[Quality control of mixtures for pediatric parenteral nutrition: Validation of sodium and potassium assay method]. / Contrôle qualité des mélanges pour nutrition parentérale pédiatriques : validation de la méthode de dosage du sodium et du potassium.

OBJECTIVES: Control of electrolyte concentration in mixtures for parenteral nutrition (MPN) for newborns is crucial before the release of the final product. We aimed to assess the validation of the electrolytes assay in MPN. METHODS: Electrolytes assay was performed with Ilyte Analyzer®. Validation of method was realized in accordance with ICH (International Conference on Harmonization) guideline Q2(R1) and the commission report of the French society of pharmaceutical science and technology. Linearity test solutions were prepared in triplicate using five levels of concentrations for sodium and potassium (60-140% of theoretical concentrations). Accuracy of the method was deducted from the same results of linearity. The intermediate precision was ensured by dosing the main electrolyte in six MPN, during three successive days. RESULTS: Linearity was assessed with correlation coefficients greater than 0.996 for both electrolytes. A non-significant result of comparison test of the intercept with zero (Student test) was obtained. A highly significant result of the test of existence of slopes (Fisher test) proved a linear regression for the 2 electrolytes (P<0.1%). Inter-day precision values were 2.68% and 2.65% respectively for sodium and potassium. CONCLUSION: The validation of sodium and potassium assay method was successfully performed with Ilyte Analyzer® allowing routine quality control in MPN.

Increased urinary type II collagen C-telopeptide levels in Tunisian patients with knee osteoarthritis.

UNLABELLED: Osteoarthritis is characterized by a progressive degeneration of articular cartilage and loss of joint function. Clinical assessment of osteoarthritis is hampered by the lack of accurate measures of disease and disease progression, especially during the early stage. BACKGROUND: To investigate urinary C-telopeptide fragments of type II collagen (CTX-II) levels in knee osteoarthritis in the Tunisian population compared with controls and to assess the association between this biomarker and radiological signs. METHODS: One hundred and twenty five female patients with knee osteoarthritis, aged 53.6 +/- 7.6 years with disease duration of 3.6 +/- 3.8 years and 57 female age-matched controls underwent Lyon Schuss X-ray exams. Two experienced readers independently measured the joint space width (JSW) and classified each knee for severity using the Kellgren/Lawrence scale. The urinary concentration of CTX-II was measured by a competitive ELISA. RESULTS: The levels of urinary CTX-II were significantly higher in knee osteoarthritis patients compared with controls (323.98 vs 218.04 microg/mol creatinine). A weak and non significant association between the CTX-II level and JSW was found. The significant correlations were observed between age and CTX-II in both groups and between BMI and CTX-II only in controls. CONCLUSIONS: Analysis of CTX-II in urine samples of Tunisian patients with knee osteoarthritis provided a sensitive method to detect increased degradation of collagen type II in patients with osteoarthritis.

Monosomy 10q26-qter and trisomy 11q13-qter as a result of de novo unbalanced translocation.

A male infant with partial monosomy 10 q and partial trisomy 11q as a result of de novo unbalanced translocation between the long arms of chromosomes 10 and 11: der(10)t(10;11)(q26;q13) is described. He had craniofacial dysmorphy, congenital heart defects, urogenital and cerebral anomalies, and severe developmental delay. To the best of our knowledge, this is the first report of this combination of chromosomal abnormalities.

Chromosomal abnormalities in liver cell dysplasia detected by comparative genomic hybridisation.

BACKGROUND/AIM: The pathogenetic relation between liver cell dysplasia and hepatocellular carcinoma (HCC) is poorly understood. The aim of this study was to determine whether there is a genetic link between liver cell dysplasia and HCC that could support the role of dysplasia as a tumour precursor lesion. METHODS: Microdissection from paraffin wax embedded sections and degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) were combined to analyse chromosomal imbalances by comparative genomic hybridisation (CGH) in nine HCCs and nodules containing liver cell dysplasia and cirrhosis adjacent to the tumours. Seven cases of large cell changes (LCC) and three cases of small cell changes (SCC) were analysed. The genetic abnormalities detected in liver cell dysplasia were then compared with those present in the corresponding HCC. RESULTS: No abnormalities were detected in LCC and cirrhotic nodules, arguing against the preneoplasic nature of these cell foci. In contrast, a subset of chromosomal alterations present in HCCs was found in the adjacent SCC. CONCLUSIONS: These findings support the preneoplastic status of SCC in human hepatocarcinogenesis.

