Cesium uptake studies on human erythrocytes

We compared the performance of second and third generation ELISA assays to detect antibodies to HIV-1 virus with conventional Western blotting (WB) and radioimmune Western blotting (RIWB). Both sera from commercial seroconversion panels and serial dilutions of a serum for HIV-1 antibodies were tested with Murex HIV Recombinant, Vidas bioMérieux HIV 1/2 (2nd generation ELISA) Murex HIV 1-2 (3rd generation ELISA), as well as with WB and RIWB. In seroconversion panels all ELISA assays were positive for the same serum with the exception of the first serum of Panel D which was negative with both sample Murex assays and borderline with Vidas assay. This serum was negative with WB but evidenced antibodies to gp160 p66, p51, p24 HIV-1 proteins when assayed by RIWB. In only two cases did WB reveal antibodies to HIV-1 proteins before ELISA assays (Panel A and E); not only did RIWB show the same sensitivity as WB in the two last panels, but it also detected antibodies to HIV-1 proteins earlier than WB, ranging from a few days (Panel C) to approximately 12 weeks (Panel D). The results obtained by testing the dilutions of the serum positive for anti HIV-1 antibodies showed the following degrees of sensitivity: Murex HIV 1-2 (the most sensitive), Murex HIV Recombinant and Vidas bioMérieux HIV 1/2. Although WB was more sensitive than the ELISA assays and picked out antibodies to gp160, gp120 and p24 HIV proteins at 1/4000 serum dilution, the most sensitive test was RIWB which at 1/20,000 serum dilution enabled detection of antibodies to gp160, p66 and p24 HIV proteins.     

Absence of pericentromeric heterochromatin (9qh-) in a patient with bilateral retinoblastoma

The diagnosis of HIV infection is based on screening of HIV antibodies and confirmed by a more specific supplementary test. The most common confirmation test is Western blot, which is expensive, time consuming and subject to technical skill. The present study was carried out to evaluate whether the anti-HIV-1 antibody titer is valid as a supplementary test for diagnosis of HIV-1 infection. Anti-HIV-1 antibody titers of 2,414 anti-HIV-1 positive sera determined by the particle agglutination (PA) method were analysed in comparison with the Western blot analysis. The Western blot negative result was found in 11 of 2,414 (0.46%) anti-HIV-1 positive sera, these sera also gave negative anti-HIV by ELISA. The PA titers of these sera were found in the range of 16 to 64. Seventeen samples (0.70%) with anti-HIV-1 in the titer range of 16 to 256 showed indeterminate Western blot analysis. The rest, 2,386 of these 2,414 sera (98.84%), were shown to be positive by Western blot. However, all of the 2,356 sera with antibody titers > or = 512 (97.6%) demonstrated positive Western blot results. Five cases among the 17 (29.4%) indeterminate sera were examples of early seroconversion of HIV infection, which were confirmed in follow up specimens. The results suggest that only the samples with antibody titers < 512 are required to be confirmed for HIV infection by Western blot. It is possible that early seroconversion may be inferred from anti-HIV titers. Therefore, in order to reduce time and cost, the PA anti-HIV titer can be used as an alternative supplementary test for diagnosis of HIV-1 infection in most positive screened anti-HIV samples. Western blot is needed for testing in only a few cases.     

Characterization of CD4-gp120 activation intermediates during human immunodeficiency virus type 1 syncytium formation

