Expression of CD69 on T-cell subsets in HIV-1 disease.

CD69 is the earliest activation marker newly synthesized and expressed during T lymphocyte activation. In this study, a whole-blood flow-cytometry-based assay was used to assess expression of the activation antigen CD69 on CD4 and CD8 T lymphocytes, and the co-expression of CD69 and CD28 on T cells. The expression of CD69 was studied in both unstimulated and in phytohaemagglutinin (PHA)- or anti-CD(3)/CD(28)-stimulated, 4-h culture, samples. The production of IL-2, IFN-gamma or both cytokines, in CD69(+) T cells, in response to Staphylococcus enterotoxin B was also tested. Fifty-three HIV-1-infected and 21 healthy volunteers participated in this study. In both PHA- and anti-CD(3)/CD(28)-stimulated cultures the percentage of CD69 on CD3(+)CD4(+) T cells was significantly lower in AIDS (and non-responders to HAART) versus healthy controls and the other HIV-1(+) groups. A decrease of CD69(+)CD28(+) T cells after PHA or MoAbs stimulation is noticed in AIDS. No difference in cytokine production was noticed between healthy volunteers and HIV-1(+) patients. Our results suggest that the expression of CD69 is affected only in the AIDS stage and in the non-responders to HAART patients.

Shear bond strength of three veneering resins to a Ni-Cr alloy using two bonding procedures.

Composite veneering materials are used as alternatives to porcelain in fixed prosthodontics. Mechanical retention of the resin on the metal framework has been associated with the formation of gaps at the resin/alloy interface, and failure of the restoration. Several chemical bonding systems have been introduced to promote resin adhesion. The purpose of the present study was to evaluate the shear bond strength of three photocured composites (Artglass, Solidex & Signum+) to a Ni-Cr alloy.72 wax disks covered with 150-mum diameter beads were cast and divided in two equal groups. In the first group, Metal Photo Primer was applied on the casting surface, while the Siloc system was used in the second. Each group was divided in three subgroups of 12 samples, in which the three composites were photocured. Half of the specimens of each subgroup were subjected to 1000 and 5000 thermal cycles (5 and 55 degrees C) respectively. All specimens were tested in shear in a universal testing machine. The Siloc-Solidex group showed the highest bond strength (17.3 +/- 3.7 MPa). No statistically significant difference was found between specimens treated with Metal Photo Primer or Siloc. Thermocycling did not significantly affect the bond strength values. Solidex showed an adhesive failure mode for both alloy surface treatments, while Artglass and Signum+ exhibited combination failures. Conclusively, the appropriate alloy surface treatment - resin combination can significantly improve the resin-alloy shear bond strength. Specifically, Solidex resin exhibited significantly higher bond values compared with Artglass and Signum+, for both surface treatments and thermocycling procedures.

Limited long-term naive CD4+ T cell reconstitution in patients experiencing viral load rebounds during HAART.

Long-term (3.5 years) immune reconstitution in relation to viral load response was determined. Plasma HIV-1 RNA was suppressed in 40 patients (full responders) up to 42 months, and 17 patients achieved partial response. The measurements of CD4(+) and CD8(+) T lymphocyte subsets (CD45RA, CD45RACD62L, CD45RO, CD28, CD38) were carried out by flow cytometry. Full responders had a significant increase of CD4(+) and all CD4(+) T subsets both up to 6 and from 6 to 42 months, while the increase for partial responders was only up to 6 months. By 6 months, higher slopes were observed in full versus partial responders in the % of CD28 on CD4(+) and the % of CD4(+) memory subset and in both naïve and memory CD4(+) subsets from 6 to 42 months. The percentage of CD8(+) and its subsets was decreased significantly in full responders both up to 6 and from 6 to 42 months (except for an increase in the CD8(+)CD45RA(+) CD62L(+) cells), while in partial responders this decrease was only up to 6 months. Lower slopes were observed in full versus partial responders from 6 to 42 months in the percentages of CD8(+), CD8(+)CD45RO(+), CD8(+)CD28(-), and CD8(+)CD38(+) T cells. In conclusion, full responders have a stronger long-term naive CD4(+) T cell subset reconstitution than partial responders.

Comparative evaluation of the diagnostic performance of the BTA stat test, NMP22 and urinary bladder cancer antigen for primary and recurrent bladder tumors.

