No expansion of the pre-B and B-cell compartments in the bone marrow of patients with multiple myeloma.

We have recently demonstrated that the CD24 antigen density of bone marrow (BM) lymphoid cells discriminates between pre-B cells and mature B-cells. Using this new method, we evaluated the B-cell lineage in the BM and peripheral blood (PB) of 18 patients with multiple myeloma (MM). First, the percentage of pre-B cells was significantly reduced by 40% in the BM of patients with MM: 2.3% +/- 2.2% versus 5.7% +/- 2.8% of normal BM lymphoid cells (P less than 0.01). This finding was associated with a significant reduction (50%) of the percentage of mature B-cells in both BM and PB, especially in patients with progressive disease (P less than 0.05). In contrast to what has been reported previously, we have not found any pre-B cells in the PB of these patients with MM. Secondly, BM pre-B and B-cell patients with MM did not express any activation markers (CD23, CD25, or CD71 antigens) and no CD5+ B-cells were found in the BM unlike in PB (8% CD5+ B-cells). Taken together, these data do not support the concept of a direct involvement (i.e, expansion or activation) of pre-B cells in MM without excluding the possibility of an early oncogenic event at the pre-B cell stage. Furthermore, our data emphasize this important reduction of the B-cell compartment (including that of pre-B cell) as a major cause of the humoral immunodeficiency in MM.

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.

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.

TT virus: a study of molecular epidemiology and transmission of genotypes 1, 2 and 3.

BACKGROUND: TT virus (TTV) is a recently discovered virus, which is not related to any other known virus infecting humans. OBJECTIVES: To investigate: (i) the world-wide distribution of the three major TTV genotypes; and (ii) the possible routes of viral transmission. STUDY DESIGN: (i) The phylogenetic distribution of 494 TTV isolates originating from 31 countries was analysed, using partial ORF1 sequences. (ii) Faeces samples (n=22) and saliva samples (n=72) from French individuals were tested for the presence of TTV DNA. (iii) Viral titres in paired serum and saliva samples were compared. RESULTS: (i) Genotypes 1, 2 and 3 were distributed world-wide, with a high proportion of type 1 in Asia (71%) and no type 3 identified in Africa to date. In the USA, 77% of isolates were grouped in four clusters only (genetic distances <10%). This was also the case of 76% of French isolates, 76% of Japanese isolates, and 89% of Hong Kong isolates. (ii) TTV DNA was detected in 18% of faeces samples and 68% of saliva samples tested. (iii) Viral titre in saliva samples was 100-1000 times higher than that of the corresponding serum. CONCLUSIONS: (i) The observed epidemiological distribution of TTV isolates is compatible with an ancient dissemination of viral ancestors belonging to the different genotypes and a slow genetic evolution in sedentary populations. (ii) Besides the possible transmission of TTV by the parental and oral-faecal routes, the high titre of TTV DNA observed in saliva raises the hypothesis of the viral transmission by saliva droplets. This route of transmission could explain the high degree of exposure to viral infection observed in the general population.

Comparison of systems performance for TT virus detection using PCR primer sets located in non-coding and coding regions of the viral genome.

BACKGROUND: the heterogeneity of the TT virus (TTV) DNA prevalence values reported from comparable human cohorts suggests that diagnostic PCR protocols still require to be optimized. OBJECTIVES: to design TTV PCR primer sets with low genotype restriction and to compare their performances with commonly used amplification systems. STUDY DESIGN: we compared full length TTV genomic sequences and identified conserved nucleotide patterns in the 5' and 3' non-coding regions of the viral genome. This permitted to design two new primer sets usable for the PCR amplification of the most divergent human isolates of TTV described to date. The performances of these amplification systems were compared with those of three other PCR systems earlier used for prevalence studies. RESULTS: the primer systems P5Bx and P3Bx exhibited higher PCR scores than the other systems tested; 14 to 34% improvement values were obtained, and divergent positive results of earlier described PCR systems were confirmed systematically by our new detection assays. CONCLUSIONS: an optimized detection of TT virus DNA is a pre-requisite for the accurate epidemiological survey of viral infection and for the realization of phylogenetic studies. Such PCR systems with low genotype restriction will be helpful in the future for a better knowledge of natural history of TT virus infection.

