PubMed Health⌕ Search

Biomedical subjects

M Carl

Publications and source records attributed to M Carl.

At least 37 records · Page 2Linked to original sources
PubMed1994

Expression of hepatitis E virus putative structural proteins in recombinant vaccinia viruses.

Hepatitis E virus (HEV) is a polyadenylated, positive-stranded RNA virus which is a major cause of enterically transmitted non-A, non-B hepatitis in many developing countries. The viral genome contains three different open reading frames (ORFs): ORF1, which is believed to encode nonstructural proteins, and ORF2 and ORF3, which are believed to encode structural proteins. The full-length putative structural proteins encoded by ORF2 and ORF3 of HEV have been cloned and expressed in recombinant vaccinia virus. Proteins encoded by ORF2 and ORF3 when expressed in vaccinia virus are recognized by pooled sera obtained from individuals with acute hepatitis E. Vaccinia-expressed viral gene products of HEV will have utility in characterizing the cell-mediated immune response to HEV.

Base Sequence↗
PubMed1993

Activation and enhanced contact of human T-lymphocytes with autologous red blood cells are required for their stable adherence at 37 degrees.

The adherence of human red blood cells (RBC) to autologous T-cells does not occur in the body, and in vitro is elicited at 4 degrees. Autologous E-rosetting at 37 degrees has not previously been described. In this work, lymphocyte-RBC adherence has been studied in mixed leukocyte-RBC cultures and in whole blood from healthy donors. Vital, cytochemical and electron microscopic studies have shown that T-cells may form stable E-rosettes with autologous RBC at 37 degrees. As in the previously reported cold-dependent reversible rosetting, stable rosetting is mediated by the erythrocyte LFA3 and lymphocyte CD2 molecules. Uniquely, this phenomenon requires both T-cell activation and an enhanced contact between the T-cell and RBC membranes. These requirements were met by exposure of cell cultures to: (1) PHAE, the erythroagglutinating component of PHAP, or (2) to either non-erythroagglutinating mitogens, PHAL, Con A, OKT3 or SEA, or to antigens of typhus group rickettsiae or salmonellae, provided that the RBC membrane was desialyted. Cultures derived from individuals seropositive to rickettsiae or vaccinated with salmonellae demonstrated the adherence phenomenon after antigen exposure when neuraminidase was present in the culture medium. The system 2 described here can be used as a diagnostic tool for defining activated T-cells and T-cell clones with the memory to antigens capable of inducing cell-mediated immunity.

Cell Adhesion↗
PubMed1993

Expression and diagnostic utility of hepatitis E virus putative structural proteins expressed in insect cells.

The full-length putative structural proteins encoded by open reading frame 2 (ORF2) and ORF3 of hepatitis E virus have been cloned and expressed in recombinant baculovirus. Sera obtained from 28 Sudanese pediatric patients with acute hepatitis and 19 pediatric control patients were analyzed for reactivity to hepatitis E virus by using the baculovirus-expressed ORF2 and ORF3 proteins in a Western blot (immunoblot) format. Seventeen of the 18 patients classified as having non-A, non-B hepatitis, without acute antibody markers for hepatitis A, B, or C viruses, Epstein-Barr virus, or cytomegalovirus, were shown to have immunoglobulin M (IgM) antibodies to the recombinant ORF2 protein, as did two patients with chronic hepatitis B, three of seven patients with acute hepatitis A, and one patient with acute hepatitis B. None of the 19 control patients had IgM antibodies against the ORF2 or ORF3 proteins. The Western blot assay using the baculovirus-expressed ORF3 protein did not appear to be as sensitive as the assay based on the ORF2 protein. Only 10 of the patients classified as having non-A, non-B hepatitis had IgM antibodies to the baculovirus-expressed ORF3 protein. We conclude that a Western blot assay which uses a baculovirus-expressed ORF2 protein is both sensitive and specific for diagnosing acute hepatitis E.

Acute Disease↗
PubMed1992

Human linear B-cell epitopes encoded by the hepatitis E virus include determinants in the RNA-dependent RNA polymerase.

