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The interpretation of cellular infiltrate from renal transplant recipients with borderline (BL) changes is still a challenging problem. To analyze the immune phenotype of such infiltrate, we quantified the mRNA expression of Foxp3 and interleukinL-10 and granzyme B (GB) in 15 kidney biopsies with BL changes. Controls were patients presenting type IA acute rejection and nonrejecting patients. Only levels of GB mRNA correlated significantly with response to antirejection therapy. Levels of Foxp3 mRNA in BL changes were intermediate between type IA acute rejection and nonrejecting controls. To determine the balance of alloagressive to graft-protecting T cells, we quantified the Foxp3/GB ratio. BL changes T cells infiltrate expressed a significantly higher Foxp3/GB ratio than that in IA acute rejection. These results suggest that T cell infiltrate from BL change exhibit a tolerogenic rather than a cytotoxic phenotype.

ABSTRACTA national evaluation study was performed in 14 specialized laboratories with the objective of assessing their capacities to provide (i) hepatitis B virus (HBV) viral loads (VL), (ii) HBV genotypes, and(iii) identification of precore/core mutants. The panel consisted of 12 HBV DNA-positive samples with VLs from 2.8 to 9.1 log10copies/ml, different HBV genotypes (A to F), and 3 mutant and 9 wild-type samples at nucleotide 1896. The coefficients of variation of the mean VLs ranged from 2.4% to 10.4% with the Cobas HBV Monitor assay, from 1.8% to 5.5% with the Cobas TaqMan 48, from 1.5 to 26.2% with RealArt HBV PCR, and from 0 to 7% with branched DNA (bDNA). The Cobas Monitor assay underestimated the VLs of genotype F samples, with differences ranging from 1.4 to 2.4 log10copies/ml. The accuracies of genotype determinations ranged from 33% to 100%, and those of precore mutant determinations ranged from 25 to 100%. This study showed some drawbacks of two widely used assays: (i) Cobas Monitor has a narrow dynamic range and underestimates genotype F sample VLs and (ii) bDNA shows poor sensitivity and may fail to identify patients with low VLs. With higher performance in terms of analytical sensitivity combined with a larger dynamic range and an ability to quantify the main genotypes equally, real-time PCR methods appear more appropriate for accurate monitoring of HBV DNA quantification. Furthermore, the clinical implications of HBV genotyping and the determination of precore/core mutants need to be clearly stated to justify the standardization of these methods.

Cornea graft recipients are exposed to viral transmission from the donor. Cadaveric donor serum is often of poor quality and frequently yields falsely positive results in serological assays that may result in the graft being needlessly discarded.

The clinical outcome and appropriate management for patients showing ‘borderline changes’ on allograft biopsy after renal transplantation is still controversial. In an attempt to identify predictive factors of clinical outcome of patients with such lesions, we reviewed the clinical course of 91 patients with borderline changes. Multivariate analysis revealed significant and independent effects of histological stage (i + t ≤ or > 2) and time to borderline changes (≤ or >3 months after transplant) on serum creatinine levels at 1 year from borderline changes episodes (respectively, p = 0.04 and p = 0.02) and only a significant effect of time to borderline changes on serum creatinine levels at 2 years (p = 0.005). Renal function at 1 year and 2 years as 5- and 8-year graft survival were not significantly different in the group of patients treated with antirejection therapy (T group, n = 49) compared with the untreated group (UT group, n = 42). This study strongly suggests that borderline changes with histological score (i + t) > 2 and late episodes of borderline changes should be considered to be of poor prognosis.

Recurrence of nephrotic syndrome (NS) after renal transplantation for primary focal segmental glomerulosclerosis (FSGS) is a frequent and still unpredictable complication. However, risk factors for recurrence have not yet been clearly identified.

The SFTPB gene indel g.1549C > GAA (121ins2) accounts for about 2/3 of the mutant alleles underlying complete surfactant protein B deficiency. It is unclear, however, whether its prevalence is due to recurrent mutation or a founder effect. The underlying mutational mechanism was therefore sought through the analysis of local DNA sequence complexity. A relatively complex two‐step process was proposed: the first step involving slipped mispairing mediated by a direct repeat and generating an AGAA micro‐insertion, the second step involving hairpin loop resolution resulting in a CA micro‐deletion. The possibility of a founder effect was then assessed by typing 8 intragenic SNPs in 17 independent 121ins2 chromosomes from 10 probands, with parental non‐121ins2 chromosomes serving as controls. The 121ins2 chromosomes were assigned to three discrete haplotypes, whilst control chromosomes were distributed between 10 of the 11 observed parental haplotypes. The 121ins2 mutation was in strong and significant linkage disequilibrium (LD) with the tightly linked marker g.1580T/C (|D'| = 1; P ≈ 0.024), although only moderate LD was found with the rest of the locus (|D'| ≈ 0.54; P ≈ 0.136). Data on haplotype structure and the locus LD pattern, obtained from 81 independent Western‐European chromosomes, were consistent with the three mutation‐bearing haplotypes having originated from a common ancestor by recombination. Interestingly, all families harboring the 121ins2 indel had ancestors from a region of Northwestern Europe populated by Frankish/Saxon migration. Taken together, these data are consistent with the view that an indel mutation occurred on a relatively common SFTPB haplotype and now accounts for the majority of (and possibly all) extant 121ins2 chromosomes. © 2005 Wiley‐Liss, Inc.

