Demonstration of human immunodeficiency virus by colorimetric in situ hybridization: a rapid technique for formalin-fixed paraffin-embedded material

A colorimetric method of in situ hybridization has been developed for the rapid detection of human immunodeficiency virus (HIV) in formalin-fixed paraffin-embedded material. Following optimization of digestion conditions, biotin-labeled DNA probes are detected with an alkaline phosphatase conjugate. The method is verified using fixed paraffin-embedded cell blocks of HIV-infected and uninfected lymphocyte cell cultures. Hybridization specifically detects both viral RNA and proviral DNA. Formalin fixation for intervals up to 21 d did not significantly hamper the signal under the appropriate digestion conditions; however, Trump's fixation for even 12 h greatly reduced the intensity of the hybridization. This technique for in situ hybridization is amenable to automation, provides results within 6 h, and results in good morphologic preservation. A key feature of the technique is the use of human placental DNA as an endogenous positive control to optimize the empirically determined conditions for protein digestion.     

Mycoplasma pneumoniae attachment: competitive inhibition by mycoplasmal binding component and by sialic acid-containing glycoconjugates.

Attachment of Mycoplasma pneumoniae to human WiDr cell culture monolayers was examined by using radiolabeled M. pneumoniae. The amount of attachment was proportional to the density of the WiDr cells and to the concentration of M. pneumoniae in the assay. Saturation of the monolayers was achieved with 40 micrograms of virulent strain M129 per assay, whereas binding of avirulent strain B176 was 70% less than that of strain M129. A competitive attachment inhibition assay was used to measure specific binding component activity. Attachment was inhibited when WiDr cells were pretreated with unlabeled virulent strain M129, whereas avirulent noncytadsorbing strain B176 did not inhibit attachment as well as the virulent strain. A protein-rich extract prepared from virulent, cytadsorbing strains of M. pneumoniae also inhibited attachment. The amount of inhibition was dependent on the amount of extract used, and units for binding component activity in the extract were calculated from the competitive attachment inhibition assays. The competitive attachment inhibition assay was also used to investigate the nature of the receptor site on the WiDr cells. Attachment was inhibited when the radiolabeled M. pneumoniae suspensions were pretreated with human sialoglycoproteins, such as orosomucoid and ceruloplasmin, and bovine gangliosides. These findings support the present concept that the mammalian receptor site for M. pneumoniae is a sialic acid-containing glycoprotein.     

FMLP-induced enzyme release from neutrophils: a role for intracellular calcium

The ability of the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) to stimulate beta-glucuronidase release and 45Ca2+ release from rabbit neutrophils was studied. FMLP stimulated enzyme release from cytochalasin B-treated cells either in the presence or the absence of extracellular calcium. Depletion of cell calcium, by exposure to either ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid or the calcium ionophore A23187, blocked the ability of FMLP to stimulate enzyme release and 45Ca2+ release in the absence of extracellular calcium. The ability of A23187 to lower the 45Ca2+ content of neutrophils, to block FMLP-stimulated 45Ca2+ release, and to inhibit FMLP-stimulated enzyme release in the absence of calcium was dose dependent over the same concentration range (10(-8) to 10(-6) M A23187) for all three actions. In contrast, FMLP stimulated enzyme release from A23187-treated cells, provided that extracellular calcium was present. This secretory response was normal as judged by cell ultrastructure and FMLP dose-response relationships. It is concluded that A23187 depletes a pool of intracellular calcium usually released by FMLP and that release of calcium from this pool is necessary for initiation of enzyme secretion in the absence of extracellular calcium.     

Molecular characterization of a serotype O121:H19 clone,a distinct Shiga toxin-producing clone of pathogenic Escherichia coli

Most illnesses caused by Shiga toxin-producing Escherichia coli (STEC) have been attributed to E. coli serotype O157:H7, but non-O157 STEC infections are now increasingly recognized as public health problems worldwide. The O121:H19 serotype is being isolated more frequently from clinical specimens and has been implicated in one waterborne outbreak. We used multilocus virulence gene profiling, a PCR-based assay, to characterize the virulence gene content of 24 isolates of serotype O121:H19 and nonmotile variants. We also performed multilocus enzyme electrophoresis and multilocus sequencing to establish the clonal relatedness of O121 isolates and to elucidate the relationship of O121 to common STEC clones. The 24 isolates were found to represent a single bacterial clone, as there was no allelic variation across 18 enzyme loci among the isolates. The complete nucleotide sequence of the intimin gene differed by four substitutions from that of the epsilon (Int- epsilon ) allele of O103:H2 strain PMK5. The typical O121 virulence gene profile was similar to the profiles of enterohemorrhagic E. coli (EHEC) clones of E. coli: it included a Shiga toxin 2 gene (stx(2)), two genes on the EHEC plasmid (toxB and ehxA), and the gene encoding intimin (eae). Despite the similarities, putative virulence genes distributed on O islands-large chromosomal DNA segments present in the O157:H7 genome-were useful for discriminating among STEC serotypes and the O121:H19 clone had a composite profile that was distinct from the profiles of the other major EHEC clones of pathogenic E. coli. On the basis of sequencing analysis with 13 housekeeping genes, the O121:H19 clone did not fall into any of the four classical EHEC and enteropathogenic E. coli groups but instead was closely related to two eae-negative STEC strains.     