Hepatitis E virus is responsible for both sporadic and epidemic hepatitis in developing countries. The nonenveloped virus is 27-34 nm in diameter and has been shown to contain a single-strand, positive-sense, polyadenylylated RNA genome of approximately 7.5 kilobases. The nucleotide sequence of the Burma strain of hepatitis E virus has been reported and three open reading frames (ORFs) have been identified. The deduced amino acid sequence from each of these ORFs was used to synthesize overlapping peptides (decamers overlapping at every fourth amino acid) on a solid phase. These peptides were then tested in an ELISA with pooled acute-phase sera from known cases of enterically transmitted non-A, non-B hepatitis collected in the Sudan. Linear B-cell epitopes were identified in all three ORFs. Epitopes were identified throughout the polyprotein encoded by ORF1, but they appeared to be particularly concentrated in the region of the RNA-dependent RNA polymerase. Distinct epitopes were identified in the presumed structural protein encoded by ORF2, and one epitope was identified close to the carboxyl terminus of the protein encoded by ORF3. These data precisely pinpoint linear B-cell epitopes recognized by antibodies from patients with acute hepatitis E and identify an antibody response directed against the RNA-dependent RNA polymerase.

Amino Acid Sequence↗
PubMed1992

Interaction of immune sera with synthetic peptides corresponding to the structural protein region of hepatitis C virus.

Comparison of the deduced amino acid sequence from the structural region of the Hutchinson strain of hepatitis C virus (HCV-H) with four other HCV isolates clearly divides the five isolates into two groups based on sequence homology. The first group includes HCV-H, HCV-1, and HC-J1, while the second includes HCV-J1 and HC-J4. Among the five isolates the first 190 residues (putative nucleocapsid) are highly conserved whereas residues 196-513 exhibit significant diversity and include a hypervariable region encompassing residues 386-404. A series of overlapping decapeptides were synthesized by solid-phase pin technology according to sequence from HCV-H (amino acids 1-513), HC-J4 (amino acids 181-513), and regions from the three other isolates which exhibited sequence variation. A modified ELISA was used to measure immunoreactivity of sera from clinical posttransfusion cases and experimentally infected chimpanzees. Comparison of pre- and postinfection samples revealed 16 clusters of immunoreactive peptides within the structural region, none of which was found in the hypervariable region. Only one cluster (amino acids 73-89) was recognized by all human and chimpanzee sera. Clear variation in the immune response was observed between individuals, although no obvious difference in reactivity between acute and chronic cases was observed. Within individual profiles, the reactivity to each peptide cluster and the total number of reactive clusters increased over time.

Amino Acid Sequence↗
PubMed1992

Genetic characterization and transovarial transmission of a typhus-like rickettsia found in cat fleas.

The identification of apparently fastidious microorganisms is often problematic. DNA from a rickettsia-like agent (called the ELB agent) present in cat fleas could be amplified by PCR with conserved primers derived from rickettsial 17-kDa common protein antigen and citrate synthase genes but not spotted fever group 190-kDa antigen gene. Alu I sites in both the 17-kDa and citrate synthase PCR products obtained with the rickettsia-like agent and Rickettsia typhi were different even though both agents reacted with monoclonal antibodies previously thought specific for R. typhi. The DNA sequence of a portion of the 17-kDa PCR product of the rickettsia-like agent differed significantly from all known rickettsial sequences and resembled the 17-kDa sequences of typhus more than spotted fever group rickettsiae. The rare stable transovarial maintenance of this rickettsia in cat fleas has important implications for the disease potential of cat fleas.

Animals↗
PubMed1992

Acute sporadic hepatitis E in children living in Cairo, Egypt.

Seventy-three pediatric patients with acute hepatitis and 19 control patients without liver disease living in Cairo, Egypt, were evaluated with a newly developed Western blot assay for IgM antibody to hepatitis E virus (IgM anti-HEV). The mean age of acute hepatitis patients was 6.4 years (range, 1-13 years); 56% were male. Among the 73 acute cases, hepatitis A was diagnosed in 30 (41%), possible acute hepatitis B in three (4%), hepatitis E in nine (12%), and by exclusion, non-A, non-B hepatitis in 29 (40%). Two additional acute cases were positive for both IgM anti-HAV and IgM anti-HEV. None of the 19 control subjects had IgM anti-HEV. Parenteral risk factors were associated with cases of non-A, non-B hepatitis but were not associated with acute hepatitis E. Contact with a family member with jaundice was associated with acute hepatitis A. In contrast to prior epidemics of enterically-transmitted non-A, non-B hepatitis, HEV was found to be a common cause of acute hepatitis in a pediatric population. This study provides additional evidence that HEV may be a frequent cause of acute sporadic hepatitis among children living in some developing countries.

