Development and Evaluation of a PCR-Based Assay for Detection of Haemobartonella felis in Cats and Differentiation of H. felis from Related Bacteria by Restriction Fragment Length Polymorphism Analysis

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set of H. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA of Eperythrozoon suis, Mycoplasma genitalium, and Bartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI and MnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felis infection in cats.     

Genomic structure of PIR-B, the inhibitory member of the paired immunoglobulin-like receptor genes in mice

Abstract: The genes encoding the murine paired immunoglobulin-like receptors PIR-A and PIR-B are members of a novel gene family which encode cell-surface receptors bearing immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and their non-inhibitory/activatory counterparts. PIR-A and PIR-B have highly homologous extracellular domains but distinct trans-membrane and cytoplasmic regions. A charged arginine in the transmembrane region of PIR-A suggests its potential association with other transmembrane proteins to form a signal transducing unit. PIR-B, in contrast, has an uncharged transmembrane region and several ITIMs in its cytoplasmic tail. These characteristics suggest that PIR-A and PIR-B which are coordinately expressed by B cells and myeloid cells, serve counter-regulatory roles in humoral and inflammatory responses. In the present study we have determined the genomic structure of the single copy PIR-B gene. The gene consists of 15 exons and spans approximately 8 kilobases. The first exon contains the 5' untranslated region, the ATG translation start site, and approximately half of the leader peptide sequence. The remainder of the leader peptide sequence is encoded by exon 2. Exons 3–8 encode the six extracellular immunoglobulin-like domains and exons 9 and 10 code for the extracellular membrane proximal and transmembrane regions. The final five exons (exons 11–15) encode for the ITIM-bearing cytoplasmic tail and the 3' untranslated region. The intron/exon boundaries of PIR-B obey the GT-AG rule and are in phase I, with the notable exception of the three boundaries determined for ITIM-containing exons. A microsatellite composed of the trinucleotide repeat AAG in the intron between exons 9 and 10 provides a useful marker for studying population genetics.     

Benefit of nasal CPAP in obstructive sleep apnea is due to positive pharyngeal pressure

The purpose of this study was to determine if the mechanism of nasal continuous positive airway pressure's (CPAP's) effectiveness is to act as a pneumatic splint or to increase functional residual capacity (FRC) and consequently, upper airway caliber. Four subjects with obstructive sleep apnea underwent 3 nights of polysomnography: night 1, control; night 2, nasal CPAP; night 3, external subatmospheric pressure (ESAP). ESAP, a negative pressure body suit, increases FRC. We measured the changes in FRC with nasal CPAP and ESAP using the weighted spirometer technique. The dose used for the ESAP night was the dose that produced the same FRC as the subject's prescribed nasal CPAP dose. The mean number of arousals and the respiratory events index were higher on ESAP and control nights. Less severe oxygen desaturation occurred during non-rapid-eye-movement sleep on the nasal CPAP and ESAP nights. These preliminary results show that increasing FRC alone does not account for the effectiveness of nasal CPAP, and splinting of the collapsible upper airway is necessary.     

Reactivity of low molecular weight material in cellular immune complex assays.

A major difference in molecular size of material reactive in the C1q binding assay and two cellular assays (Raji and L1210) for immune complexes, is reported. Elevated C1q binding of pathological sera was associated with material in the range 7-19S, as determined by Sepharose 6B chromatography of sera from patients with chronic inflammatory and neoplastic lung diseases. By contrast, reactivity of identical sera in the Raji and L1210 assays was linked predominantly with material of molecular size 7S. Dissociation of immune complexes on storage and/or in consequence of the chromatographic procedure was effectively discounted. Furthermore, differential binding of 7S IgG fractions tested at a standard concentration indicated that reactivity in either test was not attributable to non-specific binding of IgG. In a previous study, saturation of FcR (on L1210 and Raji) and C3R (on Raji only) by heat-aggregated IgG failed to distinguish whether binding directly involved these receptors or other cell surface components. In the present investigation, no firm correlations emerged between reactivity in the two tests and possible candidate antibodies reactive with cell surface components such as anti-lymphocyte and anti-nuclear antibodies. It is therefore suggested that low molecular weight binding may be attributable to more than one factor including 7S IgG (immune complex dissociated, or otherwise), autoantibodies, IgG-C3 complexes and possibly very small immune complexes (Ag1, Ab1). The assumption that Raji and L1210 and possibly other cellular assays detect only high molecular weight immune complexes is questionable and the need for further characterization of other reactive material is emphasized.     

Clinical Evaluation of the Uni-Yeast-Tek System for Rapid Presumptive Identification of Medically Important Yeasts

The results of over 400 tests for identification of clinical yeast isolates as to species using the Uni-Yeast-Tek (UYT) system in comparison with a more conventional system are reported. The conventional system utilized a total of 23 individual tests, including both fermentation and assimilation tests, whereas the UYT system included only 11 separate tests. In the initial phase of the study, coded unknown isolates were evaluated by each of two technologists using both methods independently. After this initial evaluation, the two methods were used in parallel for routine testing of yeast isolates as they were obtained from clinical specimens. A further evaluation of the UYT system was carried out by retrospectively analyzing the species reported from a clinical mycology laboratory during two separate time periods in which different approaches to yeast identification were employed. A total of 92% of the isolates tested with the UYT system were correctly reported within 72 h, 96% were correctly named after 1 week of incubation, and 97% were correctly reported after 2 weeks of incubation of UYT plates at 30 degrees C when results of the two phases of the study were analyzed together. With the conventional system, 88% of the isolates were correctly reported at 72 h, 96% at 1 week, and 98% after 2 weeks of incubation of biochemical tests. Retrospective analysis of laboratory records revealed no major changes in species reported after adoption of the UYT system for routine testing of clinical isolates. The data presented in this report suggest that the UYT system can be expected to yield rapid presumptive identification of clinical yeast isolates with reasonable confidence when certain minor limitations that are discussed in the text are taken into account.     

