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R Sahli

Publications and source records attributed to R Sahli.

At least 19 recordsLinked to original sources
PubMed2005

[Endogenous hypercortisolism and immunologically-mediated disease: three cases].

HISTORY AND CLINICAL FINDINGS: We report three women with hypercortisolism presenting with symptoms and signs of Cushing's syndrome. In two of the patients, initial symptoms of hypercortisolism were associated with spontaneous amelioration of previously known atopic dermatitis and psoriasis, respectively. DIAGNOSTIC PROCEDURES: Diagnosis was established by demonstrating both lack of responsiveness to dexamethasone (1mg) suppression test and increased 24-hour urine cortisol secretion. One patient had a low serum ACTH level indicating Cushing's syndrome of adrenal origin. In the other two patients hypercortisolism proved to be ACTH-dependent, the source being the pituitary, as demonstrated by CRH stimulation test (elevation of ACTH and cortisol by 35 % and 20 %, respectively) and sampling of the petrosus sinus. In both patients imaging confirmed the presence of a pituitary adenoma. TREATMENT AND COURSE: All three patients underwent successful surgery: the first patient had an adrenalectomy, the other two transseptal transsphenoidal hypophysectomy. As symptoms and signs of hypercortisolism improved, the previously quiescent signs of atopic dermatitis and psoriasis recurred and one patient developed Graves' disease. CONCLUSIONS: Following successful treatment of endogenous hypercortisolism, symptoms of unrelated immunologically mediated conditions, especially autoimmune thyroiditis, may occasionally appear. Furthermore, the clinical course of coexisting immunologically mediated diseases may help to diagnose Cushing's syndrome and to monitor the patients after surgical treatment.

Adult↗
PubMed2001

Involvement of connexin 43 in meiotic maturation of bovine oocytes.

In ovarian follicles, cumulus cells provide the oocyte with small molecules that permit growth and control maturation. These nutrients reach the germinal cell through gap junction channels, which are present between the cumulus cells and the oocyte, and between the cumulus cells. In this study the involvement of intercellular communication mediated by gap junction channels on oocyte maturation of in vitro cultured bovine cumulus-oocyte complexes (COCs) was investigated. The stages of oocyte maturation were determined by Hoechst 33342 staining, which showed that 90% of COCs placed in the maturation medium for 24 h progress to the metaphase II stage. Bovine COC gap junction communication was disrupted initially using n-alkanols, which inhibit any passage through gap junctions. In the presence of 1-heptanol (3 mmol l(-1)) or octanol (3.0 mmol l(-1) and 0.3 mmol l(-1)), only 29% of the COCs reached metaphase II. Removal of the uncoupling agent was associated with restoration of oocyte maturation, indicating that treatment with n-alkanols was neither cytotoxic nor irreversible. Concentrations of connexin 43 (Cx43), the major gap junction protein expressed in the COCs, were decreased specifically using a recombinant adenovirus expressing the antisense Cx43 cDNA (Ad-asCx43). The efficacy of adenoviral infection was > 95% in cumulus cells evaluated after infection with recombinant adenoviruses expressing the green fluorescence protein. RT-PCR performed on total RNA isolated from Ad-asCx43-infected COCs showed that the rat Cx43 cDNA was transcribed. Western blot analysis revealed a three-fold decrease in Cx43 expression in COCs expressing the antisense RNA for Cx43. Injection of cumulus cells with Lucifer yellow demonstrated further that the resulting lower amount of Cx43 in infected COCs is associated with a two-fold decrease in the extent of coupling between cumulus cells. In addition, oocyte maturation was decreased by 50% in the infected COC cultures. These results indicate that Cx43-mediated communication between cumulus cells plays a crucial role in maturation of bovine oocytes.

Adenoviridae↗
PubMed2000

A mutagenic PCR identifies isolates of Borrelia garinii responsible for Lyme borreliosis.

Borrelia garinii is one of the three major Borreliae responsible for Lyme borreliosis in Europe. We have characterized a protein of B. garinii (VS102) and a genomic fragment from the gene encoding this protein was cloned. The DNA sequence of the fragment showed high homology with a known gene of B. burgdorferi sensu stricto. The protein encoded by this gene in B. burgdorferi sensu stricto is a phosphocarrier protein (histidine-containing protein). A mutation T to G polymorphism at codon 57 was found to be specific to B. garinii. A PCR-based approach that allows the rapid detection of this mutation made it possible to specifically discriminate B. garinii from other B. burgdorferi genospecies with high sensitivity and specificity.

