Detection of Intimins α, β, γ, and δ, Four Intimin Derivatives Expressed by Attaching and Effacing Microbial Pathogens

Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions. A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides. Polyclonal antiserum was raised against Int280 from enteropathogenic Escherichia coli (EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic E. coli (EHEC) isolates, a rabbit diarrheagenic E. coli strain (RDEC-1), and Citrobacter rodentium. Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and C. rodentium. These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells. Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7). By using PCR primers designed on the basis of the intimin-encoding eae gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different eae gene derivatives. Accordingly, the different intimin types were designated α, β, δ, and γ, respectively.     

Modified toluidine blue O stain for Pneumocystis carinii: further evaluation of some technical factors.

A modified toluidine blue O (TBO) stain for Pneumocystis carinii cysts was evaluated with regard to the influence of (i) the age and extent of use of the sulfation reagent, (ii) the source of TBO, (iii) the TBO content of the staining solution, and (iv) the amount of TBO present in the alcohol wash solutions. All TBOs evaluated, except for a new TBO obtained from Roboz Surgical Instrument Co., Inc., Washington, D.C., produced satisfactory results. Each lot of TBO should be quality controlled before use to ensure that the P. carinii cysts stain lavender against a blue background. We have ourselves decided to use only certified TBO with a high dye content. As extensively used sulfation reagent provided less satisfactory results than did either freshly prepared or 1-week-old unused sulfation reagent, we have decided to prepare fresh sulfation reagent at least weekly and to discard used sulfation reagent after 10 slides have been processed.     

Insulin effects in clarias batrachus L.: a combined biochemical and anatomical study

The effect of mammalian insulin was studied in a freshwater fish, Clarias batrachus, at both high and low ambient temperatures. The hormone produced a significant but delayed, yet recoverable, lowering of blood glucose, a concurrent decrease in liver glycogen, and an increase in the glycogen content of muscles. The decrease in brain glycogen occurred during advanced hypoglycemia. Hypoglycemic seizures developed intermittently in most of the fishes whose plasma glucose and brain glycogen levels had been considerably depleted. Necrobiotic changes in the pancreatic islets, including degranulation and atrophy, and necrosis of B cells, were seen in the treated fish. In some cases damage to A cells and the acinar tissue was also observed. With the restitution of normal glucose and glycogen values, the islet cells also seemed to have recuperated. Changes in glycemia, glycogen, and the islets were more pronounced in the fishes held at 24 degrees C than in those at 10 degrees C, indicating that the ambient temperature plays an important role in blood glucose homeostasis as well as insulin.     

Recurrent Bacteremia Caused by a “Flexispira”-Like Organism in a Patient with X-Linked (Bruton’s) Agammaglobulinemia

Helicobacter spp., except for Helicobacter cinaedi, have only rarely been reported in cases of septicemia. A patient with X-linked (Bruton's) agammaglobulinemia was found to have persistent sepsis with a Helicobacter-like organism despite multiple courses of antibiotics. His periods of sepsis were associated with leg swelling thought to be consistent with cellulitis. The organism was fastidious and required a microaerophilic environment containing H(2) for growth. Optimal growth was observed at 35 to 37 degrees C on sheep blood, CDC anaerobe, and Bordet-Gengou agars. Serial subcultures every 4 to 5 days were required to maintain viability. The organism was strongly urease positive and showed highest relatedness to Helicobacter-like organisms with the vernacular name "Flexispira rappini" by 16S rRNA gene sequence analysis. Genomic DNA hybridization studies, however, found 24 to 37% relatedness to "F. rappini" and even less to other Helicobacter spp. Although the organism phenotypically resembles "Flexispira" and Helicobacter, it is thought to represent a new taxon. The patient's infection was eventually cleared with a prolonged (5-month) course of intravenous imipenem and gentamicin.     

Serologic analysis of white-tailed deer sera for antibodies to Borrelia burgdorferi by enzyme-linked immunosorbent assay and western immunoblotting.

White-tailed deer serum samples were collected in the Minneapolis-St. Paul, Minn., metropolitan area during the fall and winter months from 1989 to 1992 and analyzed for antibodies to Borrelia burgdorferi, the etiologic agent of Lyme borreliosis. Ninety-eight percent of the serum samples were collected from regions where currently the vector tick, Ixodes dammini, is nonexistent. Antibodies to B. burgdorferi were detected in 2.2% of 508 samples by enzyme-linked immunosorbent assay, and their presence was confirmed by Western immunoblot analysis. Western immunoblotting yielded mean numbers of reactive bands of 0.1 and 6.0 for samples that were negative and positive for antibodies by enzyme-linked immunosorbent assay, respectively. The molecular weights of the antigens in many of the reactive bands from positive samples were similar to the molecular weights of antigens reactive with samples from humans with Lyme borreliosis. An antibody response to the major outer surface proteins A and B was not detected. Serologic analysis of deer sera may provide a valuable method for surveillance programs designed to monitor the spread of B. burgdorferi in nature.     

Cytomegalovirus (CMV) antigenemia assay is more sensitive than shell vial cultures for rapid detection of CMV in polymorphonuclear blood leukocytes.