Although well documented in children with sickle cell disease (SCD), the incidence, cause, and outcome of bloodstream infection (BSI) are poorly defined in adults with SCD. Through a 5-year retrospective analysis of a cohort of 900 patients followed at our institution, we identified 56 episodes of BSI in 47 patients. The incidence rate of BSI was 1.2 episodes per 100 patient-years. As compared to the patients followed in the cohort, those with BSI were more likely to be younger (p = 0.001), to have Hb-S disease (p = 0.008), severe disease (p = 0.001), or additional immunosuppression (p = 0.05). BSI was hospital-acquired in 46% of cases and mainly associated with venous catheters (41%) and Staphylococcus aureus(34%). Pneumococci were rarely identified (10.7%). Despite an adequate duration of antibiotic therapy, the course of BSI was marked by a high frequency of associated bone-joint infection. Bone-joint infection was noted in 18 patients (32% of episodes) and occurred either during the initial BSI episode (13 patients) or 1-6 months after BSI resolution (5 patients). Factors associated with the occurrence of bone-joint infection were previous osteonecrosis (relative risk, 2.5; 95% confidence interval, 1.2-5.3) and S. aureusinfection (relative risk, 3.8; 95% confidence interval, 1.8-8.4). In conclusion, BSI is a rare event in adults with SCD compared to children. It mainly occurs in those with a severe underlying disease and a venous catheter. These patients have a high risk of associated bone-joint infection and therefore must be closely monitored.

The protein kinase C (PKC) pathway is involved in the maintenance of cell shape and cell integrity in Saccharomyces cerevisiae. Here, we show that this pathway mediates tolerance to low pH and that the Bck1 and Slt2 proteins belonging to the mitogen-activated protein kinase cascade are essential for cell survival at low pH. The PKC pathway is activated during acidification of the extracellular environment, and this activation depends mainly on the Mid2p cell wall sensor. Rgd1p, which encodes a Rho GTPase-activating protein for the small G proteins Rho3p and Rho4p, also plays a role in low-pH response. The rgd1Δ strain is sensitive to low pH, and Rgd1p activates the PKC pathway in an acidic environment. Inactivation of both genes in the double mutant rgd1Δ mid2Δ strain renders yeast cells unable to survive at low pH as in bck1Δ and slt2Δ strains. Our data provide evidence for the existence of two distinct ways, one involving Mid2p and the other involving Rgd1p, with both converging to the cell integrity pathway to mediate low-pH tolerance in Saccharomyces cerevisiae. Nevertheless, even if Rgd1p acts on the PKC pathway, it seems that its mediating action on low-pH tolerance is not limited to this pathway. As the Mid2p amount plays a role in rgd1Δ sensitivity to low pH, Mid2p seems to act more like a molecular rheostat, controlling the level of PKC pathway activity and thus allowing phenotypical expression of RGD1 inactivation.

Allogeneic hematopoietic stem-cell transplantation is a widely used, cost-intensive procedure. Our purpose was to estimate costs and determine cost predictors.

ABSTRACTBefore initiating new large-scale therapeutic trials for hepatitis C virus (HCV)-infected patients, the French Health Authorities for HCV research decided to organize an evaluation of the expertise of laboratories that could be engaged to undertake molecular biology assays in such trials; 21 experienced laboratories participated in this national evaluation of laboratory expertise, which was performed in two successive rounds. The first round evaluated the laboratories for their abilities to detect HCV RNA in serum, determine genotypes, and quantify HCV RNA loads. The results observed by qualitative assays for HCV RNA detection were 100% sensitivity and 100% specificity for all laboratories. The genotyping results were 100% concordant for 9 laboratories and greater than 90% for 10 laboratories. By contrast, large coefficients of variation were observed for quantitative determination of HCV RNA loads, leading to a second round with standardized quantitative assays only. The dispersion of the results was larger by the AMPLICOR HCV Monitor assay than by the branched-DNA assay (mean coefficients of variation, 57.4 and 16.9%, respectively). In the majority of cases, discrepancies between the results of the two tests were found for samples with high viral loads. These results indicate the usefulness of validating, by controlling for expertise, both the reliabilities of laboratories involved in multicenter work and the standardized assays chosen for use in the evaluation of the biological impacts of new therapies.

We have recently established that enhancement of intracellular calcium cycling and contraction in response to β2-adrenergic receptor (β2-AR) stimulation exclusively relies on the activation of the cytosolic phospholipase A2(cPLA2) and arachidonic acid production, via a pertussis toxin-sensitive G protein (possibly Gi), in embryonic chick cardiomyocytes. We aimed to investigate the relevance of the β2-AR/Gi/cPLA2pathway in the human myocardium. In left ventricular biopsies obtained from explanted hearts, β2-AR stimulation exerted either an inhibition of cPLA2that was insensitive to pertussis toxin (PTX) treatment, or an activation of cPLA2, sensitive to PTX treatment. In right atrial appendages from patients who were undergoing open heart surgery, we demonstrated that β2-AR-induced activation of cPLA2was favored in situations of altered β1-AR and/or β2-AR/adenylyl cyclase (AC) stimulations. Alterations were characterized by an increase in EC50value of norepinephrine and a decrease in the maximal AC activation in response to zinterol, respectively. Quantitative reverse transcription-polymerase chain reaction analyses highlighted a positive correlation between the expression of AC5 and AC6 mRNAs in human cardiac atria, which suggested that functional alterations in AC responses were unlikely to be related to changes in the AC5/AC6 mRNA ratio. In addition, the shift from the cyclic AMP to the arachidonic acid pathway was not supported at the transcriptional level by opposite regulation of AC and cPLA2mRNAs expression. This study gives the first evidence of the recruitment of cPLA2by β2-ARs in the human heart and suggests that the Gi/cPLA2pathway could substitute for a deficient Gs/AC pathway in mediating β2-AR responses.

Allocation of organs is organized on a regional basis in France. We assessed regional differences in access to organ transplant.