Animals↗
PubMed2000

Preemptive treatment approach to cytomegalovirus (CMV) infection in solid organ transplant patients: relationship between compliance with the guidelines and prevention of CMV morbidity.

Cytomegalovirus (CMV) remains a major cause of morbidity in solid organ transplant patients. In order to reduce CMV morbidity, we designed a program of routine virological monitoring that included throat and urine CMV shell vial culture, along with peripheral blood leukocyte (PBL) shell vial quantitative culture for 12 weeks post-transplantation, as well as 8 weeks after treatment for acute rejection. The program also included preemptive ganciclovir treatment for those patients with the highest risk of developing CMV disease, i.e., with either high-level viremia (>10 infectious units [IU]/106 PBL) or low-level viremia (<10 IU/106 PBL) and either D+/R- CMV serostatus or treatment for graft rejection. During 1995-96, 90 solid organ transplant recipients (39 kidneys, 28 livers, and 23 hearts) were followed up. A total of 60 CMV infection episodes occurred in 45 patients. Seventeen episodes were symptomatic. Of 26 episodes managed according to the program, only 4 presented with CMV disease and none died. No patient treated preemptively for asymptomatic infection developed disease. In contrast, among 21 episodes managed in non-compliance with the program (i.e., the monitoring was not performed or preemptive treatment was not initiated despite a high risk of developing CMV disease), 12 episodes turned into symptomatic infection (P=0.0048 compared to patients treated preemptively), and 2 deaths possibly related to CMV were recorded. This difference could not be explained by an increased proportion of D+/R- patients or an increased incidence of rejection among patients with episodes treated in non-compliance with the program. Our data identify compliance with guidelines as an important factor in effectively reducing CMV morbidity through preemptive treatment, and suggest that the complexity of the preemptive approach may represent an important obstacle to the successful prevention of CMV morbidity by this approach in the regular healthcare setting.

Adolescent↗
PubMed2000

A rapid phenotypic assay for detection of acyclovir-resistant varicella-zoster virus with mutations in the thymidine kinase open reading frame.

Susceptibility assays by cell culture methods are time-consuming and are particularly difficult to perform with varicella-zoster virus (VZV). To overcome this limitation, we have adapted a functional test of the viral thymidine kinase (TK) in TK-deficient (tdk mutant) bacteria to detect ACV-resistant VZV in clinical samples. After PCR amplification, the complete viral TK open reading frame (ORF) is purified from PCR primers, digested with two restriction enzymes, and ligated in an oriented fashion into a bacterial expression vector. The ligation products are then used to transform tdk mutant bacteria. After transformation, an aliquot of the bacteria is plated onto a plate with minimal medium containing (i) ampicillin to select for plasmids carrying the viral TK ORF and (ii) isopropyl beta-D-thiogalactopyranoside (IPTG) to induce its expression. An identical aliquot of bacteria is also plated onto a medium containing, in addition to the components described above, 5-fluorodeoxyuridine (FUdR). Compared to the number of transformants on FUdR-free medium, the number of colonies carrying TK derived from susceptible strains was reduced by 86%, on average, in the presence of FUdR. In contrast, the number of transformants carrying TK from resistant strains with a mutant TK were reduced by only 4%, on average, on FUdR-containing plates. We have assessed the validity of this assay with cell culture isolates and several clinical samples including two cerebrospinal fluid samples from which no virus could be isolated. This colony reduction assay allowed the correct identification of the TK phenotype of each VZV isolate tested and can be completed within 3 days of receipt of the sample.

Acyclovir↗
PubMed1999

Expression of human papillomavirus 16 E2 protein in Schizosaccharomyces pombe delays the initiation of mitosis.

Infection by some types of human papillomavirus (HPV) is associated with the development of cervical cancer. Analysis of viral DNA from cervical tumours shows that the E2 gene is frequently disrupted during integration into the host cell's DNA. It has therefore been suggested that loss of E2p is an important step in malignant transformation. Expression of E2p in the fission yeast Schizosaccharomyces pombe retards the G2-M transition, by delaying activation of Cdc2p kinase. In contrast, S phase progression, and commitment to cell division in late G1 are not affected. The delay is independent of the transcriptional trans-activation function of E2p, and does not result from E2p DNA binding mimicking DNA damage. Increased expression of E2p also delays mitotic initiation in mammalian cells. S. pombe may thus provide a simple model for the analysis of E2p function.

Adenosine Triphosphatases↗
PubMed1999

Impact of cerebrospinal fluid PCR on the management of HIV-infected patients with varicella-zoster virus infection of the central nervous system.