Evaluation of four PCR systems amplifying different genomic regions for molecular diagnosis of GB virus C infections.

Diagnosis of GB virus C (GBV-C) infection is based on RT-PCR methodology that detects genomic viral RNA. Four sets of primers located in different genomic regions were compared. These primers were used to amplify a reference panel of sera used by the French blood banks, including five positive sera, five negative sera and 10-fold serial dilutions of one positive serum. Three sets of primers located in the 5'UTR, NS3 and NS5a genomic regions gave comparable results with undiluted sera. When diluted sera were tested, the most sensitive protocol was that using a set of primers and a probe selected within the 5'UTR, together with a colorimetric detection based on DNA enzyme immunoassay.

Strategies for the sequence determination of viral dsRNA genomes.

The genetic study of viruses having dsRNA genomes is hampered by the technical difficulty of complete sequence determination of dsRNA. Optimised methods are described here for sequencing dsRNAs, which meet three different situations: (1) genomes that can be obtained in fairly high amounts (>20 ng per separated segment); (2) genomes with limited amounts of RNA that can be detected by electrophoretic gel separation and staining; (3) genomes that cannot be detected by electrophoretic gel separation and staining. These methods include improved Single Primer Amplification Technique protocols, an adaptation of the SMART methodology, and a new method permitting the selective enzymatic removal of dsRNA segments. Strategies permitting adaptation of these protocols to the full-length determination of dsRNA viral genomes are described. Each of the protocols is described for sequence determination of a chosen dsRNA virus.

Screening for HBV, HCV and HIV genomes in blood donations: shortcomings of pooling revealed by a multicentre study simulating real-time testing.

This study was undertaken in order to determine whether screening of viremic blood donations by testing of pooled donor samples could constitute a technically feasible transfusional safety measure. A pilot study of real-time simulation, on a day-to-day basis, of screening of three viral genomes (hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV)) was conducted by five French Blood Centers on plasma samples collected from blood donors and studied within undiluted samples and within sample pools of various sizes. This study was carried out within time conditions compatible with the release of platelets. For the detection of HCV and HIV genomes, the five laboratories achieved a sensitivity that decreased with the size of the sample pool. Four were successful in detecting all undiluted samples. In the 1/10 diluted samples, four failed to detect one HIV or HCV sample. In the 1/100 diluted samples, all laboratories failed to detect one or more HIV or HCV samples. For HBV genome, no participating laboratories detected all of the samples of the panel, even undiluted samples, and the sample pooling considerably affected sensitivity. The improvement and standardization of assays needs to be attained, and training of laboratories appears to be a step crucial for routine screening of viral genomes in blood donations.

Lessons from a multicentre study of the detectability of viral genomes based on a two-round quality control of GB virus C (GBV-C)/hepatitis G virus (HGV) polymerase chain reaction assay.

The aim of this study was to determine whether multicentre quality controls for the detectability of viral genomes could contribute to the improvement of diagnostic performance in the participating laboratories. The study was carried out during two successive rounds, during which 18 laboratories specialized in nucleic acid testing analyzed, through a polymerase chain reaction (PCR) assay, a common panel of GB virus C (GBV-C)/hepatitis G virus (HGV) RNA-positive and -negative samples. During the first round, the laboratories used either an 'in-house' PCR procedure or a partly standardized commercial test. After decoding the results of the first round, the procedures of the participating laboratories were compared in order to establish a consensus procedure deduced from those of the laboratories which provided the best results. During the second round, each participating laboratory could use the resulting consensus procedure, or its own procedure, or both. The results of this quality control study indicated that, whatever method used, even specialized and trained laboratories may give false-negative or false-positive results. The commercial assay did not guarantee a systematic high quality level of results. The striking heterogeneity of results observed among laboratories using the same commercial assay confirm that molecular biology methods need skilled technicians. The results of this quality control study suggest that full standardization of viral genome detection, including all steps of the procedure, is necessary and that the laboratories performing PCR should participate in repeated quality control studies, whatever technique is being used.