We have recently established that enhancement of intracellular calcium cycling and contraction in response to beta2-adrenergic receptor (beta2-AR) stimulation exclusively relies on the activation of the cytosolic phospholipase A2 (cPLA2) and arachidonic acid production, via a pertussis toxin-sensitive G protein (possibly Gi), in embryonic chick cardiomyocytes. We aimed to investigate the relevance of the beta2-AR/Gi/cPLA2 pathway in the human myocardium. In left ventricular biopsies obtained from explanted hearts, beta2-AR stimulation exerted either an inhibition of cPLA2 that was insensitive to pertussis toxin (PTX) treatment, or an activation of cPLA2, sensitive to PTX treatment. In right atrial appendages from patients who were undergoing open heart surgery, we demonstrated that beta2-AR-induced activation of cPLA2 was favored in situations of altered beta1-AR and/or beta2-AR/adenylyl cyclase (AC) stimulations. Alterations were characterized by an increase in EC50value of norepinephrine and a decrease in the maximal AC activation in response to zinterol, respectively. Quantitative reverse transcription-polymerase chain reaction analyses highlighted a positive correlation between the expression of AC5 and AC6 mRNAs in human cardiac atria, which suggested that functional alterations in AC responses were unlikely to be related to changes in the AC5/AC6 mRNA ratio. In addition, the shift from the cyclic AMP to the arachidonic acid pathway was not supported at the transcriptional level by opposite regulation of AC and cPLA2mRNAs expression. This study gives the first evidence of the recruitment of cPLA2by beta2-ARs in the human heart and suggests that the Gi/cPLA2pathway could substitute for a deficient Gs/AC pathway in mediating beta2-AR responses.

Posttransplant lymphoproliferative disorder (PTLD) is a well-known complication of organ transplantation that leads to death in more than 50 of cases. The aim of this work was to identify specific risk factors for lymphoproliferative disorders after liver transplantation in adults.

Depuis l’introduction des tests de dépistage, l’épidémiologie de l’hépatite C (VHC) a changé, en raison des mesures prises pour combattre l’infection et du développement de son dépistage. Si, au début des années 1990, la transfusion représentait la cause principale de contamination et la toxicomanie intraveineuse, de développement plus récent, était au second plan, l’inverse est aujourd’hui observé, et la toxicomanie représenterait 70 % des nouvelles séroconversions. Le diagnostic, qui était porté chez des sujets symptomatiques, l’est maintenant plus souvent devant des facteurs de risque, chez des sujets asymptomatiques. Si le pic de l’épidémie VHC semble passé, il reste encore de nombreux malades à diagnostiquer et à prendre en charge et l’on risque de continuer à assister à une augmentation de l’incidence des cirrhoses et des carcinomes hépatocellulaires liés au VHC. Les efforts doivent donc continuer, portant sur l’amélioration du dépistage afin d’assurer une meilleure prise en charge, dans le cadre de réseaux organisés autour des pôles de référence. La prévention doit viser prioritairement la population d’usagers de drogues et comporter des actions menées en milieu carcéral.

OBJECTIVE: To evaluate the response to treatment in a large cohort of patients with systemic lupus erythematosus (SLE) associated with autoimmune thrombocytopenia. METHODS: Response to treatment was assessed retrospectively in 59 patients with SLE, either definite (n = 44) or incomplete (n = 15), associated with frank autoimmune thrombocytopenia (defined as platelet count < 50 x 10(9)/l). Response to treatment was classified as complete (CR: platelet count > 150 x 10(9)/l), partial (PR: platelet count > 50 x 10/l), or failure (FR) in the other cases. RESULTS: Oral prednisone alone was used in 50 of the 59 patients (mean initial dose 1 mg/kg body weight/day). A response was obtained in 80% of cases (CR in 28. PR in 12) but only 11 (22%) had a sustained response (CR, n = 7; PR, n = 4). In contrast, combined treatment with prednisone and either danazol (n = 18) or hydroxychloroquine (n = 11) resulted in 50% (7 CR, 2 PR) and 64% (4 CR, 3 PR) longterm responses, respectively, allowing prednisone to be withdrawn or the dose tapered below 0.2 mg/kg body weight/day. High dose methylprednisolone pulses (n = 10) and intravenous immunoglobulin (IVIG) (n = 31) resulted in positive responses in 60% (4 CR, 2 PR) and 65% (12 CR, 8 PR) of cases, respectively, but the response was transient in each case. Splenectomy (n = 17) resulted in 65% longterm responses (10 CR, 1 PR). Only 2 longterm partial responses were obtained with the 22 immunosuppressant-containing regimens administered to 14 patients. At the end of the study. a response was observed in 52 (88%) patients [CR: 36 (61%), PR: 16 (27%)], mainly as a result of splenectomy or combined treatment with prednisone and either danazol or hydroxychloroquine. CONCLUSION: Longterm remission was obtained in the majority of patients. The major treatments inducing remission were splenectomy and prednisone combined with danazol or hydroxychloroquine.

It is thought that an increase in the adhesion of circulating reticulocytes to the vascular endothelium may initiate the vascular occlusion underlying the painful crises and organ failures typical of sickle cell disease (SCD). At least 2 receptors, usually present on reticulocytes, seem to be involved in this adhesion process: glycoprotein CD36 (glycoprotein IV) and integrin α4β1 (very late activation antigen–4). Recently, a high frequency of the platelet CD36–deficient phenotype was reported in black Africans. The frequency of this deficiency was similar in subjects with and without SCD. The role of CD36 in vaso-occlusion was then investigated by comparing the clinical course in 2 groups of black Africans homozygous for hemoglobin S, with and without CD36 deficiency, but similar in age, sex, geographical origin, number of α-globin genes, and β-globin gene haplotype. Flow cytometry showed that CD36 was absent from the circulating red blood cells and reticulocytes of platelet CD36–deficient individuals but present on those from patients with normal platelet CD36 expression, and that α4β1 integrin levels were similar on the reticulocytes of the 2 groups. Neither clinical severity, as evaluated by the frequency and characteristics of vaso-occlusive events, nor biological data differed significantly in the 2 groups of patients. Finally, although CD36 has been suggested to play a critical role in the pathogenesis of vaso-occlusion, this study, despite including only a small number of patients, supports the idea that the modulation of expression of a single type of adhesion molecule is insufficient to counteract the pathological process leading to vaso-occlusion in SCD patients.

