies in 15 of 38 patients with Guillain-Barré syndrome. In contrast, only 7 of 39 healthy control subjects, 3 of 20 patients with multiple sclerosis, and 2 of 72 patients with neuroborreliosis showed IgA or IgM antibody responses to C. jejuni. In Guillain-Barré syndrome, C. jejuni-specific antibodies were predominantly directed to outer membrane proteins of one specific serotype, Lior 11, whereas the most common serotype associated with enteritis in Germany is Lior 4. Two of 27 patients with Guillain-Barré syndrome had ganglioside-specific IgA antibodies; 1 of 32 patients, antibodies of IgM; and 4 of 31 patients, antibodies of IgG class. There was no correlation between severity, type (axonal versus demyelinating), and outcome of the disease and the presence or absence of a humoral immune response to C. jejuni or to glycoconjugates. Our findings do not support previous suggestions that a preceding C. jejuni infection heralds a poorer outcome or that antibodies to gangliosides carry prognostic significance.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Repeated detection of Borrelia burgdorferi DNA in synovial fluid of a child with Lyme arthritis.

In the pathogenesis of late Lyme borreliosis the relative importance of the causative organism, Borrelia burgdorferi, and the immune response of the host, including autoimmune phenomena, is not yet known. We describe a 7-year-old boy with Lyme arthritis from whom two synovial fluid samples were obtained 5 months apart and up to 17 months after the first appearance of arthritis. Both synovial fluid specimens were shown to contain borrelial DNA by nested polymerase chain reaction for the amplification of portions of the genes for flagellin and OspA. Thus, Borrelia burgdorferi may persist within the joint even during late stages of the disease.

Antigens, Surface↗
PubMed1993

Detection of Borrelia burgdorferi by nested polymerase chain reaction in cerebrospinal fluid and urine of children with neuroborreliosis.

Diagnosis of neuroborreliosis is often difficult since history and clinical presentation may be non-specific and serological tests may initially be negative. We therefore tested the polymerase chain reaction (PCR) for the detection of borrelial sequences in CSF and urine samples of consecutive children with neuroborreliosis seen in a single summer season. Four of eight children were negative in serum for antibodies to Borrelia burgdorferi. Two of eight children were PCR-positive in CSF and one other child was positive in urine. In two out of four children PCR was the only laboratory test confirming the clinical diagnosis. All children recovered after treatment with third generation cephalosporins. When seven of eight children were re-examined 6 months later all were healthy and antibodies to B. burgdorferi were detected in their serum. PCR may assist the paediatrician in establishing a diagnosis of neuroborreliosis; however, a negative result does not rule out neuroborreliosis. PCR is an adjunct, but no substitute for clinical judgement and serology.

Adolescent↗
PubMed1993

Demonstration of Borrelia burgdorferi infection in a child with Guillain-Barré syndrome.

A 4.5-year-old child suffered from rapidly evolving motor weakness with paresthesia and radicular pain in both legs, accompanied by an isolated elevation of protein levels in CSF. Isoelectric focusing revealed oligoclonal IgG produced in the cerebrospinal compartment only. The diagnosis of Guillain-Barré syndrome was confirmed by electrophysiological studies. High antibody titres against Borrelia burgdorferi in the serum and specific antibodies in the CSF were demonstrable by ELISA and immunoblotting. B. burgdorferi specific DNA fragments were demonstrated in urine and CSF by means of a nested polymerase chain reaction (PCR), thus providing the borrelial aetiology and indicating B. burgdorferi infection. Complete recovery was observed after treatment with high dose immunoglobulin and ceftriaxone and control urine specimens were PCR negative.

Antibodies, Bacterial↗
PubMed1993

Virulence determinants in nontoxinogenic Escherichia coli O157 strains that cause infantile diarrhea.

