Measurement of HIV-1 p24 antigen by signal-amplification-boosted ELISA of heat-denatured plasma is a simple and inexpensive alternative to tests for viral RNA

Assessment of HIV-1 RNA concentration is widely used for monitoring antiretroviral therapies. Tests are, however, expensive and require technically advanced equipment and highly trained personnel. Increasing availability of antiretroviral treatment in resource-poor settings calls for simple and inexpensive virus tests. HIV-1 p24 antigen tests were frequently used before the availability of nucleic acid tests (NAT). Two simple modifications, heat-mediated destruction of test-interfering antibodies and increased sensitivity achieved by signal amplification, have shaped the p24 antigen test into a tool that rivals NAT. This improved p24 antigen test, for which all reagents are available from Perkin Elmer Life Sciences, was evaluated in clinical studies in comparison with the most sensitive PCR methods available at a given time. In a prospective study over 4 years, HIV-1 infection among 859 samples from 307 infants born to HIV-positive mothers in Switzerland was detected as sensitively by p24 antigen assay as by PCR for viral DNA or RNA: 100% sensitivity of all methods after 10 days of age; 99.2% diagnostic specificity of p24 after neutralization (RNA, 98.6%). A study conducted in Dar es Salaam (Tanzania) found 123 of 125 samples from 76 PCR-positive infants positive for p24 antigen (sensitivity = 98.7%). In 169 infected Swiss adults with a median CD4+ T-cell count of 140 cells/microliter followed for a median of 2.7 years, p24 at baseline correlated as well as or better than HIV-1 RNA with the ensuing CD4+ T-lymphocyte decline and was independently predictive of progression to clinical AIDS (P = 0.043) and survival (P = 0.032). RNA predicted AIDS (P < 0.005), but not survival (P = 0.19). Another study of first-visit samples from 496 mostly black IVDU in the U.S. with a median CD4+ count of 518 cells/microliter showed equally strong prediction of progression to clinical AIDS for p24 antigen, HIV-1 RNA, and CD4+ T-lymphocyte concentrations at baseline. Treatment-associated changes in p24 and RNA levels in adults and children correlated well in three Swiss studies. The half-life of p24 antigen in the first phase of effective treatment was 1.6 +/- 0.4 days (RNA, 1.7 +/- 0.8). A second, slower decay phase had a half-life of 42 +/- 16 days. One study suggested that a strategy involving a somewhat more frequent testing for p24 antigen permitted detection of viral failures significantly earlier than tests for HIV-1 RNA conducted at 3-month intervals, while at the same time significantly saving on costs. Experience from three studies indicates that the p24 antigen test recognizes viruses of subtypes A-G and O, as well as some recombinant isolates, but leaves open the possibility that some non-B p24 antigens may be suboptimally detected. This improved p24 antigen test provides diagnosis of pediatric HIV infection, prediction of prognosis and treatment monitoring in quality comparable to tests for HIV-1 RNA, but at much lower costs. There is no problem with sample instability and no need for cumbersome nucleic acid extraction. The test is validated for subtype B, but requires further studies for non-B subtypes.     

