[Discontinuation of Chromium-51 for clinical use: What are the possible alternatives for radiopharmacies and nuclear medicine departments?] / Arrêt de production du chrome-51 (51Cr) a usage clinique : quelles alternatives possibles pour les services de radiopharmacie et de médecine nucléaire ?

OBJECTIVES: Last October, the nuclear medicine departments were informed of the closure of the chromium-51 production line for clinical use. This radionuclide has different diagnostic indications in nephrology and hematology. It was therefore essential to set up alternative exploration protocols to overcome this production stoppage. METHODS: Chromium-51 EDTA has been replaced by technetium-99m DTPA for the determination of glomerular filtration rates. Sodium chromate was substituted by sodium pertechnetate for the determination of globular volumes. A retrospective analysis of the chromium-51 data was performed followed by a prospective study, from January to December 2019 for technetium tracers. RESULTS: One hundred and forty-four patients were included in the study. Forty-two EDTA-51Cr and 30 DTPA-99mTc exams were conducted and compared. There were no significant differences between the methods used to assess renal function (P=0.355). For the determination of blood cell and plasma volumes, 47 tests with 51Cr and 125I and 25 tests with 99mTc and 125I were performed and compared. There were no significant differences in the determination of total (P=0.325) and globular (P=0.148) volumes. CONCLUSIONS: The study carried out shows that there is no significant difference between the results obtained with chromium-51 and technetium tracers. As a result, clinical activity was maintained in good conditions.

Reexamination of the chromium-51-labeled posttransfusion red blood cell recovery method.

BACKGROUND: The chromium-51-labeled posttransfusion recovery (PTR) study has been the gold-standard test for assessing red blood cell (RBC) quality. Despite guiding RBC storage development for decades, it has several potential sources for error. METHODS: Four healthy adult volunteers each donated an autologous, leukoreduced RBC unit, aliquots were radiolabeled with technetium-99m after 1 and 6 weeks of storage, and then infused. Subjects were imaged by single-photon-emission computed tomography immediately and 4 hours after infusion. Additionally, from subjects described in a previously published study, adenosine triphosphate levels in transfusates infused into 52 healthy volunteers randomized to a single autologous, leukoreduced, RBC transfusion after 1, 2, 3, 4, 5, or 6 weeks of storage were correlated with PTR and laboratory parameters of hemolysis. RESULTS: Evidence from one subject imaged after infusion of technetium-99m-labeled RBCs suggests that, in some individuals, RBCs may be temporarily sequestered in the liver and spleen immediately following transfusion and then subsequently released back into circulation; this could be one source of error leading to PTR results that may not accurately predict the true quantity of RBCs cleared by intra- and/or extravascular hemolysis. Indeed, adenosine triphosphate levels in the transfusates correlated more robustly with measures of extravascular hemolysis in vivo (e.g., serum iron, indirect bilirubin, non-transferrin-bound iron) than with PTR results or measures of intravascular hemolysis (e.g., plasma free hemoglobin). CONCLUSIONS: Sources of measurement error are inherent in the chromium-51 PTR method. Transfusion of an entire unlabeled RBC unit, followed by quantifying extravascular hemolysis markers, may more accurately measure true posttransfusion RBC recovery.

Intestinal permeability after Mediterranean diet and low-fat diet in non-alcoholic fatty liver disease.

BACKGROUND: In non-alcoholic fatty liver disease (NAFLD), a high-fat or high-fructose diet increases intestinal permeability and promotes derangement of the gut-liver axis. We hypothesize that, diet could be able to modulate intestinal permeability in patients with NAFLD. AIM: To detect diet-induced modification of intestinal permeability in patients with NAFLD undergoing a Mediterranean diet or a low-fat diet. METHODS: The current study was a dietary intervention for non-diabetic, patients with biopsy-verified NAFLD and increased transaminases. A crossover design was employed: participants underwent 16 weeks of Mediterranean diet, 16 wk of free wash-out, and 16 weeks of low-fat diet. Both diets were hypocaloric and no consumption of supplements was allowed. All patients were followed bimonthly by a dietitian. Evaluations of clinical and metabolic parameters were completed at baseline and at the end of each dietary period. Intestinal permeability was assessed by chromium-51 ethylene diamine tetraacetate excretion testing (51Cr-EDTA). RESULTS: Twenty Caucasian patients, 90% male, median age 43 years, body mass index (BMI) 30.9, with biopsy-verified NAFLD were enrolled. At the end of 16 weeks of a Mediterranean diet, a significant reduction in mean body weight (-5.3 ± 4.1 kg, P = 0.003), mean waist circumference (-7.9 ± 4.9 cm, P = 0.001), and mean transaminase levels [alanine aminotransferase (ALT) -28.3 ± 11.9 IU/L, P = 0.0001; aspartate aminotransferase (AST) -6.4 ± 56.3 IU/L, P = 0.01] were observed. These benefits were maintained after 16 wk of wash-out and also after 16 wk of low-fat diet, without further improvements. Fourteen of the 20 patients had intestinal permeability alteration at baseline (mean percentage retention of 51Cr-EDTA = 5.4%), but no significant changes in intestinal permeability were observed at the end of the 16 wk of the Mediterranean diet or 16 wk of the low-fat diet. CONCLUSION: Mediterranean diet is an effective strategy for treating overweight, visceral obesity and serum transaminase in patients with NAFLD. If the Mediterranean diet can improve intestinal permeability in patients with NAFLD, it deserves further investigation.

