Analysis of orthologous gene expression between human pulmonary adenocarcinoma and a carcinogen-induced murine model

Human adenocarcinoma (AC) is the most frequently diagnosed human lung cancer, and its absolute incidence is increasing dramatically. Compared to human lung AC, the A/J mouse-urethane model exhibits similar histological appearance and molecular changes. We examined the gene expression profiles of human and murine lung tissues (normal or AC) and compared the two species' datasets after aligning approximately 7500 orthologous genes. A list of 409 gene classifiers (P value <0.0001), common to both species (joint classifiers), showed significant, positive correlation in expression levels between the two species. A number of previously reported expression changes were recapitulated in both species, such as changes in glycolytic enzymes and cell-cycle proteins. Unexpectedly, joint classifiers in angiogenesis were uniformly down-regulated in tumor tissues. The eicosanoid pathway enzymes prostacyclin synthase (PGIS) and inducible prostaglandin E(2) synthase (PGES) were joint classifiers that showed opposite effects in lung AC (PGIS down-regulated; PGES up-regulated). Finally, tissue microarrays identified the same protein expression pattern for PGIS and PGES in 108 different non-small cell lung cancer biopsies, and the detection of PGIS had statistically significant prognostic value in patient survival. Thus, the A/J mouse-urethane model reflects significant molecular details of human lung AC, and comparison of changes in orthologous gene expression may provide novel insights into lung carcinogenesis.     

Evaluation of three disk tests for identification of enterococci, leuconostocs, and pediococci.

Simple rapid tests for presumptive identification of catalase-negative non-beta-hemolytic cocci (i.e., enterococci, leuconostocs, and pediococci) have not previously been available. Seven hundred thirty-four strains of aerobic and facultatively anaerobic, catalase-negative, non-beta-hemolytic gram-positive cocci were tested for susceptibility to vancomycin (Vans) by a screening procedure and production of leucine aminopeptidase (LAPase) and pyrrolidonylarylamidase (PYRase) in disk tests. Three unique patterns of activity in response to the three disks (30 micrograms of vancomycin, PYRase, and LAPase) can be used to presumptively identify the vancomycin-resistant (Vanr) enterococci (Vanr and PYRase and LAPase positive), leuconostocs (Vanr and PYRase and LAPase negative), and pediococci (Vanr, PYRase negative, and LAPase positive). The results indicate that, together with Gram stain characteristics and the catalase test, the vancomycin, LAPase, and PYRase disk tests can be used to presumptively identify Vanr strains of enterococci as well as Leuconostoc and Pediococcus strains from human infections.     

Development of a rat model for respiratory infection with Bordetella pertussis.

Considerable effort directed toward designing a safe and effective vaccine for Bordetella pertussis in which the cellular and/or acellular antigens necessary to confer immunity are known has been hampered by lack of information on the pathogenesis of the natural infection. The study presented here describes an animal model of lung infection by B. pertussis encased in agar beads in adult (200- to 220-g) male Sprague-Dawley rats. At 3 and 7 days after inoculation with phase I B. pertussis, organisms could be recovered from the lungs of rats; however, organisms were not recoverable at days 10 and 14 but reappeared in lung homogenates at day 21. Histopathological examination revealed findings similar to those seen in human disease. At day 3, a mild lymphocytic infiltrate was present in the bronchi, with progressive lymphoid hyperplasia peribronchially. By day 7, a necrotizing inflammation of the tracheobronchial mucous membranes, characterized by both mononuclear and polymorphonuclear cells, was noted. Phase III B. pertussis organisms were not recoverable from the lungs of inoculated rats at day 3 after inoculation, nor were histological changes noted in these animals. Clinical findings in phase I B. pertussis-infected rats included hypoglycemia, circulating lymphocytosis, and paroxysms in which air was forcibly expelled from the mouth or nose.     

An analysis of myeloma plasma cell phenotype using antibodies defined at the IIIrd International Workshop on Human Leucocyte Differentiation Antigens.

Fresh bone marrow from 43 cases of myeloma and three cases of plasma cell leukaemia has been phenotyped both by indirect immune-rosetting and, on fixed cytospin preparations, by indirect immunofluorescence. Both clustered and unclustered B cell associated antibodies from the IIIrd International Workshop on Human Leucocyte Differentiation Antigens were used. The results confirm the lack of many pan-B antigens on the surface of myeloma plasma cells, i.e. CD19-23, 37, 39, w40. Strong surface reactivity is seen with CD38 antibodies and with one CD24 antibody (HB8). Weak reactions are sometimes obtained with CD9, 10 and 45R. On cytospin preparations CD37, 39 and w40 are sometimes weakly positive, and anti-rough endoplasmic reticulum antibodies are always strongly positive. Specific and surface-reacting antiplasma cell antibodies are still lacking.     

Detection of smallpox virus DNA by LightCycler PCR

A 300-bp plasmid fragment of the hemagglutinin gene was used as target DNA to develop a rapid real-time LightCycler (Roche Applied Science, Indianapolis, Ind.) PCR assay for laboratory detection of smallpox virus. PCR primers and probes were designed specifically for detection of smallpox virus DNA, but all viruses of the genus Orthopoxvirus tested could be detected by use of the hemagglutinin gene target sequence. Base pair mismatches in the 204-bp amplicon allowed discrimination of cowpox virus (melting temperature [T(m)], 56.40 degrees C), monkeypox virus (T(m), 56.24 degrees C), and vaccinia virus (T(m), 56.72 degrees C), including the Dryvax vaccine strain, from smallpox virus (T(m), 62.45 degrees C) by melting curve analysis. The analytical sensitivity was 5 to 10 copies of target DNA per sample. The assay was specific for members of the genus Orthopoxvirus; the DNAs of herpes simplex virus and varicella-zoster virus were not detected by the smallpox virus LightCycler PCR.     