Ten sorbitol-fermenting Escherichia coli O157 strains that cause infantile diarrhea and are positive in the fluorescence actin staining test were determined to be negative for Shiga-like toxin (SLT) genes. We amplified their complete eae genes, contrasting them with those of SLT-producing E. coli O157 by restriction fragment length polymorphism analysis and nucleotide sequence analysis of a 400-bp stretch of the 3' end of eae. The data substantiated the presence of two eae genotypes within serogroup O157, one resembling eae of enteropathogenic E. coli (EPEC) strain E2348/69, found in nontoxinogenic E. coli O157 strains, and the other resembling eae of EHEC strain EDL 933, found in toxinogenic E. coli O157 strains. Another EPEC-specific virulence determinant was also shown to be large plasmids harboring EPEC adherence factor sequences. The SLT-negative E. coli O157 strains described here fall under the heading of EPEC, which serves as an explanation for their virulence in infants, and represent a third pathogroup within serogroup O157.

Adhesins, Bacterial↗
PubMed1993

Shiga-like toxin II-related cytotoxins in Citrobacter freundii strains from humans and beef samples.

By hybridizing colonies grown from 928 individual stool samples of patients suffering from diarrhea with oligonucleotide probes 772 and 849 complementary to Shiga-like toxin I (SLT-I) and SLT-II gene sequences, respectively, we identified two strains that hybridized with probe 849, which biochemical identification revealed as Citrobacter freundii. An additional five slt-II probe-positive isolates were screened from 81 beef samples. Polymerase chain reaction analysis and restriction of amplified products provided evidence for slt-II-related genes in all seven strains. From C. freundii LM 76, the genes encoding the A and B subunits were cloned in pUC 18 vectors and sequenced. The gene encoding the A subunit differed from that of Escherichia coli slt-IIvhc in 4 bases, resulting in two amino acid residue differences. In 11, 13, and 11 nucleotides, differentiation of slt-IIA, slt-IIcA, and vtx2haA, respectively, was found. These differences affected the predicted amino acid sequence as follows: there were six amino acid differences with SLT-IIA, five with SLT-IIcA, and four with VTx2haA. The nucleotide sequence of the gene encoding the B subunit is identical to slt-IIvhcB and differed from slt-IIcB and vtx2haB by only a single nucleotide base, but this resulted in a predicted amino acid sequence identical to that reported for these toxins. We therefore termed the toxin genes C. freundii slt-IIcA and slt-IIcB. Culture filtrates inoculated with material from the colonies from primary cultures were cytotoxic to Vero cells. Neutralization assays with antisera to E. coli SLT-I, SLT-II, and SLT-IIvhc revealed that antibodies against SLT-IIvhc reduced the C. freundii cytotoxic activity specifically and to the same degree as with the E. coli SLT-IIvhc control strain. In five of the seven strains tested, subcultivation on both a liquid or solid medium resulted in loss of cytotoxic activity. With polymerase chain reaction, we demonstrated that loss of cytotoxic activity ran parallel with the loss of slt genes. These data demonstrate the intergeneric occurrence of SLT-II-related toxins, which may well be a new marker of enteropathogenicity in C. freundii. Our findings that the toxin genes belong to the slt-II family plus their evident instability in the majority of strains should help pave the way to a better understanding of their role in diarrhea or food poisoning.

Animals↗
PubMed1993

Expression of A and B subunits of Shiga-like toxin II as fusions with glutathione S-transferase and their potential for use in seroepidemiology.