The mechanism by which cells expressing HIV envelope glycoproteins progress from binding CD4+ cells to syncytia formation is not entirely understood. The purpose of these investigations was to use physical and biochemical tools (temperature shifts, soluble CD4, protease inhibitors, and a battery of anti-CD4 monoclonal antibodies) to isolate discrete steps during syncytia formation. Previously (Fu et al., J Virol 1993;67:3818), we found that preincubation of cells stably expressing HIV-1 gp 160 (TF228.1.16) with CD4+ SupT1 cells at 16 degrees C, a temperature that is nonpermissive for syncytia formation, resulted in an increased rate of syncytia formation when the cocultures were shifted to the syncytia-permissive temperature of 37 degrees C. We have since found that syncytia formation is further enhanced by shifting the cocultures from 16 to 4 degrees C prior to incubation at 37 degrees C. Together, these data suggest that two discrete states, which we term the first and second activation intermediates (FAI and SAI), are involved in syncytia formation. We have found that acquisition of the FAI (by preincubation at 16 degree C) is sensitive to some serine protease inhibitors (PI), soluble CD4 (sCD4), shedding of gp120, and anti-CD4 monoclonal antibodies (MAb) directed toward the CDR-1/2 and CDR-3 regions of domain 1 on CD4. Expression of the FAI (formation of syncytia by shifting from 16 to 37 degrees C) remains sensitive to sCD4, shedding of gp120, and MAb directed toward CDR-1/2 but is less sensitive to MAb that bind CDR-3 and is insensitive to PI. Similarly, acquisition of the SAI (shifting cocultures from 16 to 4 degrees C), is sensitive to sCD4, shedding of gp120, and MAb directed toward CDR-1/2. In contrast, expression of the SAI (shifting cocultures from 16 to 4 to 37 degrees C) is sensitive only to MAb directed toward CDR-1/2 and cannot be blocked by sCD4, shedding of gp120, or PI. These data allow us to propose that syncytia formation, mediated by HIV-1 envelope glycoproteins, proceeds by a multistep cascade.     

Heat tolerance of vancomycin resistant Enterococcus faecium

The heat tolerance of 27 Enterococcus faecium isolates in water was studied. Stationary phase cultures including vancomycin resistant and sensitive clinical and food isolates were exposed to heat at 60 degrees, 65 degrees, 71 degrees, and 80 degrees C for one, three, 10, and 30 minutes and the log10 reductions in bacterial counts were determined. Exposure at 71 degrees and 80 degrees C resulted in > 6 log10 reduction in viable counts for all isolates. Seven (24%) isolates survived (< 5 log10 reduction) heat at 65 degrees C for 10 minutes. The E faecium isolates were more resistant to heat than the two E faecalis reference strains. No differences in heat tolerance were observed between vancomycin sensitive and resistant strains or between isolates of human or food origin.     

Expression of aquaporins in the rat ocular tissue

The current HIV pandemic is complicated by the spread of distinct types and subtypes of HIV. The currently used conventional diagnostic tests have shown limitations in the detection of antibodies against all HIV-1 subtypes, as demonstrated by recent identification of HIV-1 subtype O. To evaluate quantitatively the diagnostic potential of a double ELISA strategy for the detection and partial differentiation of HIV-1, HIV-1 subtype O and HIV-2 infections blood samples were examined at five different test centers: Blantyre, Malawi; Abidjan and Daloa, Ivory Coast; Yaoundé, Cameroon; Munich, Germany. All tests results, including ELISA extinction values and Western blot profiles, were forwarded to Munich for final interpretation. An indirect anti-HIV-1/2 ELISA and a competitive anti-HIV-1 ELISA were used in combination for the initial screening of blood specimens. All anti-HIV positive and anti-HIV negative samples were subjected to immunoblot analysis. Independent of the diversity of the extinction profiles, and of the test manufacturer, the quantitative evaluation of the ELISA extinction values could define two extinction areas with a 100% predictive value for HIV-1 seropositivity and HIV seronegativity; extinction values > 2 by the indirect ELISA and < 0.2 by the competitive ELISA for an anti-HIV-1 subtype A to I positive result; extinction values < 0.2 by the indirect ELISA and > 1.0 by the competitive ELISA for an anti-HIV negative result. Additionally, the quantitative evaluation of the extinction profile provides partial information on the HIV-1 subtype as far as the distinction in group M and group O is concerned. In conclusion, the quantitative evaluation of this double ELISA strategy can reduce the number of blood specimens that require additional confirmatory testing in developing countries and can be superior to the immunoblot method during early seroconversion.     

