Development of a flow cytometry based method for rapid and sensitive detection of a novel marine fish iridovirus in cell culture

A sensitive and accurate flow cytometry (FCM) based method has been developed to detect and quantitate a novel marine fish iridovirus (Singapore grouper iridovirus, SGIV) after amplification in cell cultures. Confluent grouper cell (GP) monolayers were infected with SGIV. When advanced cytopathic effect (CPE) appeared, the cell cultures were fixed and permeabilized, and then reacted with monoclonal antibodies specific against SGIV, followed by a second antibody conjugated with FITC (anti-mouse IgG-FITC). A Coulter EPICS Elite ESP flow cytometer was used to directly detect and analyze the percentage of virus-infected cells. Three fixation and permeabilization methods were evaluated. The kinetics of the virus infection process was determined. The FCM procedure enables large amounts of cells to be screened rapidly for infectivity, and it can also detect low levels of virus infection. As early as 8 h after inoculation with the virus, 0.34% of infected cells were detected in cell culture. The maximum level of infection was obtained at 72 h. The efficiency and reliability of the FCM procedure were compared with those of the standard methods of immunofluorescence microscopy and PCR.     

Activation-induced polarized recycling targets T cell antigen receptors to the immunological synapse; involvement of SNARE complexes

The mechanism by which T cell antigen receptors (TCR) accumulate at the immunological synapse has not been fully elucidated. Since TCRs are continuously internalized and recycled back to the cell surface, we investigated the role of polarized recycling in TCR targeting to the immunological synapse. We show here that the recycling endosomal compartment of T cells encountering activatory antigen-presenting cells (APCs) polarizes towards the T cell-APC contact site. Moreover, TCRs in transit through recycling endosomes are targeted to the immunological synapse. Inhibition of T cell polarity, constitutive TCR endocytosis, or recycling reduces TCR accumulation at the immunological synapse. Conversely, increasing the amount of TCRs in recycling endosomes before synapse formation enhanced their accumulation. Finally, we show that exocytic t-SNAREs from T cells cluster at the APC contact site and that tetanus toxin inhibits TCR accumulation at the immunological synapse, indicating that vesicle fusion mediated by SNARE complexes is involved in TCR targeting to the immunological synapse.     

Leptin effects on food and water intake in lines of chickens selected for high or low body weight

There is an association between autonomic nervous system output and obesity. The sympathetic nervous system stimulates lipid metabolism and regulates food intake and, hence, body weight. Leptin, produced by adipocytes in proportion to their size, has been shown to directly stimulate the satiety center. In the experiment reported here, food and water intake were compared after intracerebroventricular administration of human recombinant leptin to lines of chickens that had undergone divergent selection for over 45 generations from a common White Rock base population for high (HWS) or low (LWS) body weight at 8 weeks-of-age. Leptin caused a linear decrease in food intake in chickens from the LWS line whereas no effect was observed in those from the HWS line. The HWS chickens tended to have reduced water intake post leptin administration. Others reported that leptin decreased food intake in both broiler and Leghorn chickens. Leptin concentration in the central nervous system may not contribute directly to the difference of body weight between HWS and LWS chickens.     

Thyroid-stimulating antibody (TSAb) detected in sera of Graves'' patients using human thyroid cell cultures

As a homologous system is required to evaluate the effect of thyroid-stimulating antibody (TSAb) present in the serum of Graves' patients, primary cultures obtained from normal human thyroid gland have been used and the stimulatory effect measured as an increase of cAMP intracellular levels.

Monolayer cell cultures were stimulated by IgG purified from sera of Graves' patients or control subjects and compared to the effect of bovine TSH. Bovine TSH produced a dose-dependent increase in cAMP intracellular levels between 0·05 mU and 2·5 mU/ml, reaching a maximal value after 30 min with higher doses. While normal IgG had no effect, IgG prepared from untreated patients with frank Graves' disease elicited a significant increase in cAMP accumulation at a concentration between 0·05 and 0·5 mg/ml within 60 min in thirteen out of fourteen patients. A longer incubation period showed no further increase in cAMP values, even if in one case a higher concentration (5·0 mg/ml) of Graves' IgG had a delayed response. When the cAMP intracellular level modifications produced by Graves' IgG preparations in thyroid cell cultures were compared to those evoked in thyroid slices, an identical percentage (93%) of positive cases was obtained, without a coincidence of negative cases. Using thyroid slices the cAMP intracellular increase above basal levels was higher, if considered as a percentage, but in cultured cells a very low IgG concentration was sufficient to detect the presence of TSAb. No correlation between the two assays was found.

In conclusion, normal human cultured thyroid cells appeared to be a more suitable substrate when compared to human thyroid slices for detecting the presence of TSAb in Graves' disease and for studying its effect on thyroid cells. However, a 100% TSAb positivity was present in our Graves' patient series only when both assays were used.

     

Oculoauriculovertebral anomaly: Segregation analysis

Seventy-four families of probands with oculoauriculovertebral anomaly were evaluated, including 116 parents and 195 off-spring. Relatives were examined to identify ear malformations, mandibular anomalies, and other craniofacial abnormalities. For segregation analysis using POINTER, selection of the sample was consistent with single as-certainment. Different population liabilities were used for probands and relatives, because affection was narrowly defined for probands and broadly defined for relatives. The hypothesis of no genetic transmission was rejected. The evidence favored autosomal dominant inheritance; recessive and polygenic models were not distinguishable. © 1992 Wiley-Liss, Inc.     