Amplification of the oncogene MYCN in neuroblastoma has been found to correlate with aggressive tumour growth and is used as a predictor of clinical outcome. The MYCN amplicon is known to involve coamplification of extensive DNA regions. Therefore it is possible that other genes are coamplified in this amplicon and that they may play a role in the poor outcome of MYCN amplified tumours. We have implemented an approach for the two-dimensional separation of human genomic restriction fragments to detect and isolate as yet unknown amplified sequences in the MYCN amplicon in neuroblastoma. Using this approach we have recently cloned a novel gene referred to as NAG that is frequently coamplified with MYCN in neuroblastoma. We report here the identification and cloning of two ad-ditional CpG islands that are amplified in neuroblastoma. One contains a sequence that is identical to the first intron of DDX1. The other represents a novel CpG island that is associated with an as yet unidentified gene. We show that the novel CpG island is located in close proximity to the MYCN locus on chromosome 2 and is as frequently coamplified with MYCN in neuroblastoma as NAG and DDX1. Med. Pediatr. Oncol. 36:75–79, 2001. © 2001 Wiley-Liss, Inc.

After obtaining familial informed consent, between January 1996 and July 1997, 173 children (5 to 15 years old) with sickle cell disease were enrolled in a prospective multicenter study using blood screening, transcranial Doppler ultrasonography (n= 143), cerebral magnetic resonance imaging (n= 144), and neuropsychologic performance evaluation (n= 156) (Wechsler Intelligence tests WISC-III, WIPPSI-R), which were also performed in 76 sibling controls (5 to 15 years old). Among the 173 patients with sickle cell disease (155 homozygous for hemoglobin SS, 8 sickle cell β0thalassemia, 3 sickle cell β+thalassemia, 7 sickle cell hemoglobin C disease SC), 12 (6.9%) had a history of overt stroke, and the incidence of abnormal transcranial Doppler ultrasonography (defined as mean middle cerebral artery velocity > 200 cm/sec or absent) was 8.4% in the overall study population and 9.6% in patients with homozygous sickle cell anemia. The silent stroke rate was 15%. Significantly impaired cognitive functioning was observed in sickle cell disease patients with a history of stroke (Performance IQ and Full Scale IQ), but also in patients with silent strokes (Similarities, Vocabulary, and Verbal Comprehension). However, infarcts on magnetic resonance imaging were not the only factors of cognitive deficit: Verbal IQ, Performance IQ, and Full Scale IQ were strongly impaired in patients with severe chronic anemia (hematocrit ≤ 20%) and in those with thrombocytosis (platelets > 500 × 109/L). Multivariate logistic regression analysis showed that abnormal magnetic resonance imaging (odds ratio [OR] = 2.76) (P= .047), hematocrit ≤ 20% (OR = 5.85) (P= .005), and platelets > 500 × 109/L (OR = 3.99) (P= .004) were independent factors of cognitive deficiency (Full Scale IQ < 75) in sickle cell disease patients. The unfavorable effect of low hematocrit has already been suggested, but this is the first report concerning an effect of thrombocytosis and showing that silent stroke alone is not a factor of cognitive deficit when not associated with low hematocrit or thrombocytosis. The effect of hydroxyurea, which is known to increase hematocrit and decrease platelet count, on cognitive functioning of sickle cell patients should be evaluated prospectively. (JChild Neurol 2000;15:333-343).

BACKGROUND: Because GB virus type C(GBV-C)/HGV (GBV-C/HGV) is blood-borne and sexually transmitted, persons at risk of infection with such viruses have a high prevalence of GBV-C/HGV markers. However, adults with no apparent risk factors, such as blood donors, frequently are positive for GBV-C/HGV markers. Mother-to-infant transmission could explain this high prevalence, but it has been studied only through small series of GBV-C/HGV-infected mothers co-infected with HCV or HIV. STUDY DESIGN AND METHODS: To determine the rate of mother-to-infant transmission of GBV-C/HGV RNA in women who are HCV- or HIV-negative, a prospective study was performed in a cohort of 288 mothers screened for viral RNA and in the infants born to GBV-C/HGV-infected mothers. RESULTS: Thirteen mothers (4.5%) were found positive for GBV-C/HGV RNA. Of the infants in whom at least one blood sample was collected between the third and the ninth months of life, 89 percent were positive for viral RNA. The majority of these newborns were negative for GBV-C/HGV RNA at birth and positive after the third month. The viral RNA titers of infants born to GBV-C/HGV-infected mothers appeared as elevated as those of their mothers. All the GBV-C/HGV-infected infants remained positive for viral RNA during the entire study period. No clinical events possibly linked to a primary GBV-C/HGV infection were reported in infants. Serum ALT level and blood count remained within normal values throughout the follow-up of all GBV-C/HGV-infected infants. CONCLUSION: The frequency of mother-to-infant GBV-C/HGV transmission is elevated and could explain the high prevalence of GBV-C/HGV markers (viral RNA and E2 antibody) in adults at low risk for blood-borne or sexually transmitted viruses, such as blood donors.

Little is known about the natural history and the pathogenicity of the TT virus (TTV). We present our findings of a cross-sectional study based on the TTV DNA screening of 173 multiple-transfused patients and a longitudinal study based on the follow-up of TTV DNA–positive patients. Overall, 48 patients (27.7%) tested positive for TTV DNA. The influence of the number of blood donor exposures on the prevalence of blood-borne viral infection indicates that TTV, hepatitis C virus (HCV), and an RNA virus known as GB virus C/hepatitis G virus (GBV-C/HGV) share a parenteral transmission, but that TTV, in contrast to the 2 other viruses, is also transmitted by at least another efficient means. The patients having a well-defined date of TTV infection were positive for TTV DNA during a mean period of 3.1 years. A chronic infection was observed in 31 cases (86%). TTV carriage appeared clinically benign in all patients. No clinical evidence of a disease potentially linked to the TTV infection was observed in patients with TTV DNA carriage over several years. The majority of TTV carriers had no biochemical evidence of liver disease. The prevalence of elevated serum alanine aminotransferase (ALT) level was higher in the TTV DNA–positive group, even in the absence of HCV infection, but the observed peaks of ALT level were most often transient and very mild. The prevalence of TTV DNA observed in blood recipients is consistent with that of TTV infection observed in blood donors. TTV infection frequently tends to persist. (Blood. 2000;95:347-351)

