Specific detection of the genus Serpulina, S. hyodysenteriae and S. pilosicoliin porcine intestines by fluorescent rRNA in situ hybridization

PURPOSE: Our purpose was to evaluate the utility of spectral imaging for multicolor, multichromosome enumeration in human interphase cell nuclei. METHODS: Chromosome-specific probes labeled with different fluorochromes or nonfluorescent haptens were obtained commercially or prepared in-house. Metaphase spreads, interphase lymphocytes, or blastomeres cells were hybridized with either 7 or 11 distinctly different probes. Following 46 hr of hybridization, slides were washed and detected using either a filter-based quantitative image processing system (QUIPS) developed in-house or a commercial spectral imaging system. RESULTS: The filter-based fluorescence microscope system is preferred for simultaneous detection of up to seven chromosome targets because of its high sensitivity and speed. However, this approach may not be applicable to interphase cells when 11 or more targets need to be discriminated. Interferometer-based spectral imaging with a spectral resolution of approximately 10 nm allows labeling of chromosome-specific DNA probes with fluorochromes having greatly overlapping emission spectra. This leads to increases in the number of fluorochromes or fluorochrome combinations available to score unambiguously chromosomes in interphase nuclei. CONCLUSIONS: Spectral imaging provides a significant improvement over conventional filter-based microscope systems for enumeration of multiple chromosomes in interphase nuclei, although further technical development is necessary in its application to embryonic blastomeres. When applied to preconception/preimplantation genetic diagnosis, presently available probes for spectral imaging are expected to detect abnormalities responsible for 70-80% of spontaneous abortions caused by chromosomal trisomies.     

Ventilation/carbon dioxide production ratio in early exercise predicts poor functional capacity in congestive heart failure

Production of cytokines by immunocompetent cells in vitro may be assessed after stimulation with polyclonal activators. Because it mimics the natural environment, diluted whole blood (WB) culture may be the most appropriate milieu in which to study cytokine production in vitro. We tested TNF alpha production by small volume of whole blood (25 microliters) from HIV-1 positive patients by using a one-step procedure that combines WB stimulation with LPS and PHA and cytokine measurement. We studied 30 patients without secondary infection or at distance of secondary infection staged according to the classification proposed by the CDC and 12 healthy seronegative subjects. The mean values of TNF alpha from patients were not statistically different from those from normal controls however in certain patients at different stages of the disease higher values than the mean +2 SD of controls and lower values than the mean -2 SD of controls were obtained. Heparinized blood from 5 control subjects had been collected sequentially during a period of 5 months. The individual variation of TNF alpha production were limited for all these individuals. For each of 6 HIV-1 patients, whole blood samples were collected sequentially during a period of 5 months and in most of patients large variations of levels of TNF alpha were observed from one sample to another one. Our method can detect abnormal cytokine production in HIV-1 positive individuals and can become a useful tool to investigate the role of cytokines in the outcome of HIV-1 infection.     

Pulmonary sequestration: the setting sun sign

Acquired immune deficiency syndrome (AIDS) has become a topic of increasing public concern. Several conditions have been described in patients who are afraid that they have acquired AIDS, including depression, somatization disorders, hypochondriasis, adjustment disorders and various psychoses. This paper presents a case study of a young man with persistent fear of having AIDS. In spite of negative HIV test results, the patient still insisted that he suffered from AIDS. The psychiatric diagnosis was major depressive disorder with delusion of having AIDS. The patient was treated with fluoxetine 40 mg qd and sulpiride 400 mg qn. After two weeks on medication, his fear of AIDS subsided and he improved remarkably. The most important intervention in patients with delusion of having AIDS is to identify and treat the underlying psychopathology. The following case is representative of our experience.     

CD56+ NK lymphomas: clinicopathological features and prognosis

Dosing regimen is an important determinant of both drug cost and patient compliance. This retrospective analysis evaluated dosing regimens and drug acquisition costs for 101 patients identified from medical records in a large metropolitan hospital as having hypertension and/or benign prostatic hyperplasia and receiving alpha-blocker therapy with either doxazosin or terazosin. Although once-daily administration is generally recommended for both drugs, 25 (38%) of 66 patients receiving terazosin were treated twice daily compared with 6 (17%) of 35 patients treated twice daily with doxazosin. This difference was statistically significant. The average (mean +/- SD) daily treatment cost per patient for all individuals receiving terazosin during the period of the record review was $1.68 +/- 0.60. For patients treated with doxazosin, the average was $0.96 +/- 0.65-a highly statistically significant result. If all 66 patients receiving terazosin had been converted to doxazosin at the beginning of the study, annual savings would have been $17,345.00. These results demonstrate the importance of reviewing actual dosing regimens. The fact that doxazosin could be administered to a significantly higher percentage of patients once daily rather than twice daily substantially decreased its cost relative to terazosin. A once-daily treatment regimen may also enhance patient compliance, thereby improving the chances of therapeutic success.     

