Resequencing microarray method for molecular diagnosis of human arboviral diseases.

BACKGROUND: Resequencing DNA microarray (RMA) technology uses probes designed to identify a panel of viral sequences. It can be used for detecting emerging viruses by revealing the nucleotide polymorphisms within the target of interest. OBJECTIVES/STUDY DESIGN: As a new tool for molecular diagnosis of arbovirus infection, high density PathogenID v2.0 RMA (PID2-RMA) was assessed for the detection and genetic analysis of dengue, West Nile, and Chikungunya viruses in spiked blood samples or sera from individuals infected with dengue virus. Viral RNAs extracted from biological samples were retrotranscribed into cDNA and amplified using the Phi 29 polymerase-based method. This amplified cDNA was used for hybridization on PID2-RMA. RESULTS: A good specificity of RMA-based detection was demonstrated using a panel of arboviruses including Dengue, West Nile and Chikungunya viruses. This technology was also efficient for the detection and genetic analysis of the different serotypes of dengue virus in sera of infected patients. Furthermore, the mixing of dengue, West Nile and Chikungunya prototype viruses within a single sample of human blood did not interfere with the sensitivity of PID2-RMA. CONCLUSIONS: Our data show that high density PID2-RMA was suitable for the identification of medically important arboviruses. It appears to be particularly adapted to the genetic analysis of dengue, West Nile, and Chikungunya viruses in urgent clinical situations where the rapid identification and characterization of the pathogen is essential.

Características generales y factores predictores de mortalidad hospitalaria en pacientes con asistencia ventilatoria mecánica / General characteristics and predictive factors of Hospital mortality in patients with assistance mechanical ventilation

Objetivo: Estudiar características generales y factores predictores de mortalidad hospitalaria en pacientes internados en Unidad de Terapia Intensiva (UTI) que requirieron Asistencia ventilatoria mecánica (AVM).Diseño: Estudio prospectivo, observacional, 36 meses de duración. Ámbito: Terapia intensiva polivalente. Hospital universitario. Pacientes: AVM ≥24 horas. Variables de interés: Datos demográficos; tipo de patología de ingreso a UTI; tiempo de internación en UTI y hospital; escores al ingreso, PCR (mg/dl); AVM: motivo de inicio, permanencia y complicaciones asociadas; evolución. Resultados: 372 pacientes requirieron AVM. Edad media 52 años (r 18-93), 67% eran varones. Estadía en UTI fue 12,59 (±13,52) días y Hospital de 20,31 (±25,20). 53,2% fallecieron en UTI. Se realizó comparación de variables entre sobrevivientes y fallecidos. Análisis de regresión logística múltiple se incluyeron: edad, PCR, APACHE II, SAPS II, SOFA, patología médica, insuficiencia respiratoria y utilización de vasopresores. Mayor valor predictivo fueron: edad, patología médica, uso de vasopresores y PCR. (D de Sommer = 0,59). Sensibilidad: 68,15% (60,18 - 75,22), especificidad: 75,35% (68,93 - 80,84), VPP 66,88% (58,94 - 73,99), VPN 76,42% (70,01­ 81,84). La curva COR presenta un AUC de 0,793 (IC 95%: 0,747-0,839) p = 0,000.Conclusiones: Factores predictores de mortalidad fueron: edad (p=0,007), utilización de vasopresores (p=0,000) y patología médica (p=0,002). PCR, mostró un valor de p cercano al valor de significación (p=0,065).(AU)

Objective: Examining the general characteristics and predictive factors of inhospital mortality in patients in Intensive Care Unit (ICU) who needed mechanical ventilatory support (MVS). Design: Prospective and observational study, 36 months duration. Area: Multipurpose intensive therapy. University hospital. Patients: MVS≥24 hours. Variables of interest: Demographic data, type of pathology on admission to ICU, hospitalization time, scoring, C - reactive protein (CRP) (mg/dl), MVS: reason for initiation, duration and complications associated; evolution. Results: 372 of the patients required MVS, average age was 52 years old (r 18-93), 67% were men. Intensive care unit length of stay was 12, 59 (±13, 52) days and hospital length of stay was 20, 31 (±25, 20). 53,2% died in ICU. A comparison of variables between survivors and deceased was made. Multiple logistic regression (MLR) analysis included: age, PCR, APACHE II, SAPS II, SOFA, medical condition, respiratory failure and use of vasopressors. The greatest predictive value was: age, medical condition, use of vasopressors and PCR. (Sommer´s D = 0,59). Sensitivity 68,15% (60,18 - 75,22), specificity: 75,35% (68,93 - 80,84), VPP 66,88% (58,94 - 73,99), VPN 76,42% (70,01­ 81,84). AUC of ROC was 0,793 (IC 95%: 0,747-0,839) p = 0,000. Conclusion: Predictive factors of mortality were: age (p=0,007), use of vasopressors (p=0,000) and medical condition (p=0,002). CRP showed a p-value close to a significance value of (p=0,065).(AU)

