A new AQP1 null allele identified in a Gypsy woman who developed an anti-CO3 during her first pregnancy.

BACKGROUND AND OBJECTIVES: The Colton blood group antigens are carried by the AQP1 water channel. AQP1(-/-) individuals, also known as Colton-null since they express no Colton antigens, do not suffer any apparent clinical consequence but may develop a clinically significant alloantibody (anti-CO3) induced by transfusion or pregnancy. Identification and transfusion support of Colton-null patients are highly challenging, not only due to the extreme rarity of this phenotype, the lack of appropriate reagents in most laboratories, as well as the possibility of confusing it with the recently described CO:-1,-2,3,-4 phenotype where AQP1 is present. This study investigated a new Colton-null case and evaluated three commercially available anti-AQP1s to identify Colton-null red blood cell samples. METHODS: The Colton-null phenotype was investigated by standard serological techniques, AQP1 sequencing, immunoblot and flow cytometry analyses. RESULTS: We identified and characterized the Colton-null phenotype in a Gypsy woman who developed an anti-CO3 during her first pregnancy. After developing a simple and robust method to sequence AQP1, we showed that she was apparently homozygous for a new AQP1 null allele, AQP1 601delG, whose product is not expressed in her red blood cells. We also established the Colton specificity of three commercially available anti-AQP1s in immunoblot and/or flow cytometry analyses. CONCLUSION: This Gypsy woman represents the sixth Colton-null case characterized at the serological, genetic and biochemical levels. The validation here of new reagents and methods should facilitate the identification of Colton-null individuals.

Legionella pneumophila inhibits superoxide generation in human monocytes via the down-modulation of alpha and beta protein kinase C isotypes.

Legionella pneumophila may subvert monocyte defenses by several mechanisms including the inhibition of phagosome-lysosome fusion or the impairment of oxidative metabolism. We have investigated the effect of L. pneumophila Knoxville 1, a virulent strain that does not inhibit phagosome-lysosome fusion, on the oxidative responsiveness of human monocytes. Infection of monocytes with L. pneumophila for 48 h resulted in marked inhibition of superoxide generation stimulated by phorbol myristate acetate (PMA) but not by zymosan, a particulate agonist. Evidence is provided that L. pneumophila interfered with the transductional pathway (i.e., protein kinase C, PKC) leading to activation of the NADPH oxidase in monocytes. The phosphorylation of 34-, 48-, 62-, 68-, and 80-kDa proteins stimulated by PMA was markedly inhibited in infected monocytes. In addition, the expression of both alpha and beta PKC isotypes was partially inhibited in infected monocytes. Taken together, our data suggest that the down-modulation of PKC isotypes plays a role in the inhibition of PMA-stimulated superoxide generation.

Q fever and HIV infection.

OBJECTIVE: To study the frequency of Q fever in HIV-infected individuals. DESIGN: A seroprevalence study. SETTING: French National Reference Centre for Rickettsial Agents, Marseille, France. PATIENTS AND METHODS: Five out of the 68 hospitalized cases of Q fever diagnosed in 1987-1989 were also HIV-infected and are described here. Sera from a blood-donor bank (n = 925) and from HIV-positive individuals selected at random, irrespective of clinical or immunological status (n = 500) were tested for Q fever. RESULTS: Comparisons of the two groups showed a statistically significant difference (2.4 versus 0.8%; Fisher's exact test) at the diagnostic dilution 1:200 and at the dilution considered positive for seroprevalence study (1:1000). CONCLUSIONS: Using the estimated incidence of HIV infection in Marseille, the number of Q fever cases in 1987-1989 was 13 times higher and the clinical expression more frequently symptomatic in the HIV-positive population than in the general one. The prevalence:seroprevalence ratio for Q fever was 1.37% in the HIV-positive group and 0.36% in the blood-donor group. Sera positive for Q fever were confirmed by Western blot analysis in order to minimize cross-reaction. Transmission of Q fever appears to be more frequent in HIV-positive individuals than in the general population; this is not surprising, since Coxiella burnetii lives in the phagolysosome, like other micro-organisms described in immunocompromised hosts. Q fever should be added to the spectrum of diseases that occur more frequently during HIV infection.