Human immunodeficiency virus (HIV) antigen testing to detect HIV infection in female sex workers in Singapore

Human immunodeficiency virus (HIV) infection is characterised by seroconversion after a ?window? period of 2 to 3 months. After this period antibodies are usually detectable by screening tests (enzyme immunoassay or particle agglutination) confirmed by Western blot analysis. We studied 1000 newly enrolled female sex workers who had not been previously tested for HIV to assess the usefulness of HIV antigen testing to improve the efficacy of HIV infection detection. Blood was taken at enrollment for HIV antigen and HIV antibody testing. The Abbott HIVAG-1 test was used to detect antigen; antibody detection was by the Abbott recombinant HIV-1/HIV-2 3rd generation enzyme immunoassay (EIA) test, the Fujirebio Serodia-HIV particle agglutination (PA) test for screening, and the Diagnostic Biotechnology HIV Blot 2.2 Western blot (WB) test for antibody confirmation. Of the 1000 samples, 26 were positive for HIV antibody testing (26/26 for EIA, 25/25 for PA, 26/26 for WB), giving a prevalence rate of 2.6%, Of these 26 seropositive samples 1 was positive on HIV antigen testing. There were no samples which were antigen-positive and antibody-negative. HIV antigen testing does not add to increased efficacy of HIV detection among female sex workers in Singapore.     

Using focus groups to identify psychosocial issues of urban black individuals with diabetes

BACKGROUND: Human serum represents an important barrier to the entry of most mucosal organisms into tissues and to the systemic circulation. If at all present, Helicobacter pylori within gastric tissue is rare, and bacteremia for this organism has been described only once. METHODS: To assess the susceptibility of H. pylori to the bactericidal activity present in normal human serum (NHS), we examined 13 H. pylori isolates. To assess the contributions of the classical and alternative complement pathways to killing, we added either C2-deficient or factor B-deficient serum, respectively, to heat-inactivated NHS. Also we assessed the ability of the strains to bind 125I-C3. RESULTS: After incubation for 60 minutes at 37 degrees C, all 13 H. pylori strains were killed by NHS; heating to 56 degrees C for 30 minutes ablated killing, indicating complement dependence for this phenomenon. In the absence of an antibody source, there was no killing when either an alternative or classical complement pathway source was used. Adding B-deficient serum to heat-inactivated normal human serum did not restore killing, but adding C2-deficient serum permitted partial killing. All of the 13 strains bound 125I-C3. Although the kinetics varied from strain to strain, C3 bound was significantly correlated (r = 0.61, p = 0.03) with serum susceptibility. CONCLUSIONS: H. pylori are susceptible to complement, alternative pathway activation appears critical, and C3 binding is a major locus of variability.     

Bronchiolitis obliterans organizing pneumonia and chronic graft-versus-host disease in a child after allogeneic bone marrow transplantation

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) subtype O infections are not reliably detected by commonly used anti-HIV-1/2 screening assays. Therefore, anti-HIV-1/2 assays have been modified to increase their sensitivity in detecting antibodies to HIV-1 subtype O. STUDY DESIGN AND METHODS: Two new anti-HIV-1/2 enzyme-linked immunosorbent assays (ELISAs) (Abbott Plus and Ortho Enhanced) were compared with a currently used anti-HIV-1/2 ELISA (Abbott Recombinant) in various serum panels: 91 Western blot-confirmed anti-HIV-1-positive samples, 20 samples from Western blot-confirmed HIV-1-infected patients in log3 serial dilutions, and 1463 samples from consecutive, volunteer, nonremunerated blood donors. RESULTS: Among 91 anti-HIV-1 Western blot-positive samples, 2 (2.2%) were missed by the Abbott Recombinant ELISA, but all 91 were detected by the Abbott Plus and Ortho Enhanced ELISAs. In contrast, two discrepant samples were found to react in viral lysate-based assays. In serial dilutions, Ortho Enhanced ELISA was significantly less sensitive than the Abbott Recombinant and Abbott Plus ELISAs, with the latter two being of comparable sensitivity. The specificities of Abbott Recombinant, Abbott Plus, and Ortho Enhanced ELISAs in 1463 blood donors were 100, 99.93, and 99.86 percent, respectively. Routine testing of 29,102 donations with the enhanced Abbott Plus ELISA revealed a specificity of 99.93 percent. CONCLUSION: Two Western blot-confirmed anti-HIV-1-positive samples were missed by the Abbott Recombinant ELISA but detected by the Abbott Plus and Ortho Enhanced ELISAs. The analytic sensitivity of the Ortho Enhanced ELISA was inferior to that of both Abbott ELISAs. The specificities of the Abbott Recombinant, Abbott Plus, and Ortho Enhanced ELISAs were comparable.     

