[Interobserver agreement on diagnoses classified according the International Classification of Primary Care]. / Concordance inter-observateurs des diagnostics posés selon la classification CISP.

OBJECTIVE: To measure interobserver agreement on diagnoses classified and coded by family physicians using manual or computerized input modes. METHOD: Used increasingly in a variety of information management systems, the International Classification of Primary Care is the system best adapted to primary care. Ten physicians independently viewed 44 taped medical visits. Five physicians were randomly assigned to manual coding and five to computer coding. The study of reproducibility explored three aspects: written diagnoses, manually coded diagnoses, and diagnoses coded using a software program. The K statistic was calculated in order to compare interobserver agreement. RESULTS: Descriptive analysis of interobserver agreement in the written diagnoses revealed an agreement rate of 70.5% (+/- 6.3). Among physicians using manual coding, the agreement rate was 70.2% (+/- 7.2). In the group using the software program, the agreement rate was 75.0% (+/- 8.7). The K coefficients were low, but three were significant with critical ratios (z) above 1.96. CONCLUSION: Results suggest that input method has no bearing on interobserver agreement and that agreement is more a function of clinical presentation of health problems than of coding process.

The putative tumour suppressor EXT1 alters the expression of cell-surface heparan sulfate.

Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by the formation of cartilage-capped tumours (exostoses) that develop from the growth plate of endochondral bone. This condition can lead to skeletal abnormalities, short stature and malignant transformation of exostoses to chondrosarcomas or osteosarcomas. Linkage analyses have identified three different genes for HME, EXT1 on 8q24.1, EXT2 on 11p11-13 and EXT3 on 19p (refs 6-9). Most HME cases have been attributed to missense or frameshift mutations in these tumour-supressor genes, whose functions have remained obscure. Here, we show that EXT1 is an ER-resident type II transmembrane glycoprotein whose expression in cells results in the alteration of the synthesis and display of cell surface heparan sulfate glycosaminoglycans (GAGs). Two EXT1 variants containing aetiologic missense mutations failed to alter cell-surface glycosaminoglycans, despite retaining their ER-localization.

[Prevalence of positive tuberculin test in a population of patients requiring long-term care in a hospital setting]. / Prévalence du test à la tuberculine positif dans une population de patients en hébergement intra-hospitalier.

OBJECTIVE: To determine the prevalence of positive tuberculin tests in a population of patients requiring long-term care in a hospital setting. DESIGN: Prevalence study: to evaluate reaction to the test among patients who agreed to be included in the study. SETTING: The study took place in two units of the Enfant-Jésus hospital where patients are admitted for chronic care. PARTICIPANTS: A total of 108 patients were eligible for the study; 56 agreed to take part. One patient died before the study was completed. INTERVENTIONS: The tuberculin test consisted of an injection of PPD-T and reading the reaction 48 to 78 hours later. A reaction > 10 mm was considered significant. Patients with insignificant reactions were injected again 2 weeks later in order to evaluate positive response secondary to reactivation of the immune system (booster effect). MAIN OUTCOME MEASURES: Indications of previous tuberculosis, risk factors for tuberculosis, immunosuppressive medication, length of stay in hospital, size of reaction. RESULTS: Seventeen tuberculin tests were positive (30.9%); of these, six were positive after the second injection. CONCLUSION: The prevalence of positive tuberculin tests was high in our elderly population; this finding is comparable to the findings of American studies.

[Utilization of computerized classification system of primary care: three years of experience]. / Utilisation d'un système informatisé de classification pour les soins de première ligne: trois années d'expérience.

OBJECTIVE: To develop and implement a computerized version of the International Classification of Primary Care. To create a data bank and to conduct a descriptive study of our clinic's clientele. DESIGN: Testing a software program and creating a data bank. SETTING: Family Medicine Unit at Enfant-Jésus Hospital, Quebec City. PARTICIPANTS: All Family Medicine Unit doctors and patients seen between July 1, 1990, and June 30, 1993. MAIN OUTCOME MEASURE: Description of our clientele's health problems using the ICPC. RESULTS: During the study, 48,415 diagnostic codes for 33,033 visits were entered into the bank. For close to 50% of these visits, two or more health problems were coded. There was good correlation between the description of our clientele and descriptions in other studies in the literature. CONCLUSION: This article describes the development of a data bank in a family medicine unit using a software program based on the ICPC. Our 3-year experiment demonstrated that the method works well in family physicians' daily practice. A descriptive study of our clientele is presented, as well as a few examples of the many applications of such a data bank.

