Gene transfer of RANTES and MCP-1 chemokine antagonists prolongs cardiac allograft survival.

Vascularized organ allografts are rapidly destroyed by host immune cells that are recruited along chemokine gradients. Among chemokines, Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES) CC chemokine ligand (CCL5) and monocyte chemoattractant protein (MCP)-1 (CCL2) are upregulated in rejecting cardiac allografts. To antagonize these chemokines, we constructed adenoviral vectors expressing NH(2)-terminal deletion (8ND) mutants of the respective genes. Using the F344-to-LEW rat model, intragraft gene transfer of chemokine analogs prolonged cardiac allograft survival from 10.1+/-0.7 and 10.4+/-0.7 days using non-coding adenovirus and vehicle alone, respectively, to 17.0+/-0.7 days for 8ND-RANTES (P<0.001) and 14.2+/-0.8 days for 8ND-MCP-1 (P<0.01). 8ND-RANTES reduced graft infiltration by monocytes/macrophages, cluster of differentiation (CD) 8alpha(+) and T-cell receptor alphabeta(+) cells, while 8ND-MCP-1 reduced monocytes/macrophages. In mixed leukocyte reactions in vitro, proliferation of host lymphocytes from regional lymph nodes in response to donor splenocytes was unaffected by 8ND-RANTES gene transfer. Using a two-gene approach, the contribution of 8ND-MCP-1 was negligible, consistent with available evidence that 8ND-RANTES inhibits both RANTES and MCP-1 activities. 8ND-RANTES gene transfer and a short course of low-dose cyclosporine A synergistically prolonged graft survival to 37.8+/-5.5 vs 15.4+/-0.5 days with cyclosporine alone (P<0.001). These results suggest a role for anti-chemokine gene therapy as an adjuvant therapy in heart transplantation.

Association between the A-2518G polymorphism in the monocyte chemoattractant protein-1 gene and insulin resistance and Type 2 diabetes mellitus.

AIMS/HYPOTHESIS: The molecular mechanisms of obesity-related insulin resistance are incompletely understood. Macrophages accumulate in adipose tissue of obese individuals. In obesity, monocyte chemoattractant protein-1 (MCP-1), a key chemokine in the process of macrophage accumulation, is overexpressed in adipose tissue. MCP-1 is an insulin-responsive gene that continues to respond to exogenous insulin in insulin-resistant adipocytes and mice. MCP-1 decreases insulin-stimulated glucose uptake into adipocytes. The A-2518G polymorphism in the distal regulatory region of MCP-1 may regulate gene expression. The aim of this study was to investigate the impact of this gene polymorphism on insulin resistance. METHODS: We genotyped the Ludwigshafen Risk and Cardiovascular Health (LURIC) cohort ( n=3307). Insulin resistance, estimated by homeostasis model assessment, and Type 2 diabetes were diagnosed in 803 and 635 patients respectively. RESULTS: Univariate analysis revealed that plasma MCP-1 levels were significantly and positively correlated with WHR ( p=0.011), insulin resistance ( p=0.0097) and diabetes ( p<0.0001). Presence of the MCP-1 G-2518 allele was associated with decreased plasma MCP-1 ( p=0.017), a decreased prevalence of insulin resistance (odds ratio [OR]=0.82, 95% CI: 0.70-0.97, p=0.021) and a decreased prevalence of diabetes (OR=0.80, 95% CI: 0.67-0.96, p=0.014). In multivariate analysis, the G allele retained statistical significance as a negative predictor of insulin resistance (OR=0.78, 95% CI: 0.65-0.93, p=0.0060) and diabetes (OR=0.80, 95% CI: 0.66-0.96, p=0.018). CONCLUSIONS/INTERPRETATION: In a large cohort of Caucasians, the MCP-1 G-2518 gene variant was significantly and negatively correlated with plasma MCP-1 levels and the prevalence of insulin resistance and Type 2 diabetes. These results add to recent evidence supporting a role for MCP-1 in pathologies associated with hyperinsulinaemia.

Long-term kinetics of T cell production in HIV-infected subjects treated with highly active antiretroviral therapy.

The long-term kinetics of T cell production following highly active antiretroviral therapy (HAART) were investigated in blood and lymph node in a group of HIV-infected subjects at early stage of established infection and prospectively studied for 72 wk. Before HAART, CD4 and CD8 T cell turnover was increased. However, the total number of proliferating CD4(+) T lymphocytes, i.e., CD4(+)Ki67(+) T lymphocytes, was not significantly different in HIV-infected (n = 73) and HIV-negative (n = 15) subjects, whereas proliferating CD8(+)Ki67(+) T lymphocytes were significantly higher in HIV-infected subjects. After HAART, the total body number of proliferating CD4(+)Ki67(+) T lymphocytes increased over time and was associated with an increase of both naive and memory CD4(+) T cells. The maximal increase (2-fold) was observed at week 36, whereas at week 72 the number of proliferating CD4(+) T cells dropped to baseline levels, i.e., before HAART. The kinetics of the fraction of proliferating CD4 and CD8 T cells were significantly correlated with the changes in the total body number of these T cell subsets. These results demonstrate a direct relationship between ex vivo measures of T cell production and quantitative changes in total body T lymphocyte populations. This study provides advances in the delineation of the kinetics of T cell production in HIV infection in the presence and/or in the absence of HAART.

Equal distribution of congenital blood cell chimerism in dizygotic triplets after in-vitro fertilization.