Comparison of a new rapid test (TestPack Rotavirus) with standard enzyme immunoassay and electron microscopy for the detection of rotavirus in symptomatic hospitalized children.

We compared a new, rapid, qualitative test for rotavirus (TestPack Rotavirus; Abbott Laboratories, North Chicago, Ill.) with another enzyme immunoassay (Pathfinder Rotavirus; Kallestad Laboratories, Inc., Austin, Tex.) and electron microscopy to determine its clinical utility in a population of symptomatic hospitalized children. In the first part of the study, 100 frozen stool samples were tested. The results after resolution with a blocking reagent showed a sensitivity of only 50% and a specificity of 88% for TestPack Rotavirus. In the second part of the study, we tested TestPack Rotavirus on 100 fresh, unfrozen samples. The results (sensitivity/specificity) were as follows: TestPack Rotavirus, 95/90%; Pathfinder Rotavirus, 84/98%; direct electron microscopy, 63/100%. Although it was not as sensitive or specific as immune electron microscopy, TestPack Rotavirus was more sensitive than direct electron microscopy or Kallestad Pathfinder Rotavirus. TestPack Rotavirus represents a rapid, qualitative method for the detection of rotavirus in stools of symptomatic children.     

Antimicrobial susceptibility testing of porcine Brachyspira (Serpulina) species isolates

No standardized method for susceptibility testing of Brachyspira spp. is currently available. A broth dilution procedure was evaluated and used to test the activities of six antimicrobial agents for 108 isolates of Swedish porcine Brachyspira spp. representing biochemical groups I, II, and III. Group I corresponds to Brachyspira hyodysenteriae, group II corresponds to B. intermedia, and group III corresponds to B. murdochii and B. innocens. A panel was designed with the antimicrobial agents dried in tissue culture trays with wells that allowed a liquid volume of 0.5 ml in each and agitation of the broth when incubated on a shaker. The MICs were determined by using brain heart infusion broth with 10% fetal calf serum. For 10 isolates, the results obtained in broth were compared to the MICs obtained on two different types of agar. Different inoculum densities and incubation times were also compared. The concentrations at which 90% of the B. hyodysenteriae isolates (n = 72) were inhibited in the broth dilution test by tiamulin (0.25 micro g/ml), tylosin (>256 micro g/ml), erythromycin (>256 micro g/ml), clindamycin (>4 micro g/ml), virginiamycin (4 micro g/ml), and carbadox (0.06 micro g/ml) were determined. The MICs tended to be lower in broth than on agar. Differences in inoculum densities and incubation times had little influence on the MICs. The evaluated broth dilution test was simple to perform, the end points were easily read, and the results were reproducible and reliable. No isolates with decreased susceptibility to tiamulin were found among the Swedish isolates tested.     

Serodiagnosis of Helicobacter hepaticus infection in mice by an enzyme-linked immunosorbent assay.

Helicobacter hepaticus is a newly recognized bacterium associated with chronic active hepatitis, hepatic carcinoma, and inflammatory bowel disease in mice. Currently, fecal or tissue PCR, fecal culture, or histologic examination of silver-stained liver sections is used to diagnose H. hepaticus infection. In this report, we describe an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of H. hepaticus infection in mice with a membrane digest preparation of H. hepaticus as the antigen. Sera from mice positive for H. hepaticus by PCR or histologic examination (n = 88), positive for Helicobacter bilis by PCR (n = 13), positive for other helicobacters (not identifiable to species level) by PCR (n = 25), or negative for all Helicobacter species by PCR (n = 162) were used to evaluate the ELISA. Results indicated that ELISA provided 93.2% sensitivity, 94% specificity, 87.2% positive predictive value, and 96.9% negative predictive value. Cross-reactive antibodies were detected in some mice infected with helicobacters not identifiable to species level. To further define ELISA sensitivity and specificity, groups of 10 C57BL/6 mice were inoculated per os with H. hepaticus, Helicobacter muridarum, or H. bilis. Sera were collected and examined by the ELISA. H. hepaticus-infected mice seroconverted by 2 weeks and maintained ELISA reactivity throughout the 18-week study, while mice infected with H. muridarum and H. bilis were negative by ELISA. These results indicate that this reported ELISA is highly sensitive and specific for the serodiagnosis of H. hepaticus infection in mice.     

Assessment of fetal-maternal haemorrhage in mothers with hereditary persistence of fetal haemoglobin.

Kleihauer examination of peripheral blood cannot be used reliably to detect transplacental fetal-maternal haemorrhage in mothers with hereditary persistence of fetal haemoglobin (HPFH). In Rh(D) negative pregnancies diagnostic confusion with a large fetal-maternal haemorrhage could result in the administration of inappropriately excessive amounts of anti-D immunoglobulin, and the inability to diagnose and quantify transplacental haemorrhage in maternal HPFH by current methods could result in insufficient anti-D administration and subsequent Rh(D) sensitisation. Accordingly, a method to detect and quantify fetal-Rh(D) positive maternal haemorrhage using erythrocyte fluorescent immunocytometry was developed. An indirect immunofluorescence method with IgG anti-D immunoglobulin as the primary antibody was used, combined with quantitative analysis on a fluorescence activated cell sorter. The method was accurate, specific, and sensitive and could detect a contaminating population of 0.1% Rh(D) positive cells in Rh(D) negative blood--a level of fetal-maternal haemorrhage well covered by a single dose of 500 IU of anti-D immunoglobulin.