Are Canadian Inuit at increased genetic risk for coronary heart disease?

The Keewatin Inuit of the Northwest Territories of Canada have a very low age-adjusted mortality rate from coronary heart disease. We hypothesized that this apparent protection from disease has a genetic basis. We determined the prevalence of the disease-associated alleles of five candidate genes for atherosclerosis-related phenotypes. Surprisingly, four of the five alleles studied, namely AGT T235, FABP2 T54, PON R192 and APOE E4, were significantly more frequent in a sample of 175 Keewatin Inuit than among a representative control sample of whites living in the region. The high frequencies of these disease-associated alleles suggests either that they have no relationship with disease susceptibility in the Inuit, or that some unmeasured genetic and/or environmental factors mitigate disease susceptibility that is associated with these alleles. This highlights the difficulty in extrapolating findings from one population to another. Also, very modest genotype-phenotype associations were observed between APOE genotype (P = 0.016) and plasma low-density lipoprotein cholesterol concentration and between FABP2 genotype and plasma 2-h postprandial, glucose concentration (P = 0.048). The relationship between APOE alleles and plasma low-density lipoprotein cholesterol was the same as has been previously reported in many study samples. However, the relationship between FABP2 alleles and plasma 2-h postprandial glucose concentrations was the opposite to that reported in other studies. This suggests that differences in environment, such as the type of fatty acid consumed, interacts with functional differences in gene products involved in candidate metabolic pathways to produce phenotypic differences.     

The gastrin-releasing peptide receptor is differentially coupled to adenylate cyclase and phospholipase C in different tissues

A magnetization-prepared sequence, T2-Prep-IR, exploits T1, T2, and chemical shift differences to suppress background tissues relative to arterial blood. The resulting flow-independent angiograms depict vessels with any orientation and flow velocity. No extrinsic contrast agent is required. Muscle is the dominant source of background signal in normal volunteers. However, long-T2 deep venous blood and nonvascular fluids such as edema also contribute background signal in some patients. Three sets of imaging parameters are described to address patient-specific contrast requirements. A rapid, spiral-based, three-dimensional readout is utilized to generate high-resolution angiograms of the lower extremities. Comparisons with x-ray angiography and two-dimensional time-of-flight angiography indicate that this flow-independent technique has unique capabilities to accurately depict stenoses and to visualize slow flow and in-plane vessels.     

Polymerase chain reaction amplification of Trichomonas vaginalis DNA from Papanicolaou-stained smears

We describe 3 cases of Hodgkin's disease (HD) of unusual suppurative type, which were diagnosed on fine-needle aspirates. The smears were dominated by neutrophils, macrophages, and cellular debris. Only a few large, atypical cells of Hodgkin and Reed-Sternberg type were observed. The differential diagnoses of such smears include infectious mononucleosis, tuberculosis, metastatic lymph node involvement, non-Hodgkin's large-cell anaplastic Ki-1-positive lymphomas, T-cell-rich B-cell lymphomas, and peripheral T-cell lymphomas of mixed type. Immunocytochemistry identified the large atypical cells as CD 30 (BerH2)-positive and negative for CD 45 (LCA) in cytospin material from 2 patients, which allowed a conclusive diagnosis of HD.     