Detection of gastrin mRNA in fresh human colonic carcinomas by reverse transcription-polymerase chain reaction.

To investigate the hypothesis that gastrin might be synthesized by tumour tissues in cancer of the colon, samples from six human colon tumours, one hepatic metastasis, four normal colonic mucosal samples and two antral and one fundic gastric mucosal samples from nine patients were analysed to determine whether gastrin mRNA was present. RNA was extracted from surgical specimens by ultracentrifugation on a CsCl cushion, purified using the guanidinium thiocyanate method, reverse-transcribed and amplified by polymerase chain reaction. Gastrin mRNA was detected in each colonic carcinoma sample (including the hepatic metastasis), while no such signal was observed in normal colon biopsies. Positive and negative controls (gastric antrum and fundus respectively) gave the expected results. In each of the positive samples, the chemiluminescent revelation of amplified products after Southern blotting corresponded to gastrin mRNA without the intron. These findings demonstrate the ability of primary and metastatic human colonic tumours to produce gastrin mRNA. Since malignant cell lines have been reported to produce gastrin peptide, and since gastrin receptors were present in some cases, our results support the idea that gastrin may be involved in an autocrine mechanism.

Cytofluorometric detection of human endothelial cells in whole blood using S-Endo 1 monoclonal antibody.

It has long been debated whether endothelial cells are present at very low frequency in peripheral blood. Elevated concentrations of such circulating cells may represent a good marker of vascular injury. We have therefore designed an immunocytometric assay for the detection of rare endothelial cells in whole blood. This assay is based on a new monoclonal antibody (MAb) S-Endo 1, made against human umbilical vein endothelial cells (HUVEC) and specific for endothelial cells of various origins without detectable reactivity with blood cells. First, the sensitivity of the assay was established by using normal blood samples with admixed HUVEC as an in vitro model. A good correlation was obtained between added and counted endothelial cells; the recovery was greater than 90% and the minimum detectable concentration of HUVEC was about 0.2 cells/microliters whole blood. Using this rapid counting technique, no detectable levels of endothelial cells were found in the blood of normal individuals (CE less than or equal to 0.1 cells/microliters) while elevated concentrations (up to 8 cells/microliters) were detected in a human model of vascular injury corresponding to a traumatic venepuncture. Thus, this new whole blood immunocytometric assay using S-Endo 1 MAb may be useful in determining the levels of circulating endothelial cells in vascular disorders.

Epidemiologic features and clinical presentation of acute Q fever in hospitalized patients: 323 French cases.

PURPOSE: To contribute to the knowledge of epidemiologic and clinical features of patients hospitalized with Q fever in France. METHODS: We conducted a retrospective analysis of 22,496 sera submitted between 1982 and 1990 to the French National Reference Center for Rickettsial Diseases (NRC). The diagnosis of acute Q fever was based on an IgG titer greater than or equal to 1:200 and an IgM titer greater than or equal to 1:25 against phase II Coxiella burnetii antigen on an indirect immunofluorescence test (IFA). Fifteen cases prior to 1985 were diagnosed on the basis of a complement fixation titer greater than or equal to 1:8. A serosurvey of blood donors from Marseille was also conducted in 1988 on 924 sera, using IFA with a cutoff titer of 1:25. RESULTS: The serosurvey conducted in 1988 showed a seroprevalence of 4.03%, without age or sex prediction. The incidence rate of acute Q fever detection at the NRC was 0.58 per 100,000 inhabitants over the 9-year period. Three hundred twenty-three clinical cases were diagnosed, rising from 1 in 1982 to 107 in 1990. In patients hospitalized for acute Q fever, there was a significantly higher sex ratio of males to females (2.3), which, coupled with the age distribution, indicated that elder males, who are overrepresented due to our recruitment bias, are more susceptible to C. burnetii infections. The mean age of the patients was 45.5 years, while the risk was increased in the 30 to 39 age group as well as in the 60 to 69 age group. Usual epidemiologic risk factors were found in 20.1% of the cases. Hepatitis (61.9%) was a more common clinical presentation in our patients with Q fever than pneumonia (45.8%). This might reflect differences in strains of C. burnetii or the biology of the host. However, French farmers and stock breeders commonly drink unpasteurized raw milk from their cattle, which might indicate a relationship between hepatitis and infection via the digestive tract. CONCLUSION: Our results indicate that many cases of acute Q fever are undiagnosed. A greater awareness of the disease and more extensive serologic testing of patients with symptoms compatible with Q fever may improve the situation.