Spherical lesion phantoms for testing the performance of elastography systems

A set of three cubic one-litre phantoms containing spherical simulated lesions was produced for use in comparing lesion detection performance of different elastography systems. The materials employed are known to be stable in heterogeneous configurations regarding geometry and elastic contrast identical with (storage modulus of lesion material) / (storage modulus of background material), and regarding ultrasound and NMR properties. The materials mimic soft tissues in terms of elastic, ultrasound and NMR properties. Each phantom has only one value of elastic contrast (3.3, 4.6 or 5.5) and contains arrays of 1.6 mm, 2 mm, 3 mm and 4 mm diameter spherical simulated lesions. All the spheres of a given diameter are arranged in a regular array with coplanar centres. Elastograms of an array made with ultrasound allow determination of the depth range over which lesions of that diameter and elastic contrast can be detected. Two phantoms are made from agar-plus-gelatin-based materials, and one is made from oil-in-gelatin dispersions. The methods for producing the phantoms are described in detail. Lesion detection performances for two ultrasound systems, both operating at about 7.5 MHz and focused at about 5 cm, were quantified with distinctions between the two systems demonstrated. Neither system was capable of detecting any of the 1.6 mm lesions. Phantoms such as these should be useful in research labs that are refining hardware and/or software for elastography.     

The rising trend of invasive zygomycosis in patients with uncontrolled diabetes mellitus.

Zygomycosis is an emerging infection worldwide. A study was conducted to understand its spectrum in the Indian scenario. All patients diagnosed for invasive zygomycosis at a tertiary care center in north India from 2000-2004, were retrospectively analyzed. A total of 178 cases (mean average of 35.6 cases/year) of zygomycosis were diagnosed. Rhino-orbito-cerebral type (54.5%) was the commonest presentation followed by cutaneous (14.6%), disseminated (9.0%), and gastrointestinal (8.4%) zygomycosis. Renal and pulmonary zygomycosis were seen in 6.7% patients each. Uncontrolled diabetes mellitus (in 73.6% of cases) was the significant risk factor in all types (Odds Ratio 1.5-8.0) except renal zygomycosis. Breach of skin was the risk factor in 46.2% patients with cutaneous zygomycosis. However, no risk factor could be detected in 11.8% patients. Antemortem diagnosis was possible in 83.7% cases. The commonest (61.5%) isolate was Rhizopus oryzae followed by Apophysomyces elegans in 27% patients. Combination of debridement surgery and amphotericin B therapy was significantly better in survival of the patients (P<0.005) than amphotericin B alone (79.6% vs. 51.7% survival). Thus, a rising trend of invasive zygomycosis was observed in patients with uncontrolled diabetes mellitus in India. Consistent diagnosis of renal zygomycosis in apparently healthy hosts and the emergence of A. elegans in India demand further study.     

Formation of rabbit reaginic antibodies to protein and hapten—protein conjugates

The capacities of BSA and DNP—protein conjugates to evoke reagin formation in rabbits were compared. Reagins to DNP generally appeared earlier and disappeared more rapidly from the circulation than did anti-BSA reagins. Initial formation of reagins proceeded with a logarithmic phase indicating a doubling time of 7–8 hours. Booster antigen injections resulted in some cases in a reagin response after a shorter latent phase than that observed after primary immunization. A secondary reagin response was more readily evoked in rabbits with low titres of agglutinating antibodies than in those with high titres. Anti-DNP reagins were demonstrable in a higher percentage of the injected rabbits than were anti-BSA reagins. The two types of reagins were equally sensitive to heat and 2-mercaptoethanol. A positive correlation between serum levels of anti-DNP but not anti-BSA reagins and agglutinating antibodies was demonstrated. Some evidence that a low antigen dose was more efficient than a high dose in evoking reagin formation was obtained. Treatment of rabbits with 6-mercaptopurine during the 1st week following antigen injection resulted in an increased latent phase and an enhancement of the production of anti-BSA reagins and some suppression of the formation of anti-DNP reagins.     

Kinesiophobia and symptomatology in chronic fatigue syndrome: A psychometric study of two questionnaires

Objectives. The aims of the study were to examine the reliability of the Dutch and French versions of the Tampa scale kinesiophobia (TSK) version chronic fatigue syndrome (CFS), and to examine the reliability and validity of the Dutch and French versions of the CFS symptom list. Design. Repeated‐measures design. Methods. Native Dutch speakers (N=100) and native French (N=48) speakers fulfilling the diagnostic criteria for CFS were asked to list the five most important symptoms and to complete the TSK–CFS, the CFS symptom list, and the Short Form 36 Health Status Survey or SF‐36. A modified version of the TSK–CFS and the CFS symptom list was filled in within 24 hours of the first assessment. Results. The French and Dutch version of the TSK–CFS and CFS symptom lists displayed good reliability (ICC≥.83). The CFS symptom list was internally consistent (Cronbach's α≥.93) and concurrently valid with the SF‐36. For the native Dutch and French speakers, respectively, 82 and 78% of the self‐reported symptoms matched the content of CFS symptom list. Conclusions. The results are in support of the psychometric properties of the French and Dutch versions of both the TSK–CFS and the CFS symptom list for assessing kinesiophobia and symptom severity, respectively.