Evaluation of MALDI-TOF mass spectrometry for the identification of bacteria growing as biofilms.

We evaluated MALDI-TOF mass spectrometry to identify bacteria from biofilms. We compared three sample preparation procedures on biofilms grown in vitro. The extended direct transfer method was able to identify 13 isolates out of 18 (72%) at the species level and 15 out of 18 (83%) at the genus level.

Rate of emergence of cytomegalovirus (CMV) mutations in leukocytes of patients with acquired immunodeficiency syndrome who are receiving valganciclovir as induction and maintenance therapy for CMV retinitis.

The emergence of mutations conferring ganciclovir resistance was evaluated in an open-label randomized clinical trial that compared oral valganciclovir with intravenous ganciclovir as induction therapy, followed by maintenance with valganciclovir, for newly diagnosed cytomegalovirus (CMV) retinitis in 148 patients with acquired immunodeficiency syndrome. The presence of CMV mutations was directly assessed in patient leukocytes by polymerase chain reaction, followed by restriction fragment-length polymorphism (RFLP) for detection of the most common UL97 mutations associated with ganciclovir resistance and by sequencing of the viral UL97 gene. The cumulative percentages of patients with UL97-mutant viruses at 3, 6, 12, and 18 months (based on the number of patients on treatment at each time point) was 2.2%, 6.5%, 12.8%, and 15.3%, respectively. Of the 20 relevant UL97 mutations found by sequencing in 14 patients, 14 (70%) were detected by RFLP analysis. The rate of emergence of ganciclovir-resistant viruses with use of oral valganciclovir is no greater than that reported with use of intravenous ganciclovir.

Evaluation of the human herpesvirus 8 DNA load in blood and Kaposi's sarcoma skin lesions from AIDS patients on highly active antiretroviral therapy.

OBJECTIVES: To evaluate the human herpesvirus 8 (HHV-8) DNA load in peripheral blood mononuclear cells (PBMC) and Kaposi's sarcoma (KS) skin lesions of subjects with AIDS and to correlate these measures with the tumour load. DESIGN: Assessment of the HHV-8 DNA load was performed every 3 months in PBMC and every 6 months in KS skin lesions from seven subjects with AIDS who were receiving highly active antiretroviral therapy (HAART). METHODS: The HHV-8 DNA load was determined by a quantitative-competitive PCR using 0.2 microg of DNA from PBMC or KS skin biopsies. Staging of KS was performed by evaluating the number and type of cutaneous KS lesions. RESULTS: The three subjects with the most extensive and active (nodular) KS had the highest amounts of HHV-8 DNA in KS skin lesions and the lowest CD4 T cell counts (< 200 x 10(6)/l). In contrast, the four other subjects with regressing KS while on HAART had a low viral load in their KS lesions. All but one subject who also had multicentric Castleman's disease had low amounts of HHV-8 DNA in PBMC. CONCLUSION: There is a strong relationship between the tumour burden and the HHV-8 viral load in KS skin lesions of subjects with AIDS, reinforcing the causal link between this herpesvirus and AIDS-related KS.

Human herpesvirus 8 DNA load in leukocytes of human immunodeficiency virus-infected subjects: correlation with the presence of Kaposi's sarcoma and response to anticytomegalovirus therapy.

Specific human herpesvirus 8 (HHV-8) DNA sequences were found in leukocytes of 12 of 29 (41.4%) AIDS subjects with Kaposi's sarcoma (KS), whereas they were found in 4 of 43 (9.3%) AIDS subjects without KS (P = 0.003), although the peak HHV-8 DNA load in PCR-positive subjects with KS (mean, 425 copies per 0.2 microgram of DNA) did not significantly differ from the one found in PCR-positive patients without KS (mean, 218 copies). The use of intravenous ganciclovir or foscarnet therapy to treat cytomegalovirus disease did not affect the HHV-8 DNA load in seven patients for whom serial samples were analyzed.