Detection of gastrin mRNA in paraffin-embedded samples of normal antral mucosae using polymerase chain reaction technique.

Gastrin, a peptide hormone produced by the G cells of the gastric antrum, plays a major role in the regulation of digestive mucosal growth. Although some light has been shed on the peptidic aspects of this hormone's mode of action and the co-regulatory activity in which it is involved along with the other gastrointestinal hormones, little is known at present about the modes of expression of its mRNA at the tissue level. A few attempts have been made so far to detect the transcript, mostly using molecular hybridization techniques. Here it was proposed to detect gastrin mRNA using a RT-PCR technique on a series of paraffin-embedded samples of normal human antrum processed with various fixatives commonly used in histology. The transcript was detectable in all the 7-microns sections of the samples treated with either formalin or Carnoy's solution, whereas Bouin's solution, which is also used as a fixative in histology, was found to have inhibitory effects on the method described here.

Mediterranean spotted fever in Marseille, France: correlation between prevalence of hospitalized patients, seroepidemiology, and prevalence of infected ticks in three different areas.

We report the results of a comparison of several epidemiologic and ecologic parameters affecting the incidence and seroprevalence of Mediterranean spotted fever (MSF) in northern, central, and southeastern Marseille, an area endemic for this disease. In northern Marseille, the incidence of hospitalized patients with MSF was 24.2/100,000 persons compared with 9.8/100,000 and 8.8/100,000 for the central and southeastern regions, respectively. The seroprevalence in sera from blood donors, determined by microimmunofluorescence and confirmed by Western blot assays, was higher in the northern region than in the other two areas (6.7% versus 3.6% and 2.4%, respectively). This higher prevalence of MSF in the northern part of the city may be related to a greater tick exposure due to a higher number of dogs (32.6/100 inhabitants versus 28.4/100 and 27.2/100 in the central and southeastern regions, respectively) and a higher rate of infection of dogs in the northern region (51.4% versus 43.5% and 39.9%, respectively). The ratio of spotted fever group rickettsia-infected ticks was similar in both the northern and southeastern areas (14.8% and 13.4% respectively), but lower in the central area of the city (8.9%), leading to a higher risk of having MSF after a tick bite in the northern and southeastern parts of Marseille.

Is hepatitis C virus-RNA detection by nested polymerase chain reaction clinically relevant in hemodialysis patients?

We have prospectively studied in hemodialysis (HD) patients the evolution of hepatitis C virus (HCV) viremia and the putative relationships between the viremia and the biological markers of liver disease. For each of 22 HD patients having detectable antibodies to HCV (anti-HCV+), we looked four times for serum HCV-RNA by nested PCR (N-PCR), in April and November 1992, November 1993 and May 1994. We checked the transaminases (Trans) and the gamma glutamyl transpeptidase (gamma(GT)) levels on the same day as blood tests for the N-PCR. Abnormal Trans or gamma(GT)++ values were considered if they exceeded the upper limit of the normal level for our laboratory. Fifteen patients (68%) were intermittently N-PCR positive (N-PCR+): 3 patients were N-PCR+ at three determinations, 7 were N-PCR+ at two determinations and 5 only one time. Two patients (9%) were always N-PCR+ and five (23%) always negative. No correlation between an abnormal value of either Trans or gamma(GT) and viremia was evidenced at successive determinations. In conclusion, the majority (68%) of the anti-HCV+ patients had intermittent HCV N-PCR+. Among the anti-HCV+ patients, 77% were viremic. Since HCV viremia is often transitory and since there is no correlation between N-PCR positivity and the increase in Trans or gamma(GT) activities, HCV-RNA detection by N-PCR is probably not clinically relevant in anti-HCV+ HD patients.