Adolescent↗
PubMed1992

Mapping of monoclonal antibody binding sites on CNBr fragments of the S-layer protein antigens of Rickettsia typhi and Rickettsia prowazekii.

The 120 kDa surface protein antigens (SPAs) of typhus rickettsiae lie external to the outer membrane in regular arrays and chemically resemble the S-layer proteins of other bacteria. These proteins elicit protective immune responses against the rickettsiae. In order to study the immunochemistry of these proteins, purified SPAs from Rickettsia typhi and Rickettsia prowazekii were fragmented with CNBr. The fragments were separated by SDS-PAGE and were recovered on PVDF membrane following electroblotting. The origin of eight major fragments from R. prowazekii and seven major fragments from R. typhi was determined by automated N-terminal amino acid sequencing and by comparison with the DNA sequence encoding R. prowazekii SPA. The cleavage patterns and protein sequences of the two proteins differed significantly. CNBr fragments corresponding to the C-terminus (amino acid 1372-1612 of the deduced sequence from encoding gene spaP) were not present in both SPAs. This suggests that the corresponding C-terminal region was not synthesized or was removed during SPA translocation to the cell surface. Modified amino acids were detected in each protein. Eighteen monoclonal antibodies selected for varied reactivity with both native and denatured SPA proteins could be classified into eight different types based on western blot analysis of the CNBr fragments. Six of the monoclonal antibody types reacted predominantly with a single region of the SPAs. Two types of antibodies bound to several CNBr fragments which contained both limited sequence similarity and modified amino acids either of which might account for the multisite binding of these antibodies.

Amino Acid Sequence↗
PubMed1992

Association of mycoplasma and human immunodeficiency virus infection: detection of amplified Mycoplasma fermentans DNA in blood.

A cross-sectional study was undertaken to determine the prevalence of Mycoplasma fermentans infection in patients with human immunodeficiency virus (HIV) infection using polymerase chain reaction methodology. Targeted M. fermentans DNA sequences could be amplified from the DNA extracted from the blood of 6 (11%) of 55 HIV-seropositive patients but from none of 26 HIV-seronegative subjects at low risk for HIV infection (P = .17). There was no correlation between M. fermentans infection and HIV clinical stage. There was a nonsignificant trend toward an association between M. fermentans infection and a history of syphilis. Infection with M. fermentans may occur more commonly in HIV-infected patients; however, a role as a copathogen or opportunistic infection was not established in this study.

Adult↗
PubMed1992

Acute sporadic hepatitis E in Sudanese children: analysis based on a new western blot assay.

A newly developed Western blot assay for antibody to hepatitis E virus (anti-HEV) was used to evaluate 39 cases of acute pediatric hepatitis and 39 control patients in Khartoum, Sudan. The mean age of cases was 6.5 years (range, 2-14); 64% were male. Acute hepatitis A (IgM anti-HAV-positive) was diagnosed in 13 cases, acute hepatitis B (IgM anti-HBc-positive) in 1, and acute hepatitis E (positive for IgM anti-HEV) in 23 (59%). None of the cases with IgM anti-HAV or IgM anti-HBc had IgM anti-HEV; 3 controls had IgM anti-HEV. Acute hepatitis E was associated with recent contact with a family member or acquaintance with jaundice and the presence of indoor plumbing. The newly developed hepatitis E assay appeared to be specific for the diagnosis of acute icteric non-A, non-B hepatitis. Hepatitis E was found to be the most common cause of acute sporadic hepatitis in children living in an urban area of Africa.