Bioavailability of chromium(III)-supplements in rats and humans.

Chromium(III) is long regarded as essential trace element but the biochemical function and even basic transport ways in the body are still unclear. For a more rational discussion on beneficial as well as toxic effects of Cr(III), we re-investigated the bioavailability of the most important oral Cr supplements by using radiolabeled compounds and whole-body-counting in rats and in the first time also in humans. The apparent absorption of (51)Cr(III) from Cr-picolinate, Cr-nicotinate, Cr-phenylalaninate, Cr-proprionate, or Cr-chloride was generally low (0.04-0.24 %) in rats with slightly higher values for Cr-chloride and -phenylalaninate. Taking a fast urine excretion into account, the true absorption of (51)Cr was clearly higher for CrPic(3) (0.99 %), probably indicating a different uptake mechanism of this rather stable organic Cr complex. The bioavailability of CrPic(3) and Cr(D: -Phen)(3), the leading compounds in actual investigations, was analysed also in human volunteer by intraindividual comparison. The apparent absorption (=Cr bioavailability) of (51)Cr from both compounds was substantially higher in humans (0.8-1 %) than in rats. Again, most of freshly absorbed CrPic(3) was excreted into the urine resulting in the same low whole-body retention after 7 days for both compounds. In summary, the bioavailability of Cr from pharmaceutical Cr compound is lower than hitherto assumed. Importantly, humans absorb Cr(III) clearly better than rats. The absorption mechanism of CrPic(3) seems to be different from ionic Cr(III) but, as only the same low amount of Cr is retained from this compound, it is also not more bioavailable than other Cr compounds.

Blood loss comparison during transurethral resection of prostate and high power GreenLight(™) laser therapy using isotopic measure of red blood cells volume.

BACKGROUND: Transurethral resection of the prostate (TURP) remains the gold standard in the operative management of symptomatic benign prostatic hyperplasia (BPH). The main morbidity of TURP is bleeding, which leads to clot retention and blood transfusion. Newer techniques have appeared, and photovaporization of the prostate (PVP) with the GreenLight™ laser has been developed to reduce the morbidity of bladder outflow surgery. Isotopic measurements of total red cell volume and total blood volume (BV) are a recommended reference technique to evaluate bleeding occurring during endoscopic ablation of the prostate. Here, we compare blood loss during PVP and TURP using an isotopic method. METHODS: Eighteen patients underwent PVP, and 20 patients underwent a TURP for symptomatic BPH by one surgeon. The two groups were comparable in demographic data; however, prostate volume was significantly higher in the PVP group. BV was measured pre- and postoperatively using the isotope technique. RESULTS: The total BV was measured to have increased by 362 mL in PVP group compared with a loss of 315 mL in TURP group (p=0.001). The difference in total red cell volume increased by 148 mL in PVP group compared with a loss of 216 mL in TURP group (p=0.005). CONCLUSIONS: Using the isotope method, we have shown a significant difference in postoperative blood loss between TURP and PVP. Our study is the first to use an isotopic method to measure the blood loss during PVP. This technique needs further standardization before being introduced into routine clinical practice.

Red blood cell survival in long-term dialysis patients.

