Molecular Approaches to Diagnosis of Pulmonary Diseases Due to Mycoplasma pneumoniae

In this prospective study, the use of a culture-enhanced PCR assay for the detection of Mycoplasma pneumoniae, followed by hybridization with a specific probe (MP-HPCR) or without hybridization (MP-PCR), and the use of a nested PCR (MP-NPCR) were evaluated. Clinical samples (190 specimens) from 190 patients with respiratory complaints were incubated in culture broth overnight and then subjected to PCR. The results of the PCR were compared to those obtained by culture, the direct antigen test, and serologic testing by microparticle agglutination and by immunoblotting in unclear cases. The sensitivities were 19 CFU for MP-PCR, 1.9 CFU for MP-HPCR, and 0.019 CFU for MP-NPCR. PCR amplification of the β-globin gene was possible in 98% of cases: after dilution of the β-globin-negative samples, all samples were reactive. Correlation between negative MP-NPCR results and negative serology results was found in 89% of cases; a positive correlation was found with 10% of the patients. Samples from three immunocompromised patients were MP-NPCR positive but serologically negative. High respiratory colonization by M. pneumoniae (>10⁵ CFU/ml) in patients with acute respiratory disease could be detected by culture, MP-PCR, and MP-NPCR. These results indicate that MP-PCR and MP-NPCR are reliable methods for the detection of M. pneumoniae in respiratory tract samples of patients with respiratory complaints.     

Effects of actinomycin D analogs on nucleolar phosphoprotein B23 (37,000 daltons/pI 5.1)

Localization of protein B23 in HeLa cells after treatment with actinomycin D and its analogs was studied using indirect immunofluorescence. Bright nucleolar fluorescence was observed in control HeLa cells. After treatment with actinomycin D (250 ng/ml) for 2 hr, a uniform nucleoplasmic fluorescence was observed. Similar results were obtained with the actinomycin analogs, actinomycin Z5 and actinomycin K2T. Only after a much longer incubation (24 hr) with actinomycin 4-4'-gly was nucleoplasmic fluorescence observed. Actinomycin D, actinomycin Z5, and actinomycin K2T inhibited [3H]uridine incorporation into the trichloroacetic acid insoluble fraction of HeLa cells with IC50 values of 9.5 +/- 3.2, 59.1 +/- 19.6 and 1423.3 +/- 212.2 ng/ml respectively. No inhibition of [3H]uridine incorporation was observed using actinomycin 4-4'-gly (2000 ng/ml, 2-hr incubation). The order of potency for the loss of nucleolar fluorescence and the concurrent increase in nucleoplasmic fluorescence was actinomycin D greater than actinomycin Z5 greater than actinomycin K2T greater than actinomycin 4-4'-gly, which correlated with the order of their IC50 values for inhibition of [3H]uridine incorporation. Studies of the effects of actinomycin D and its analogs on RNA synthesis and localization of protein B23 indicated that there is a direct relationship between the B23 "translocation" from nucleolus to nucleoplasm and the inhibition of RNA synthesis. At 45-55% inhibition of RNA synthesis, both nuclear and nucleolar B23 immunofluorescence were observed. At 75-85% inhibition, only a uniform nucleoplasmic fluorescence was observed.     

Single-cell fate mapping reveals widespread clonal ignorance of low-affinity T cells exposed to systemic infection

T cell ignorance is a specific form of immunological tolerance. It describes the maintenance of naivety in antigen-specific T cells in vivo despite the presence of their target antigen. It is thought to mainly play a role during the steady state, when self-antigens are presented in absence of costimulatory signals and at low density or to T cells of low affinity. In how far antigen-specific T cells can also remain clonally ignorant to foreign antigens, presented in the inflammatory context of systemic infection, remains unclear. Using single-cell in vivo fate mapping and high throughput flow cytometric enrichment, we find that high-affinity antigen-specific CD8⁺ T cells are efficiently recruited upon systemic infection. In contrast, most low-affinity antigen-specific T cells ignore the priming antigen and persist in the naïve state while remaining fully responsive to subsequent immunization with a high-affinity ligand. These data establish the widespread clonal ignorance of low-affinity T cells as a major factor shaping the composition of antigen-specific CD8⁺ T cell responses to systemic infection.     