AbstractObjective:To document the costs and the benefits (both in terms of costs averted and of injuries averted) of education sessions and replacement of phlebotomy devices to ensure that needle recapping did not take place.Design:The percentage of recapped needles and the rate of needlestick injuries were evaluated in 1990 and 1997, from a survey of transparent rigid containers in the wards and at the bedside and from a prospective register of all injuries in the workplace. Costs were computed from the viewpoint of the hospital. Positive costs were those of education and purchase of safer phlebotomy devices; negative costs were the prophylactic treatments and follow-up averted by the reduction in injuries.Setting:A 1,050-bed tertiary-care university hospital in the Paris region.Results:Between the two periods, the proportion of needles seen in the containers that had been recapped was reduced from 10% to 2%. In 1990, 127 needlestick (12.7/100,000 needles) and 52 recapping injuries were reported versus 62 (6.4/100,000 needles) and 22 in 1996 and 1997. When the rates were related to the actual number of patients, the reduction was 76 injuries per year. The total cost of information and preventive measures was $325,927 per year. The cost-effectiveness was $4,000 per injury prevented.Conclusion:Although preventive measures taken to ensure reduction of needlestick injuries appear to have been effective (75% reduction in recapping and 50% reduction in injuries), the cost of the safety program was high.

The aim of this study was to establish the mean plasma GBV‐C/ hepatitis G virus (HGV) RNA load in groups of GBV‐C/HGV‐infected individuals with varied immune status and to determine the most frequent patterns of evolution of the plasma GBV‐C/HGV RNA load over time during the natural history of infection. The mean plasma GBV‐C/HGV RNA load observed was, from the lowest to the highest: 5.21 log in immunodepressed multiply‐transfused patients, 6.45 log in HIV‐positive individuals, 6.66 log in immunocompetent multiply‐transfused patients, and 6.71 log in blood donors. The difference was significant between the four groups (P< 0. 0001). The most frequent pattern of evolution of the plasma GBV‐C/HGV RNA load was as follows: after the primary GBV‐C/HGV infection, the viral load was elevated from the onset; then, a high, persistent and relatively steady viral RNA level was the rule; and when it occurred, the loss of viremia was not preceded by a decrease before recovery from GBV‐C/HGV infection. J. Med. Virol. 59:32–37, 1999. © 1999 Wiley‐Liss, Inc.

The aim of this study was to describe the features of posttransplantation tumors observed in a series of liver transplant recipients with special reference to patients receiving a transplant for alcoholic cirrhosis.

Despite a decade of human immunodeficiency virus (HIV) seropositivity, a few individuals termed as long-term nonprogressors (LTNPs) maintain a stable CD4+ T-cell count for a period of time. The aim of this study was to establish, through the sequential determination of all known predictors of HIV disease, the proportion of such patients having stringent criteria of true long-term nonprogression. Among 249 individuals who were HIV-infected and prospectively followed up over a 10-year period (1985 to 1995), 12 having a CD4+ T-cell count greater than 500/µL (LTNP I group) and 9 having a CD4+ T-cell count less than 500 but stable over time (LTNP II group) after at least 10 years of infection without intervention of antiviral therapy, were studied over the entire follow-up period. The plasma HIV RNA copy number and the serum concentrations of p24 antigen, each anti-HIV antibody, neopterin, ß-2-microglobulin, Immunoglobulin (Ig) G and IgA were determined every 18 months over the study period. Cellular and plasma viremias were cross-sectionaly assayed in all 21 patients. Only two patients had strictly no marker of progression over the follow-up period. They were the only ones who had, over the 10-year period, a viral copy number too low to be detected. The other patients had a viral copy number higher than 400/mL at at least one visit and increasing over the follow-up period, and they evidenced one or more markers of virological or immunological deterioration. Cellular viremia was positive in all patients but two, while plasma viremia was negative in all but one. The population of individuals termed as LTNPs is not virologically and immunologically homogeneous. The majority present biological signs of HIV disease progression. A new pattern of true LTNP can be drawn through stringent criteria based on the whole known predictors. This pattern appears to be rare in HIV-positive population.

Myofibroblasts that express α‐smooth muscle actin(α‐SMA) are detected in many chronic inflammatory diseases. Transforming growth factor‐β (TGF‐β) is a potent inducer of myofibroblast accumulation in tissues. In this study, scattered myofibroblasts and TGF‐β were quantified and localized in nasal polyps (NPs) and normal nasal mucosa(NM). NPs were sampled in 16 patients during ethmoidectomy and NM was obtained from 10 control subjects during rhinoplasty. α‐SMA and TGF‐β were detected using immunohistochemistry and the numbers of labeled cells were quantified (α‐SMA and TGF‐β indices) and compared between NPs and NM. In eight NPs, in which the pedicle was preserved, α‐SMA and TGF‐β were evaluated and compared in the pedicle, central, and tip areas. Finally, TGF‐β expression was compared between low (zone 1), moderate(zone 2), and high (zone 3) zones of α‐SMA positivity. α‐SMA and TGF‐β indices were significantly higher in NPs than in NM. In the eight selected NPs, α‐SMA‐positive cells were significantly more abundant in the pedicle than in the central and tip areas, whereas TGF‐β‐positive cells were significantly more numerous in the pedicle than in the tip area. The number of TGF‐β‐positive cells was significantly higher in zone 3 than in zone 1 of α‐SMA positivity. Myofibroblasts, which are abundant in NPs but rare in NM, could be involved in the growth of NPs by inducing extracellular matrix accumulation. The local development of myofibroblasts in NPs could be controlled by TGF‐β, locally produced by inflammatory cells.