Sequential isolation of proteoglycan synthesis mutants by using herpes simplex virus as a selective agent: evidence for a proteoglycan-independent virus entry pathway.

A novel mouse L-cell mutant cell line defective in the biosynthesis of glycosaminoglycans was isolated by selection for cells resistant to herpes simplex virus (HSV) infection. These cells, termed sog9, were derived from mutant parental gro2C cells, which are themselves defective in heparan sulfate biosynthesis and 90% resistant to HSV type 1 (HSV-1) infection compared with control L cells (S. Gruenheid, L. Gatzke, H. Meadows, and F. Tufaro, J. Virol. 67:93-100, 1993). In this report, we show that sog9 cells exhibit a 3-order-of-magnitude reduction in susceptibility to HSV-1 compared with control L cells. In steady-state labeling experiments, sog9 cells accumulated almost no [35S]sulfate-labeled or [6-3H]glucosamine-labeled glycosaminoglycans, suggesting that the initiation of glycosaminoglycan assembly was specifically reduced in these cells. Despite these defects, sog9 cells were fully susceptible to vesicular stomatitis virus (VSV) and permissive for both VSV and HSV replication, assembly, and egress. HSV plaques formed in the sog9 monolayers in proportion to the amount of input virus, suggesting the block to infection was in the virus entry pathway. More importantly, HSV-1 infection of sog9 cells was not significantly reduced by soluble heparan sulfate, indicating that infection was glycosaminoglycan independent. Infection was inhibited by soluble gD-1, however, which suggests that glycoprotein gD plays a role in the infection of this cell line. The block to sog9 cell infection by HSV-1 could be eliminated by adding soluble dextran sulfate to the inoculum, which may act by stabilizing the virus at the sog9 cell surface. Thus, sog9 cells provide direct genetic evidence for a proteoglycan-independent entry pathway for HSV-1, and results with these cells suggest that HSV-1 is a useful reagent for the direct selection of novel animal cell mutants defective in the synthesis of cell surface proteoglycans.

Evidence for an interaction of herpes simplex virus with chondroitin sulfate proteoglycans during infection.

In a previous study, a mouse L cell mutant was isolated which is 90% resistant to HSV-1 infection (S. Gruenheid, L. Gatzke, H. Meadows, and F. Tufaro. J. Virol. 67, 93-100, 1993). This cell line, termed gro2C, failed to express heparan sulfate (HS)-glycosaminoglycans on the cell surface, which normally act as initial receptors for HSV-1 attachment to cultured cells. In this report, we extended the characterization of gro2C cells to explore the possibility that cell-surface chondroitin sulfate (CS) facilitates virus attachment to gro2C cells in the absence of HS. We found that soluble CS types A, B, and C strongly interfere with adsorption of HSV-1 to the surface of gro2C cells in a dose-dependent manner, and CS type B (dermatan sulfate) inhibited adsorption to parental (control) L cells by up to 10%. Moreover, gro2C cell infection was hypersensitive to inhibition by HS in comparison to control L cell infection. In all cases, a decrease in adsorption resulted in a decrease in infection. By contrast, the highly-sulfated glycosaminoglycan analog dextran sulfate was a relatively poor inhibitor of gro2C cell infection, indicating that the inhibitory effects of CS were related to its carbohydrate structure and not solely to its strong negative charge. By using a mutant virus strain which does not express the heparin-binding glycoprotein gC, we show that gC was not required for infection of gro2C cells, and was not required for the inhibition by HS or CS. Thus, the characterization of gro2C cell infection has revealed that one or more components of the HSV-1 particle can interact with cell-surface CS as well as HS to mediate infection of susceptible cells.

Examination of conditions affecting the efficiency of HVS-1 amplicon packaging.