The inhibitory effect of the antipsychotic drug haloperidol on HERG potassium channels expressed in Xenopus oocytes

1. The antipsychotic drug haloperidol can induce a marked QT prolongation and polymorphic ventricular arrhythmias. In this study, we expressed several cloned cardiac K⁺ channels, including the human ether-a-go-go related gene (HERG) channels, in Xenopus oocytes and tested them for their haloperidol sensitivity.
2. Haloperidol had only little effects on the delayed rectifier channels Kv1.1, Kv1.2, Kv1.5 and I_sK, the A-type channel Kv1.4 and the inward rectifier channel Kir2.1 (inhibition <6% at 3 μM haloperidol).
3. In contrast, haloperidol blocked HERG channels potently with an IC₅₀ value of approximately 1 μM. Reduced haloperidol, the primary metabolite of haloperidol, produced a block with an IC₅₀ value of 2.6 μM.
4. Haloperidol block was use- and voltage-dependent, suggesting that it binds preferentially to either open or inactivated HERG channels. As haloperidol increased the degree and rate of HERG inactivation, binding to inactivated HERG channels is suggested.
5. The channel mutant HERG S631A has been shown to exhibit greatly reduced C-type inactivation which occurs only at potentials greater than 0 mV. Haloperidol block of HERG S631A at 0 mV was four fold weaker than for HERG wild-type channels. Haloperidol affinity for HERG S631A was increased four fold at +40 mV compared to 0 mV.
6. In summary, the data suggest that HERG channel blockade is involved in the arrhythmogenic side effects of haloperidol. The mechanism of haloperidol block involves binding to inactivated HERG channels.

     

Daytime sleep in myotonic dystrophy is not caused by sleep apnoea.

Daytime sleepiness is common in myotonic dystrophy and might be attributed to disturbed nocturnal breathing. Seventeen out of 22 patients complained of excessive daytime sleepiness, resembling "idiopathic hypersomnolence". Sleep apnoea might have contributed to daytime sleepiness in only three of 17 patients. Treatment with the central stimulant methylphenidate produced sustained benefit in seven out of 11 patients tested. Daytime sleepiness in myotonic dystrophy is usually caused by dysfunction of central sleep regulation and not by disturbed nocturnal breathing.     

Primary colorectal non-Hodgkin's lymphoma

Background: The gastrointestinal tract is the most common site of extranodal involvement in non-Hodgkin's lymphoma (NHL). Primary colorectal NHL comprises 13–18% of all gastrointestinal NHL but is not commonly reported as a separate entity. Methods: This was a retrospective review of the medical records of 19 patients over a 16-year period to evaluate the clinical features and behavior of colorectal NHL. Results: A pediatric group of seven male patients presented at an early stage with acute symptomatology. The primary tumor was located in the ileocecum in all cases and intussusception was common. An adult group of 12 patients presented at a later stage with chronic symptomatology. Staging study results were positive by bone marrow biopsy in four of 16 patients (25%), by lymphangiography in six of 11 patients (54.5%), and by gallium scan in eight of 10 patients (80%). Seven patients relapsed a median of 8 months after treatment. Three other patients died during treatment, one died of other causes, and one died without receiving treatment. The remaining seven patients are alive from 41 to 231 months without evidence of disease. Five of these patients are in the pediatric group, where the median survival was >72 months. The overall median survival was 45 months. Conclusion: Colorectal NHL is a disease that affects both the pediatric and adult population. Although pediatric patients have an excellent prognosis with anticipated long-term survival after treatment, long-term survival can be expected in 50% of adult patients. In both groups of patients, multimodality therapy with surgery, chemotherapy, and radiation is the treatment of choice.Presented at the 46th Annual Cancer Symposium of The Society of Surgical Oncology, Los Angeles, California, March 18–21, 1993.     

Unsuspected idiopathic mediastinal fibrosis complicating coronary bypass operation

A 66-year-old woman was referred for coronary bypass operation to the left anterior descending and the circumflex arteries. She had a systemic inflammatory process of unknown origin. On opening the pericardium, fibrotic plaques encircling the entire ascending aorta were found. Aortic cannulation and proximal anastomosis of the planned vein graft were impossible. The right internal mammary was anastomosed to the left anterior descending artery, the left to the obtuse marginal after femoral arterial cannulation. Pathologic examination revealed idiopathic mediastinal fibrosis.