Chronic fatigue syndrome: a novel disorder with cutaneous manifestations

Effects of heating and 60cobalt (60Co) irradiation to hepatitis C virus (HCV) RNA in sera were studied. HCV-RNA can not be detected by PCR in 10 positive serum samples after being heated at 56 degrees C for 10 hours. Whereas, HCV-RNA can still be detected in positive sera after being lyophilized and then heated at 60 degrees C for 20 hours. It suggested heating at 56 degrees C for 10 hours can inactivate HCV in sera and the sera should be in liquid status when thermal inactivation is taken. Lyophilized sera with thermal inactivation for HCV should be inactivated first and then lyophilized. But the safety of sera with heat-treatment should be further investigated. Inactivation of HCV in sera with 60Co irradiation needed extra large dose of it, and protein components in sera would obviously be altered. Therefore, inactivation of HCV in sera with 60Co irradiation is impractical.     

Detection of anti-HIV-1 IgG antibodies in whole saliva by GACELISA and Western blot assays

The present study, based on 158 HIV seropositives and 167 HIV seronegatives, demonstrates that saliva collected with the Omni-SAL device and tested with GACELISA (an IgG antibody capture ELISA) is an effective non-invasive alternative to serum for anti-HIV IgG antibody screening. The study also shows that a conventional serum Western blot kit can be used, with slight modifications, for confirmatory testing of saliva specimens. Collecting saliva with the Omni-SAL device had a very good acceptance rate among Tanzanian subjects, and although this diagnostic method is not yet known by the general public, 65% of the study participants preferred to give saliva instead of blood for HIV testing.     

Evaluation of a new HTLV-I/II polymerase chain reaction

AIM: Evaluation of a qualitative HTLV-I/II DNA polymerase chain reaction (PCR) test for the detection of HTLV-I/II DNA (Roche Diagnostic Systems, Branchburg, N.J., USA) in various panels. METHODS: The panels consisted of fresh EDTA blood samples from blood donors who were anti-HTLV-I/II ELISA repeatably reactive: 53 were Western blot (WB) positive, 228 were WB indeterminate and 15 were WB negative. Elevent ELISA-negative blood donors were used as negative controls. Furthermore, specimens from 1 HTLV-II-infected intravenous drug user and from 1 HTLV-II-infected blood donor were included in the panel. Peripheral blood lymphocytes were prepared by red blood cell lysis with the Roche washing solution and stored at < -23 degrees C until processing. Amplification products were analyzed with the HTLV-I/II detection kit. RESULTS: All 53 anti-HTLV-I/II ELISA- and WB-positive samples and both HTLV-II-positive samples tested positively by PCR. All 228 anti-HTLV-I/II ELISA-positive and WB-indeterminate, all 15 ELISA-positive and WB-negative and all II ELISA-negative control samples tested negative by PCR. CONCLUSION: The Roche Amplicor HTLV-I/II test is a simple test, suitable for the confirmation of HTLV-I and-II infection in individuals with indeterminate or positive WB patterns.     

Diagnosis of paratuberculosis in dairy cattle, using enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in milk

