An in vitro study to compare the influence of different all-ceramic systems on the polymerization of dual-cure resin cement.

AIM: The aim of the study is to compare the effect of composition of three different all-ceramic systems on the polymerization of dual-cure resin cement, using different curing cycles and evaluated immediately within 15 min and after 24 h. MATERIALS AND METHODS: Resin cement disc samples were fabricated by polymerization through three different all-ceramic disc, namely: lithium disilicate discs - IPS e.max (Group B), leucitereinforced discs - IPS Empress (Group C), zirconia discs - Cercon (Group D), and without an intervening ceramic disc, as control (Group A). A total of 80 resin cement disc samples were fabricated for fur groups (n = 20). Each group further consisted of two subgroups (n = 10), t10 and t20 according to two different exposure times of 10 and 20 s, respectively. Each of the 80 resin disc samples was evaluated for their degree of polymerization achieved, by measuring the microhardness(Vickers hardness number) of the samples immediately within 15 min and after 24 h, giving us a total of 160 readings. Oneway analysis of variance test, ttest, and paired ttest were used for multiple group comparisons followed by Tukey's post hoc for groupwise comparison. RESULTS: Direct activation of the resin cement samples of control (Group A) showed statistically significant higher mean microhardness values followed by Groups C then B and D, both immediately and after 24 h. The mean microhardness for immediate post-activation was always inferior to the 24 h post-activation test. For both 10 and 20 s curing cycle, there was a significant increase in the microhardness of the resin cement discs cured for 20 s through the different ceramics. CONCLUSION: Ceramic composition affected the polymerization of dual cured resin cement. Doubling the light irradiation time or curing cycle significantly increased mean microhardness value. Greater degree of conversion leading to an increase in hardness was observed when the resin cement discs were evaluated after 24 h.

Sustained Release of Diltiazem Hydrochloride from Cross-linked Biodegradable IPN Hydrogel Beads of Pectin and Modified Xanthan Gum.

Interpenetrating polymer network hydrogel beads of pectin and sodium carboxymethyl xanthan were prepared by ionotropic gelation with Al(+3) ions and covalent cross-linking with glutaraldehyde for sustained delivery of diltiazem hydrochloride. Fourier transform infrared spectroscopy, X-ray diffraction, differential scanning colorimetry and scanning electron microscopy were used to characterise the hydrogel beads. The swelling of the hydrogel and the release of drug were relatively low in pH 1.2 buffer solutions. However, higher swelling and drug release were observed in pH 6.8 buffer solutions. The carboxyl functional groups of hydrogels undergo ionisation and the osmotic pressure inside the beads increases resulting in higher swelling and drug release in higher pH. The release of drug depends on concentration of polymer, amount and exposure time of cross-linker and drug content in the hydrogel matrices. The present study indicated that the hydrogel beads minimised the drug release in pH 1.2 buffer solutions and to prolong the drug release in pH 6.8 buffer solutions.

Protein S Thr103Asn mutation associated with type II deficiency reproduced in vitro and functionally characterised.

Protein S functions as a cofactor to activated protein C (APC) in the degradation of FVa and FVIIIa. In protein S, the thrombin sensitive region (TSR) and the first EGF-like domain are important for expression of the APC cofactor activity. A naturally occurring Thr103Asn (T103N) mutation in the first EGF-like domain of protein S has been associated with functional (type II) protein S deficiency. To elucidate the functional consequences of the T103N mutation, recombinant protein S mutant was expressed in mammalian cells and functionally characterised. The expression level of protein S T103N from transiently transfected COS 1 cells was equal to that of wild type protein S. The mutant protein S and wild type protein S were also expressed in 293 cells after stable transfection, and the recombinant proteins purified. In APTT- and PT-based coagulation assays, the mutant protein demonstrated approximately 50% lower anticoagulant activity as compared to wild type protein S. The functional defect was further investigated in FVa- and FVIIIa-degradation assays. The functional defect of mutant protein S was attenuated at increasing concentrations of APC. The results demonstrate the region around residue 103 of protein S to be of functional importance, possibly through a direct interaction with APC.

Deficient APC-cofactor activity of protein S Heerlen in degradation of factor Va Leiden: a possible mechanism of synergism between thrombophilic risk factors.