Defective herpes simplex virus type 1 vectors (HSV amplicons) have been used as vehicles for efficiently delivering foreign genes into non-dividing cells such as neutrons in vitro and in vivo. This system is useful for studying neuronal physiology and may have potential for human gene therapy of neuronal disorders. The preparation of infectious amplicon particles is normally achieved by transfecting amplicon plasmid DNA, which contains the HSV replication origin and packaging signal, into mammalian cell lines followed by infection of the cells with HSV helper virus. This allows for replication and packaging of both viral and amplicon plasmid DNA. To improve the packaging efficiency of amplicons, several parameters involved in the packaging process were investigated. By introducing the SV40 DNA replication origin into an amplicon plasmid and prereplicating it before HSV infection, it was demonstrated that the existing amount of amplicon DNA prior to infection in the cells is not a rate-limiting step during HSV packaging. In addition, it was shown that the yield of the packaged amplicon particles can be improved by: (1) using a relatively small amount of HSV helper virus up to multiplicity of infection (m.o.i.) equal to 0.1 at infection; (2) infecting with HSV helper virus at 2 or 3 days post-transfection; and (3) passaging the initial packaged amplicon stocks 1-2 times on fresh host cells.

Immunoelectron microscopical distribution of poly(ADP-ribose)polymerase in the mammalian cell nucleus.

The ultrastructural distribution of poly(ADP-ribose)polymerase has been studied using a specific antibody and immunocytochemistry with immunogold markers. In situ localization in synchronized Chinese hamster ovary cells reveals the antibody associated with mitotic chromosomes, and later with condensed chromatin and perichromatin regions in G1 phase. During S and G2, the label occurs mostly on perichromatin regions where perichromatin fibrils are also observed. In the nucleolus, the label appears especially on the dense fibrillar component and to a minor extent on the granular component. Immunolabeled spread active chromatin preparations from exponentially growing cultured mouse P815 cells indicate preferential association of the antibody with nascent nonribosomal RNP fibrils compared to inactive chromatin. The results, suggesting a relationship between the poly(ADP-ribose)polymerase occurrence and RNA (or RNP) formation, are discussed in view of the present knowledge about possible relations between poly(ADP-ribosylation) and synthesis of RNA and DNA.

Structural and functional analysis of poly(ADP ribose) polymerase: an immunological study.

Poly(ADP ribose) polymerase (EC 2.4.2.30) was studied using monoclonal antibodies for three different epitopes on the enzyme. The epitopes were mapped in relation to the functional domains of the protein and the inhibitory properties of the antibodies. The intranuclear and interspecies immunoreactivity of the enzyme was also investigated. The epitope of antibody 2 was mapped to the 17 kDa fragment generated by chymotryptic digestion of the C-terminal 54 kDa NAD-binding domain. Antibody 9 binds to the N-terminal 29 kDa fragment of the DNA binding domain and inhibits the enzyme activity by 80%. This antibody was used to purify poly(ADP ribose) polymerase by immunoaffinity chromatography. The third antibody binds to a central 36 kDa fragment that possesses part of the DNA-binding domain and the automodification domain. This antibody increases the enzymatic activity by 30%. An analysis of the species cross-reactivity of the antibodies was carried out by immunoblot analysis of nuclear proteins. Antibody 10 binding was detected in rat FR3T3 cells, Chinese hamster ovary cells (CHO) and epidermoid carcinoma lung human cells (CALU-1). The other two antibodies are specific for the human and bovine enzymes. Western blot analysis showed the association of poly(ADP ribose) polymerase with residual nuclear material obtained after nuclease treatment and high-salt extraction. Immunofluorescence studies with the three different monoclonals demonstrated that accessibility of the epitopes varies in the nucleus.

Cell cycle regulation of poly(ADP-ribose) synthetase in FR3T3 cells.