An ELISA containing lipoarabinomannan (LAM) antigen was used to detect antibodies in milk and serum for diagnosis of Mycobacterium paratuberculosis infection in dairy cattle. In experiment 1, milk and serum samples were obtained from 25 cows, and subjected to LAM ELISA testing immediately, and after 1 year of storage at -70 C. Milk samples, with and without a commonly used chemical preservative, were tested. There was no significant difference in LAM ELISA results between fresh and frozen samples or between preserved and unpreserved milk samples. In experiment 2, milk samples were collected daily from 30 cows over a 14-day period. The day-to-day coefficient of variation was 0.19 for milk LAM ELISA and was 0.15 for serum LAM ELISA, with no statistically significant time effect detected. In experiment 3, single milk, serum, and fecal samples were obtained from 764 cows. The fecal samples were cultured for M paratuberculosis to identify infected cows, and the serum and milk samples were subjected to LAM ELISA testing. Results were compared, using the area under the receiver operating characteristic curves. The milk LAM ELISA had specificity (+/- 95% confidence limits) of 87 +/- 8.1% when the cutoff was set at 50% sensitivity, and specificity of 83 +/- 9.1% when sensitivity was set at 60%. The area under the receiver operating characteristic curve was 0.85 +/- 0.03 for the milk ELISA and 0.75 +/- 0.02 for the serum ELISA. In this population of cattle, the milk LAM ELISA had comparable accuracy to serum LAM ELISA, although the milk LAM ELISA was slightly less reproducible (higher coefficient of variation).     

The effect of dry heat on mite, cat, and dog allergens

BACKGROUND: Various techniques have been tried in an attempt to reduce allergen levels in homes. This study investigated the effect of dry heat on mite, cat, and dog allergens. METHODS: Samples (50 mg) of Dermatophagoides pteronyssinus and D. farinae cultures, and of house dust rich in the major cat and dog allergens Fel d 1 and Can f 1 were heated for 5, 10, 15, 30, and 60 min at 60 degrees, 80 degrees, 100 degrees, 120 degrees, and 140 degrees C. Control samples remained at room temperature. Extracts were assayed with the appropriate two-site mono- or mono/polyclonal sandwich ELISA. RESULTS: For Der p 1, the breakdown was proportional to temperature and heating time; after 30 min at 120 degrees C, allergen levels were reduced to < 1% of control. Der p 2 was more heat stable, requiring 140 degrees C for 30-60 min to achieve > 99% reduction. D. farinae groups 1 and 2 allergens showed results similar to those obtained with D. pteronyssinus. In contrast, Can f 1 and Fel d 1 were considerably more thermostable, with 50% and 70%, respectively, of allergen remaining after 60 min at 140 degrees C. CONCLUSIONS: The effect of dry heat on allergens increased with increasing time and temperature, cat and dog allergens demonstrating greater heat resistance than mite allergens. Dry heating methods may represent an alternative technique for removal of mite allergens; however, the greater stability of Fel d 1 and Can f 1 suggests that this procedure may not be appropriate for pet allergens.     

The use of multiple antigenic peptide (MAP) in the immunodiagnosis of human immunodeficiency virus infection

In order to develop an ELISA system for the antibody detection of HIV-1 or HIV-2 infections, MAPs for HIV-1 gp41(584-618) and HIV-2 gp36(574-602) corresponding to the immunodominant regions of HIV-1 gp41 and HIV-2 gp36 were used as coating antigens in the ELISA. The MAPs were synthesized by the solid phase method using Fmoc-Lys(Fmoc)-OH and their molecular weights were confirmed by tricine gel electrophoresis. The MAPs reacted with all HIV positive sera (64 samples), but did not react with HIV negative sera (48 samples) obtained from healthy blood donors. The MAPs showed high sensitivity and specificity in anti-HIV 1/2 combo panel and anti-HIV-1 seroconversion panels. The results indicated that the ELISA system using synthetic MAPs of gp41(584-618) and gp36(574-602) as coating antigens can be used as an analytical system for the immunodiagnosis of HIV-1 or HIV-2 infections.     