In protein S Heerlen, an S-to-P (single-letter amino acid codes) mutation at position 460 results in the loss of glycosylation of N458. This polymorphism has been found to be slightly more prevalent in thrombophilic populations than in normal controls, particularly in cohorts of patients having free protein S deficiency. This suggests that carriers of the Heerlen allele may have an increased risk of thrombosis. We have now characterized the expression in cell cultures of recombinant protein S Heerlen and investigated the anticoagulant functions of the purified recombinant protein in vitro. Protein S Heerlen was synthesized and secreted equally well as wild-type protein S by transiently transfected COS-1 cells. The recombinant protein S Heerlen interacted with conformation-dependent monoclonal antibodies and bound C4b-binding protein to the same extent as wild-type protein S. Protein S Heerlen displayed reduced anticoagulant activity as cofactor to activated protein C (APC) in plasma-based assays, as well as in a factor VIIIa-degradation system. In contrast, protein S Heerlen functioned equally well as an APC cofactor in the degradation of factor Va as wild-type protein S did. However, when recombinant activated factor V Leiden (FVa:Q506) was used as APC substrate, protein S Heerlen was found to be a poor APC cofactor as compared with wild-type protein S. These in vitro results suggest a possible mechanism of synergy between protein S Heerlen and factor V Leiden that might be involved in the pathogenesis of thrombosis in individuals carrying both genetic traits. (Blood. 2000;96:523-531)

In vitro characterisation of two naturally occurring mutations in the thrombin-sensitive region of anticoagulant protein S.

The molecular consequences of two naturally occurring mutations in the thrombin-sensitive region of protein S were investigated using a combination of recombinant protein expression, functional analysis and molecular modelling. Both mutations (R49H and R70S) have been found in thrombosis patients diagnosed as having type I protein S deficiency. Molecular modelling analysis suggested the R49H substitution not to disrupt the structure of thrombin-sensitive region, whereas the R70S substitution could affect the 3D structure mildly. To elucidate the molecular consequences of these substitutions experimentally, site directed mutagenesis of protein S cDNA and expression in mammalian cells created the two mutants. The secretion profiles and functional anticoagulant activities of the protein S mutants were characterised. Secretion of the R49H mutant was similar to that of wild type protein S, whereas the R70S mutant showed moderately decreased expression. Neither of the mutants showed any major functional defects as cofactors to activated protein C (APC) in an APTT-based assay or in degradation of factor Va. However, both mutants demonstrated decreased activity in a factor VIIIa degradation assay, which in addition to APC and protein S also included factor V as synergistic APC cofactor. In conclusion, the R49H substitution did not produce a quantitative abnormality in vitro, raising doubts as to whether it caused the type I deficiency. In contrast, the experimental data obtained for the R70S mutant agrees well with the observed type I deficiency. Our study illustrates that in vitro experimental characterisation together with computer-based structural analysis are useful tools in the analysis of the relationship between naturally occurring mutations and clinical phenotypes.

Topological studies of the amino terminal modules of vitamin K-dependent protein S using monoclonal antibody epitope mapping and molecular modeling.

Protein S is an important anticoagulant protein acting as cofactor to activated protein C (APC) in the degradation of membrane-bound factors Va and VIIIa. Binding of protein S to the membrane depends on the Gla-domain, whereas sites for APC-interaction are located in the thrombin-sensitive region (TSR) and the first EGF domain. The aims of the present investigation were to localize the sites on protein S which are involved in APC-cofactor function and to elucidate possible orientations of the TSR in relation to the membrane. For these purposes, we determined the epitope for a calcium-dependent monoclonal antibody (HPS67) against the TSR, which inhibits APC cofactor activity even though it does not impede protein S binding to the membrane. HPS67 did not recognize wild-type mouse protein S but gained reactivity against a recombinant mouse protein in which G49 and R52 were mutated to R and Q (found in human protein S), respectively, suggesting these two residues to be part of a surface exposed epitope for HPS67. This information helped in the validation and refinement of the structural model for the Gla-TSR-EGF1-modules of protein S. The X-ray structure of a Fab-fragment mimicking HPS67 was docked onto the protein S model. The observation that HPS67 did not inhibit phospholipid binding of protein S has implications for the possible orientation of protein S on the membrane surface. In the proposed model for membrane-bound protein S, there is no contact between the TSR and the membrane. Rather, the TSR is free to interact with membrane-bound APC.