The properties of poly(ADP-ribose) synthetase were studied throughout the cell cycle progression of non-synchronized rat FR3T3 fibroblasts using an immunological and biochemical approach. Cells in the various phases of the cell cycle were sorted from an asynchronously growing population by using flow cytofluorometry. G1, S and G2 + M fractions were used for enzymatic assays in the presence of saturating concentrations of DNAase I for the analysis of total poly(ADP-ribose) synthetase; maximal enzyme activity was found in the G2 + M phase. Purified IgG, specific for the FR3T3 poly(ADP-ribose) synthetase were used for the labelling of endogenous synthetase in order to quantify the enzyme immunologically. Localization of nuclear immunofluorescence was observed and analysis of poly(ADP-ribose) synthetase content throughout the cell cycle were carried out using double fluorescent staining and cytofluorometry. Poly(ADP-ribose) synthetase content as measured immunologically was found to increase from G1 to S and G2 + M phases. Quiescent cells showed a lower content as measured immunologically of poly(ADP-ribose) synthetase than cells in the G1 phase. In exponentially growing cells, the ratio between enzyme activity of poly(ADP-ribose) synthetase over the amount of enzyme measured immunologically was found to be higher in the G2 + M phase. These results show that a cell-cycle specific event activates poly(ADP-ribose) synthetase in the G2 + M phase.

Production and characterization of monoclonal antibodies specific for the functional domains of poly(ADP-ribose) polymerase.

Monoclonal antibodies were developed against poly(ADP-ribose) polymerase and analyzed for their reactivity against the NAD+- and DNA-binding fragments. Two fusions were performed to obtain hybridomas and the resulting anti-poly(ADP-ribose) polymerase antibodies were further screened by characterization of their immunoglobulin light chains. Five different hybridomas were isolated which produced different immunoglobulin light chains, all of which were specific for poly(ADP-ribose) polymerase. The specificities of these antibodies were determined by immunoblotting against the purified poly(ADP-ribose) polymerase, its autodegradation fragments, and the fragments prepared by limited proteolysis with chymotrypsin and papain. These fragments have been suggested to contain the NAD+-binding site, the DNA-binding site, and the automodification site, respectively. All the monoclonal antibodies reacted with the 116 kdalton (kDa) band corresponding to the purified enzyme. Four antibodies reacted exclusively with antigenic site(s) on the 46-kDa fragment which contains the DNA-binding site. A fifth antibody reacted exclusively with a clearly different antigenic site on the 74- and 54-kDa fragments which possess the NAD+ (substrate) binding site. The immunoreactivity with the major autodegradation products (69- and 46-kDa fragments) of the purified enzyme confirms this difference between the two groups of antibodies. The 22-kDa fragment corresponding to the auto-modification site does not show any immunoreactivity with the antibodies.

Visualization of poly(ADP-ribose) synthetase associated with polynucleosomes by immunoelectron microscopy.

Antibodies showing a high specificity for poly(ADP ribose) synthetase have been purified. A fraction binding nonspecifically to histones present in antiserum and non-immune serum has been demonstrated by immunoblotting and separated by histone-Sepharose chromatography. The antibody without the nonspecific binding fraction was analyzed by Western blot with calf thymus protein extract and was found to react only with a band at 116 kDa. There was no reaction with purified topoisomerase I, this weak activity was copurified with poly(ADP-ribose) synthetase preparation. The specific IgG fraction has been used for the visualization of the interaction of poly(ADP-ribose) synthetase with chromatin by indirect gold-labelling. This immunomicroscopic study suggests that the synthetase is located in the inner part of polynucleosomes and would be associated preferentially with the core nucleosome.

Non-histone chromosomal protein acceptors for poly(ADP)-ribose in phorbol-12-myristate-13-acetate treated mouse embryo fibroblasts (C3H10T1/2).

The tumor promoter phorbol-12-myristate-13-acetate (PMA) increases the level of poly ADP-ribosylation of chromosomal proteins in mouse embryo fibroblasts C3H10T1/2. The poly ADP-ribosylated nuclear proteins fall into the following molecular weight classes: 40, 48, 61, 77, 92, 158, 200 kd. Preincubation with catalase reduced the poly ADP-ribose (ADPR) substitution of all these proteins essentially to control levels. Western blot analysis with antibody directed against ADPR transferase indicates that the major acceptors are ADP-ribose transferase (116 kd) itself and its proteolytic degradation products of 20-25, 45 and 72-95 kd. Poly ADP-ribosylation of these proteins is suppressed by cycloheximide, 3-aminobenzamide, antipain and catalase. The latter three inhibitors possess anti-promotional activities in certain in vitro cell culture systems. Auto-poly ADP-ribosylation of ADPR transferase and its proteolytic cleavage as well as the poly ADP-ribosylation of other chromosomal proteins may play a role in the modulation of gene expression by PMA.