Molecular characterization and phylogenetic analysis of a human T cell leukemia virus type 2 strain from French Guiana.

Extensive studies have been carried out on native Amerindian populations living in French Guiana in an attempt to detect human T cell leukemia virus type 2 (HTLV-2). However, the first strain of this virus identified in this region was not detected in these populations, but in a Brazilian woman of Amerindian origin. Comparative analyses of the nucleotide sequences of 589 bp of the gp21 env gene and of 625 bp of the long terminal repeat (LTR) showed that this new HTLV-2 strain (HTLV-2 GUY) was of subtype A. Sequence comparison and phylogenetic analyses demonstrated that HTLV-2 GUY was closely related to a group of distinct variants of HTLV-2 subtype A strains originating mostly from Brazilian inhabitants and formerly called HTLV-2 subtype C. As there is a high level of immigration from Brazil in French Guiana, we carried out a seroepidemiological study of 175 Brazilians, mostly women (obtained from a serum databank) and 72 female Brazilian prostitutes living in French Guiana to determine whether HTLV-2 is likely to become an emerging infection in this area. No HTLV-2 infection was detected, indicating that this virus is unlikely to become prevalent in the near future.

Distinct chromosomal abnormality pattern in primary liver cancer of non-B, non-C patients.

To discriminate among the chromosomal abnormalities associated with the etiology of hepatocellular carcinoma (HCC), we performed a comparative genomic hybridization (CGH) analysis on 34 HCCs resected on non-cirrhotic livers from patients serologically negative for both hepatitis B (HBV) and C (HCV) viruses. The results were compared to those of a previous analysis of 50 HCCs selected on the basis of their positivity for HBV infection. The majority of the abnormalities found in the HBV positive cases (losses of chromosome arms 1p, 8p, 6q, 13q and 14q and gains of 1q, 8q, 6p and 17q) were similarly detected in the virus negative specimens. In contrast, a significant decrease (40% on average) was observed for losses at 4q, 16q and 17p in non-viral HCC samples, suggesting that these abnormalities are tightly associated with HBV infection. Thus, in addition to a common pathway towards malignancy, a subset of alterations may preferentially contribute to virus-induced carcinogenesis. In a parallel CGH study of 10 fibrolamellar carcinomas, a rare subtype of HCC, we found in six out of the seven informative cases, gains of chromosome arm 1q. This region, which is also preferentially amplified in non fibrolamellar tumors (58%), may contain an essential proto-oncogene commonly implicated in liver carcinogenesis.

Effect of TT virus infection on hepatocellular carcinoma development: results of a Euro-Asian survey.

A small percentage of persons with hepatocellular carcinoma (HCC) lack identifiable causes of liver pathology. The single-stranded DNA virus, TT virus (TTV), has been found in persons with acute and chronic liver injury. Nested polymerase chain reaction was used to search for both TTV and parvoviruses in 293 HCC samples from Asia and Europe. TTV was found in >30% of Chinese and Italian samples but in only 13% of French samples. No clinicopathologic differences were found between TTV-positive and -negative populations. A significant association was found between TTV infection and hepatitis B virus (P<.01) and herpesviruses (P<.02) in HCC patients, suggesting that factors promoting these infections are associated with enhanced TTV positivity. Parvovirus B19 and adeno-associated virus were found in only 7.5% of the tumors. Taken together, these data suggest that TTV infection is unlikely to be associated with the induction or acceleration of the hepatocarcinogenic process in humans.

Gain of chromosome arm 17q and adverse outcome in patients with neuroblastoma.