PURPOSE: We compared overall sensitivity and specificity of the urinary bladder cancer antigen enzyme-linked immunosorbent assay (UBC, IDL Biotech, Sollentuna, Sweden), BTA stat test (Bion Diagnostic Sciences, Inc., Redmond, Washington) and NMP22 test kit (Matritech, Newton, Massachusetts), and the differential sensitivity regarding the histological pattern of tumors. MATERIALS AND METHODS: A total of 213 patients with clinical and/or imaging signs of bladder cancer provided a single voided urine sample for the bladder cancer antigen, BTA stat test and NMP22 before cystoscopy. Of these patients 95 were monitored for superficial bladder cancer, while the remaining 118 had no history of bladder cancer. All detected bladder tumors or suspicious lesions were resected transurethrally. A group of 21 age and sex matched healthy volunteers were also evaluated with the same tests. RESULTS: Bladder cancer was confirmed histologically in 118 patients, of whom primary and recurrent tumors were in 68 and 50, respectively. The optimal cutoffs calculated with receiver operating characteristics curves were 8 units per ml. for NMP22 and 12 microg./l. for bladder cancer antigen. Overall sensitivity and specificity were 72.9% and 64.6% for the BTA stat test, 63.5% and 75.0% for NMP22, and 80.5% and 80.2%, respectively, for bladder cancer antigen. Bladder cancer antigen proved significantly more sensitive than NMP22 for detecting bladder cancer (p = 0.001) but not more than the BTA stat test, while the specificity of it was significantly higher than that of the BTA stat test (p = 0.009). Bladder cancer antigen had a sensitivity of 80.7% for stage Ta tumors, which was significantly higher than NMP22 (52.6%, p = 0.001) and the BTA stat test (57.9%, p = 0.01). In grade I tumors the sensitivity of bladder cancer antigen (70%) did not differ significantly than that of the BTA stat test (50%) and NMP22 (50%, p = 0.14). Bladder cancer antigen had the least false-positive results in patients with a history of bladder cancer and negative cystoscopy, and those with urological disease other than bladder cancer. CONCLUSIONS: Our data indicate that bladder cancer antigen may be a more potent diagnostic marker for bladder cancer than NMP22 and the BTA stat test based on the higher sensitivity for detecting low stage and low grade tumors, and the higher specificity. The contribution of these tests for detection of bladder cancer should still be considered adjunctive to cystoscopy.

Flow-cytometric detection of minimal residual disease with atypical antigen combinations in patients with de novo acute myeloid leukemia.

The immunophenotypic features in adult de novo acute myeloid leukemia (AML) patients at diagnosis using flow cytometry double marker analysis and the detection of minimal residual disease with atypical leukemia-associated antigen combinations during remission were investigated. Fifty adult patients with de novo AML at diagnosis were studied. Bone marrow samples from 21 patients with AML were analyzed upon achievement of complete remission and during continuous complete remission. Ten bone marrow samples of normal donors were also studied. CD34/CD13, CD34/CD33, CD33/CD7, CD33/CD10, CD33/CD19 and CD33/TdT are the leukemia-associated antigen combinations used for the detection of minimal residual disease. The outcome of 19 patients has been evaluated. Of these 19 patients, 10 were found to be in immunophenotypic remission (median follow-up after the study: 837 days, range 620-1343 days). Only one patient in this group has relapsed so far. In the other nine patients residual disease was detected. Seven of these patients developed systemic relapse following a median follow-up time of 86 days after the study (range 34-273 days), one received allogeneic bone marrow transplantation 70 days after the study, and another has been in complete remission and off chemotherapy for 36 months. The presence of cells with atypical antigen combinations identified at diagnosis in certain patients is valuable for monitoring the disease in remission. The persistence of such a population in remission has indicated the impending relapse in this study.

Comparative evaluation of the BTAstat test, NMP22, and voided urine cytology in the detection of primary and recurrent bladder tumors.

OBJECTIVES: This prospective study was undertaken to evaluate the diagnostic efficacy of the BTAstat test and nuclear matrix protein (NMP22) compared with voided urine cytology (VUC) in the detection of primary and recurrent bladder cancer. METHODS: A total of 147 patients provided a single voided urine sample for the BTAstat test, NMP22, and cytology prior to cystoscopy. Eighty-five of them had no bladder cancer history, whereas the remaining 62 were monitored for superficial bladder cancer. A group of 21 healthy age-matched volunteers were also enrolled in the study. RESULTS: Bladder cancer was confirmed histologically in 99 patients, of which 62 had primary tumors and 37 had recurrent ones. The overall sensitivity and specificity were 71.7% and 56.5% for the BTAstat test, 62.6% and 73. 9% for NMP22, and 38.4% and 94.2% for VUC. The optimal threshold value for NMP22 calculated with receiver operating characteristics curve, was 8 U/mL. BTAstat test was significantly more sensitive than VUC in detecting bladder cancer in all stage and grade subgroups, except GIII. On the contrary, NMP22 was significantly more sensitive than VUC only in stage Ta, grade I and II patients. BTAstat test had higher but not significantly different sensitivity than NMP22. CONCLUSIONS: Our data indicate a superiority of both BTAstat test and NMP22 over VUC in the detection of bladder cancer. Comparing BTAstat test with NMP22, the former proved to be more sensitive, whereas the latter was more specific. Ruling out diseases with potential interference can increase the overall specificity of both tests. False-positive results of either test in patients followed up for bladder cancer seem to correspond to future recurrences.