BACKGROUND: Shortening of red blood cell (RBC) survival contributes to the anemia of chronic kidney disease. The toxic uremic environment accounts for the decreased RBC life span. The contribution of mechanical damage caused by hemodialysis to the shortened life span is unclear. Reductions up to 70% in RBC survival have been reported in uremic patients. To date, no accurate well-controlled RBC survival data exist in dialysis patients treated using different dialysis modalities and receiving erythropoiesis-stimulating agent (ESA) therapy. The aim of this study was to determine RBC survival in hemodialysis (HD) and peritoneal dialysis (PD) patients compared with healthy persons. STUDY DESIGN: Observational study. SETTING & PARTICIPANTS: 14 HD patients and 5 PD patients were recruited from the dialysis unit. Healthy volunteers (n = 14) age- and sex-matched to HD participants were included. All dialysis patients received either ESA therapy or regular iron supplementation. PREDICTOR: Dialysis patients versus age- and sex-matched healthy controls. OUTCOMES: RBC survival. MEASUREMENTS: RBC survival was determined using radioactive chromium labeling. RESULTS: More than 85% of dialysis patients were anemic (hemoglobin, 12.0 ± 1.1 g/dL); hemoglobin concentrations were not significantly different between HD and PD patients. Median RBC survival was significantly decreased by 20% in HD patients compared with healthy controls: 58.1 (25th-75th percentile, 54.6-71.2) versus 72.9 (25th-75th percentile, 63.4-87.8) days (P = 0.02). No difference was shown between the PD and HD groups: 55.3 (25th-75th percentile, 49.0-60.2) versus 58.1 (25th-75th percentile, 54.6-71.2) days (P = 0.2). LIMITATIONS: Label loss from RBCs associated with the chromium 51 labeling technique needs to be accounted for in the interpretation of RBC survival data. CONCLUSIONS: Despite current ESA therapy, decreased RBC survival contributes to chronic kidney disease-related anemia, although the reduction is less than previously reported. There does not appear to be net mechanical damage associated with HD therapy resulting in decreased RBC life span.

Blood loss during transurethral resection of the prostate as measured by the chromium-51 method.

PURPOSE: To evaluate blood loss during transurethral resection of the prostate (TURP), and its predictive factors, using the chromium 51 (51Cr) labeling method. PATIENTS AND METHODS: From January to June 2008, 41 patients who underwent TURP for symptomatic benign prostatic hyperplasia (BPH) at four French urology centers were included in the analysis. Red cells volume was measured by the 51Cr method 1 day before TURP, and on postoperative day 3. Overall blood loss was estimated by multiplication of red cells volume loss and preoperative venous hematocrit value. RESULTS: Mean preoperative red cells volume was 1997 mL. Mean loss of red cells volume was 209 ml, which corresponds to an estimated blood loss of 507 mL. Mean delta of hematocrit and hemoglobin were 1.4% and 0.71 g/dL, respectively. In univariate analysis, prostate volume, weight of resected tissue, preoperative red cells volume, and resection time were significantly and directly associated with loss of red cells volume (P = 0.038, P = 0.004, P = 0.002, and P = 0.039, respectively). Bipolar and monopolar TURP did not lead to significant difference of red cells loss. In multivariate analysis, both preoperative red cells volume and weight of resected tissue were independent predictors of red cells loss (P = 0.017 and P = 0.048 respectively). CONCLUSION: We present the first study to measure blood loss secondary to TURP using the 51Cr method. This technique allowed evaluating blood loss not only during the surgical procedure but also during the postoperative period. We learned from this study that, on average, blood loss from the procedure until postoperative day 3 was more than 500 mL, which is larger than previously reported amounts as measured by other methods. Because significant blood loss might occur during the postoperative period, the 51Cr method should be used to measure blood loss when evaluating new emerging techniques to manage BPH.

Hemolysis and survival of autologous red blood cells salvaged after cemented and uncemented total hip arthroplasty.