A comparison of freezing and thawing methods for the cryopreservation of human semen

The purpose of this study was twofold. In the first part are compared cryosurvival rates for human semen following two different freezing and three different thawing techniques. In the second part a larger number of samples processed by the best of the six methods described were examined to ensure reproducibility. Eighteen human semen samples with initial motility greater than 60% and density greater than 20 X 10(6)/ml were mixed with a cryopreservative medium at 35 degree C and vacuumed into 0.5 ml straws. Six aliquots were prepared from as many specimens as possible. Three straws were frozen by a programmed method (P) and three by a rapid freezing technique (V). All six straws were stored in liquid nitrogen vapor (-196 degree C) for 1 week. The straws were thawed by 1 of 3 methods: (1) room temperature for 10 minutes and 37 degree C hot plate for 10 minutes, (2) ice water for 10 minutes and 37 degree C hot plate for 10 minutes, (3) 35 degree C water for 12 seconds and 37 degree C hot plate for 10 minutes. Postthaw motility was assessed for each aliquot. The freeze/thaw method P/1 was judged optimal. In the second part of the study a larger number of samples were processed by P/1. The mean +/- standard deviation postthaw motility o 57 semen specimens processed by this method was 61.4 +/- 12.1.     

Temperature determines the pattern of anaerobic microbial dechlorination of Aroclor 1260 primed by 2,3,4,6-tetrachlorobiphenyl in Woods Pond sediment

Reductive dechlorination of the Aroclor 1260 residue in Woods Pond (Lenox, Mass.) sediment samples was investigated for a year at incubation temperatures from 4 to 66 degrees C. Sediment slurries were incubated anaerobically with and without 2,3,4,6-tetrachlorobiphenyl (2346-CB; 350 microM) as a primer for dechlorination of the Aroclor 1260 residue. Dechlorination of the Aroclor residue occurred only in live samples primed with 2346-CB and only at 8 to 34 degrees C and 50 to 60 degrees C. The extent and pattern of polychlorinated biphenyl (PCB) dechlorination were temperature dependent. At 8 to 34 degrees C, the dechlorination resulted in 28 to 65% decreases of the hexathrough nonachlorobiphenyls and corresponding increases in the tri- and tetrachlorobiphenyls. At 12 to 30 degrees C, 30 to 40% of the hexa- through nonachlorobiphenyls were dechlorinated in just 3 months. The optimal temperature for overall chlorine removal was 20 to 27 degrees C. We observed four different microbial dechlorination processes with different but partially overlapping temperature ranges, i.e., Process N (flanked meta dechlorination) at 8 to 30 degrees C, Process P (flanked para dechlorination) at 12 to 34 degrees C, Process LP (unflanked para dechlorination) at 18 to 30 degrees C, and Process T (a very restricted meta dechlorination of specific hepta- and octachlorobiphenyls) at 50 to 60 degrees C. These temperature ranges should aid in the development of strategies for the enrichment and isolation of the microorganisms responsible for each dechlorination process. The incubation temperature determined the relative dominance of the four PCB dechlorination processes and the extent and products of dechlorination. Hence, understanding the effects of temperature on PCB dechlorination at contaminated sites should assist in predicting the environmental fate of PCBs or planning bioremediation strategies at those sites.     

Cerebral metabolic effects of sequential periods of hypothermic circulatory arrest

During repair of congenital heart defects, extended periods of hypothermic circulatory arrest (CA) have been shown to cause short-term cerebral metabolic and flow abnormalities as well as long-term neuropsychologic dysfunction. Occasionally, a second period of CA is required during the same operative setting to revise a complicated repair. However, the metabolic effects of two consecutive periods of CA on the brain are unclear. In this study, we compared the recovery of cerebral metabolism after 60 minutes of CA with that after two sequential 30-minute periods of CA separated by a brief period of rewarming (30'SEQ). Fifteen neonatal piglets (2 to 3 kg) were placed on cardiopulmonary bypass at 100 mL.kg-1 x min-1 and cooled to 18 degrees C. Each animal then underwent either 60 minutes of uninterrupted cardiopulmonary bypass at 18 degrees C, 60 minutes of CA, or two 30-minute periods of CA separated by a brief period of rewarming. After these experimental periods, animals were rewarmed to 37 degrees C and weaned from cardiopulmonary bypass. Data were obtained before cardiopulmonary bypass and after cardiopulmonary bypass at 37 degrees C and included measurements of cerebral blood flow by xenon 133 clearance, arterial and sagittal sinus blood gases, and cerebral metabolism (mL O2.100 g-1 x min-1). Our results demonstrated that acute recovery of cerebral metabolism was significantly impaired after 60 minutes of CA and that recovery of cerebral metabolism after two sequential 30-minute periods of CA was significantly better than after 60 minutes of continuous CA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of neuroprotection from excitotoxicity by moderate and profound hypothermia in cultured cortical neurons unmasks a temperature-insensitive component of glutamate neurotoxicity