A new direct, fast and quantitative enzyme-linked ligandsorbent assay for measurement of free protein S antigen.

A new method to determine the concentration of free protein S in plasma is described. It is an enzyme-linked ligandsorbent assay (ELSA) which utilises the protein S binding capacity of the natural ligand C4b-binding protein (C4BP) to capture the free protein S from plasma samples. The use of C4BP as ligand in the assay is possible due to the high affinity (Kd = 0.1 nM) of the interaction between protein S and C4BP and to a slow rate of complex dissociation. A monoclonal antibody (HPS 54) was conjugated with horseradish peroxidase and used as target antibody. This antibody recognises a Ca2+ dependent epitope in the first EGF-like domain of protein S and does not interfere with C4BP binding sites of protein S. Addition of calcium in the assay helped prevent dissociation of the C4BP-protein S-HPS 54 complex. Three different experiments demonstrated the assay to be specific for free protein S. First, near-identical dose response curves were obtained with protein S in plasma and with purified protein S. Second, addition of purified C4BP to normal plasma resulted in loss of free protein S. Third, protein S depleted plasma gave zero values and around 80% of purified protein S added to protein S depleted plasma, and approximately 70% of protein S added to protein S deficient plasma samples, was recovered with the assay. The assay is fast (involves only a single incubation step of 30 min), sensitive and the range of measurement is 3% to 200% of free protein S when plasma dilution 1:20 represents 100%. Intra- and inter-assay coefficients of variation at two levels were 2.3-4.3% and 5.1-7.4%, respectively. In a large protein S deficient family, the assay showed 100% sensitivity and specificity for the causative mutation. Moreover, free protein S levels in anticoagulated protein S deficient patients were completely separated from those obtained in non-anticoagulated controls. The new assay for free protein S is suitable for automation and it provides a useful means for routine clinical purposes to detect protein S deficiencies.

Spectrum of clinical and laboratory characteristics of HIV infection in northern India.

To define the impact of HIV infection in India, the clinical and laboratory profile and the correlation of CD4 count to the likely opportunistic infection in a cohort of 134 HIV positive patients in Northern India was analysed. Majority of the patients, 72% and 67.8% (children and adults respectively) were asymptomatic, having been detected during routine screening and maintained that status for a median follow-up period of 3 years. Among the symptomatic patients, oropharyngeal candidiasis was the most common opportunistic infection followed closely by tuberculosis (both pulmonary and extra pulmonary) around 3.6-4.0 years from probable HIV infection with a median CD4 of 420-578 per cmm. Infection with Cryptococcosis, Cryptosporidiosis and cytomegalovirus occurred only after a significant fall in CD4 to < 100/cmm usually around 8-10 years from probable HIV infection. Pneumocystis carinii pneumonia was the terminal event among the 12 deaths at a mean CD4 count of 6/cmm. Non specific constitutional symptoms like fever, prolonged diarrhoea and significant weight loss were frequent. In general, the clinical profile of Indian patients with HIV bears much resemblance to African countries owing perhaps to the similar background of poverty, malnutrition and endemic infection.

Sociodemographic characteristics of HIV infection in northern India.

134 patients testing positive for HIV antibody during the period 1986-1993 were included in the present study. An in-depth analysis of the subjects revealed that the adult males seemed to have the highest propensity for HIV infection in this part of the country. Marital status had no bearing on incidence and route of seropositivity. This was not so in females. Extramarital heterosexual contact was the mode of HIV acquisition in adults in contrast to blood transfusion in children. Clinically, most of these patients were still asymptomatic. At presentation, oral Candidiasis was common. Pneumocystis carinii pneumonia (PCP) was the leading cause of death.