Treatment of microfilaraemia in asymptomatic brugian filariasis: the efficacy and safety of the combination of single doses of ivermectin and diethylcarbamazine

BACKGROUND: Nitric oxide (NO) seems to play an important role in modulating tissue injury during reperfusion of the liver. In this study, we have evaluated and compared the effects of FK409 (FK), a potent spontaneous NO releaser, and L-arginine in ischemia-reperfusion injury of the rat liver. METHODS: Male Sprague-Dawley rats underwent 90 min of hepatic ischemia followed by reperfusion. FK or L-arginine was used (intravenously) in two different doses for each drug (group I, 3.2 mg/kg FK; group II, 1.6 mg/kg FK; group IV, 100 mg/kg L-arginine; and group V, 300 mg/kg L-arginine). Saline was used in control animals (group III). Hepatic enzyme status, microcirculation, serum nitrite (NO2-) and nitrate (NO3-) and tissue injury score were evaluated at predetermined times. RESULTS: Serum NO2-/NO3- was elevated immediately by FK treatment dose-dependently but not by L-arginine. However, L-arginine caused late (6-24 hr) elevation of the NO metabolites dose-dependently. The elevation of serum aspartate aminotransferase and alanine aminotransferase was suppressed and hepatic microcirculation was improved in the FK-treated groups dose-dependently. L-Arginine also improved the microcirculation, but hepatic enzymes at 24 hr of reperfusion were significantly higher in group V than in the control group. These findings were well reflected by the extent of tissue injury in respective groups. CONCLUSION: FK treatment in the immediate reperfusion period improves hepatic microcirculation and confers a significant protective effect on hepatic ischemia-reperfusion injury in the rat.     

Sandwich-type deoxyribonucleic acid hybridization assays based on enzyme amplified time-resolved fluorometry

We report microtiter well-based sandwich-type DNA hybridization assays using enzyme amplified time-resolved fluorometry of Tb3+ chelates. The target DNA was hybridized with two adjacent and non-overlapping oligonucleotide probes, one oligonucleotide serving as the capture probe and the other as the detection probe. Two ligand-specific binding protein pairs were used alternately for capture of the hybrids to the solid phase and detection; the biotin-streptavidin and the digoxigenin-anti-digoxigenin interaction. In both cases, alkaline phosphatase was used as a reporter molecule and diflunisal phosphate as a substrate. The catalytic hydrolysis of the substrate produces diflunisal which forms ternary fluorescent complex with Tb(3+)-EDTA. Furthermore, we studied the effect of the probe labeling method and the position of the label on the sensitivity of the assays. The data suggest that capture of the hybrids through biotin-streptavidin and detection via digoxigenin-anti-digoxigenin offer 2-3 times higher sensitivity than the reverse configuration. The highest sensitivity was achieved with enzymatic labeling of capture and detection probes at the 3' end. A signal-to-background ratio of 4 was achieved for 0.2 fmol of target DNA. The RSD were better than 4%.     

Pharmacology and intracellular signaling mechanisms of the native human orphan receptor BRS-3 in lung cancer cells

Neither the native ligand nor the cell biology of the bombesin (Bn)-related orphan receptor subtype 3 (BRS-3) is known. In this study, we used RT-PCR to identify two human lung cancer lines that contain sufficient numbers of native hBRS-3 to allow study: NCI-N417 and NCI-H720. In both cell lines, [DPhe6,betaAla11,Phe13, Nle14]Bn(6-14) stimulates [3H]inositol phosphate. In NCI-N417 cells, binding of 125I-[DTyr6,betaAla11,Phe13,Nle14]Bn(6-14) was saturable and high-affinity. [DPhe6,betaAla11,Phe13,Nle14]Bn(6-14) stimulated phospholipase D activity and a concentration-dependent release of [3H]inositol phosphate (EC50 = 25 nM) and intracellular calcium (EC50 = 14 nM); the increases in intracellular calcium were primarily from intracellular stores. hBRS-3 activation was not coupled to changes in adenylate cyclase activity, [3H]-thymidine incorporation or cell proliferation. No naturally occurring Bn-related peptides bound or activated the hBRS-3 with high affinity. Four different bombesin receptor antagonists inhibited increases in [3H]inositol phosphate. Using cytosensor microphysiometry, we found that [DPhe6,betaAla11,Phe13, Nle14]Bn(6-14) caused concentration-dependent acidification. The results show that native hBRS-3 receptors couple to phospholipases C and D but not to adenylate cyclase and that they stimulate mobilization of intracellular calcium and increase metabolism but not growth. The discovery of human cell lines with native, functional BRS-3 receptors, of new leads for a more hBRS-3-specific antagonist and of the validity of microphysiometry as an assay has yielded important tools that can be used for the identification of a native ligand for hBRS-3 and for the characterization of BRS-3-mediated biological responses.