Autotransfusion is widely used after total hip arthroplasty (THA), but there are concerns about damage of red blood cells (RBCs) collected after surgery. In this study, we wanted to determine the damage and survival of RBCs salvaged after cemented THA and after uncemented THA and to compare the results. In this prospective study of 60 patients-30 who underwent cemented THA and 30 who underwent uncemented THA-postoperative autotransfusion systems (BIODREN; B.E.R.C.O., Modena, Italy) were used. Levels of potassium and free hemoglobin in the postoperative blood samples were analyzed. Before transfusion, salvaged RBCs were labeled with radioactive chromium-51, and their survival was measured. In blood salvaged after cemented THA, medium potassium level was 4.1 mmol/L (range, 3.2-5.6 mmol/L), and mean free hemoglobin level was 327 mg% (range, 120-410 mg%). In blood salvaged after uncemented THA, mean potassium level was 4.2 mmol/L (range, 3.1-5.5 mmol/L), and mean free hemoglobin level was 296 mg% (range, 130-402 mg%). In the cemented group, RBC survival was 73% at 48 hours after transfusion (range, 61%-79%), and mean time from 100% activity to 50% activity was 21 days (range, 14.2-28.2 days). In the uncemented group, RBC survival was 75% at 48 hours after transfusion (range, 68%-82%), and mean time from 100% to 50% activity of radio-labeled RBCs was 22 days (range, 16.2-29.4 days). There were no statistically significant differences in potassium levels, free hemoglobin levels, or RBC survival between the cemented and uncemented groups. Blood salvaged after surgery was not significantly damaged. Our study results confirmed that washing blood collected after surgery is not necessary. Not washing this blood is safe and decreases allogeneic transfusion in orthopedic procedures.

Chronic kidney disease--a common and serious complication after intestinal transplantation.

BACKGROUND: Chronic kidney disease after organ transplantation is a serious complication that negatively impacts on long-term patient survival. We describe long-term renal function after intestinal transplantation by serial measurements of glomerular filtration rates (GFR) with Chromium EDTA clearance. MATERIALS AND METHODS: Ten patients with at least 6 months survival form the basis of this report. Glomerular filtration rate measurements were performed at baseline, 3 months posttransplantation, and yearly thereafter. Median follow-up time for the cohort was 1.5 years (0.5-7.8 years). Tacrolimus (Prograf) was discontinued in four patients because of impaired renal function. These four patients were switched to sirolimus (Rapamune) at 11, 18, 24, and 40 months posttransplantation. RESULTS: Median baseline GFR was 67 (22-114) mL/min/1.73 m. In the adult patients, GFR 3 months posttransplantation had decreased to 50% of the baseline. At 1 year, median GFR in the adult patients was reduced by 72% (n=5). Two patients developed renal failure within the first year and required hemodialysis. One of the pediatric patients fully recovered her renal function, the second pediatric patient lost 20% of her baseline GFR at 6 months posttransplantation. Glomerular filtration rate calculated with the modified diet in renal disease formula consistently overestimated GFR by approximately 30% compared with measured GFR. CONCLUSION: Chronic kidney disease and renal failure are common after intestinal transplantation. These two factors significantly contribute to poor long-term survival rates. Measurements of GFR may help to identify those individuals at risk for developing chronic kidney disease to implement renal sparing strategies.

Decline in 51Cr-labelled EDTA measured glomerular filtration rate following lung transplantation.

BACKGROUND: The nephrotoxity of calcineurin inhibitors in lung-transplanted patients is well described, but previous studies have estimated rather than directly measured glomerular filtration rate (GFR). This study describes the decline of measured GFR in a large cohort of lung-transplanted patients from a national centre, and the correlation between measured and calculated GFR. METHODS: All lung-transplanted patients 1992-2004 (n = 390) were included in a longitudinal analysis. Seven patients were excluded due to retransplantation. Pre- and post-transplant parameters included (51)Cr-labelled EDTA clearance (mGFR) and the Cockcroft-Gault calculated clearance (cGFR). Trough cyclosporine levels (C0) and demographic and transplant information were also included in the analysis. RESULTS: A total of 66959 C0 and serum creatinine and 1945 mGFR measurements pertaining to 383 patients were included in the analysis. Pre-transplant mGFR was significantly lower with respect to recipient age over 60 years; and patients with a referral diagnosis of pulmonary hypertension had a lower mGFR and higher baseline serum creatinine levels than patients with emphysematous disease (P < 0.05). There were linear correlations between log(10) mean interval serum creatinine and log(2) mGFR at all time points pre- and post-transplantation (P < 0.0001, Spearman correlation coefficient = -0.81) and between log(2) cGFR and log(2) mGFR (P < 0.0001, Spearman correlation coefficient = 0.81), however, the agreement between mGFR and cGFR was poor (-2.7 +/- 38.6 ml/min). A simplified repeated measure ANOVA model describing post-transplant GFR over time demonstrated a 54% decline in mGFR within the first 6 months post-transplant. Pre-transplant mGFR was an important determinant of 6 month post-transplantation mGFR. Increasing mean C0, body mass index and early acute renal failure were independent risk factors for a more rapid decline in post-transplant mGFR. CONCLUSION: mGFR decreases dramatically during the first 6 months after lung-transplantation. Avoidance of high dose calcineurin inhibition may postpone the onset of post-transplant end-stage renal failure.