Over a 2 year period, we identified five HIV-infected patients who presented with central nervous system infection caused by varicella-zoster virus, three with myelitits, and two with meningoencephalitis. All five patients were profoundly immunocompromised. Clinical presentation of these patients overlapped to a significant extent with diseases caused by other viruses, e.g. CMV. Indeed, in one case, a dual infection with CMV was diagnosed, but the respective role of each virus was ascertained by in situ hybridisation. At the time of CNS involvement, only one patient had active VZV cutaneous lesions, which were instrumental in diagnosing her condition. In contrast, PCR for VZV DNA in the CSF was helpful in making a diagnosis in the four other cases, one of which was confirmed by a post mortem. Of these five patients, two patients developed VZV disease while receiving oral acyclovir and had foscarnet treatment initiated when MRI demonstrated widespread lesions. They did not respond to antiviral therapy. The three other patients had intravenous acyclovir initiated at a time when no or limited parenchymal lesions were observed by MRI. Two of these three patients had VZV infection diagnosed solely on the basis of PCR: all three responded to treatment. Our data show that reactivation of VZV involving the central nervous system occurs frequently in the absence of cutaneous lesions. PCR of cerebrospinal fluid may help in making an early diagnosis which is probably a prerequisite for successful treatment of VZV infection of the CNS.

AIDS-Related Opportunistic Infections↗
PubMed1998

The nef gene controls syncytium formation in primary human lymphocytes and macrophages infected by HIV type 1.

nef, the 3'-most open reading frame of HIV, has been reported to enhance HIV replication in various host cell types and to promote in vivo replication and pathogenesis. The mechanism underlying the increased in vivo viral replication is still unclear. We have examined the effect of a nef deletion on the infection of primary human CD4+ T lymphocytes and macrophages, using clones with nef and env sequences derived, respectively, from T cell- and macrophage-tropic viruses. The deletion of nef enhanced the formation of syncytia in CD4+ T lymphocytes infected with macrophage-tropic clones, despite a severalfold reduced viral production. No such enhancement of syncytium formation was observed in CD4+ T lymphocytes infected with a T cell line-tropic clone, but in this clone, the deletion of nef imparted a more severe replication defect. A similar increase in syncytium formation was observed in primary human macrophages infected with nef-deleted clones compared with wild-type counterparts, except under conditions in which the deletion of nef markedly reduced viral replication. We could not demonstrate an enhanced cell surface expression of HIV-1 envelope in lymphocytes infected with nef-deficient clones to explain the increased syncytium formation. In enhancing the HIV-1 cytopathic effect, the deletion of nef might curtail virus production by infected cells, and thus explain in part the reduced viral load observed in vivo in hosts infected with nef-deficient viruses.

Animals↗
PubMed1998

Single tube competitive PCR for quantitation of CMV DNA in the blood of HIV+ and solid organ transplant patients.

Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality in transplant and HIV-infected patients. However, CMV can also cause asymptomatic infection. An elevated blood viral load as assessed by various methods appears to be a predictor for symptomatic infections, and can be used to identify patients at the highest risk of developing CMV disease. We developed a single tube competitive quantitative PCR assay for CMV DNA, using as a competitor a plasmid carrying the target sequence for amplification with an internal deletion. The analysis of data from repeated extractions and amplifications of samples showed that the coefficient of variation of the assay was typically less than 20%. Clinical samples from 14 HIV-infected and 13 solid organ transplant patients were analyzed. Widely varying CMV DNA levels were found in leukocytes, with a positive correlation with the measure of infectivity in the leukocytes by quantitative culture on fibroblasts. The highest CMV DNA content in leukocytes was found in two patients with presumptive CMV disease. In HIV patients, the amount of DNA in leukocytes was much larger than in solid organ transplant recipients, when standardized for infectivity. Although based on a very limited number of patients, this observation probably points to a difference in the biology of CMV infection in these two categories of susceptible individuals. CMV DNA was also found in the plasma of some of the patients with a high CMV DNA leukocyte load. The present test should be useful for identifying patients at high risk of developing CMV disease, for monitoring therapeutic efficacy of antiviral regimens and to improve the understanding the pathogenesis of CMV infection.

AIDS-Related Opportunistic Infections↗
PubMed1997

Human papillomavirus type 16 virus-like particles expressed in attenuated Salmonella typhimurium elicit mucosal and systemic neutralizing antibodies in mice.