Analysis of ELISA hepatitis C virus-positive blood donors population by polymerase chain reaction and recombinant immunoblot assay (RIBA). Comparison of second and third generation RIBA.

A new RIBA-3 (Chiron-Ortho Diagnostic System) was performed for discriminating uninterpretable results of RIBA-2. Recognition of antibodies to hepatitis C virus by RIBA-2 and RIBA-3 was compared among 95 ELISA-2 (second generation ELISA) positive blood donors and correlated with alanine-aminotransferase (ALAT) levels and viremia, using polymerase chain reaction (PCR). These studies led to three important conclusions. First, all ELISA-2-positive, RIBA-2-positive and ALAT-positive samples were found viremic compared with 73% of ELISA-2-positive, RIBA-2-positive and ALAT-negative samples. Then, the comparison of the different RIBAs allowed to conclude that RIBA-3 was more sensitive but less specific than RIBA-2. RIBA-3 was interesting to discriminate undetermined RIBA-2, owing to an improved specificity of C100-3 antigen. In fact, most of the C100-3 positive, RIBA-2 undetermined samples became RIBA-3 negative whereas C22-3 positive, RIBA-2 undetermined samples became RIBA-3 positive or undetermined. Finally, a significant correlation was found between the presence of antibodies against C33-c antigen and viremia.

[New developments of antiplatelet drugs]. / Nouveaux développements des médicaments antiplaquettaires.

A better knowledge of platelet activation mechanisms has made it possible to develop antiplatelet agents that are capable of inhibiting primary haemostasis at very precise levels. Many of these agents block the synthesis or receptor of an hemostasis I agonist. Thus, the thromboxane A2 receptor can be blocked, or its synthesis can be interrupted, by thromboxane synthetase inhibitors, by cyclooxygenase inhibitors, or by omega 3 fatty acids which are competitive inhibitors. Inhibitors of thrombin (hirudin), PAF acether and serotonin (ketanserin) also are available. Other antiplatelet agents secreted by endothelial cells act as haemostasis I antagonists by elevating platelet cAMP or cGMP levels (prostacyclins and analogues, nitrate derivatives). Monoclonal antibodies and RGD peptides directly inhibit the glycoproteins that are responsible for platelet adhesion or aggregation, but their users are faced with problems of cost and route of administration. Of all these new antiplatelet agents, only ticlopidine, which has an imperfectly known mode of action, has proved effective in multiple situations, but its use is limited by its side-effects.

[Exploration of fetal erythropoiesis]. / Exploration de l'érythropoïèse foetale.

Rat embryos (8-14 days) were fixed, included and cut. Others in all cases from the same litter, were crushed on smears, in toto or after dissection. A comparative study of the two types of samples is necessary to obtain good results. Primitive erythropoietic stem cells seem to arise by the extra-endothelial way in the yolk sac mesodermic layer. They disappear when blood islands are connected to the vascular net. Primitive red cells, Perls-positive, are always intravascular in hepatic buds, normoblasts always extravascular. When grown on a medium containing hematopoietic factors, primitive (megaloblastic) cells remain megaloblastic and mature, as do the (normoblastic) definitive red cells. On a poor culture medium (Parker 199) normoblasts do not become megaloblastic. The two series, yolk sac primitive and hepatic definitive red cells, seem to proceed from individual cellular clones.

[HLA-DRB1 and HLA-DQB1 polymorphism in Algerians from Algiers]. / Polymorphisme HLA-DRB1 et HLA-DQB1 dans la population algérienne originaire d'Alger.