The effect of oral glutamine on 5-fluorouracil/leucovorin-induced mucositis/stomatitis assessed by intestinal permeability test.

BACKGROUND & AIMS: Systemic chemotherapy may damage gastrointestinal epithelium. Mucositis is associated with increased intestinal permeability (IP). It is known that IP test with chromium 51-ethylene diaminetetra-acetate (51Cr-EDTA) is a useful tool to assess the mucositis. Oral glutamine supplements (OGS) may have a role in the prevention of chemotherapy-induced mucositis/stomatitis. The aim of this study was to characterize the relationship between the urinary excretion of 51Cr-EDTA and the severity of mucositis, and the effect of OGS on 5-fluorouracil/leucovorin (FU/LV)-induced mucositis/stomatitis. METHODS: Fifty-one patients with advanced or metastatic cancer received FU/LV chemotherapy. The control group included 18 healthy volunteers. IP was assessed via the measurement of 51Cr-EDTA urinary excretion after oral challenge, on days 7 after the discontinuation of chemotherapy. Of the 51 patients, 22 patients received OGS (30 g/day) and 29 received only best supportive care (BSC). Glutamine supplementation continued for 15 days. It was initiated at least 3 days before the beginning of chemotherapy. Mucositis/stomatitis was graded according to version 3.0 of the Common Terminology Criteria for Adverse Events. RESULTS: In the chemotherapy group, the median (25 percentile, 75 percentile) IP test score was significantly higher than those of the control group [6.78% (4.63, 10.66) vs. 2.17% (1.38, 2.40), P<0.001]. The severity of stomatitis was significantly correlated with IP test scores (r=0.898, P<0.001). In the OGS group, the median IP test score was significantly lower than that of the BSC group [4.69% (3.10, 6.48) vs. 8.54% (6.48, 15.31), P<0.001]. A mucositis/stomatitis of grade 2-4 was observed in two patients of the OGS group (9%), and in 11 patients (38%) in the BSC group (P<0.001). CONCLUSIONS: The IP test may be a useful tool in the evaluation of mucositis/stomatitis. OGS may exert a protective effect on FU/LV-induced mucositis/stomatitis. Further studies, however, will be necessary to define the role of glutamine supplementation in FU/LV-induced mucositis/stomatitis.

Effect of wheat gluten hydrolysate on the immune system in healthy human subjects.

Nine healthy volunteers were divided into a test group (n = 5) and a control group (n = 4). The test group consumed 3 grams per d of wheat gluten hydrolysate for 6 d, and their NK cell activity and hematological parameters were measured: The same assessments were performed in the control group, which did not receive wheat gluten hydrolysate. In the test group, NK cell activity increased significantly (P = 0.018) after wheat gluten hydrolysate intake. No adverse effects were observed in either group.

Susceptibility of glomerular filtration rate estimations to variations in creatinine methodology: a study in older patients.

BACKGROUND: It is recommended that measurement of serum creatinine should be supplemented with a creatinine-based estimation of glomerular filtration rate (GFR). The influence of creatinine methodology on these estimates is not always appreciated. We have studied differences in creatinine methods and their influence on GFR estimation specifically in older people. METHODS: In all, 46 older patients (mean age 80 y, range 69-92 y) with predominantly mild or moderate kidney disease were studied. Serum creatinine was measured using a rate Jaffe method and two different enzymatic methods. Isotope dilution mass spectrometry served as the reference creatinine method. GFR was estimated using both the Modification of Diet in Renal Disease (MDRD) and Cockcroft and Gault formulae: a 51Cr-EDTA GFR estimation served as the reference GFR method. RESULTS: Both enzymatic methods produced creatinine results that were significantly different (P<0.001) from the reference method. The Jaffe method over- and underestimated creatinine at low and high concentrations, respectively. The most likely explanation for these differences relates to standardization of the assays. Irrespective of creatinine method, the Cockroft and Gault formula tended to underestimate GFR, and the MDRD formula to overestimate GFR. Use of the differing creatinine methods to estimate GFR produced predictable biases of the estimate, with mean GFR estimates varying by 14% across the creatinine methods. CONCLUSION: Estimates of GFR depend critically upon the accuracy and precision of the creatinine measurement used in their calculation.