Up-regulation of cathepsin X in Helicobacter pylori gastritis and gastric cancer

Recently, we identified increased cathepsin X expression in H. pylori-infected gastric mucosa. Here, we describe further up-regulation in gastric cancer and report on the role of inflammatory cytokines required for cathepsin X up-regulation in H. pylori-infected gastric mucosa, as well as on consequences for cellular invasion. Biopsy specimens were taken from the antrum, corpus and cardia of H. pylori-infected and non-infected patients. Gastric cancer samples were obtained from patients undergoing gastric surgery. Cathepsin X was detected in gastric mucosa by quantitative real-time RT-PCR, western blotting and immunohistochemistry. Induction of cathepsin X expression in epithelial and inflammatory cells caused by H. pylori infection was tested in in vitro contact and non-contact co-cultures of AGS cells and monocytic cells. Patients with H. pylori gastritis showed significantly higher cathepsin X mRNA (2.5-fold) and protein (1.6-fold) expression than H. pylori-negative patients. Cathepsin X was also up-regulated in gastric cancer (3-12-fold) compared to non-neoplastic mucosa. Cathepsin X was predominantly expressed by macrophages in the mucosal stroma and in glands of the antral mucosa. In addition, tumour cells stained for cathepsin X in 26 (68%) patients with gastric carcinoma. In general, staining was significantly more common (20 vs. 6 patients) and more intense (3.55 vs. 0.83) in intestinal type gastric cancer than in the diffuse type. In vitro cell culture experiments revealed that intercellular signalling between pathogenicity island (PAI)-positive H. pylori-infected epithelial cells and macrophages via soluble factors in the culture medium seems to be responsible for increased expression of cathepsin X in monocytes. Using antisense oligonucleotides, cathepsin X up-regulation was directly associated with higher invasiveness in vitro. Although no correlation of cathepsin X expression and TNM stage was found, our study demonstrates that cathepsin X plays a role not only in the chronic inflammation of gastric mucosa but also in the tumourigenesis of gastric cancer.     

Health education for pregnant women: the role of background characteristics

This was a pilot study with the purpose of determining the effect of health education in enhancing the self-care agency of pregnant women and to define the role of their background characteristics in the success of this education. Subjects of this study were 30 pregnant women who visited a university hospital for check. The success of the given education was measured by pre- and post-tests that were applied before and after health education using "self-care agency scale". Data were evaluated according to t-test and variance analysis. After health education, the self-agency scores of the pregnant women increased significantly (P < 0.05). It was defined that pregnant women with the least self-care agency scores before health education, displayed the best progress after the education.     

Neuroinvasion of prions: insights from mouse models

The prion was defined by Stanley B. Prusiner as the infectious agent that causes transmissible spongiform encephalopathies. A pathological protein accumulating in the brain of scrapie-infected hamsters was isolated in 1982 and termed prion protein (PrPSc). Its cognate gene Prnp was identified more than a decade ago by Charles Weissmann, and shown to encode the host protein PrP(C). Since the latter discovery, transgenic mice have contributed many important insights into the field of prion biology, including the understanding of the molecular basis of the species barrier for prions. By disrupting the Prnp gene, it was shown that an organism that lacks PrP(C) is resistant to infection by prions. Introduction of mutant PrP genes into PrP-deficient mice was used to investigate the structure-activity relationship of the PrP gene with regard to scrapie susceptibility. Ectopic expression of PrP in PrP knockout mice proved a useful tool for the identification of host cells competent for prion replication. Finally, the availability of PrP knockout mice and transgenic mice overexpressing PrP allows selective reconstitution experiments aimed at expressing PrP in neurografts or in specific populations of haemato- and lymphopoietic cells. The latter studies have allowed us to clarify some of the mechanisms of prion spread and disease pathogenesis.     

Structural evidence for counter-current flow in proximal tubules versus pertitubular capillaries in the rat kidney. Evaluation of the counter-current mechanism between the proximal convoluted tubules and the peritubular capillaries in the rat nephron

BACKGROUND: In spite of the very high exchange of water and solutes between the proximal tubules and the peritubular capillaries, very little is known about flow directions in these two interrelated structures. We therefore developed a morphological technique suitable for the quantitative evaluation of a counter-current system between the proximal convoluted tubules and the peritubular capillaries in rat renal cortex. METHODS: In male pentothal-anesthetized Wistar rats (body weight 200-250 g), India ink was injected into the aorta above the renal arteries, followed by instant freezing of the right kidney in isopentane at -165 degrees C, and subsequent freeze-substitution in alcohol. In microscopic slides from kidneys in which only 20-55% of the cortical peritubular capillary loops was filled with ink--representing the arterial end of the capillaries--and in which the proximal tubular segmentation could be identified in PAS-stained sections, the segments of the convoluted proximal tubules were quantitatively compared with regard to the presence of ink-stained and unstained peritubular capillaries in nephrons from the whole renal cortex. RESULTS: In the microscopic specimens of the five animals used both the loops from the first segment (P1) of the proximal convoluted tubule and those of the second segment (P2) were systematically packed closely together, the transitional segment (P1-2) being interposed between the groups. Around the loops of P1, 8%+/-2% of the capillaries was stained with India ink. In contrast, surrounding the P2 loops 67%+/-5% of the capillaries contained ink, significantly exceeding that for P1 (p<0.01). CONCLUSION: Throughout the rat renal cortex, the most proximal fraction of the peritubular capillaries surrounds the second segments of the proximal convoluted tubules, while the first tubular segments are surrounded by the more distal fraction of the peritubular capillaries. Consequently, the flows in the peritubular capillaries and in the proximal convoluted tubules in the rat renal cortex are systematically arranged as a counter-current system. This feature was previously identified only in superficial nephrons.     