BACKGROUND: Gain of genetic material from chromosome arm 17q (gain of segment 17q21-qter) is the most frequent cytogenetic abnormality of neuroblastoma cells. This gain has been associated with advanced disease, patients who are > or =1 year old, deletion of chromosome arm 1p, and amplification of the N-myc oncogene, all of which predict an adverse outcome. We investigated these associations and evaluated the prognostic importance of the status of chromosome 17. METHODS: We compiled molecular cytogenetic analyses of chromosome 17 in primary neuroblastomas in 313 patients at six European centers. Clinical and survival information were collected, along with data on 1p, N-myc, and ploidy. RESULTS: Unbalanced gain of segment 17q21-qter was found in 53.7 percent of the tumors, whereas the chromosome was normal in 46.3 percent. The gain of 17q was characteristic of advanced tumors and of tumors in children > or =1 year of age and was strongly associated with the deletion of 1p and amplification of N-myc. No tumor showed amplification of N-myc in the absence of either deletion of 1p or gain of 17q. Gain of 17q was a significant predictive factor for adverse outcome in univariate analysis. Among the patients with this abnormality, overall survival at five years was 30.6 percent (95 percent confidence interval, 21 to 40 percent), as compared with 86.0 percent (95 percent confidence interval, 78 to 91 percent) among those with normal 17q status. in multivariate analysis, gain of 17q was the most powerful prognostic factor, followed by the presence of stage 4 disease and deletion of 1p (hazard ratios, 3.4, 2.3, and 1.9, respectively). CONCLUSIONS: Gain of chromosome segment 17q21-qter is an important prognostic factor in children with neuroblastoma.

Overrepresentation of 7q31 and 17q in renal cell carcinomas.

Xenografts from four metastatic renal cell carcinomas (RCCs) were established in immunodeficient mice. All tumors exhibited cytogenetic features specific for the papillary subtype, namely, partial or total polysomy of chromosomes 7 and 17 and integrity of 3p. Cytogenetic analysis of the initial and xenografted tumors indicated that although clonal characteristics were consistently maintained in xenografts derived from metastases, a minor clone had been selected for in the xenografts derived from the primary tumors. Reverse painting and comparative genomic hybridization (CGH) allowed us to localize minimal overrepresented genomic regions to 7q31, where the MET protooncogene is located, and to 17q. Other overrepresented regions were 8q in all xenografts and Xq22-qter in three of them. The gain of genetic material from these regions may be a key factor ensuring the papillary nature of RCCs and their survival in xenografts.

Comparative genomic hybridization analysis of sporadic neuroendocrine tumors of the digestive system.

Little information is available on the molecular mechanisms underlying neuroendocrine tumorigenesis. To obtain an overview of the genomic imbalances characterizing these tumors, we studied 20 benign or malignant sporadic endocrine gastroenteropancreatic tumors by comparative genomic hybridization. Chromosomal imbalances were found in all tumors. Gains of chromosomal material were more frequent than losses. The most frequent gains were of chromosomes and chromosome arms 5 (55%), 14 (55%), 17q (55%), and 7 (50%). Losses were most frequent from 11q (30%) and 16p (30%). Gains of chromosome 5 did not occur in nonmetastatic tumors, whereas losses of 9p were observed exclusively in intestinal tumors. In addition, we found two high-level amplifications, of 17q11-21 and 19q13. A complementary FISH analysis revealed that the gain in 17q11-21 included amplification of the protooncogene HER2/neu. As in multiple endocrine neoplasia type-1-associated tumors, deletions of chromosome band 11q13 appear to be involved in the development of sporadic digestive tract neuroendocrine tumors, but our results suggest that other chromosomal regions are also involved.

Recurrent chromosomal abnormalities in hepatocellular carcinoma detected by comparative genomic hybridization.

Comparative genomic hybridization (CGH) was used to evaluate and map genomic aberrations in 50 hepatocellular carcinomas (HCCs) from patients chronically infected with hepatitis B virus (HBV). CGH clearly detected nonrandom genomic imbalances. Losses were most prevalent on chromosome regions 4q (70%), 8p (65%), 16q (54%), 17p (51%), 13q and 6q (37% each), and lp (30%). The most frequent gains occurred on 8q (60%), 1q (58%), and 6p and 17q (33% each). In a few cases, sequence amplifications were detected that were mapped to bands 11q12, 12p11, 14q12, and 19q13.1. This study represents the first analysis of primary liver cancers by CGH, and it confirms the presence of previously known chromosomal aberrations in HCC and highlights new quantitative abnormalities and sequence amplifications. These findings should lead to the characterization of new loci involved in liver cancer pathogenesis.

Emergence and scattering of multiple neurofibromatosis (NF1)-related sequences during hominoid evolution suggest a process of pericentromeric interchromosomal transposition.

