Evaluation of the proficiency of trained non-laboratory health staffs and laboratory technicians using a rapid and simple HIV antibody test

In Cambodia, nearly half of pregnant women attend antenatal care (ANC), which is an entry point of services for prevention of mother-to-child transmission of HIV (PMTCT). However, most of ANC services are provided in health centres or fields, where laboratory services by technicians are not available. In this study, those voluntary confidential counselling and testing (VCCT) counsellors involved in PMTCT were trained by experienced laboratory technicians in our centre on HIV testing using Determine (Abbot Laboratories) HIV1/2 test kits through a half-day training course, which consisted of use of a pipette, how to process whole blood samples, and how to read test result. The trained counsellors were midwives working for ANC and delivery ward in our centre without any experience on laboratory works. The objective of this study was to assess the feasibility of the training by evaluating the proficiency of the trained non-laboratory staffs. The trained counsellors withdrew blood sample after pre-test counselling following ANC, and performed the rapid test. Laboratory technicians routinely did the same test and returned reports of the test results to counsellors. Reports by the counsellors and the laboratory technicians were compared, and discordant reports in two groups were re-tested with the same rapid test kit using the same blood sample. Cause of discordance was detected in discussion with both groups. Of 563 blood samples tested by six trained VCCT counsellors and three laboratory technicians, 11 samples (2.0%) were reported positive in each group, however four discordant reports (0.7%) between the groups were observed, in which two positive reports and two negative reports by the counsellors were negative and positive by the laboratory technicians, respectively. Further investigation confirmed that all the reports by the counsellors were correct, and that human error in writing reports in the laboratory was a cause of these discordant reports. These findings lead us the conclusion that the half-day training using the rapid and simple test was feasible for non-laboratory staffs to attain enough proficiency to implement VCCT services for PMTCT in resource-limited settings, and that human error was more likely to occur in laboratory before giving reports to counsellors.     

Immunohistochemical localization of glomerular basement membrane antigens in various renal diseases

Summary The immunofluorescent localization of glomerular basement membrane (GBM) antigens was examined in 52 specimens from normal kidneys and in various renal diseases using antisera to human GBM HGBM), IV type collagen (IV Col) and P3 antigen, a rat nephritogen. Anti-HGBM serum normally stained the GBM and the mesangium in a restrictive pattern, anti-IV Col serum stained the GBM and the mesangium in a wider pattern and anti-P3 serum stained only the GBM. In mesangial proliferative glomerulonephritis, including IgA nephropathy pathy and Henoch-Schönlein nephritis, the widened mesangial areas were stained with anti-HGBM and anti-IV Col sera. In membranous nephropathy, the punched-out lesions of thickened GBM were demonstrated with the three antisera in moderate cases and a double linear distribution with fine granulation with anti-HGBM and anti-IV Col sera were revealed in one severe case. In membranoproliferative glomerulonephritis, the expanded mesangium and thickened capillary walls were stained with anti-HGBM and anti-IV Col sera, while the outer line of glomerular capillary walls was only positive with anti-P3 serum. In crescentic glomerulonephritis, the collapsed glomerular tufts were stained normally with anti-HGBM and anti-P3 sera and weakly with anti-IV Col serum. In diabetic nephropathy, anti-HGBM serum stained the GBM in a double linear distribution without reacting with the expanded mesangium; anti-IV Col serum stained the mesangium and the GBM in a less clear double linear fashion while anti-P3 serum stained the GBM as single line. Thin membrane disease and Alport's syndrome had normal reactivity with all antisera. However, in one case of Alport's syndrome anti-HGBM and anti-P3 sera stained the GBM in a focal and segmental pattern, while normal staining with anti-IV Col serum was found. In lesions with adhesions and crescents the staining was positive for HGBM and IV Col and negative for P3; obsolescent glomeruli were stained with anti-HGBM and anti-P3 sera, and had diminished staining with anti-IV Col serum.The identification of the various structural glomerular antigens is useful in the classification of certain types of glomerular diseases. Further insight into the mechanisms underlying these conditions may be obtained in this way.     