First evidence for a restriction-modification system in Leptospira sp.

The LE1 leptophage exhibited a host range restricted to the saprophytic Leptospira biflexa [Saint Girons et al., Res. Microbiol. 141 (1990) 1131-1133] and mainly to the Patoc 1 strain (hereafter called PFRA) kept in the Paris, France collection. Results of titration of LE1 lysates indicated the presence of a host-controlled modification and restriction system within PUSA (Patoc 1 strain maintained in the Morgantown, WV, USA collection) that was absent in PFRA. Because genomic DNA of PITAL (Patoc 1 strain maintained in Trieste, Italy) appeared smeared in pulsed field gel electrophoresis (PFGE), this strain is likely to contain nucleases that are activated upon DNA isolation. Moreover, comparative NotI digestions of PUSA and PFRA DNAs, as visualized by PFGE, indicated that PUSA belonged to a different serovar than PFRA. Finally, 16S ribosomal sequence analysis indicated that PUSA belonged to the saprophytic Leptospira meyeri species, while PITAL and PFRA appertained to L. biflexa. The evolutionary significance and the importance of the restriction and modification enzymes or non-specific nucleases within strains for genetic experiments are discussed.

The LE1 bacteriophage replicates as a plasmid within Leptospira biflexa: construction of an L. biflexa-Escherichia coli shuttle vector.

We have discovered that LE1, one of the plaque-forming phages previously described as lytic for the Leptospira biflexa saprophytic spirochete (I. Saint Girons, D. Margarita, P. Amouriaux, and G. Baranton, Res. Microbiol. 141:1131-1138, 1990), was indeed temperate. LE1 was found to be unusual, as Southern blot analysis indicated that it is one of the few phages to replicate in the prophage state as a circular plasmid. The unavailability of such small endogenous replicons has hindered genetic experimentation in Leptospira. We have developed a shuttle vector with DNA derived from LE1. Random LE1 DNA fragments were cloned into a pGEM 7Zf(+) derivative devoid of most of the bla gene but carrying a kanamycin resistance marker from the gram-positive bacterium Enterococcus (Streptococcus) faecalis. These constructs were transformed into L. biflexa strain Patoc 1 by electroporation, giving rise to kanamycin-resistant transformants. A 2.2-kb fragment from LE1 was responsible for replication of the vector in L. biflexa. However, a larger region including an intact parA gene homologue was necessary for the stability of the shuttle vector. Direct repeats and AT-rich regions characterized the LE1 origin of replication. Our data indicate that the replicon derived from the LE1 leptophage, together with the kanamycin resistance gene, is a promising tool with which to develop the genetics of Leptospira species.

CIITA B-cell-specific promoter suppression in MHC class II-silenced cell hybrids.

In this study, various sets of somatic cell hybrids, generated by the fusion of epithelial cell lines with B-lymphoblastoid cell lines, were analyzed for the expression of major histocompatibility complex (MHC) class II antigens. We first demonstrate, in human and mouse intraspecies hybrids, the coordinate suppression of MHC class II, Ii (invariant chain) and HLA-DM gene transcription, and the release of the silencing by the addition of interferon gamma. Using interspecies hybrids, the segregation of human chromosomes allowed us to establish that MHC class II extinction is linked to the presence in the hybrids of the chromosomes from the epithelial fusion partner. Moreover, our data provide evidence that the expression pattern of MHC class II mRNA is correlated with that of the class II transactivator (CIITA), suggesting that CIITA is the actual target of the silencing. To gain further insight into the suppression phenomenon we performed luciferase assays which show that silencing affects the activity of the B-cell-specific promoter of CIITA. These results therefore demonstrate that the MHC class II gene silencing in somatic cell hybrids is due to an active suppression of one of the promoters of the CIITA gene, mediated by the epithelial cell fusion partner.