Human growth hormone and prolactin secreting pituitary adenomas analyzed by in situ hybridization

Acidophilic pituitary adenomas commonly produce growth hormone (GH) or prolactin (PRL), according to studies employing immunohistochemical and ultrastructural methods. To examine this question, in situ hybridization with oligonucleotide probes was done on routinely processed tissues received in the pathology laboratory to analyze for the presence of GH and PRL messenger RNA (mRNA) in 4 normal pituitaries, 10 prolactinomas, and 16 GH-secreting adenomas. Most acidophilic cells in normal pituitaries expressed either GH or PRL hormone and the respective mRNAs, but GH mRNA and PRL hormone were also detected in some of the same cells. Patients with a clinical diagnosis of prolactinoma had cells with only PRL mRNA in their tumors, while most (14 of 16) patients with a clinical diagnosis of acromegaly or gigantism had both GH and PRL mRNAs in their tumors. The GH adenomas varied in these studies. In situ hybridization was helpful in characterizing the adenoma from a patient with acromegaly who had immunoreactive PRL, but no immunoreactive GH in the resected tumor; in situ hybridization analysis revealed mRNAs for both GH and PRL in the same tumor cells. Our findings indicate that pituitary adenomas from patients with acromegaly commonly express PRL mRNA. It is concluded that in situ hybridization provides new information about the clinical biology and the histopathologic classification of pituitary adenomas.     

Interstitial deletion of 20q in a patient with Waldenström macroglobulinemia following chemotherapy

A 76-year-old male with a history of renal insufficiency was found to have anemia, an IgM kappa paraprotein on serum immunofixation studies, absence of lytic bone lesions, and findings in the bone marrow consistent with Waldenstr?m macroglobulinemia (WM). Cytogenetic studies including fluorescence in situ hybridization (FISH) on the post-treatment bone marrow revealed the karyotype 46,XY,del(20)(q13.1q13.3). Less than 70 cases of karyotypic abnormalities in patients with WM have been reported, which have shown no abnormalities specific to WM. Monosomy or trisomy of chromosome 20 has been reported in approximately eight cases, but to our knowledge this is the first case report of an interstitial deletion of 20q, confirmed by FISH using chromosome 20 subtelomeric specific probes. Interstitial deletions of 20q are known to occur in polycythemia vera and other hematological malignancies, especially those of myeloid origin.     

Levels and specificity of antibody in bronchoalveolar lavage (BAL) and serum in an animal model of trimellitic anhydride-induced lung injury

A study was undertaken to characterize the antibody response in rats exposed to trimellitic anhydride (TMA) by inhalation. Total antibody levels directed to trimellitic rat serum albumin (TM-RSA) from TMA-exposed rats were assayed by an ammonium sulfate technique. Total antibody levels in bronchoalveolar lavage (BAL) and the matched serum were compared by correction for the albumin content of each. An ELISA was developed to detect IgG, IgA, and IgM directed toward TM-RSA in BAL and serum and to compare class-specific antibody levels in BAL and serum by normalizing for albumin content. The specificity of the rat IgG response was determined by ELISA inhibition with TM-RSA and TM-human serum albumin (TM-HSA) and compared with reciprocal inhibition studies with serum from TMA-exposed workers. The levels of total antibody in BAL were three to 15 times greater than the levels found in the matched serum pair. IgG, IgA, and IgM antibodies were detected in the BAL and the serum of TMA-exposed rats but not in control rats. In each of the four rats tested, all antibody classes were present in equal or greater amounts in the BAL than in the serum. Complete inhibition of the rat IgG binding in ELISA was observed when TM-RSA or TM-HSA were added as inhibitors. Human IgG was inhibited in ELISA only by TM-HSA. In an animal model of human lung disease, the levels of total antibody as well as class-specific antibodies directed against TM-RSA were greater in BAL than in serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exposure of bovine cornea to different strains of Moraxella bovis and to other bacterial species in vitro

A collection of strains of Moraxella bovis, some pathogenic and some non-pathogenic in cattle, together with other M. bovis preparations, Neisseria ovis, Staphylococcus aureus and Moraxella non-liquefaciens were studied by scanning electron microscopy for their affinity to bovine corneal preparations in vitro. The in vitro procedure provides a convenient method for studies on host-pathogen interactions at the early stage of pathogenesis. The results corresponded well with the pathogenicity of the respective strains and species in cattle. It is considered that the pathogenicity of M. bovis is associated with at least two factors, piliation and the ability to produce pit-like depressions in corneal epithelial cells. The other bacterial species, which are not thought to play an important role in infectious bovine keratoconjunctivitis, had the ability to adhere to the bovine cornea but did not produce pits. The pitting factor of M. bovis is of interest in relation to studies on vaccination against infectious bovine keratoconjunctivitis.     

The biopsychosocial approach: Clinical examples from a consultation-liaison service

In this second of three articles exploring Engel's biopsychosocial model, five case histories illustrate how psychological factors, sometimes related to organic trauma or illness, may precipitate psychiatric conditions. The patients’ disorders, and their management by a consultation-liaison team, demonstrate the interaction of biologic, psychological, and social systems in assessment and treatment of disease.