Although profound hypothermia has been used for decades to protect the human brain from hypoxic or ischemic insults, little is known about the underlying mechanism. We therefore report the first characterization of the effects of moderate (30 degrees C) and profound hypothermia (12 degrees to 20 degrees C) on excitotoxicity in cultured cortical neurons exposed to excitatory amino acids (EAA; glutamate, N-methyl-D-aspartate [NMDA], AMPA, or kainate) at different temperatures (12 degrees to 37 degrees C). Cooling neurons to 30 degrees C and 20 degrees C was neuroprotective, but cooling to 12 degrees C was toxic. The extent of protection depended on the temperature, the EAA receptor agonist employed, and the duration of the EAA challenge. Neurons challenged briefly (5 minutes) with all EAA were protected, as were neurons challenged for 60 minutes with NMDA, AMPA, or kainate. The protective effects of hypothermia (20 degrees and 30 degrees C) persisted after rewarming to 37 degrees C, but rewarming from 12 degrees C was deleterious. Surprisingly, however, prolonged (60 minutes) exposures to glutamate unmasked a temperature-insensitive component of glutamate neurotoxicity that was not seen with the other, synthetic EAA; this component was still mediated via NMDA receptors, not by ionotropic or metabotropic non-NMDA receptors. The temperature-insensitivity of glutamate toxicity was not explained by effects of hypothermia on EAA-evoked [Ca2+]i increases measured using high- and low-affinity Ca2+ indicators, nor by effects on mitochondrial production of reactive oxygen species. This first characterization of excitotoxicity at profoundly hypothermic temperatures reveals a previously unnoticed feature of glutamate neurotoxicity unseen with the other EAA, and also suggests that hypothermia protects the brain at the level of neurons by blocking, rather than slowing, excitotoxicity.     

Plasma levels of soluble CD30, tumour necrosis factor (TNF)-alpha and TNF receptors during primary HIV-1 infection: correlation with HIV-1 RNA and the clinical outcome

OBJECTIVES: The immunological and virological events associated with primary HIV-1 infection have a major impact on the course of HIV-1 disease, and the identification of early predictors during primary HIV infection is critical for the therapeutic strategy. DESIGN AND METHODS: Eighteen consecutive patients with primary HIV infection were followed for a median of 398 days. Clinical status, CD4+ T-cell counts, and plasma samples were obtained weekly from enrollment until week 6, then at weeks 12, 24 and 52, and every 6 months thereafter. Seroconversion was assessed by anti-HIV-1/2 antibodies and Western blot analysis. HIV-1 RNA in plasma was quantified by Amplicor HIV Monitor test. Samples were assayed for immune complex-dissociated p24 antigen, tumour necrosis factor (TNF)-alpha, soluble TNF receptor (sTNFR)-1, sTNFR-II, sCD30 and sCD8 by enzyme immunoassays. Outcome was defined as entering clinical category B or C according to the Centers for Disease Control and Prevention criteria. As a control group, we included 23 HIV-1-negative healthy blood donors. RESULTS: Plasma levels of sCD30, TNF-alpha and sTNFR were significantly higher in HIV-1-infected patients than in controls, and were positively correlated with each other and with values of HIV-1 RNA. Patients who developed an outcome (n = 4) had significantly higher levels of sCD30, TNF-alpha and sTNFR compared with those who did not. Multivariate logistic regression analysis showed that sCD30 and TNF-alpha were the best predictors of outcome independently of CD4+ T-cell counts. CONCLUSIONS: During primary HIV infection, a persistent immune activation may be associated with a poor clinical outcome. The identification of sCD30 and TNF-alpha levels in plasma as early predictors of outcome in primary HIV infection, may direct the implementation of early therapeutic strategies in patients with elevated risk of disease progression.