We compared the cytomegalovirus (CMV) antigenemia assay with shell vial cultures of polymorphonuclear leukocyte (PMNL)-enriched blood fractions for rapid diagnosis of CMV viremia. PMNL fractions of 280 blood specimens from 171 patients (170 solid-organ transplant recipients and 1 patient undergoing pretransplant evaluation) were inoculated in shell vial and conventional CMV cultures. A commercially available kit (CMV-vue kit; INCSTAR Corp.) was used for the CMV antigenemia assay, in which PMNL preparations were stained with monoclonal antibodies directed against the CMV protein pp65. Mixed-leukocyte blood fractions from the same blood specimens were inoculated in parallel shell vial and conventional cultures. CMV viremia (defined by the isolation of CMV in conventional cultures) was detected in 32 (13%) of 245 PMNL fractions included in the final analysis. Twenty-eight (87.5%) were also positive in the CMV antigenemia assay, whereas 22 (69%) were positive in shell vial cultures. Ten (4%) additional PMNL fractions positive only in the CMV antigenemia assay were from eight patients with active CMV infections (six patients), who had previous or subsequent episodes of CMV viremia (seven patients), or in whom CMV was isolated in cultures of simultaneously obtained mixed-leukocyte fractions (three patients). Overall, the CMV antigenemia assay was significantly more sensitive than shell vial cultures for detection of CMV in the PMNL fraction of blood leukocytes (P < 0.01, McNemar's test), and we recommend it as the method of choice for rapid diagnosis of CMV viremia.     

Microbiology subsystem of a total, dedicated laboratory computer system.

The computer system used by the Microbiology Service of the Clinical Pathology Department, Clinical Center, National Institutes of Health is discussed. This microbiology subsystem is a part of a dedicated on-line laboratory computer system used by the entire department. The laboratory computer is connected on-line to a hospital computer which provides patient admission, transfer, and discharge data. Mark sense worksheets and cathode ray tube terminals are used for result entry and correction. Cumulative patient reports are printed. Results for both active and completed accessions can be easily retrieved on cathode ray terminals in the laboratory. All laboratory data are archived on magnetic tape from which a research data base and microfiched laboratory records are generated. The manner in which the system is integrated in the routine operation of the microbiology laboratory is emphasized. In addition, some of the costs, benefits, liabilities, and pitfalls associated with the introduction of the computer in the laboratory are reviewed. Finally, we have presented our concept of some of the future enhancements to our present system and some of the directions in which any future microbiology system might develop.     

AIDS-associated Kaposi''s sarcoma-derived cells in long-term culture express and synthesize smooth muscle alpha-actin.

Spindle-shaped cells from Kaposi's sarcoma lesions (AIDS-KS cells) were cultured for long periods in the presence of conditioned medium from activated CD4-positive T cells (HTLV-II infected transformed nonvirus producer) and characterized under in vitro conditions. To investigate a possible vascular origin, AIDS-KS cells were analyzed for the presence of smooth muscle alpha-actin, a differentiation marker for vascular smooth muscle cells. Immunofluorescence studies using a monoclonal antibody for smooth muscle alpha-actin demonstrated positive staining of the AIDS-KS cells (KS-3 and KS-4) but not by endothelial cells or fibroblasts. Northern blot analysis using an oligonucleotide probe unique for human smooth muscle alpha-actin indicated the expression of this gene by AIDS-KS cells. Similar analysis of biopsies from the KS lesion showed that in addition to the staining of smooth muscle cells associated with the blood vessels, the tumor-related spindle cells also stained positively. These cells were also analyzed for the expression of different growth factor genes. The platelet-derived growth factor (PDGF) A-chain gene was expressed at a moderate level. The insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-2 (IGF-2) genes were not overexpressed in relation to control cells. These data suggest that the analyzed AIDS-KS cells may be smooth muscle-like cells and therefore of vascular origin. Based on these results as well as previous reports, we speculate that cells of the immune system may regulate growth of cells in the vascular wall by a novel pathway.     

B cell chronic lymphocytic leukaemia cells have reduced capacity to upregulate expression of MHC class I in response to interferon-gamma

AIMS: An important consideration in the design of a tumour vaccine is the ability of tumour-specific cytotoxic T lymphocytes (CTL) to recognise unmanipulated tumour cells in vivo. To determine whether B-CLL might use an escape strategy, the current studies compared B-CLL and normal B cell MHC class I expression. METHODS: Flow cytometry, TAP allele PCR and MHC class I PCR were used. RESULTS: While baseline expression of MHC class I did not differ, upregulation of MHC class I expression by B-CLL cells in response to IFN-gamma was reduced. No deletions or mutations of TAP 1 or 2 genes were detected. B-CLL cells upregulated TAP protein expression in response to IFN-gamma. Responsiveness of B-CLL MHC class I mRNA to IFN-gamma was not impaired. CONCLUSIONS: The data suggest that MHC class I molecules might be less stable at the cell surface in B-CLL than normal B cells, as a result of the described release of beta(2)m and beta(2)m-free class I heavy chains from the membrane. This relative MHC class I expression defect of B-CLL cells may reduce their susceptibility to CTL lysis in response to immunotherapeutic approaches.