Histological features of early mycosis fungoides (MF) can simulate numerous inflammatory lesions and histological confirmation of MF is often delayed, compared with clinical diagnosis. Recently, using molecular techniques, the detection of a dominant T‐lymphocyte clone has been reported in cutaneous lesions of MF. The aim of the present study was to determine the diagnostic value of a dominant T‐lymphocyte clone as assessed by PCR‐DGGE in early MF. Histopathological and molecular analyses were performed on cutaneous lesions from 104 patients clinically suspected as having MF. In this population, the positive predictive value of a PCRγ(+) was 0·86. In addition, four of six patients whose lesions were PCRγ(+) (detectable dominant T‐cell clone) but not histologically MF progressed to MF within 2–48 months. In order to evaluate the relevance of PCRγ–DGGE in MF follow‐up, serial biopsies were performed in 24 patients. In 89 per cent of cases, the presence or absence of a PCRγ(+) was constant during the course of the disease. When present, the DGGE imprint of PCR products was case‐specific. These data demonstrate the diagnostic value in MF of T‐lymphocyte clonality assessed by PCRγ–DGGE on cutaneous lesions and show that the technique can be used in MF follow‐up to evaluate residual disease with high specificity. © 1998 John Wiley & Sons, Ltd.

The crystal structure of the enol silyl ether compound, C28H33NO3Si, 1, has been established by direct methods and refined to a final Rvalue of 0.053 for 1825 observed reflections. The crystals belong to the monoclinic system, a = 8.683(5), b = 12.689(5), c = 24.585(11) Å, β = 105.33(4)°, and the space group is P21/c. The C(sp2)—C(sp2) bond distance is normal but the Si(1)—O(1)—C(1) angle with a value of 132.5(4)° is larger than usual. This is attributed to an interaction between Si(1) and O(2) of the N-acetate group, d(Si … O) = 3.320(4) Å. There is a distortion of the tetrahedral coordination around the silicon atom since the bond angles involving Si vary from 102.8(4) to 112.6(5)°. All three phenyl rings and the N-acetate group are planar. The two phenyls at C(3) are inclined by with respect to the ethylenic group constituted of the two C(sp2) atoms and their four substituents. The molecules are held in the crystal by van der Waals forces only.

The crystal structure of 3′,3″,5′,5″-tetrabromophenolphthalein ethyl ester (TBPE) has been determined by X-ray diffraction at room temperature. This compound crystallizes in space group , with a = 12.361(4), b = 12.533(4), c = 9.986(2) Å, α = 99.57(3), β = 112.76(2), γ = 100.73(3) °, and Z = 2. The structure was solved by direct methods and refined by full-matrix least-squares calculations to a residual of 0.034 for 1862 observed reflections. TBPE is propeller shaped about the central carbon and crstallizes with one molecule of benzene solvent per asymmetric unit. In 1:1 CD2Cl2:CDCl3solution complete 1H and 13C NMR signal assignments have been made via 1H1H COSY and 1H13C HETCOR experiments. The quinoidal and phenolic integrities are retained on the NMR timescale. By contrast for an acetone-d6solution only 14 13C resonances are found and the colour of the solution changed from green to dark blue. These changes in acetone solution are attributed to the presence of a trace of base which induces the loss of the phenolic proton of TBPE and the existence of resonance forms which render the phenolic and quinoidal rings equivalent. Keywords: crystal structure, NMR, 3′3″5′5″-tetrabromophenolphthalein ethyl ester.

Early recommendations on prophylactic transfusion of thrombocytopenic patients involved a standard platelet dose of about 0.5 × 1011/10 kg body weight. Given the lack of data supporting this dose, we prospectively studied the dose response to platelet transfusions in adults and children with hematologic malignancies. Each patient received, in similar clinical conditions, a medium, high, and very high dose of fresh (< 24 hours old) ABO-compatible platelets, in the form of apheresis platelet concentrates (APC). For the adults, the medium dose was defined as APC containing between 4 and 6 × 1011 platelets, the high dose between 6 and 8 × 1011, and the very high dose > 8 × 1011; for the children, the three doses corresponded to 2 to 4, 4 to 6, and > 6 × 1011 platelets. The end points were the platelet increment, platelet recovery, and the transfusion interval, and the results were compared with a paired t-test. Sixty-nine adults and 13 children could be assessed. Recoveries in the adults were similar with the three doses (from 28% to 30%), but the high and very high doses led to a significantly better platelet increment (52 and 61 × 109/L, respectively) than the medium dose (33 × 109/L, P < .01). The main difference was in the transfusion interval, which increased with the dose of platelets transfused, from 2.6 days with the medium dose to 3.3 and 4.1 days with the high and very high doses, respectively (P< .01). The positive effect of the high dose was observed regardless of pretransfusional clinical status, but was more marked in patients with no clinical factors known to impair platelet recovery. In these patients, a platelet dose of 0.07 × 1011 per kg of body weight led to a transfusion interval of more than 2 days in 95% of cases. In patients with clinical factors favoring platelet consumption, the proportion of transfusions yielding an optimal platelet increment and transfusion interval increased with the dose of platelets.The platelet dose-effect was also significant in the children, in whom the high and very high doses led to 1.5-fold to twofold higher posttransfusion platelet counts and transfusion intervals. We conclude that transfusion of high platelet doses can reduce the number of platelet concentrates required by thrombocytopenic patients and significantly reduce donor exposure. © 1998 by The American Society of Hematology.

The preparation of o-nitrophenylethylene glycol is described along with its application as a photolabile protective group for aldehydes and ketones. Formation of the acetals and ketals is achieved in good to high yields in the usual manner and deprotection is carried out in fair to high yield, by photolysis at 350 nm in an inert solvent such as benzene. Because of the particular nature of the present protective group, its stability to basic and acidic conditions has been examined and is reported to complete the scope and limitations aspect. From a mechanistic point of view, the isolation and characterization of o-nitroso-α-hydroxyacetophenone as the spent reagent demonstrates a mechanistic link with the known o-nitrobenzaldehyde to o-nitrosobenzoic acid photorearrangement.

5-Fluoro-3-[3-[4-(5-methoxy-4-pyrimidinyl)-1-piperazinyl]propyl]-1H-indole dihydrochloride (1) facilitates 5-HT neurotransmission and was an antidepressant drug candidate. The development of a safe, rugged process for the large-scale, chromatography-free preparation of this compound is described. The main areas of optimization included a Fischer indole synthesis, preparation and chlorination of a monohydroxypyrimidine, and coupling of the resultant fragments to prepare the drug substance.