Kinetic analysis of amyloid fibril polymerization in vitro

We investigated the polymerization kinetics of murine senile amyloid fibrils (fASSAM) in vitro. When sonicated murine senile amyloid fibrils was incubated with its constituent monomer protein, the extension of amyloid fibrils was observed in an electron microscopic analysis. Quantitative fluorometric analysis with thioflavine T (Naiki H, Higuchi K, Hosokawa M, Takeda T: Anal Biochem 177:244, 1989) revealed that (a) extension of amyloid fibrils occurred by a pseudo-first-order exponential increase in the fluorescence of thioflavine T; (b) the rate of extension was maximal around pH 7.5, and was inhibited with the increase in KCl or NaCl concentration in the reaction mixture; (c) the rate of polymerization was proportional to the product of the murine senile amyloid fibrils number concentration and the constituent monomer protein concentration; (d) the net rate of extension was the sum of the rates of polymerization and depolymerization with the equilibrium association constant K of 5 x 10(7) M-1. These results show that amyloid fibril formation can apparently be explained by a first-order kinetic model: that is, extension of amyloid fibrils proceeds by consecutive association of precursor proteins onto the ends of existing fibrils.     

Cardiovascular changes associated with decreased aerobic capacity and aging in long-distance runners

Summary Fifty-five male runners aged between 30 to 80 years were examined to determine the relative roles of various cardiovascular parameters which may account for the decrease in maximal oxygen uptake ( ) with aging. All subjects had similar body fat composition and trained for a similar mileage each week. The parameters tested were , maximal heart rate (HR _max), cardiac output (Q), and arteriovenous difference in oxygen concentration (C _a —C _ˉv) O₂ during graded, maximal treadmill running. Average body fat and training mileage were roughly 12% and 50 km·week⁻¹, respectively. The average 10-km runtime slowed significantly by 6.0%·decade⁻¹ {[10-km run-time (min)=0.323 x age (years)+24.4] (n=49,r=0.692,p<0.001)}. A strong correlation was found between age and {[ (ml·kg⁻¹·min⁻¹)=- 0.439xage+76.5] (n=55,r=-0.768, p<0.001)}. Thus, decreased by 6.9%·decade⁻¹ along with reductions ofHR _max (3.2%·decade⁻¹, p<0.001) andQ (5.8%·decade⁻¹, p<0.001), while no significant change with age was observed in estimated (C _a —C _ˉv) O₂. It was concluded that the decline of with aging in runners was mainly explained by the central factors (represented by the decline ofHR andQ in this study), rather than by the peripheral factor (represented by (C _a —C _ˉv) O₂). This study was supported, in part, by a Research Grant on Aging and Health, Ministry of Health and Welfare, Japan, and by a Research Grant for young researchers, Meiji Life Foundation of Health and Welfare, Japan.     

Short-latency trigemino-cervical ref

We describe a reflex evoked in neck muscles by stimulation of afferent fibres in the trigeminal nerve. The clearest responses were seen in averaged, unrectified, monopolar surface electromyographic (EMG) recordings from active sternocleidomastoid muscles after stimulation of the infraorbital nerve. They consisted of a bilateral positive/negative (p19, n31) wave with a mean onset latency of 12.9 ms which corresponded to a period of inhibition in the underlying motor unit activity. Responses also could be seen in splenius and trapezius, but not in arm muscles. Stimuli to other branches of the trigeminal nerve (supraorbital or mental) did not produce such clear effects. The threshold for the reflex was relatively low (2–4 times perceptual threshold) and its size scaled with the level of background EMG in an approximately linear fashion. Responses to infraorbital stimulation did not interact with other short-latency inhibitory responses in the sternocleidomastoid muscle evoked by loud acoustic clicks or stimulation of the median nerve at the wrist. We suggest that the infraorbital response is part of a head withdrawal reflex involving an oligosynaptic trigemino-cervical system similar to that described in the cat.     