The 24-hour posttransfusion survival of baboon red blood cells preserved in citrate phosphate dextrose/ ADSOL (CPD/AS-1) for 49 days.

The purpose of this study was to evaluate the baboon as an animal model for evaluating red blood cell (RBC) preservation by comparing the 24-h posttransfusion survival of baboon RBCs preserved in citrate phosphate dextrose/ADSOL (CPD/AS-1) solution at 4 degrees C for 49 days to that of human RBCs preserved under similar conditions. CPD/AS-1 originally was approved by the Food and Drug Administration for 49-day storage of RBCs, but this period subsequently was reduced to 42 days. Adult male baboons (Papio anubis and P. cynocephalus) were autotransfused with RBCs that had been harvested using CPD and that had been resuspended and stored in AS-1 solution at 4 degrees C for as long as 49 days. The 24-h posttransfusion survival was measured using the 51Cr/125I-albumin method. The 24-h posttransfusion survival (mean +/- standard deviation) was 74% +/- 7% for seven units of CPD/AS-1-treated RBCs stored for 35 days, 65% +/- 15% for 12 units stored for 42 days, and 43% +/- 16% for seven units stored for 49 days. The mean 24-h posttransfusion survival rate for autologous baboon RBCs stored in CPD/AS-1 at 4 degrees C for 35 days (74%) was similar to that for autologous human RBCs stored in a similar manner. Further storage for 42 and 49 days resulted in lower values for baboon RBCs compared with human RBCs.

Role of inducible bronchus associated lymphoid tissue (iBALT) in respiratory immunity.

Bronchus-associated lymphoid tissue (BALT) is occasionally found in the lungs of mice and humans; however, its role in respiratory immunity is unknown. Here we show that mice lacking spleen, lymph nodes and Peyer's patches generate unexpectedly robust primary B- and T-cell responses to influenza, which seem to be initiated at sites of induced BALT (iBALT). Areas of iBALT have distinct B-cell follicles and T-cell areas, and support T and B-cell proliferation. The homeostatic chemokines CXCL13 and CCL21 are expressed independently of TNFalpha and lymphotoxin at sites of iBALT formation. In addition, mice with iBALT, but lacking peripheral lymphoid organs, clear influenza infection and survive higher doses of virus than do normal mice, indicating that immune responses generated in iBALT are not only protective, but potentially less pathologic, than systemic immune responses. Thus, iBALT functions as an inducible secondary lymphoid tissue for respiratory immune responses.

ISCOMATRIX adjuvant: an adjuvant suitable for use in anticancer vaccines.

Human Papillomavirus type 16 (HPV16) E6 and E7 oncoproteins are associated with cervical cancer development and progression and can therefore be used as target antigens for cancer immunotherapy. In this study we evaluated the immunogenicity in mice, of different vaccine formulations using recombinant HPV16 derived E6E7 or E7GST fusion proteins. When co-administered with ISCOMATRIX adjuvant, these E6E7 proteins consistently induced E7 specific CTL, in vivo tumor protection, antibody and DTH responses. ISCOMATRIX adjuvant has been developed for use in the formulation of novel human vaccines and has been evaluated for safety and toxicity in human trials. A formulation containing aluminum hydroxide (Al(OH)3) gave a lesser degree of E7 specific antibody, and no local E7 specific CTL response but similar DTH and tumor protection. These findings demonstrate the potential of ISCOMATRIX adjuvant to stimulate both cellular and humoral immune responses to endogenously processed target antigens, and hence is the preferred adjuvant when CTL responses are desirable.

Human airway epithelial cells present antigen to influenza virus-specific CD8+ CTL inefficiently after incubation with viral protein together with ISCOMATRIX.