CD28 costimulation and CD28 expression in T lymphocyte subsets in HIV-1 infection with and without progression to AIDS.

In a prospective study of 152 HIV-1 patients (with and without progression to AIDS) we examined CD28 MoAb costimulation and CD3 MoAb response using whole blood culture at baseline and up to either the time of AIDS diagnosis or the end of the observation period. CD28 antigen expression on both CD4+ and CD8+ T lymphocytes was also studied in both groups of patients. In patients who progressed to AIDS, CD28 MoAb costimulation was found to be decreased. Univariate time-dependent analysis showed that decreases in (i) absolute numbers of either CD4+, CD4+CD28+, CD8+CD28+ T cells, (ii) CD28 MoAb costimulation, and (iii) CD3 MoAb response, and an increase in CD8+CD28- %, are significant predictors for progression to AIDS. In addition, multivariate time-dependent analysis demonstrated that a decrease in CD28 MoAb costimulation (but not a decrease in CD3 MoAb response) was predictive for progression to AIDS, as were decreases in the percentage of CD4+ T cells and the absolute number of CD4+CD28+ T cells. Thus, CD28 MoAb costimulation can be considered a useful assay for monitoring HIV-1 infection. Furthermore, apart from the early increase in the percentage of CD8+CD28- T cells and an increase in the percentage of CD28- on CD8+ T cells in both groups of patients at baseline compared with normal controls, a negative correlation was found to exist between the percentages of CD4+ or CD4+CD28+ T cells and the percentage of CD8+CD28- T cells; this suggests that these cells are probably mutually regulated.

Standardisation and quality assurance of lymphocyte proliferation assays for use in the assessment of immune function. European Concerted Action on Immunological and Virological Markers of HIV Disease Progression.

Lymphocyte proliferation is a widely used technique to assess immune competence. However, the technique is subject to a large degree of variation, some biological and some technical. In this study, the components of variation in whole blood proliferation assays were analysed over time, using both antibody and mitogenic stimulants. The levels of variation within individual samples, between individuals and between groups of individuals over time were examined. A method of transforming the data is proposed which reduces the coefficients of variation to an acceptable level, and which expresses individual results as a standardised count. This method overcomes the problem of different levels of absolute counts, it corrects for time sensitive errors and allows data from multiple laboratories to be pooled.

Pesticide poisoning of animals of wild fauna.

Poisoning of rare birds of prey (7 Aegipius monachus and 1 Aquila chrysaetus) and 11 foxes by carbofuran is reported. The poisoning is an ecological disaster because of the death of A monachus, which is a rare species. Identification, confirmation and distribution of the toxic substance was performed by TLC and HPLC techniques.

Reversed-phase high-performance liquid chromatography--photodiode-array analysis of alkaloid drugs of forensic interest.

A reversed-phase high-performance liquid chromatography-photodiode-array method for the analysis of 23 drugs of forensic interest is presented. The separation method development was based on mobile-phase optimisation, temperature control and use of three ODS stationary phases. Multiwavelength detection and quantitation was performed at 225, 232, 239, 254, 275 and 289 nm. Absorbance rationing proved to be very helpful for the identification of these drugs. Recognition of the analysed compounds was achieved by means of correlation of retention time and absorbance ratios. The method was directly applied to the analysis of illicit heroin and cocaine samples and to the analysis of pharmaceutical preparations containing codeine.

CD28 expression on T cell subsets in vivo and CD28-mediated T cell response in vitro in patients with rheumatoid arthritis.