The human gastrin/cholecystokinin receptors: type B and type C expression in colonic tumors and cell lines.

The CCK-type B receptors are recognized by gastrin, which is known to be possibly involved in the development of gastro-intestinal cancers; alternate splicing of exon 4 of the human CCK-B receptor gene gives 2 different mRNA isoforms, the exact significance of which still remains to be elucidated. The recently described CCK-type C receptors recognize gastrin but do not discriminate between mature and immature forms of the hormone. A series of healthy and tumoral colon samples, the associated hepatic metastases and four colonic cell lines were examined for gene expression of the 2 isoforms of the CCK-B receptor and the CCK-C receptor using reverse transcription-polymerase chain reaction. Gastrin mRNA expression was also investigated. The short isoform of the CCK-B was detected in 80% of the normal colon tissues, 76.5% of the colon tumors, 100% of the metastasis samples and 75% of the colonic cell lines; whereas the long isoform, which is presumably more strongly activated by gastrin, was expressed in 50% of the normal colon samples, 23% of the colon tumors, 43% of the hepatic metastases and 1 cell line (Sk-Co15). However, although CCK-C transcript was detected in 100% of the tumors tested and gastrin mRNA in 86.5%, only 16.5% also expressed the long isoform of the CCK-B receptor. The gastrin/CCK-B receptor might therefore be involved in an hypothetic autocrine proliferative loop only in some colonic tumors, and the receptor mainly involved in this loop may well be the CCK-C receptor, since its mRNA is expressed as often as gastrin mRNA in tumors and cell lines.

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.

Inactivation of a European strain of West Nile virus in single- donor platelet concentrate using the INTERCEPT blood system.

BACKGROUND AND OBJECTIVE: In order to prevent West Nile virus (WNV) contaminations by transfusion, the French National Blood Service decided to evaluate the INTERCEPT Blood System's efficiency on a European strain. MATERIALS AND METHODS: Culture supernatant of WNV was used to infect six platelets concentrates. Viral titre was determined by plaque reduction neutralization test before and after viral inactivation using the INTERCEPT Blood System. RESULTS: In all assays, the absence of plaque forming unit was observed after viral inactivation. The log reduction observed ranged between > 5.1 logs to > 5.2 logs. CONCLUSION: INTERCEPT Blood System is a commercially viral inactivation method potentially useful in order to prevent WNV transmission by blood products in France during re-emerging outbreaks.

[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.

High prevalence of GB-C/hepatitis G virus in a Brazilian population with helminth infection.

A study of GB-C virus/Hepatitis G virus (GBV-C/ HGV) infection was carried out in a rural population of Northeastern Brazil, in which the prevalence of schistosomiasis is 80-90%. Despite the absence of parenteral risk exposure, the prevalence of GBV-C/HGV markers of infection was found to be unusually increased: viremia, 16.4%; specific antibody, 18.3%. It is therefore suspected that helminth infection influenced the immune response to GBV-C/HGV infection by shifting the balance of cytokine responses from Th1 to Th2, resulting in a delayed viral clearance. Phylogenetic analysis of viral isolates did not provide evidence for high rates of sexual or mother-to-infant viral transmission. The study revealed that viral strains belonged to types 1 and 2 only (predominant in Africa and Europe, respectively), suggesting that GBV-C/HGV was introduced into the New World by white conquerors and black slaves since the 16th century.

Complete sequences of two highly divergent european isolates of TT virus.

TT virus is a virus distantly related to the Circoviridae family. We report here the complete genome characterization of two European human isolates (T3PB and TUPB) using a new and simple protocol for sequencing GC-rich genomic regions. Sequence analysis confirmed the existence of two major ORFs, of a CAV-like VP2 motif in ORF2 and of potential stem-loop structures in non-coding regions. Phylogenetic analyses based on complete genomic sequences of human isolates suggested that three different lineages exist at least. The first lineage includes genotypes 1, 2, and 3, and two other lineages include viruses related to the Japanese SANBAN and to the North American TUS01 isolates respectively. Sequence comparison made it possible to assign strain T3PB to genotype 3, and strain TUPB to the TUS01 group. Consequently, this study reports the first full-length sequence of a genotype 3 isolate and demonstrates that viruses belonging to the TUS01 lineage are present in the Old Word.