We used the plasmid vector pGEX-2T for the expression of recombinant subunits of Shiga-like toxin II (SLT-II). The 5' terminus of the genes that code for either the SLT-IIA or SLT-IIB subunits was genetically fused to the 3' terminus of the gene coding for the enzyme glutathione S-transferase, which serves as a carrier in this expression system. The subunit genes were constructed synthetically by polymerase chain reaction, with appropriate restriction sites to permit in-frame downstream insertion of the genes. The resulting plasmids containing the A and B subunit genes were designated pFG1 and pFG2, respectively. Induction of Escherichia coli laboratory strains harboring pFG1 with isopropyl-beta-D-thiogalactopyranoside (IPTG) yielding only small quantities of SLT-IIA fusion proteins. Since IPTG induction was lethal for cells harboring pFG2, we constructed the recombinant plasmid pFG4, which contained a subgenic fragment of slt-IIB but without the 5' signal sequence. With this construct we were able to express very large quantities of a 33.5-kDa fusion protein, which was purified by affinity chromatography on immobilized glutathione and used as an antigen in immunoblot analysis. Rabbit serum against native SLT-II, as well as all of 12 serum samples with high neutralizing activity against SLT-II, reacted with SLT-IIB purified from an E. coli pFG4 expression system, whereas only 3 of 208 human serum samples with low neutralization titers and none of 54 serum samples with no SLT-II-neutralizing capability reacted. Failure of specific reactivity with the SLT-IIB fusion protein in the majority of human serum samples with low neutralizing activity suggests that serum factors other than immunoglobulins may be responsible for neutralizing activity in these cases. The immunoblot assay with recombinant SLT-IIB as the antigen can be recommended for use in a diagnostic setting as a simple and reliable approach to detect specific human serum antibodies to SLT-II.

Animals↗
PubMed1993

Clonal structure and pathogenicity of Shiga-like toxin-producing, sorbitol-fermenting Escherichia coli O157:H-.

We compared a collection of sorbitol-fermenting (SF) Escherichia coli O157:H- strains with SF E. coli O157:H45 and non-SF E. coli O157:H7 and E. coli O157:H- strains by pulsed-field gel electrophoresis. The SF E. coli O157:H- strains had identical or closely related XbaI patterns that differed markedly from those for the other E. coli O157 strains. Plasmid content and the presence of Shiga-like toxin-converting phages were determined for the SF E. coli O157:H- strains, indicating that these strains harbor a single 90-kb plasmid. They are lysogenized by toxin-converting phages and harbor the eae gene. Nonmotile E. coli O157 strains were observed to adhere more efficiently to HEp-2 cells than the motile strains. From their phenotypic and genotypic features, the SF E. coli O157:H- strains may well represent a new clone with non-SF E. coli O157:H7 pathogenic characteristics.

Bacterial Adhesion↗
PubMed1993

Phenotypical and genotypical characterization of epidemic clumping factor-negative, oxacillin-resistant Staphylococcus aureus.

A total of 50 oxacillin-resistant Staphylococcus aureus (ORSA) strains that were clumping factor negative (CFN) and protein A negative by latex agglutination were collected from patients in six different hospitals at different locations in Germany during 1991 and 1992. Antibiograms, bacteriophage typing, and plasmid analysis were performed. The antibiograms showed that, besides oxacillin, all CFN ORSA strains were resistant to gentamicin, clindamycin, erythromycin, ciprofloxacin, and fosfomycin. All these isolates were nontypeable with an international set of phages, and an additional experimental phage set indicated that the strains were phage type 16, 192. Moreover, all isolates possessed a single plasmid of 30 kb, and restriction analysis of those plasmids revealed identical patterns. For genotyping, these 50 isolates were also analyzed by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction (PCR) of the coagulase and protein A genes and then by restriction enzyme digestion and analysis of restriction fragment length polymorphisms (RFLPs). With 49 strains, electrophoresis of SmaI-digested chromosomal DNA revealed identical PFGE patterns regarding the number and size of the DNA fragments, which could be differentiated from those of clumping factor-positive ORSA strains. Typing for the coagulase gene by PCR revealed PCR products of identical sizes. The AluI restriction digestion patterns of the PCR products were identical. PCR with primers derived from the region of that part of the protein A gene that encodes the immunoglobulin G-binding domains showed a PCR product that was about 170 bp smaller than that of the protein A gene from strains that were positive in the protein A latex agglutination test. Since it is precisely this size that is required in order to encode one immunoglobulin G-binding region, we assume that this is not present in the CFN ORSA strains. The phenotypical and genotypical features identify these very unusual CFN ORSA stains as being of clonal origin.

Base Sequence↗
PubMed1992

Cloning and sequencing of a Shiga-like toxin II-related gene from Escherichia coli O157:H7 strain 7279.