The polymorphism of HLA-DRB1 and HLA-DQB1 genes in 100 unrelated Algerians from Alger was investigated using PCR amplification and oligonucleotide typing. Compared to western Europeans, this population shows a higher haplotypic frequency of HLA DRB1*03-DQB1*0201 (21.5%) and a lower haplotypic frequency of DRB1*0101-DQB1*0501 (2%). Two unexpected haplotypes are observed: DRB1*07-DQB1*0301 and DRB1*0406-DQB1*0402. DRB1*0402 is the most common subtype in the DRB1*04 group. Furthermore, we detected a rare DQB1*0305 allele, only found in a Sardinian subject until today.

Neonatal thrombocytopenia in HLA-DR, -DQ, -DP-typed mother due to rare anti-HPA-1b (PLA2) (Zwb) fetomaternal immunization.

A new case of rare neonatal alloimmune thrombocytopenia, due to an IgG anti-HPA-1b in a mother HPA-1 (a+, b-), was diagnosed using monoclonal antibody-specific immobilization of platelet antigens. Clinically, it was similar to the 2 previously reported observations and confirmed that, in this particular case of anti-HPA-1b, the treatment with random platelet pools may be as effective as selected single-donor platelet units when maternal platelets are unusable. The HLA-DR, -DQ, -DP genotypes of the family were obtained by PCR-SSO. The mother's typing, compared to the HLA-DR of the 6 similar cases reported in Europe, suggests that a combined effect of two rare HLA haplotypes might enhance this immunization.

Detection of hepatitis C infection by polymerase chain reaction among hemodialysis patients.

One hundred forty-five patients on regular hemodialysis (HD) at our institution were evaluated for the presence of hepatitis C virus (HCV) infection. Forty-three patients (29%) were found to have detectable antibodies to HCV using second-generation enzyme-linked immunosorbent and recombinant immunoblot assays. Forty positive patients (anti-HCV+) and 10 negative patients (anti-HCV-) were tested for direct detection of the HCV genome by the polymerase chain reaction (PCR). Twenty-one anti-HCV+ patients (52%) had detectable RNA HCV in plasma (PCR+). No anti-HCV- patient had viremia. In addition, we compared the 43 anti-HCV+ patients with the 102 anti-HCV- patients for duration of HD, history of blood transfusion, serologic markers of hepatitis B virus, and acute and chronic liver disease. On retrospective univariate analysis, statistically significant associations with anti-HCV+ were duration of HD (P = 0.0001), blood transfusions (P = 0.0005), co-infection with hepatitis B virus (P = 0.01), and acute and chronic liver disease (P = 0.06 and 0.01, respectively). Three significant variables (duration of HD, chronic hepatitis, and blood transfusions) of the multivariate analysis permit the classification of 65% of anti-HCV+ patients and 81% of anti-HCV- patients. In the anti-HCV+ group, when the same parameters were compared in PCR+ or PCR- patients, no statistical difference appeared. These results reveal that 52% of anti-HCV+ HD patients have HCV infection. The clinical consequences of HCV infection in that population are not characterized since no difference has been documented between PCR+ and PCR- results.

Combination of standard cytology and immunocytology with BL2-10D1 monoclonal antibody for monitoring-treated bladder cancer patients.

Monoclonal antibody BL2-10D1 directed against a tumor-associated antigen of bladder carcinoma was used for monitoring 11 intravesically treated patients. Thirty-three bladder washout specimens were used for standard cytology and immunological staining. Prior to treatment, 9 of 11 cytologic specimens examined with standard cytology were found to be positive. Using BL2-10D1 alone, only 6 were positive but 1 patient negative with standard cytology was positive with the antibody and corresponded to a positive histological control. Thus, before treatment, an increase in positive rate was observed using the combination of the 2 methods from 82 to 91%. At the end of treatment, 9 washout specimens remained positive with standard cytology, whereas 1 case negative in standard cytology was positive in immunocytology. Thus, the positive rate increased from 82 to 91%. One month after the end of treatment, of 11 washout specimens tested, 3 false-negative standard cytologies and 4 false-negative immunocytologies were shown. However, used in combination, the two methods lead to an increase in positive rate from 67 to 89%. In view of these results, BL2-10D1 may be considered as a useful reagent in combination with the standard cytology for the confirmation of the presence of tumor cells before and after immunotherapy.