Type 1 neurofibromatosis (NF1) gene encodes for a member of the GTPase activating protein family and is considered to be a tumor suppressor gene. Its very high rate of de novo mutation in humans led us to study a specific feature of this gene: the presence of numerous NF1-related sequences. According to our results, the human genome contains at least 11 NF1-related sequences, nine of which are scattered near centromeric sequences of seven different chromosomes. These NF1-related sequences, whose extent is quite varied according to loci, are unprocessed copies of the NF1 gene, and bear numerous mutations. A phylogenetic analysis of the six largest sequences indicates that they are all derived from a common ancestor, which would have appeared 22-33 million years ago, and was subsequently duplicated several times during hominoid evolution. The most recent duplication and interchromosomal transposition occurred in the last million years suggesting that the process could still be ongoing. Intriguing similarities between the evolution of alpha-satellite DNA and NF1-related sequences suggest the involvement of a common genetic mechanism for the generation and pericentric spreading of these NF1 partial copies.

Additional copies of a 25 Mb chromosomal region originating from 17q23.1-17qter are present in 90% of high-grade neuroblastomas.

Neuroblastoma shows remarkable heterogeneity, ranging from spontaneous regression to progression toward highly malignant tumors. In search of genetic abnormalities that could explain this variability, we have characterized neuroblastoma tumors by using multiple fluorescent hybridizations. Our results indicate that chromosome 17 is rearranged very frequently in the form of unbalanced translocations with numerous chromosomal partners, all leading to the presence of supernumerary copies of a 25 Mb chromosomal region originating from 17q23.1-qter. Additional 17q material was detected in more than 90% of untreated high-grade neuroblastomas and, along with 1p36 deletion, should represent the most frequent genetic abnormality of neuroblastoma observed until now.

Assignment of the NTRK4 (trkE) gene to chromosome 6p21.

Reverse transcriptase-polymerase chain reactions using foetal brain RNA with reverse and forward primers of the first, second and third NTRK4 region allowed us to obtain three amplified NTRK4 fragments. The specificity of amplified fragments was checked by digestion with restriction endonucleases AvrII, HindIII and PspII for the first, second and third regions, respectively. Each restriction site was specific for each amplified fragment. The fragment of the NTRK4 first region was also sequenced and the sequence determined was identical to the human NTRK4 sequence. The three amplified fragments were cloned in pBS. For the Southern technique, plasmid pBS-NTRK4a (with an insert of 1052 bp) detected a human 9-kb HindIII sequence which was localised unambiguously on chromosome 6. For fluorescence in situ hybridisation, the three plasmids, pBS-NTRK4a, pBS-NTRK4b (insert 924 bp) and pBS-NTRK4c (insert 1114 bp) were pooled and used as a probe. This NTRK4 probe was localised on 6p21. Of 50 metaphases analysed, 49 contained twin spot signals on both sister chromatids.

MDM2 amplification in a primary alveolar rhabdomyosarcoma displaying a t(2;13)(q35;q14).

This report describes a case of rhabdomyosarcoma associated with a 2;13 translocation and multiple double minute chromosomes. The origin of the amplified DNA was identified using comparative genomic hybridization, which pinpointed a unique spot at 12q13-->q14. Band 12q13 has been shown to contain several genes that are occasionally amplified in other sarcomas. Fluorescene in situ hybridization to tumor metaphases with probes specific for this region indicated that the double minutes contained the MDM2 gene but not the CDK4 gene. MDM2 amplification was further quantified by Southern hybridization, which showed a mean value of 25 copies per haploid genome. This is the first example of MDM2 amplification in a rhabdomyosarcoma.

Characteristics of the antibiotic resistance plasmid in Salmonella typhi isolated in Tunis in 1990.

A multiresistant Salmonella typhi (S typhi, strain 302) was isolated from a blood culture of a patient in the Infectious Diseases department of Rabta Hospital in Tunis. The following tests were carried out: antibiotic susceptibility testing by the agar diffusion method; determination of the minimum inhibitory concentration against four beta-lactam antibiotics (amoxicillin, ceftriaxone, ceftazidime, imipenem), chloramphenicol, gentamicin, and amikacin by the agar dilution method; conjugation with E coli K12 for study of transferability of resistance markers; and electrophoresis of plasmid DNA extracts on agarose gel. S typhi 302 was resistant to amoxicillin, streptomycin, tetracycline, chloramphenicol, and sulfamide-trimethoprim, and this resistance was transferable in toto with a frequency of 10(-4). The MICs of amoxicillin and chloramphenicol were, respectively, 1024 (due to the production of TEM-1 beta-lactamase) and 256 mg/l. These resistance markers were carried by a plasmid of about 40 kb, similar to the Salmonella wien plasmid. The easy acquisition of a multiresistance plasmid by S typhi suggests that epidemiological monitoring of this serovar should be carried out.