OBJECTIVE: To detect, quantify, and compare respiratory epithelial cell proliferation in nasal mucosa and polyps from patients with nasal polyposis. DESIGN: Cohort study. SETTING: Patients and samples were selected at the Hôpital Intercommunal de Créteil (France). Flow cytofluorometry and immunohistochemistry were performed at Hôpitaux Tenon and Mondor (Université Paris [France] VI et XII). PATIENTS: Twenty-one patients undergoing endoscopic ethmoidectomy for treatment of nasal polyposis. METHODS: In 10 cases, epithelial cells were removed from frozen inferior turbinate mucosa and polyps by mechanical disaggregation and were then analyzed by flow cytofluorometry, providing the cell DNA content (propidium iodide labeling) and the percentage of S-phase cells. In 11 cases, inferior turbinate mucosa and polyps were fixed in formaldehyde and embedded in paraffin. Proliferating cell nuclear antigen expression in the epithelium was quantified by immunohistochemistry: a proliferating cell nuclear antigen index was calculated for each sample in the basal area, suprabasal area, and full height of the epithelium. RESULTS: All cell populations studied were diploid, and percentages of S-phase cells were significantly higher in nasal polyps than in mucosa. Proliferating cell nuclear antigen indexes were significantly higher in nasal polyps than in the suprabasal area and full height of the mucosal epithelium. CONCLUSION: Cell proliferation is increased in epithelium from nasal polyps. Epithelial damage caused by inflammatory mediators could induce this increased cell proliferation via epithelial repair processes. Inflammatory cells could up-regulate epithelial cell proliferation by secreting growth factors.(Arch Otolaryngol Head Neck Surg. 1996;122:432-436)

The two-dimensional (2-D) separation of genomic digests has provided the means to analyze over 2000 unique restriction fragments simultaneously in a single gel, for genetic variation as well as for genomic alterations in cancer. By utilizing different combinations of restriction enzymes or different electrophoretic conditions, the number of analyzable fragments in multiple 2-D patterns can be augmented. We have previously shown the feasibility of distinguishing between spot intensities representing fragments from one allele and from two alleles and have implemented approaches for the cloning of fragments of interest in 2-D gels. In this study, the 2-D separation and cloning of chromosome 1NotI–EcoRV-derived genomic fragments was performed. Three hundred forty-sixNotI fragments in whole genomic preparations were assigned to chromosome 1. To verify the reliability of the assignment, two of theNotI fragments attributed to chromosome 1 were cloned and sequenced. The fragments that contained CpG islands were mapped by FISH to 1p35–p36.1 and to 1p13.3–p21, respectively. Our study indicates the feasibility of analyzing 2-D separations of whole genomic digests for the detection of alterations in specific chromosomes. The large number of restriction fragments attributed to chromosome 1 provides the means to screen 2-D patterns for chromosome 1 deletions and amplifications with a high marker density.

To assess the effects of endothelin 1 (ET) on the pulmonary and systemic vascular beds simultaneously, we examined the hemodynamic responses to ET in awake calves implanted with a Jarvik total artificial heart (TAH), a device that maintains constant cardiac output (CO). During basal conditions, successive incremental intravenous (i.v.) injections of 1, 3, and 10 μg ET caused a dose-dependent decrease in pulmonary arterial pressure (PAP), (from 24 ± 3 to 15 ± 1 mm Hg, p < 0.05) while having no effect on systemic arterial (SAP), left atrial (LAP), and right atrial (RAP) pressures. Administration of 30 μg ET i.v. also decreased PAP, had no effect on LAP and RAP, but increased SAP from 100 ± 6 to 118 ± 4 mm Hg (p < 0.05). The decrease in PAP was rapid, occurring within seconds and lasting 10 min, whereas the increase in SAP occurred after 2–5 min and was prolonged for ≥20 min. As compared with injection in the right atrium, administration of 30 μg ET into the left atrium reduced PAP to a similar extent, but induced a greater increase in SAP (32.5 ± 4 vs 17.5 ± 2 mm Hg, p < 0.05). ET also dose-dependently reversed the acute pulmonary vasoconstriction induced by inhalation of an hypoxic gas mixture. In all cases, pulmonary vasodilation occurred without evidence of short-term tolerance. The results demonstrate that ET is a potent in vivo pulmonary vasodilator. In calves, the predominant hemodynamic response to ET is pulmonary vasodilation, with systemic vasoconstriction apparent only at higher concentrations of the peptide.

OBJECTIVE: To determine the relationship of p53 mutations in advanced laryngeal carcinomas to p53 immunohistochemistry, organ preservation, and patient survival. DESIGN: Paraffin-embedded tumor specimens were obtained from patients enrolled in the Department of Veterans Affairs Laryngeal Cancer Cooperative Study, a multi-institutional randomized clinical trial comparing induction chemotherapy (cisplatin and fluorouracil) plus radiation therapy with surgery plus postoperative radiation therapy. Tumor specimens were analyzed for p53 mutations in exons 5 through 8 by using single-strand conformational polymorphism (SSCP) analysis followed by DNA sequencing of all variants. Fiveyear follow-up data were available for all patients studied. SUBJECTS: Forty-four patients enrolled in the Department of Veterans Affairs Laryngeal Cancer Cooperative Study from whom paraffin-embedded tumor specimens were readily available. RESULTS: p53 immunostaining did not correlate with p53 SSCP and DNA sequencing results. More than half (62% [16/26]) of the tumors that overexpressed p53 immunohistochemically did not have a detectable p53 gene mutation. Similarly, 39% (7/18) of tumors that did not overexpress p53 did have a p53 gene mutation. p53 mutations were present in 39% of tumors tested. Mutations within exon 5 made up 41% of p53 gene mutations in laryngeal carcinomas. Transitions were the most common type of mutation in this study (92% of mutations). CONCLUSIONS: The presence of a p53 mutation as detected by SSCP is associated with decreased patient survival. Further study is required to confirm this relationship and to determine whether specific p53 mutations predict organ preservation.Arch Otolaryngol Head Neck Surg. 1997;123:605-609