Rapid B cell apoptosis induced by antigen receptor ligation does not require Fas (CD95/APO-1), the adaptor protein FADD/MORT1 or CrmA-sensitive caspases but is defective in both MRL-+/+ and MRL-lpr/lpr mice

Antigen receptor ligation-induced apoptosis is thought to play a role in self-tolerance by deleting autoreactive lymphocytes. Antigen receptor ligation-induced apoptosis of mature T cells and T cell lines requires autocrine or paracrine activation of Fas (CD95/APO-1). Whether B cell antigen receptor (BCR)-mediated apoptosis requires Fas or related molecules is unclear. Here we demonstrate that expression of either CrmA, the cowpox virus serpin, or an inhibitor of the adapter protein FADD/MORT1 blocks Fas-mediated apoptosis but has no effect on BCR ligation-induced apoptosis of the B cell line WEHI-231. In contrast, expression of Bcl-2 blocks BCR-mediated but not Fas-induced apoptosis in WEHI-231 cells. These results indicate that BCR ligation activates an apoptotic signaling pathway distinct from Fas-mediated apoptosis in WEHI-231 cells, and that BCR-mediated apoptosis of WEHI-231 cells does not require Fas or related molecules such as DR3, DR4 and DR5, as all of these death receptors require FADD/MORT1 and/or CrmA-sensitive caspases for induction of apoptosis. Moreover, extensive BCR ligation induces death of mature B cells from C57BL/6-lpr/lpr mice as efficiently as those from C57BL/6 mice, indicating that Fas is not essential for BCR-mediated apoptosis of mature B cells. In contrast, BCR ligation-induced apoptosis is reduced in mature B cells from MRL mice and this is not affected by the lpr mutation. Since MRL-lpr/lpr mice but not C57BL/6-lpr/lpr mice develop severe autoimmune disease, defects in BCR-mediated apoptosis in the MRL background, together with lpr mutation, may contribute to the development of severe autoimmune disease in MRL-lpr/lpr mice by allowing survival of self-reactive B cells.     

Dandy-Walker variant in Coffin-Siris syndrome

We describe a five-month-old male infant with Coffin-Siris syndrome, the so-called Dandy-Walker variant (hypoplasia of the cerebellar vermis with cystic dilatation of the fourth ventricle, but without enlargement of the posterior fossa), and partial agenesis of the corpus callosum. Dandy-Walker malformation and mega cisterna magna, but not Dandy-Walker variant, have been reported in Coffin-Siris syndrome. The presence of Dandy-Walker variant in the infant we described confirms that the full continuum of the Dandy-Walker complex can occur in Coffin-Siris syndrome. The yet unidentified gene(s) for the syndrome may be related to the development of the hindbrain.     

Cytofluorometric study on lectin binding of isolated guinea pig keratinocytes

Lectin binding was cytofluorometrically measured on fractionated keratinocytes of guinea pig. Free keratinocytes were obtained by treatment of EDTA and trypsin. After the treatment, they were separated into 3 fractions by centrifugation on a continuous colloidal silica (Percoll) density gradient. Cells in each fraction were stained by biotinyl lectins and avidin-FITC, and fluorescence intensity was measured by cytofluorometry. Results obtained indicate that little cell surface glycoconjugate is lost during the preparation of free keratinocytes.     

Analysis of intestinal HLA-DR bound peptides and dysregulated immune responses to enteric flora in the pathogenesis of inflammatory bowel disease

Isolation of antigenic peptides from the MHC-groove has contributed to the understanding of T cell responses. However, these MHC-associated peptides have been isolated from various murine and human cell lines. The specific antigen responsible for the pathogenesis of inflammatory bowel disease is unknown. We examined antigenic peptides bound to the class II major histocompatibility complex (MHC) groove in human intestine by ion-trap tandem mass spectrometry equipped with online reverse-phase high performance liquid chromatography. We detected 55 parent proteins from 4 controls, 9 patients with ulcerative colitis, and 9 patients with Crohn's disease. The calculated molecular masses (m/z) of these peptides ranged from 874.4 to 2727.4, representing 10-26 amino acid residues. Fifty-one of these 55 parent proteins were exogenous proteins. Escherichia coli-, Saccharomyces cerevisiae-, and Caenorhabditis elegans-derived peptides were found frequently in patients with inflammatory bowel disease. The present results suggest that in vivo antigen processing by antigen-presenting cells and T lymphocytes in human intestine participate with exogenous antigen presentation. Increased immune responses against E. coli, S. cerevisiae and C. elegans found in patients with inflammatory bowel may participate as dysregulated immune responses to enteric flora in the pathogenesis of inflammatory bowel disease.