In the present paper, an in vitro model was established in which the interaction between influenza virus-specific CD8+ T cells and human airway epithelial cells can be studied. To this end, the human lung epithelial cell line A549 was transduced with the HLA-A*0201 gene. This MHC class I allele is involved in the presentation of the immunodominant M158-66 cytotoxic T lymphocyte (CTL) epitope of the influenza A virus matrix protein. The A549-HLA-A2 cells and a CD8+ T cell clone specific for the M158-66 epitope were used to evaluate ISCOMATRIX (IMX), which is considered a potential mucosal adjuvant for influenza vaccines, for its capacity to activate virus-specific CTL after incubation with epithelial cells. It was found that virus infected epithelial cells activated virus-specific CTL efficiently. However, incubation of epithelial cells with ISCOMATRIX and recombinant M1 protein activated CD8+ T cells inefficiently, unlike the incubation of C1R cells expressing a HLA-A2 trans gene or HLA-A2+ B-lymphoblastoid cells with these reagents. It was concluded that this lack of antigen presentation by epithelial cells indicate that these cells are not subject to killing by virus-specific CTL upon instillation with ISCOMATRIX-based vaccines, which may be a favorable property of mucosal vaccines.

Selenium has a protective role in caspase-3-dependent apoptosis induced by H2O2 in primary cultured pig thyrocytes.

OBJECTIVE: Hydrogen peroxide (H2O2), necessary for thyroid hormonogenesis, is produced at the apical surface of the thyroid follicular epithelium. Excess H2O2 is potentially cytotoxic and may contribute to the development of hypothyroidism, e.g. in severe selenium deficiency. Yet it is unclear how H2O2 contributes to thyroid cell death. DESIGN AND METHODS: H2O2-induced apoptosis and necrosis were studied in primary cultured pig thyroid cells. Glutathione peroxidase (GPx) activity was altered by culture in low serum with or without selenite substitution. Apoptosis was evaluated by spectrofluorometric measurement of caspase-3-specific substrate cleavage, and by analysis of DNA fragmentation by agarose gel electrophoresis. Necrosis was detected by 51Cr release from prelabeled cells. RESULTS: Exogenous H2O2 dose-dependently (100-400 micromol/l) activated caspase-3 within 3-12 h, and DNA degradation was observed after 24 h. The potency of H2O2 to induce apoptosis was low compared with that of staurosporine, a strong proapoptotic agent. H2O2-treated cells with reduced GPx activity showed increased caspase-3 activation. Incubation of serum-starved cells with selenite (10-100 nmol/l) normalized the GPx activity and reduced the activation of caspase-3 by H2O2. High H2O2 concentrations (400-800 micromol/l) were required to obtain necrosis. The H2O2-induced necrosis was exaggerated by both low GPx activity and catalase inhibition. CONCLUSIONS: Cytotoxic effects of H2O2 on thyroid cells include caspase-3-dependent apoptosis that occurs at H2O2 concentrations insufficient to induce necrosis. Selenium deficiency aggravates the apoptotic response, probably due to impaired capacity of GPx to degrade H2O2.

Co-immunization with plasmid IL-12 generates a strong T-cell memory response in mice.

Plasmid encoded exogenous IL-12 delivered as a DNA vaccine adjuvant has been shown to improve vaccine-induced immunity. In particular, pIL-12 greatly improves antigen (Ag)-specific cytotoxic tlymphocyte (CTL) activity in immunized mice. The longevity of this response has not previously been studied in detail. We have studied the effect of co-immunization with pIL-12 on HIV gp160 and Influenza A Hemeagglutinnin-specific memory immune responses. Mice co-immunized with pIL-12 and plasmid encoded antigens maintained a greater memory response than those immunized with the plasmid antigen alone which could be measured at least 6 months after vaccination. Further, this translated to an improved outcome after challenge of long term rested mice that were previously immunized. The strength of the immune response as well as the number of Ag-specific T-cells is proportional to the number of Ag-specific cells primed by the vaccination regimen.

Construction and characterisation of a candidate HIV-1 subtype C DNA vaccine for South Africa.

A candidate DNA vaccine pTHgagC expressing the immunodeficiency virus-1 (HIV-1) gag gene from South African isolate Du422 was constructed and characterised. The isolate was selected on the basis of being the closest to the South African subtype C consensus sequence. Sequence analysis of cytotoxic T lymphocyte (CTL) epitopes showed that HIV subtype C-infected individuals have CTL responses to a number of epitopes present in the vaccine, but also revealed a more limited presence of subtype A- and any B-derived epitopes. A high level of expression of the immunogen was demonstrated in human cells and a potent, long-lived CTL response to a single inoculation of the DNA vaccine was elicited in BALB/c mice, which could be significantly increased by a boost vaccination at 4 weeks. This is the first candidate HIV-1 DNA vaccine employing the South African subtype C sequences, and constitutes a part of a vaccine scheduled to enter a clinical evaluation in South Africa in 2004.