Escherichia coli O157:H7 strains are newly recognized pathogens associated with haemorrhagic colitis and haemolytic-uremic syndrome. In addition to Shiga-like toxin types I and II known to be produced by E. coli O157 isolates, we have identified in E. coli O157:H7 strain 7279 a third toxin, designated SLT-IIvhc, that is neutralized neither by anti-SLT-I nor by anti-SLT-II antibodies. The genes for this toxin were isolated by using a PCR-mediated cell-free cloning technique. DNA sequence analysis revealed a high degree of homologies to SLT-II and several SLT-II variants. The predicted amino acid sequence of the A subunit of SLT-IIvhc differed from that of the O91:H7 toxin VT2ha and SLT-IIc from E. coli O157:H- strain E2511 by 2 and 3 amino acids, respectively. The amino acid sequence of its B subunit was identical to VT2ha and SLT-IIc but different from SLT-II and SLT-IIv. Immunological differences of SLT-IIvhc and SLT-II as well as their different toxicity to HeLa cells presumably resulted from the small deviations within the primary structure of the B subunit.

Amino Acid Sequence↗
PubMed1992

Serological and biochemical properties of Shiga-like toxin (verocytotoxin)-producing strains of Escherichia coli, other than O-group 157, from patients in Germany.

During 1987-1990, 62 infections with Shiga-like toxin (Verocytotoxin)-producing E. coli, other than O group 157, were identified. The isolates belonged to 26 serovars within 20 O groups; of them, 10 serovars belonged to 6 O groups of traditional enteropathogenic E. coli (EPEC). The serovars O2:H5 and O91:H14 were exclusively associated with ulcerative colitis. Eighteen patients (children under 4 years of age) developed a haemolytic uraemic syndrome. All together, 31 strains produced SLT I, 26 strains, SLT II, and 5 strains, both toxins. All strains produced beta-glucuronidase and fermented, with one exception, D-sorbitol. The phenotypic diversity of SLT-producing E. coli implies that the only means of their identification is by indirect (genetic) or direct (phenotypic) demonstration of toxin production.

Adolescent↗
PubMed1992

A biotyping scheme for Shiga-like (Vero) toxin-producing Escherichia coli O157 and a list of serological cross-reactions between O157 and other gram-negative bacteria.

In this study, 41 strains of E. coli O157 were investigated and divided into 4 serovars: O157:H7 (19 strains), O157:H- (20 strains), O157:H43 (1 strain) and O157:H45 (1 strain). However, a formation of SLT cytotoxins was only associated with the serovars O157:H7 and O157:H-. The strains of O157:H7 produced either SLT I and SLT II (the vast majority of 10 isolates), SLT II (7 isolates), or SLT II vhc human variant (2 isolates) which had been isolated in Germany for the first time. The majority of 16 strains of serovar O157:H- produced only the SLT II cytotoxin and 4 strains, both toxins, SLT I and SLT II. The pathogens were identified in connection with watery and bloody diarrhoea, with HUS and TTP sequelae. All age groups, in particular that of infants from 0 to 5 years, were affected by these infections. Ten strains (25.6%) out of a total of 39 SLT-producing strains were sorbitol-positive and not recognized on the sorbitol MacConkey plate during isolation. A scheme of 11 biotypes has been suggested for the biotyping of E. coli O157 and the antigenic relationships between O and OK antigens of E. coli O157 and those of other E. coli serogroups and other Gram-negative bacteria which were tested. An antigenic relationship was proved to exist between the O157 antigen and the twelve O antigens of other E. coli serogroups as well as between the O157 antigen and the antigens O:IB of Y. pseudotuberculosis, O:30 of Salmonella group (N) and O:9 of Y. enterocolitica.

Adolescent↗
PubMed1992

Inflammatory signs, antibody response and antigen detection in cerebrospinal fluid over the course of neuroborreliosis.