Endothelin and endothelin receptors A and B in the human testis

 Human testicular capillaries interconnect Leydig cells and seminiferous tubules. Microcirculation and blood flow are therefore essential for the maintenance of spermatogenesis. The expression and the localisation of ET (endothelin) and its receptors in testicular tissue, in seminiferous tubules and in human testicular capillaries were studied. ET-1 mRNA was detected in whole testicular tissue and in seminiferous tubules whereas isolated testicular capillaries were negative. Big ET-1 (Big endothelin 1) and ET peptides were localised in Leydig and Sertoli cells whereas interstitial and intramural capillaries (within the lamina propria) remained unstained. ET was also found in mature spermatids. ET-A (endothelin receptor A) mRNA was detected in seminiferous tubules and whole testicular tissue whereas testicular blood vessels were negative. ET-A immunostaining was displayed in Leydig and Sertoli cells and in spermatids. ET-B (endothelin receptor B) mRNA was detected in whole testicular tissue, seminiferous tubules and in testicular capillaries. ET-B peptide was prominent in Leydig cells, peritubular cells, endothelial cells and pericytes of interstitial and intramural capillaries as well as in vascular endothelial and smooth muscle cells. From these results we conclude that ET produced in Leydig and Sertoli cells can act in a paracrine manner via ET-B on the human testicular microvasculature and the peritubular cells. The presence of both ET-A and ET-B in Leydig cells and of ET-A in Sertoli cells leads to the assumption that ET could influence these cells as an autocrine factor. Accepted: 9 October 1998     

Biochemical identification of Aeromonas genospecies isolated from humans

One hundred phenotypic characteristics were determined for 138 clinical and environmental Aeromonas strains. Cluster analysis revealed three major phenons equivalent to the A. hydrophila, A. caviae, and A. sobria groups, each of which contained more than one genospecies and more than one named species. An excellent correlation was found between phenotypic identification and classification based on DNA relatedness. DNA hybridization groups within each of the phenotypic groups were also separable by using a few biochemical characteristics. Key tests were production of acid from or growth on D-sorbitol (which separated DNA hybridization group 3 from groups 1 and 2 within the A. hydrophila phenogroup), growth on citrate (which essentially separated DNA hybridization group 4 from groups 5A and 5B within the A. caviae phenogroup), and growth on DL-lactate (which separated DNA hybridization group 1 from groups 2 and 3 within the A. hydrophila phenogroup as well as group 5A from groups 4 and 5B within the A. caviae phenogroup). All except one strain in the A. sobria phenogroup belonged to DNA hybridization group 8. DNA hybridization groups were not equally distributed among clinical and environmental isolates, suggesting that strains of certain DNA hybridization groups might be less virulent than others.     

Massive haemorrhagic necrosis of the liver after liver transplantation.

Six of the first 85 patients who received the first 100 liver transplantations carried out in Birmingham developed a syndrome of fulminant liver failure with distinctive clinical and pathological features. The typical clinical presentation was of an uneventual initial postoperative period, followed by a sudden deterioration in graft function, progressing rapidly to graft failure. All six patients died. The characteristic pathological changes were those of massive haemorrhage and hepatocyte necrosis with only mild inflammation and without occlusive lesions in large arteries or veins. These distinctive features differed from other recognised patterns of graft damage and seemed to comprise a specific post-transplant syndrome. The pathogenesis was not clear and in the absence of any definite aetiology it is suggested that the term "massive haemorrhagic necrosis" be used to describe these cases. Additional findings seen in five of the six cases were venoocclusive lesions (n = 4) and a combination of ductopenia and foam cell arteriopathy (n = 2). The presence of these associated lesions suggests that there may be an overlap with other types of graft damage.     