Isolation of a B-cell-specific promoter for the human class II transactivator.

The class II transactivator (CIITA) is essential for the expression of major histocompatibility complex (MHC) class II antigens. The tissular patterns of CIITA and MHC class II gene expression are tightly correlated: CIITA mRNA is highly expressed in B cells, and is induced by interferon gamma (IFN-gamma) in macrophage and epithelial cell lines. We first isolated two overlapping cosmids encoding human CIITA which, when co-transfected, are able to restore MHC class II expression in a B-lymphoblastoid cell line (B-LCL) defective for CIITA. Subsequently, a 1.8 kilobase (kb) fragment of the CIITA promoter was isolated and sequenced. A motif presenting a strong similarity to an initiator was detected, as well as putative binding sites for Sp1, GATA-2, LyF-1, ets-1, AP1, and MZF1 transcription factors, and two GAS motifs. When introduced in front of a luciferase reporter gene, this promoter is able to direct a high luciferase activity in a human B-LCL. In contrast, luciferase expression was not stimulated after IFN-gamma treatment when the construct was transfected in macrophage or in epithelial cell lines. However, an induction of the human CIITA gene was observed in mouse macrophage and fibrosarcoma cell lines, when the cells were transfected with a cosmid containing the human CIITA gene, but lacking the 1.8 kb promoter described above. Taken together, these data suggest the existence of an intragenic promoter driving an IFN-gamma-inducible expression of CIITA.

The RAG cell line defines a new complementation group of MHC class II deficiency.

We previously described RAG, a mouse adenocarcinoma cell line, as deficient for the induction of major histocompatibility (MHC) class II antigens by IFN-gamma, but responding normally for MHC class I antigen stimulation and anti-viral protection. We had established that the fusion of RAG with various human cell lines restored the induction of MHC class II antigens, whenever the human chromosome 16 was present in somatic cell hybrids. Here we show that the RAG cell line does not exhibit any induction by IFN-gamma of DMA, DMB, and the invariant chain (Ii) mRNAs, and that the induction is restored in somatic cell hybrids containing human chromosome 16. In order to define the gene (designated F16) affected in the RAG cells, we performed a complementation analysis by fusing RAG with previously described human cell lines defective for MHC class II antigen expression (e.g., BLS cell lines), and which belong to five different complementation groups. Our data show that the resulting somatic cell hybrids present an inducible expression of mouse MHC class II antigens, Ii, DMA, and DMB. Therefore, the RAG cell line represents a yet undescribed cellular mutant affected in the expression of MHC class II antigens. In addition, we demonstrate that MHC class II antigens can be constitutively expressed in the RAG cell line when transfected with the cDNA encoding human CIITA driven by the RSV LTR promoter. Since the complementation analysis assessed that F16 and CIITA are distinct, our data suggest that F16 is required for the expression of CIITA.

Transfer of yeast artificial chromosomes into mammalian cells and comparative study of their integrity.

Yeast artificial chromosomes (YACs) from the CEPH MegaYAC library (Paris, France) ranging in size from 350 to 1600 kb and mapping to the q22.1 and q22.2 regions of human chromosome 21 were transferred into mammalian cells by spheroplast fusion. The integrity of the YACs from two adjacent parts of the region was compared after retrofitting and stable transfer into mammalian cells. We found that large YACs could easily be manipulated to allow transfer of the YAC material into mammalian cells and that the size of the YAC did not appear to be limiting for fusion. However, we show that there was great variability in the integrity of the YACs from the two regions, which was not related to the size of the YACs. Four YACs in region I from sequence-tagged site (STS) G51E05 up to STS LL103 showed, in general, no loss of material and correct gene transfer into mammalian cells. In contrast, the three YACs in the more centromeric region II (from STS G51B09 up to G51E05) frequently showed a loss of human material during handling, retrofitting and transfer. As a YAC from another library covering region II was also found to be unstable, we propose that the integrity of the YACs is highly dependent on the incorporated human chromosomal DNA.