Comparative sequence analysis of American, European and Asian isolates of viruses in the genus Coltivirus.

In this study, the basis for the classification of virus isolates grouped within the genus Coltivirus, family Reoviridae, is discussed. Sequences of dsRNA segments from American (segments 9-12), European (segment 12) and Asian (segments 7-12) isolates were characterized and polythetic criteria were defined for their taxonomic classification. These criteria (including sequence analysis) permitted the different species to be distinguished and classified into two groups. In both groups, subgroups were defined according to the degree of homology between the genomic sequences. American and European isolates are classified within group A, which includes subgroups A1 (Colorado tick fever virus species) and A2 (Eyach virus species). Asian isolates are classified in group B, which includes subgroups B1 (JKT-7075 virus species) and B2 (JKT-6423 virus species). The proteins encoded by the sequenced genomic segments were analysed. This allowed the identification of dsRNA binding domains in the proteins encoded by segment 8 of subgroup B1 isolates and segment 12 of subgroup B2 isolates. A conserved pattern of amino acids in segment 7 of group B isolates matched sequences found in the catalytic domains of protein kinases.

Sequence determination and analysis of the full-length genome of colorado tick fever virus, the type species of genus Coltivirus (Family Reoviridae).

The Colorado tick fever virus (CTFV) is the type species of genus Coltivirus, family Reoviridae. Its genome consisting of 12 segments of dsRNA was completely sequenced. It was found to be 29,174 nucleotides long (the longest of all Reoviridae genomes characterized to date). Conserved sequences at the 5' end (SACUUUUGY) and at the 3' end (WUGCAGUS) of the 12 segments were identified. The analysis of the putative proteins deduced from the nucleotide sequences permitted to identify functional motifs. In particular, the VP1 was identified unambiguously as the viral RNA dependent RNA pylmerase (RDRP) (VP1pol), with a GDD located at a similar position to Reoviridae RDRPs. In other genes, RGD cell-binding, NTPAse, single strand binding protein and kinase motifs were identified. Comparison with Reoviridae proteins showed significant similarities to RDRPs (CTFV-VP1) and sigma C protein of orthoreovirus (CTFV-VP6). Similarities to nonviral enzymatic proteins, such as methyltransferases, NTPAses, RNA replication factors, were also identified.

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.

High prevalence of TT virus infection in French blood donors revealed by the use of three PCR systems.

BACKGROUND: The purpose of this study was to determine the prevalence of TT virus (TTV) infection in voluntary blood donors in Southeastern France. STUDY DESIGN AND METHODS: The sera of 289 blood donors were tested for the presence of TTV DNA by two PCR systems detecting genes located in the 5' UTR (primer set A [Set A]) and the open reading frame (ORF2) (primer set B [Set B]) of the viral genome. A randomized sample of 40 blood donors was also tested by a nested-PCR system in the ORF1 by use of primer set C (Set C). Donors were questioned for possible risk factors for virus transmission. RESULTS: In the entire population studied, 30.8 percent of blood donors tested positive with both Sets A and B, and 70.6 percent with at least one set. In the sample tested with three sets of primers, 27.5 percent of blood donors were positive in testing with all PCR systems and 80 percent with at least one system. The specificity of TTV DNA amplification was confirmed by sequencing 10 PCR products obtained with each set of primers. Statistical analysis revealed that the prevalence of TTV reactivity increased with age. CONCLUSION: The high prevalence of TTV reactivity and the absence of a pathologic condition or risk factors obviously associated with the infection in blood donors suggest that there is no need for systematic detection of TTV infection before blood donation. Further studies are required to determine if TTV isolates can be responsible for a pathologic condition in humans after blood transfusion.