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BACKGROUND & AIMS: Dual infection by hepatitis GB virus type C (GBV-C) and hepatitis C virus (HCV) is common. To assess the histopathologic impact of GBV-C infection on liver lesions, liver biopsy specimens of 105 patients chronically infected with HCV, 17 of whom (15%) were also infected with GBV-C, were reviewed. METHODS: Semiquantitative histopathologic assessment of liver lesions was performed using the Knodell's score and the METAVIR grading system. RESULTS: Hepatitis activity was mild, moderate, or severe in 3 (18%), 11 (64%), and 3 (18%) patients, respectively, infected with GBV-C and HCV vs. 26 (29%), 56 (64%), and 6 (7%) patients, respectively, infected with HCV alone (no significant difference). Cirrhosis was present in 4 (24%) coinfected patients vs. 19 (22%) HCV-positive patients (no significant difference). No significant difference in fibrosis, presence of portal lymphoid aggregates, steatosis, and hemosiderosis was observed between the two groups. There was no significant difference in the evaluation of each item of the Knodell's score. CONCLUSIONS: This detailed histopathologic evaluation of GBV-C infection in chronic hepatitis C shows that GBV-C infection does not affect histopathologic severity and characteristics of chronic hepatitis C, thus suggesting a minor role of GBV-C infection in liver disease. (Gastroenterology 1997 Jan;112(1):188-92)

Sixty-six adults with chronic autoimmune thrombocytopenic purpura AITP and platelet count &lt;50 x109 l were treated with dapsone (75-100 mg orally). A response was observed in 33 patients. The median duration of treatment required to obtain a response was 21 d (range 8-90). The median maximal platelet count on treatment was 130 x 109 l (range 71-355). Dapsone was continued in 20/33 responders for a median of 12.5 months (range 1-48) and the response persisted in 19. Treatment was intentionally withdrawn in the other 13 responders and thrombocytopenia immediately recurred in 12. Reversible side-effects required cessation of treatment in seven patients. These results demonstrate that dapsone is an effective, inexpensive, and well-tolerated treatment for chronic AITP.

To limit the ordering of serum tumour marker tests that are clinically irrelevant, we designed a new request form. This is a matrix of boxes, rows being individual markers and columns body organs. The box at the intersection of line and column is colour-coded to indicate whether the test is appropriate or not. These ratings were allocated on the basis of published data and local consensus. This new form replaced the form in use at our hospital at the beginning of 1992, and resulted in a 25% decrease in the ordering of tumour markers. Adapting the design of the test request form is a low-cost intervention that combines both economic and clinical requirements in reducing the prescription of tests that are useless in a given clinical situation.

Background/Aims:Because the effects of interferon in the presence and absence of cirrhosis are still debated in chronic active hepatitis type non-A, non-B, C (NANB/ C), the aim of this study was to determine to what extent the presence of cirrhosis influences the response to interferon. Methods:We compared the response to interferon alfa in 108 patients with chronic active hepatitis NANB/C with or without cirrhosis. The patients were randomly assigned to one of the two regimens: one group received 6 months of interferon 3 MU 3 times weekly, while the second group received a 12-month course, 3 MU three times weekly during the first 6 months, 2 MU for the following 3 months, and 1 MU for the last 3 months. Results:In both regimens, the proportion of patients with normal alanine aminotransferase at the end of treatment was significantly lower in the cirrhotic than in the noncirrhotic patients. In males, abnormal serum alkaline phosphatase levels and gamma glutamyl transpeptidase were also related to a lesser response independent of cirrhosis. The response at the end of treatment was not significantly different between the two regimens in either the cirrhotic or the noncirrhotic patients. However, the 12-month regimen gave a significantly higher rate of sustained response 6 months after the end of treatment in patients without cirrhosis. Conclusions:It is suggested that the presence of cirrhosis markedly reduces the rate of response to interferon and that in noncirrhotic patients, a 1-year treatment regimen could improve the beneficial effect of interferon.

BACKGROUND:The first epidemiologic evidence of GB virus type C (GBV‐C)/hepatitis G virus (HGV) infection showed a high prevalence of asymptomatic carriers in blood donors and in populations at risk for blood‐borne viruses. However, by using only viral RNA polymerase chain reaction, those studies underestimated the true spread of GBV‐C/HGV infection. The combined detection of GBV‐C/HGV RNA and of anti‐E2 (which reflects recovery from infection) is necessary to define accurately the prevalence of GBV‐C/HGV.

We have implemented an approach for the detection of DNA alterations in cancer by means of computerized analysis of end-labeled genomic fragments, separated in two dimensions. Analysis of two-dimensional patterns of neuroblastoma tumors, prepared by first digesting DNA with the methylation-sensitive restriction enzyme Not I, yielded a multicopy fragment which was detected in some tumor patterns but not in normal controls. Cloning and sequencing of the fragment, isolated from two-dimensional gels, yielded a sequence with a strong homology to a subtelomeric sequence in chimpanzees and which was previously reported to be undetectable in humans. Fluorescence in situ hybridization indicated the occurrence of this sequence in normal tissue, for the most part in the satellite regions of acrocentric chromosomes. A product containing this sequence was obtained by telomere-anchored PCR using as a primer an oligonucleotide sequence from the cloned fragment. Our data suggest demethylation of cytosines at the cloned Not I site and in neighboring DNA in some tumors, compared with normal tissue, and suggest a greater similarity between human and chimpanzee subtelomeric sequences than was previously reported.

We constructed an avian leukosis virus-based packaging cell line, pHF-g, containing Rous-associated virus DNA with several alterations to abolish RNA packaging. One of them is a 52-base-pair deletion encompassing the putative encapsidation signal in the leader region. The 3' long terminal repeat was also removed and replaced by the polyadenylation sequence from the herpes simplex virus thymidine kinase gene. When pHF-g cells were transfected by an avian leukosis virus-based vector, they produced replication-defective virus at high titer but they did not release any replication-competent particles. Proviral DNA was shown to be correctly integrated as well as correctly expressed. Viral RNAs were shown to be correctly translated into gag-related polypeptides.