Attenuated strains of Salmonella are attractive live vaccine candidates for eliciting mucosal as well as systemic immune responses. The ability to induce immune responses in the reproductive tract may be critical for the effectiveness of a prophylactic vaccine against genital human papillomaviruses (HPV), which are important etiologic agents in the development of cervical cancer. To examine the potential of a live Salmonella-based vaccine to prevent genital HPV infection, the L1 major capsid protein from HPV type 16 (HPV16) was constitutively expressed in the PhoPc strain of Salmonella typhimurium. As demonstrated by electron microscopy, the L1 protein expressed in these bacteria assembled into virus-like particles (VLPs) that resemble authentic papillomavirus virions. This is the first demonstration that papillomavirus VLPs can self-assemble in prokaryotes. BALB/c mice were immunized with the HPV16 L1 recombinant PhoPc strain by the oral and nasal routes. Despite a low stability of the L1-expressing plasmid in vivo, a double nasal immunization was effective in inducing L1-specific serum antibodies that recognized mainly native, but not disassembled, VLPs. These antibodies effectively neutralized HPV16 pseudotyped virions in an in vitro infectivity assay. Conformationally dependent anti-VLP immunoglobulin A (IgA) and IgG were also detected in oral and vaginal secretions, indicating that potentially protective antibody responses were elicited at mucosal sites. Recombinant attenuated Salmonella expressing HPV capsids may represent a promising vaccine candidate against genital HPV infection.

Animals↗
PubMed1995

The mismatched nucleotides in the 5'-terminal hairpin of minute virus of mice are required for efficient viral DNA replication.

The 5'-terminal sequence in the DNA of the parvovirus minute virus of mice (MVM) is a palindrome. It can form a hairpin, the stem of which is entirely base-paired except for three consecutive unpaired nucleotides which form a bubble. Since this structure is well conserved among different parvoviruses, we examined its importance for viral replication by generating MVM mutants with alterations in this region. A clone of MVMp DNA which contained the entire 3' end and more than half of the 5' palindrome was made. Although it lacked the sequence information to form a wild-type bubble, this DNA was infectious. On transfection into A9 fibroblasts, it gave rise to a virus (MVMs) which had a bubble in its 5' palindrome. The bubble consisted of four mismatched nucleotides in the same location as the unpaired nucleotides of the wild-type palindrome. Apparently, neighboring plasmid sequences were incorporated into the viral DNA, enabling formation of the mismatch. This observation suggested that a bubble is critical for growth of MVM but that its sequence is not. To find out whether MVM lacking a bubble in the 5' palindrome is viable, we made a second clone in which the plasmid sequences incorporated in MVMs were removed. Transfection of this DNA gave rise to a virus (MVMx) in which the nucleotides unpaired in the wild-type hairpin are now fully base-paired. Although MVMx can be propagated, it is defective in comparison with wild-type MVMp; it exhibited about a 50-fold-lower ratio of plaque-forming units to DNA content. In mixed infections, MVMp consistently outgrew the bubbleless MVMx. The rate of accumulation of DNA replication intermediates was lower for MVMx than for the wild-type virus. Quantitative analysis of the 5' termini of replicative form DNA suggested that the ability of MVMx to convert hairpin 5' termini to extended termini is impaired. In contrast, the virus with the altered bubble, MVMs, behaved like the wild-type MVMp in all the assays. We conclude that MVM lacking a bubble in its 5'-terminal DNA hairpin is less infectious than and has a selective disadvantage compared with wild-type MVM. The nucleotide sequence of the bubble is not critical. We provide evidence that the presence of a bubble is necessary for efficient viral DNA replication.

Animals↗
PubMed1994

[Identification of genital papillomavirus using PCR].

Over 20 different types of human papillomaviruses (HPV) are associated with tumoral lesions of the anogenital tract. Some of these lesions may become cancerous at a frequency which depends, among other risk factors, upon the type of HPV. Genital papillomaviruses with high oncogenic risk (HPV16, 18, 31, 33, 35 ...) are associated with the majority of cervical cancers and to a high proportion of high grade anogenital lesions, while HPV6 and HPV11 are most often associated with benign condylomas. The recently introduced PCR (Polymerase Chain Reaction) has distinctly simplified and improved detection and typing of genital HPVs. PCR detection of HPV has its place in epidemiologic studies and basic research, its application in daily clinical practise is, however, still controversial. For routine analysis, PCR serves more as a complementary tool to cytology.

Base Sequence↗
PubMed1994

[Bilateral conjunctivo-palpebral tumor in an immunocompetent man cause by human papilloma virus].