Phenotypic and genotypic characterization of acyclovir-resistant herpes simplex viruses from immunocompromised patients.

Phenotypic and genotypic analyses were done on 30 acyclovir-resistant and 5 acyclovir-susceptible herpes simplex virus (HSV) isolates (22 HSV type 1 and 13 HSV type 2) recovered from 24 subjects. All isolates were susceptible to foscarnet. The phenotypes of the acyclovir-resistant HSV isolates were as follows: 17 were thymidine kinase (TK) deficient, 12 had decreased TK activity (produced low amounts of viral TK) or TK with altered substrate specificity, and 1 was undetermined. Sequencing analysis of the HSV TK gene revealed that 14 (46.7%) of 30 acyclovir-resistant isolates had an insertion or deletion of 1 or 2 nucleotides, especially in homopolymer runs of Gs, Cs, and rarely in As. On the other hand, 16 (53.3%) of 30 acyclovir-resistant isolates had point mutations in conserved or nonconserved regions of the TK gene. In conclusion, HSV can develop multiple strategies to exhibit acyclovir resistance, including, in about half of the cases, frameshift mutations in homopolymer nucleotide stretches of the TK gene.

Modulation of chromatin superstructure induced by poly(ADP-ribose) synthesis and degradation.

It has been demonstrated recently by Poirier et al. (Poirier, G. G., de Murcia, G., Jongstra-Bilen, J., Niedergang, C., and Mandel, P. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3423-3427) that poly(ADP-ribosyl)ation of pancreatic nucleosomes causes relaxation of the chromatin superstructure through H1 modification. The in vitro effect of poly(ADP-ribose) synthesis and degradation on calf thymus chromatin was investigated by the time course incorporation of ADP-ribose, electron microscopy, analytical ultracentrifugation, and autoradiography of the protein acceptors. Purified calf thymus poly(ADP-ribose) polymerase and partially purified bull testis poly(ADP-ribose) glycohydrolase were used. Degradation of ADP-ribose units on hyper(ADP-ribosyl)ated H1 by poly(ADP-ribose) glycohydrolase restores the native condensed chromatin superstructure. This reversible conformational change induced by poly(ADP-ribosyl)ation on nucleosomal arrangement could be one of the mechanisms by which the accessibility of DNA polymerases and/or excision-repair enzymes is favored, the native structure being fully restorable.

Poly(ADP-ribose) accessibility to poly(ADP-ribose) glycohydrolase activity on poly(ADP-ribosyl)ated nucleosomal proteins.

Hydrolysis of protein-bound 32P-labelled poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase shows that there is differential accessibility of poly(ADP-ribosyl)ated proteins in chromatin to poly(ADP-ribose) glycohydrolase. The rapid hydrolysis of hyper(ADP-ribosyl)ated forms of histone H1 indicates the absence of an H1 dimer complex of histone molecules. When the pattern of hydrolysis of poly(ADP-ribosyl)ated histones was analyzed it was found that poly(ADP-ribose) attached to histone H2B is more resistant than the polymer attached to histone H1 or H2A or protein A24. Polymer hydrolysis of the acceptors, which had been labelled at high substrate concentrations (greater than or equal to 10 microM), indicate that the only high molecular weight acceptor protein is poly(ADP-ribose) polymerase and that little processing of the enzyme occurs. Finally, electron microscopic evidence shows that hyper(ADP-ribosyl)ated poly(ADP-ribose) polymerase, which is dissociated from its DNA-enzyme complex, binds again to DNA after poly(ADP-ribose) glycohydrolase action.

Sequential ADP-ribosylation pattern of nucleosomal histones. ADP-ribosylation of nucleosomal histones.