CSF and serum specimens were consecutively obtained from three patients with neuroborreliosis (stage I, II and III), CSF protein content, cell counts and differential, IgG index, oligoclonal bands and anti-B. burgdorferi antibodies were measured. Cerebrospinal fluid (CSF) was tested for Borrelia-DNA being present prior to and after antibiotic treatment. While DNA could be identified before ceftriaxone was administered, there were no more amplification products afterwards. The goal of this study was to compare the usefulness of serodiagnostic methods and the detection of Borrelia burgdorferi-DNA in patients with clinically confirmed neuroborreliosis to test the efficiency of antibiotic therapy.

Aged↗
PubMed1992

Frequent loss of Shiga-like toxin genes in clinical isolates of Escherichia coli upon subcultivation.

Forty-five consecutive patients with various gastrointestinal disorders were identified as having Shiga-like toxin (SLT)-producing Escherichia coli infections. This was shown by the cytotoxic effect of stool extracts in Vero cell cultures which was neutralizable by antibodies to SLTs and by isolation of E. coli that hybridized with DNA probes complementary to SLT-I and SLT-II sequences. When we tested the same strains for SLT genes after subcultivation, the isolates from 15 patients became negative by colony hybridization and polymerase chain reaction and failed to produce SLTs. The instability of SLT genes warrants direct screening methods for clinical material and the development of new culture methods to prevent the loss of SLT genes.

Bacterial Toxins↗
PubMed1992

Ovalbumin blocking improves sensitivity and specificity of immunoglobulin M immunoblotting for serodiagnosis of patients with erythema migrans.

To improve the serodiagnosis of erythema migrans, we evaluated how sensitivity and specificity of immunoblotting are influenced by antigen concentration and blocking conditions. We found that an antigen concentration of 0.5 micrograms per lane in concert with ovalbumin blocking of the nitrocellulose provided the best results. In this case, 81% of the erythema migrans had positive immunoglobulin M tests, whereas only 33% were positive in a flagellum enzyme-linked immunosorbent assay (ELISA) and 28% were positive in a sonicate ELISA.

Antibody Specificity↗
PubMed1992

Indirect hemagglutination assay for diagnosis of Escherichia coli O157 infection in patients with hemolytic-uremic syndrome.

An indirect hemagglutination assay consisting of sheep erythrocytes coated with lipopolysaccharide (LPS) from Shiga-like toxin-producing Escherichia coli O157 was used for the serological diagnosis of E. coli O157 infections in children with classical (enteropathic) hemolytic-uremic syndrome (HUS). One week after the onset of diarrhea (acute phase of the disease), the E. coli O157 antibody titer was greater than or equal to 1:4,096 in 22 of 27 patients with HUS, compared with 4 of 249 controls, the majority of whom had O157 antibody titers of between 1:4 and 1:256. This antibody response was observed in HUS patients with stool cultures positive and negative for E. coli O157. Selective absorption with homologous LPS and heterologous LPS showed that the antibody response was specific for E. coli O157. Because of its simplicity and ease of interpretation, the indirect hemagglutination assay described in this paper is recommended for the serological diagnosis of E. coli O157 infections in patients with HUS.

Antibodies, Bacterial↗
PubMed1992

Molecular detection of sorbitol-fermenting Escherichia coli O157 in patients with hemolytic-uremic syndrome.

Shiga-like toxin-producing Escherichia coli strains of serogroup O157 were identified in 26 of 104 patients with hemolytic-uremic syndrome and in 18 of 668 patients with diarrhea. All strains were identified by colony hybridization with DNA probes complementary to Shiga-like toxin I and Shiga-like toxin II gene sequences and characterized by biochemical tests and serotyping. Seventeen of these 44 patients had E. coli O157 strains which were unusual because they fermented sorbitol within 24 h of incubation and were positive for beta-glucuronidase activity. Culture filtrates of these sorbitol-fermenting strains were highly toxic to Vero cells in culture. Serological tests and DNA analysis performed by restriction endonuclease digestion of B-subunit toxin genes revealed that all 17 isolates produced Shiga-like toxin II. Although by using molecular probes we established a high frequency of sorbitol-fermenting E. coli O157 strains in the patients we examined, further studies on the prevalence of such isolates in other areas of endemic disease are clearly warranted.

Adolescent↗