Homozygosity for autosomal dominant Marfan syndrome.

Marfan syndrome is an autosomal dominant condition with varying phenotypic manifestations. Affected persons are usually heterozygotes. A family is presented in which the gene for this syndrome is segregating in a large number of members. Two sibs suffered from unusually severe, identical, and fatal manifestations from birth, their parents having mild cardiovascular and somatic symptoms common in Marfan syndrome. Investigation of collagen biosynthesis in fibroblasts revealed no abnormalities in fibronectin and procollagen I and III synthesis and secretion or in the procollagen to collagen conversion. We suggest that these two sibs are examples of homozygosity for the Marfan syndrome gene, based on the large number of affected members, the absence of additional consanguinity, manifestation of the syndrome in both parents, and the severity of the disease in the two sibs.     

Visceral pleural invasion in lung cancer: recognizing histologic parameters that impact staging and prognosis

Visceral pleural involvement (VPI) is a critical component in the staging of non-small cell lung carcinoma (NSCLC). Tumors < or =3 cm that involve the visceral pleura are classified as T2 lesions, underscoring the prognostic significance of this histologic parameter. Accurate staging of small NSCLCs depends on appropriately assessing the presence or absence of VPI. Elastic stains can be instrumental in detecting disruptions of the visceral pleural elastic layer by tumor, a finding that has prognostic and staging implications similar to tumor that is present on the visceral pleural surface.     

Processing of radical prostatectomy specimens for correlation of data from histopathological, molecular biological, and radiological studies: a new whole organ technique

AIMS: To develop a method of processing non-formalin fixed prostate specimens removed at radical prostatectomy to obtain fresh tissue for research and for correlating diagnostic and molecular results with preoperative imaging. METHODS/RESULTS: The method involves a prostate slicing apparatus comprising a tissue slicer with a series of juxtaposed planar stainless steel blades linked to a support, and a cradle adapted to grip the tissue sample and receive the blades. The fresh prostate gland is held in the cradle and the blades are moved through the cradle slits to produce multiple 4 mm slices of the gland in a plane perpendicular to its posterior surface. One of the resulting slices is preserved in RNAlater. The areas comprising tumour and normal glands within this preserved slice can be identified by matching it to the haematoxylin and eosin stained sections of the adjacent slices that are formalin fixed and paraffin wax embedded. Intact RNA can be extracted from the identified tumour and normal glands within the RNAlater preserved slice. Preoperative imaging studies are acquired with the angulation of axial images chosen to be similar to the slicing axis, such that stained sections from the formalin fixed, paraffin wax embedded slices match their counterparts on imaging. CONCLUSIONS: A novel method of sampling fresh prostate removed at radical prostatectomy that allows tissue samples to be used both for diagnosis and molecular analysis is described. This method also allows the integration of preoperative imaging data with histopathological and molecular data obtained from the prostate tissue slices.     

New classification for mental disorders with management guidelines for use in primary care: ICD-10 PHC chapter five.

A primary care version of the International classification of diseases (10th revision) chapter five for mental and behavioural disorders (ICD-10 PHC chapter five) has been developed. This provisional version focuses on 24 conditions which are frequently seen in primary care and which can be managed effectively by general practitioners. The classification is accompanied by a flipcard for each of the conditions. The cards have diagnostic guidelines on one side and management guidelines on the other. The latter provide information which should be given to the patient, advice on the content of counselling, the available treatment methods, and indications for specialist referral. This classification system is also supported by diagnostic decision making aids, medication cards, and patient leaflets to facilitate the recognition and management of patients with mental disorders in primary care settings. The draft version of ICD-10 PHC chapter five will be finalized after field trials which will test the applicability and usefulness of the system in different primary care settings in various countries.     

Correlation of flunisolide plasma levels to eosinopenic response in humans

Plasma levels of flunisolide were measured in healthy male volunteers after the administration of single doses of the drug by the intravenous, oral, intranasal, and bronchial inhalation routes. The systemic availability of a 1-mg dose orally was only 21%. After a single dose of approximately 0.117 mg intranasally plasma levels ranged up to 1 ng/ml. When 1 mg was administered by bronchial inhalation, peak or near peak plasma levels were recorded at 2 min and remained near this level throughout the first hour before declining at a rate similar to that observed after flunisolide intravenously (plasma ). Gargling with an alcoholic mouthwash immediately after inhalation reduced plasma levels at 30 and 60 min but not earlier, suggesting rate-limiting dissolution of flunisolide in bronchial fluids or rate-limiting diffusion across the mucociliary blanket or pulmonary membrane. The systemic availabilities of the inhaled-mouthwash and inhaled-no mouthwash doses were 32% and 39%, respectively. Systemic potency of flunisolide, measured by eosinopenic response, was oral < inhaled < intravenous and correlated with the systemic availability of flunisolide after drug administration by these three routes. These pharmacokinetic properties of flunisolide are clinically advantageous in that relatively small doses are delivered topically to the target organs, i.e., the nasal mucosa and lungs, whereas a large portion of the dose is swallowed and subsequently extensively metabolized to relatively inactive metabolites.