Acute Disease↗
PubMed1992

Detection of spores of Bacillus anthracis using the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to identify spores of Bacillus anthracis. By using an assay capable of amplifying a 1247-bp fragment from the gene that encodes the edema factor of B. anthracis, as few as 10(3) copies of a plasmid containing the edema factor gene and as few as 2 x 10(4) spores were detected. Subjecting the product of this PCR to a second PCR designed to amplify a 208-bp fragment nested within the 1247-bp product improved detection to a single plasmid copy per PCR and to two spores of B. anthracis per PCR.

Bacillus anthracis↗
PubMed1990

Characterization of the gene encoding the protective paracrystalline-surface-layer protein of Rickettsia prowazekii: presence of a truncated identical homolog in Rickettsia typhi.

The DNA sequence of the gene encoding the protective surface protein antigen (SPA) of Rickettsia prowazekii has been determined. The open reading frame of 4836 nucleotides with promoter and ribosome-binding site is present on a 10.1-kilobase EcoRI fragment. The encoded carboxyl terminus of the 169-kDa protein contains a potential transmembrane region and hydrophilic regions with many lysine and arginine residues potentially accessible to proteolytic cleavage. Because the rickettsia-derived SPA has an estimated molecular mass of only 120 kDa and does not contain several predicted large carboxyl-region CNBr fragments, the SPA product appears to be processed by the rickettsiae. Eight other CNBr fragments were identical in sequence to those predicted from the encoded gene. A complementary 8.7-kilobase EcoRI fragment of Rickettsia typhi DNA was cloned. This fragment lacked a 1433-base-pair region that included the promoter, ribosome-binding site, and the initial 1162 base pairs of the open reading frame encoding the R. prowazekii SPA but had a 3674-base-pair region identical with the remainder of the R. prowazekii SPA gene sequence.

Amino Acid Sequence↗
PubMed1990

Diagnosis of acute typhus infection using the polymerase chain reaction.

The first use of the polymerase chain reaction (PCR) for the diagnosis of an acute rickettsial infection is described. A primer pair derived from the 17-kDa antigen sequence of Rickettsia rickettsii gave specific amplification of a 434-base pair DNA fragment from the genome of Rocky Mountain spotted fever and endemic and epidemic typhus. The assay could detect as few as 30 rickettsiae. Detection of PCR-amplified DNA with a nonradioactive DNA probe confirmed an acute infection with Rickettsia prowazekii.

Acute Disease↗
PubMed1990

Diagnosis of acute typhus infection using the polymerase chain reaction.

We used the polymerase chain reaction (PCR) for the diagnosis of an acute rickettsial infection. A primer pair derived from the 17-kDa antigen sequence of Rickettsia rickettsii amplified specifically a 434-bp DNA fragment from the genome of Rocky Mountain spotted fever and endemic and epidemic typhus. The assay was able to detect as few as 30 rickettsiae. Detection of PCR-amplified DNA with a digoxigenin-labeled DNA probe confirmed an acute human infection with Rickettsia prowazekii.

Acute Disease↗
PubMed1990

Detection of Rickettsia tsutsugamushi by gene amplification using polymerase chain reaction techniques.

Scrub typhus is commonly undiagnosed in endemic areas due, in part, to dependence on retrospective serodiagnosis. Since the etiologic agent, R. tsutsugamushi, will not grow in cell-free systems, a rapid direct-agent detection system such as provided by polymerase chain reaction (PCR) methodology is needed. Genes coding for the variable 56-kDa antigen of R. tsutsugamushi were amplified through 35 cycles using 20-mer oligonucleotide primers and Taq polymerase. Amplification of 1-ng samples of DNA extracted from purified prototype R. tsutsugamushi Karp, Gilliam, and Kato strains was detected by direct visual inspection of the electrophoresed, ethidium bromide-stained, specific bands. Specificity of the PCR was shown when PCR amplification of various non-scrub typhus rickettsial DNAs was unsuccessful. R. tsutsugamushi DNA extracted from the blood of infected mice could be PCR amplified and the 1477-base pair product detected by either direct visualization or by specific hybridization with amplified non-radioactive digoxigenin-11-dUTP-labeled Karp 56-kDa DNA probe.

Animals↗