The pattern of nucleosomal histones poly(ADP-ribosyl)ation is changed under conditions which affect the poly(ADP-ribosyl)ation state of the enzyme. At low NAD concentrations the enzyme can poly(ADP-ribosyl)ate histones H1 and H1, H2A, A2A, and H2B. However at NAD concentrations above 10 microM the enzyme preferentially poly(ADP-ribosyl)ates histone H1 to a hyper ADP-ribosylated form. Furthermore we have observed hyper ADP-ribosylation of histone H2B at NAD concentrations of 10 microM suggesting that histone H2B can undergo the same type of ADP-ribosylation pattern as histone H1. Also at higher NAD concentrations an elongation of the polymer attached to the enzyme and other nuclear proteins takes place.

Familial articular chondrocalcinosis in Quebec.

The existence of articular chondrocalcinosis was documented in 9 members of 3 generations of a Quebec family. No associated or secondary forms of the disease were found. The clinical manifestations appeared early in life, and extensive radiologic involvement was apparent. We determined that genetic transmission was dominant, either autosomal or sex-linked, and not related to the HLA system.

[Renal histology in 44 patients with specific antibodies of soluble nuclear antigens]. / Etude de l'histologie rénale chez 44 malades porteurs d'anticorps spécifiques d'antigènes nucléaires solubles.

The authors studied the correlations between renal histology and specific antinuclear antibodies of soluble nuclear antigens (anti-Sm, anti-RNP, anti-protein) in 44 patients with such auto-antibodies. They were mostly patients with lupus erythematosus (35/44), more rarely mixed collagen disease or Sjögren's disease. The presence of any one of the specific antibodies of nuclear antigens is not associated with any special renal prognosis; thus the presence of anti-RNP does not mean that there are no histological renal lesions. The renal prognosis depends in fact on the presence of anti-ADN native antibodies. Among the other laboratory parameters (rheumatoid factors, complement levels, cryoglobulinemia) only hypocomplementemia seems to be associated with a poor renal prognosis, the presence of rheumatoid factor has perhaps a protective role.

Clinical significance of antibodies to soluble extractable nuclear antigens (anti-ENA).

Clinical and biological manifestations have been studied in 134 patients whose serum had antibodies to soluble extractable nuclear antigens (ENA). 85 of the patients had anti-RNP antibodies, 18 had anti-Sm antibodies, and 31 had antibodies to one or more soluble nuclear antigen. In all groups, the predominant clinical manifestations were polyarthritis, Raynaud's phenomenon, fever, and skin involvement. Renal disease was less common in those patients with anti-RNP antibodies than in the other patients. Most patients with definite renal disease (13 out of 15) also had circulating anti-DNA antibodies. The final diagnoses in these 134 patients were well defined connective tissue disease in 59; overlap syndromes in 34; a limited clinical syndrome made up of polyarthritis Raynaud's phenomenon--often with swollen fingers--and/or hypergammaglobulin-aemia in 31, and various other clinical conditions in 10.

[The clinical significance of soluble nuclear antigen specific antibodies (author's transl)]. / Signification clinique des anticorps spécifiques d'antigènes nucléaires solubles.

Anti-ENA antibodies have been found in 176 sera which nearly all contained antinuclear antibodies giving a speckled pattern of nuclear fluorescence. The charts of 134 of these 176 patients were available for a thorough clinical study. Among these 134 patients, 59 had a well defined Connective Tissue Disease including 40 SLE, 31 had a limited clinical syndrome made of Raynaud's phenomenon, inflammatory polyarthritis, swollen fingers and hyperglobulinemia and 34 had a complex clinical picture associating signs of more than one connective tissue disease. Some of the patients in this third group could be considered as-having the Mixed Connective Tissue Disease (MCTD) described by Sharp et al. Anti-RNP antibodies were more common in this series than the other anti-ENA antibodies. However, no narrow specificity could be assigned to any of these antibodies. This is true of the non anti-RNP antibodies, the anti-Sm in particular, which were found in 49 patients of whom 32 had SLE existing alone or in association with features of other connective tissue diseases and 17 had another connective tissue disease or the afore-mentioned limited clinical syndrome. In any case, the anti-ECT antibodies never reach the diagnostic value of the anti-DNA antibodies.