OBJECTIVE: In view of the critical importance of the CD28-CD80 (B7/BB1) costimulatory pathway in antigen-specific T cell activation and clonal expansion, we examined CD28 surface molecule expression in vivo, and T cell receptor/CD3-mediated and B7/BB1-costimulated T cell proliferation in vitro, in rheumatoid arthritis (RA). METHODS: Two-color immunofluorescence analyses of peripheral blood and synovial fluid-derived T cells, as well as 3H-thymidine incorporation assays, were performed. RESULTS: In the peripheral blood of 31 patients with active, untreated RA, a mean of 91% (range 48-100%) of CD4+ and 46% (range 13-82%) of CD8+ T cell subsets were CD28+, which was not significantly lower than normal. Although an overall decrease in the number of T cells was not observed, the numbers of CD28+CD8+ T cells were significantly lower in RA patients (mean 233/microliters, versus 292/microliters in controls), and this decrease was more pronounced in patients with severe disease (mean 172/microliters). CD28 expression on peripheral CD8+ T cells in RA patients, but not in healthy individuals, correlated inversely with T cell activation as assessed by HLA-DR antigen expression. In contrast to the peripheral blood, RA synovial fluid T cells were almost exclusively CD28+, suggesting that migration of CD28+CD8+ T cells to active sites of inflammation may occur. In vitro proliferative responses of peripheral blood T cells to B7/BB1 costimulation in the presence of mitogenic doses of anti-CD3 monoclonal antibody were identical in patients with RA and healthy individuals. CONCLUSION: Functionally intact CD28+ T cells may contribute to the abnormal immunoregulation and joint inflammation in RA.

Selected heavy metal concentrations in goat liver and kidney.

Concentrations of copper, zinc, lead and cadmium in liver and kidney of goats was determined. Goats were intended for human consumption and bred in the rural area of Arnea and in the metal contaminated area of Olympias. A total of 104 specimens were measured by flame atomic absorption spectroscopy. Zinc and lead concentrations were within acceptable limits. Copper levels were deficient in animals from the Olympias area. Cadmium levels from Olympias were higher than those from Arnea. Liver and particularly kidney specimens from Olympias had cadmium levels exceeding tolerance limits and should be avoided for human consumption.

Downregulation of CD28 surface antigen on CD4+ and CD8+ T lymphocytes during HIV-1 infection.

A progressive significant decrease of CD28 surface antigen expression on CD4+ (mean, 90, 86, 79, 68% in stages I, II, III, and IV, respectively, versus 96% in normals), as well as on CD8+ T lymphocytes (mean, 38, 32, 31, and 29% in stages I, II, III, and IV, respectively, versus 47% in normals) was observed during HIV-1 infection. The increase of cytotoxic/suppressor T cells, in both percentage and absolute numbers, that was observed in almost all HIV-1 patients, was associated with an increase of the CD8+ cells lacking the CD28 surface antigen. The loss of CD28 antigen expression was parallel to the increase of CD38, human leukocyte antigen (HLA)-DR, and CD45RO antigen expression on T lymphocytes throughout the disease. Furthermore, a positive significant correlation within the CD4+ but not the CD8+ subset was observed between the percentage of cells lacking the CD28 antigen and the percentage of cells expressing the HLA-DR and CD38 antigens, a finding suggesting that the loss of CD28 antigen expression on CD4+ lymphocytes may be associated with T-lymphocyte activation. Patients treated with zidovudine showed no significant differences in the percentages of either CD4+CD28+ or CD8+CD28+ T-cell subsets when compared to untreated patients. These phenotypic changes may be associated with the functional defects of T lymphocytes in HIV-1 infected individuals.

Determination of chloramphenicol residues in eggs by high performance liquid chromatography (HPLC).

A rapid and sensitive HPLC method for the determination of chloramphenicol (CAP) residues in albumin and yolk of eggs is described. Conditions of isolation and identification are presented in detail. The detection limit is 10 ppb and the linearity is very good up to 2500 ppb CAP. The recovery of the method is 82 and 77%, and the precision is 6.6 and 11.1% for the albumin and yolk determinations respectively. The method was tested to quantitate CAP residues in eggs of 5 laying hens to which CAP had been administered in feed at a concentration of 800 mg/kg. CAP was detected in albumin and yolk of eggs laid 3 and 8 days after the CAP administration, respectively.

Magnesium, calcium and zinc fluctuations on skin induced injuries in correlation with time of induction.

In the present study, fluctuations of trace elements Mg, Ca and Zn concentrations with time on skin-induced injuries was investigated. To accomplish this, 144 animals (pig) aged between 5-6 months scheduled for food provision process (slaughter) were used. At the gluteus area, injuries were induced prior to slaughter at intervals of 30 s, 30 min, 1, 2, 4, 8 h. Local anaesthesia and cold therapy for prompt relief of pain (ethylchloride, C2H5Cl) was applied. Postmortem tissue excision in and around the injured site was promptly performed. The tissue obtained was segmented into three zones of equal distances (2 cm) in between and weighed 3 g wet weight. Tissue specimens were analysed by atomic absorption spectroscopy for the three elements. The results were correlated with time of injury. Suggestive alterations in trace elements mean concentrations with time were confirmed. The ratios of the mean in twos (Ca/Zn, Ca/Mg, Mg/Zn) versus time were graphed. Effectively, the curves achieved by analysing postmortem tissues, serve to estimate the time of an injury induced in vivo.