A 46-year-old white male patient in good general health presented with a bilateral conjunctival and eyelid tumor and a wart at the tip of the fourth right finger. Impression cytology showed dysplasic cells with loss of cohesion and up to 4 nucleoli per nucleus. The excisional biopsy of the conjunctiva revealed a dysplasic epithelial tissue that allowed the diagnosis of papilloma with parts of conjunctival intraepithelial neoplasia (CIN). Papilloma has been associated with Human Papillomavirus (HPV). The molecular hybridization analysis using PCR revealed that HPV was present in the eye tumor and in the wart, but the viral type was not the same. Treatment of the eye tumor with excision and low temperature cryocoagulation was successful.

Biopsy↗
PubMed1993

Defect in entry and altered pathogenicity of a polyoma virus mutant blocked in VP2 myristylation.

We have examined effects of mutations of the minor capsid proteins VP2 and VP3 of polyoma virus. Infection of cells by mutants blocked in expression of either VP2 or VP3 by alterations of their initiator methionine codons led to the rapid selection of wild-type revertants. This reversion suggests a strong, if not absolute, requirement for both proteins in virus growth. A mutant virus, VP2*, in which alanine was substituted for glycine-2, expressed a nonmyristylated form of VP2 that was incorporated into virions. Studies of VP2* revealed a 15- to 20-fold lower specific infectivity and a delay in growth in both primary and established mouse cells compared to wild-type virus. Analysis of the growth delay indicated a defect in an early step of infection, at or prior to uncoating. Synthesis of viral DNA and VP1 was delayed by about 9 hr in mutant compared to that in wild-type infected cells. No evidence was obtained for an effect on either virus assembly (encapsidation) or stability of virions. Infection of mice by the VP2* mutant virus resulted in attenuated virus replication and tumor induction which were much more severely affected following intranasal inoculation than intraperitoneal inoculation.

3T3 Cells↗
PubMed1993

Ribotyping of Pseudomonas aeruginosa: discriminatory power and usefulness as a tool for epidemiological studies.

Restriction fragment length polymorphism of ribosomal DNA regions (ribotyping) of Pseudomonas aeruginosa was evaluated as a tool for epidemiological purposes. Fifty-five epidemiologically unrelated isolates from three geographic areas of Switzerland and 11 isolates obtained during an outbreak of P. aeruginosa infections in a burn unit were typed by this method. Typeability and reproducibility of the method reached 100%. With four selected restriction enzymes (BamHI, ClaI, EcoRI, and PstI), the 55 unrelated isolates could be classified into 33 ribotypes. To assess the value of this method for the interpretation of epidemiological data, we calculated an index of discrimination (ID) which takes into consideration both the number of types defined by the typing method and their relative frequencies. Our ribotyping system obtained a high ID of 0.958 with only four restriction enzymes, comparing well with other different typing schemes for which ID values could be calculated from published data. All clinical isolates of the outbreak belonged to the same ribotype, whereas environmental isolates, initially thought to be the source of the epidemic, belonged to a different ribotype. Thus, the typeability, reproducibility, and discriminatory power of our method as well as its value established in an epidemiological investigation were found to be appropriate for further epidemiological studies of P. aeruginosa.

Bacterial Typing Techniques↗
PubMed1993

Molecular analysis of the LMP (latent membrane protein) oncogene in Hodgkin's disease.

Molecular analysis of the LMP (latent membrane protein) oncogene was performed in 21 Epstein-Barr virus (EBV) positive cases of Hodgkin's disease (HD) with proven LMP gene expression. In each case, viral DNA of the LMP gene was assessed for polymorphism (deletions, insertions, mutations) by polymerase chain reaction (PCR) amplification with selected primers. Specificity of the amplified targets was proven by internal oligonucleotide hybridisation and nested primer PCR. Homogeneity of the 5' LMP gene region coding for the amino terminal, transmembrane, and short extracytoplasmic domains of the protein was identified in all cases. However, deletions or insertions of small DNA sequences within the coding region for the intracytoplasmic LMP domain were observed in about 20% of cases. In one of them, a 30-base-pair deletion was precisely localized by DNA sequencing. A particularly high frequency of DNA polymorphism (30% of cases) was found in the 3' untranslated LMP region. However, when analysing the LMP gene in seven benign conditions, no DNA polymorphism was found. These data suggest conservation of oncogenic LMP regions coding for the protein domains known to be associated with transforming capacities and immunogenic functions. They also show a considerable genomic heterogeneity of the coding region for the intracytoplasmic domain and the 3' untranslated mRNA region. This LMP DNA polymorphism identified within a localized (Swiss) population suffering from HD is unexpected. Its eventual clinical significance remains to be determined.

Antigens, Viral↗