Visual discrimination learning in dwarf goats and associated changes in heart rate and heart rate variability

We studied visual discrimination learning in a group of Nigerian dwarf goats using a computer-based learning device which was integrated in the animals' home pen. We conducted three consecutive learning tasks (T1, T2 and T3), each of which lasted for 13 days. In each task, a different set of four visual stimuli was presented on a computer screen in a four-choice design. Predefined sequences of stimulus combinations were presented in a pseudorandom order. Animals were rewarded with drinking water when they chose the positive stimulus by pressing a button next to it. Noninvasive measurements of goats' heartbeat intervals were carried out on the first and the last 2 days of each learning task. We analysed heart rate (HR) and heart rate variability (HRV) of resting animals to study sustained physiological effects related to general learning challenge rather than acute excitement during an actual learning session. The number of trials to reach the learning criterion was 1000 in T1, when visual stimuli were presented to the goats for the first time, but decreased to 210 in T2 and 240 in T3, respectively. A stable plateau of correct choices between 70% and 80% was reached on Day 10 in T1, on Day 8 in T2 and on Day 6 in T3. We found a significant influence of the task and of the interaction between task and day on learning success. Whereas HR increased throughout T1, this relationship was inverted in T2 and T3, indicating different effects on the HR depending on how familiar goats were with the learning task. We found a significant influence of the task and the interaction between task and time within the task on HRV parameters, indicating changes of vagal activity at the heart. The results suggest that changes in HR related to learning were predominantly caused by a withdrawal of vagal activity at the heart. With regard to nonlinear processes in heartbeat regulation, increased deterministic shares of HRV indicated that the animals did not really relax until the end of T3. Comparing changes of HR and HRV in T3 and in a subsequent postexperiment (PE), we assume a positive effect of such cognitive challenges once the task had been learned by the animals.     

Prediction of the average skin temperature in warm and hot environments

The prediction of the mean skin temperature used for the Required Sweat Rate index was criticised for not being valid in conditions with high radiation and high humidity. Based on a large database provided by 9 institutes, 1999 data points obtained using steady-state conditions, from 1399 experiments and involving 377 male subjects, were used for the development of a new prediction model. The observed mean skin temperatures ranged from 30.7 °C to 38.6 °C. Experimental conditions included air temperatures (T _a) between 20 and 55 °C, mean radiant temperatures (T _r) up to 145 °C, partial vapour pressures (P _a) from 0.2 to 5.3 kPa, air velocities (v _a) between 0.1 and 2 m/s, and metabolic rates (M) from 102 to 620 W. Rectal temperature (T _re) was included in the models to increase the accuracy of prediction. Separate models were derived for nude (clothing insulation, I_cl, ≤0.2 clo, where 1 clo=0.155 m² · °C · W⁻¹, which is equivalent to the thermal insulation of clothing necessary to maintain a resting subject in comfort in a normally ventilated room, air movement=10 cm/s, at a temperature of 21 °C and a humidity of less than 50%) and clothed (0.6 ≤ I_cl ≤ 1.0 clo) subjects using a multiple linear regression technique with re-sampling (non-parametric bootstrap). The following expressions were obtained for nude and clothed subjects, respectively: T _sk=7.19 + 0.064T _a + 0.061T _r + 0.198P _a− 0.348v _a + 0.616T _re and T _sk=12.17 + 0.020T _a + 0.044T _r + 0.194P _a − 0.253v _a + 0.0029M + 0.513T _re. For the nude and clothed subjects, 83.3% and 81.8%, respectively, of the predicted skin temperatures were within the range of ±1 °C of the observed skin temperatures. It is concluded that the proposed models for the prediction of the mean skin temperature are valid for a wide range of warm and hot ambient conditions in steady-state conditions, including those of high radiation and high humidity. Accepted: 7 February 2000