PIP: Data are analyzed from 134 HIV positive individuals who were referred to the National AIDS Control Organization of the Indian Government for clinical management during June 1986-June 1993. The center was a major referral center for northern India. HIV was determined by enzyme linked immunosorbent assay (ELISA). Retesting was conducted. The population was grouped as under and over 13 years of age. Laboratory testing was performed in order to determine the absolute lymphocyte count (ALC), the absolute and percentage of CD4+ and CD8+ lymphocyte counts and CD4/CD8 ratios, immunoglobulins, and delayed-type cutaneous hypersensitivity (DTH). Findings indicated an increase in HIV positive cases over time and a greater number of adults who were HIV positive. The mean age was 27.2 years for males and 22.2 years for females. The youngest age was 1.5 years. 116 HIV positive people were Indians, and most lived in metropolitan areas of northern India. 25 were children. 25 lived in neighboring villages of Haryana, Punjab, and around Delhi. Marital status appeared to be unrelated to HIV status. 51 men were single and 46 were married and seropositive due to sexual contacts. 4 women were single and 8 were married. Of the 4 single women, 2 were sexually very active with multiple partners. 6 of the 8 married females acquired HIV infection through their spouses. The other 2 received HIV infected blood transfusions. 39.5% of men and 75% of women acquired HIV infections from heterosexual contacts. 29% of transmission was due to contaminated blood and blood products. The HIV infected male population comprised mainly businessmen and defense personnel. HIV infected persons came mainly from the Bombay-Pune area. 66.6% of persons infected from contaminated blood were from Delhi. Asymptomatic PGL and ARC screenings were the common reason for referral to the center. 13 of the 134 have already died. The most common cause of death was Pneumocystis carinii pneumonia. The most common opportunistic infection was candidiasis.

Profile of HLA-B27-related 'unclassifiable' seronegative spondyloarthropathy in females and its comparison with the profile in males.

Unclassifiable seronegative spondyloarthropathy (SSA) syndrome is primarily considered to be an affliction of males. In this report from northern India, 25 HLA-B27 antigen positive females with this condition are described and compared with 39 HLA-B27-positive males with the same disease. All these patients presented with typical features of spondyloarthropathy such as predominantly lower limb synovitis, enthesopathy and inflammatory spinal pain. The onset was insidious in 56% of the females and in 64% of the males. The mean age of onset as also the mean duration of symptoms prior to diagnosis were significantly higher in females (26.2 vs 19.4 yr and 8 vs 2 yr, respectively). A mono- or oligo-arthritis was seen in 52% of the females and in 53% of the males, but the average number of joints involved was less in females (4.8 vs 7.7). Lower limb joints alone were involved in 56% of the females and 49% of the males, with the knees, ankles and hips being most commonly involved, often asymmetrically. The mean degree of symmetry was significantly lower in females (62 vs 76). Ninety-two per cent of females and 74% of males had inflammatory spinal pain. Radiographic sacroiliitis was demonstrable in 56% females and 74% males. It is concluded that 'unclassifiable' SSA syndrome is not infrequent in females but is diagnosed late. Fewer joints tend to be involved and there is greater tendency towards asymmetry in females.

Surveillance of STD patients for AIDS using World Health Organisation criteria.

PIP: The World Health Organization (WHO) criteria for HIV clinical disease were tested among individuals with high-risk behavior in northern India. A questionnaire, based upon history and physical examination alone, standardized by the WHO to include both major and minor signs necessary for the clinical diagnosis of AIDS in adults was applied to 165 consecutive patients attending the STD clinic of Dr. R.M.L. Hospital, New Delhi. All patients were screened for the presence of STDs by the dermatologist in charge of the clinic, with patients fulfilling two major and at least two minor WHO criteria eventually classified as having clinical AIDS based upon the WHO case definition. Each of those patients was subjected to serological confirmation of the clinical suspicion using ELISA and Western blot commercial tests. Of the 165 patients screened, a definite diagnosis of STD was possible in 85. These patients were 20-45 years old (mean age, 30.59 years). All were male and chancroid was the most common STD in the cohort. Of the 85, only one satisfied the WHO clinical criteria for AIDS. Serological investigations, ELISA, and Western blot confirmed the subject's HIV-seropositive status. These results indicate that in northern India, clinical HIV disease remains rare even among individuals with high-risk behavior. The low prevalence of clinical HIV disease in that part of the country makes it difficult to assess the specificity and sensitivity of the WHO clinical criteria for AIDS.^ieng