The special situation of multiple pregnancies following IVF has led to a growing interest in the assessment of embryonal development by means of molecular genetics. We report a case of congenital blood chimerism in dizygotic triplets (two boys, one girl) present in erythrocytes and leukocytes in both sexes. Routine pre-operative blood serology of the 6 year old female triplet revealed chimerism of the red cells. Flow cytometry of the erythrocytes and DNA analysis of the leukocytes demonstrated that all three children had the same proportions of male and female cells. Fluorescent in-situ hybridization (FISH) analyses revealed Y chromosomes in 84% of the girl's leukocytes and in 89/92% of the two boys' leukocytes. The true genetic lines were determined by analysing polymorphism of serum groups (glycoprotein, transferrin, protease inhibitor and plasminogen) secreted by non-haematopoetic tissue, by blood group typing of hair roots and by DNA analysis of endothelial cells. Evidently placental anastomoses allowed a reciprocal intra-uterine transfusion of blood stem cells in the triplets.

Further study on the specificity and incidence of neutralizing antibodies to interferon (IFN) in relapsing remitting multiple sclerosis patients treated with IFN beta-1a or IFN beta-1b.

The development of neutralizing antibodies (NAbs) to interferon (IFN) is a common phenomenon of IFN beta therapy for relapsing-remitting multiple sclerosis (RRMS) patients. Here we examine the specificity of NAbs developed during therapy for RRMS with recombinant interferon (rIFN) beta-1a or rIFN beta-1b, and study the effect of switching from rIFN beta-1a to rIFN beta-1b on the incidence and specificity of NAbs. The relative ability to neutralize rIFN beta-1a and beta-1b was assayed in sera positive for NAbs derived from RRMS patients treated with either rIFN beta-1a (N=9) or rIFN beta-1b (N=16), while the incidence and specificity of NAbs to IFN beta developed during therapy were studied in 50 RRMS patients who were treated for two years with rIFN beta-1a followed by a further year either switching to rIFN beta-1b (N=34) or continuing treatment with rIFN beta-1a (N=16). The results show that all positive sera, independent of the source, may recognize both forms of rIFN beta and that a further year of treatment does not significantly affect the incidence and specificity of the NAbs developed during the first two years of treatment even if treatment is switched to a different type of IFN beta. The data then suggests that it is unlikely that the administration of rIFN beta-1b to anti-rIFN beta-1a NAbs-positive patients can overcome the inhibitory effect exerted by the serum antibodies (and vice versa), and that a further period of treatment with IFN beta-1b in patients previously treated with rIFN beta-1a does not significantly change the pattern of antibody response to IFN beta.

Antigenic characterization of recombinant, lymphoblastoid, and leukocyte IFN-alpha by monoclonal antibodies.

To gain more insight into similarities of different interferon-alpha (IFN-alpha) species, we evaluated neutralization and immunoactivity of a variety of IFN preparations with various monoclonal antibodies (IFN-alpha mAb). Nine IFN-alpha mAb obtained through immunization with recombinant IFN-alpha (rmAb), lymphoblastoid IFN-alpha (LY mAb), and leukocyte IFN-alpha (LE mAb) were tested. The IFN-alpha mAb were evaluated for their ability to neutralize the antiviral activity of 11 recombinant IFN-alpha subtypes, two recombinant IFN-alpha hybrids, and lymphoblastoid and leukocyte IFN-alpha preparations. The same IFN-alpha mAb were also used in immunoblotting, and some of them were used in immunoaffinity chromatography. The results of the neutralization assay reveal that the IFN-alpha mAb significantly differ in their ability to neutralize the individual IFN-alpha species. Interestingly, none of the IFN-alpha mAb was able to neutralize all the IFN-alpha species. In particular, rmAb were unable to neutralize LE-IFN-alpha or LY-IFN-alpha, whereas LE mAb and LY mAb efficiently neutralized rIFN-alpha2. In some cases, the epitopes to which IFN-alpha mAb are directed were identified through the use of synthetic fragments of IFN-alpha2 or by evaluating the selectivity in binding to IFN-alpha subtypes.

Correlation of interferon-induced expression of MxA mRNA in peripheral blood mononuclear cells with the response of patients with chronic active hepatitis C to IFN-alpha therapy.

MxA, a protein with selective activity against certain viruses, is an accepted specific indicator of type I interferon (IFN) activity. We have developed an internally controlled quantitative-competitive PCR to measure the amounts of MxA mRNA expressed in peripheral blood mononuclear cells (PBMC). This assay is more sensitive, quantitative, and easily applied to serial clinical samples than previously described methods. We have applied this assay retrospectively to 27 patients with chronic active hepatitis C given IFN-alpha2. Most such patients gain no sustained benefit but nevertheless suffer from the side effects, expense, and inconvenience of the treatment. Fourteen of the 27 had been classified on clinical grounds as responders and 13 as nonresponders at the end of a 6 month treatment period. We measured MxA mRNA in PBMC obtained before and after 8 weeks of IFN-alpha2 treatment. All the patients expressed some level of mRNA before treatment began, and after 8 weeks of treatment, the level rose in 19. This increase was significant (p < 0.001) only in patients classified as responders. This strongly suggests that hepatitis C virus (HCV) patients who express increased amounts of MxA mRNA in their PBMC during IFN-alpha treatment are most likely to obtain long-term benefit. If this finding is confirmed in future prospective studies, it will provide an extremely important predictive marker for managing IFN-alpha therapy in patients with HCV.

Development of neutralizing antibodies in patients with relapsing-remitting multiple sclerosis treated with IFN-beta1a.

Sixty-eight patients with relapsing-remitting multiple sclerosis (RRMS) were treated with 3 million or 9 million i.u. of recombinant interferon-beta1a (recIFN-beta1a) s.c. three times a week for 2 years. Their sera were tested for antibodies neutralizing the IFN (NAb) in a bioassay. Sera with titers > or = 1:20 were considered positive. We detected NAb in 3.2%, 13.8%, and 15.9% of the patients in sera obtained at 3, 6, and 24 months, respectively. The incidence was not related to the IFN dose. Interestingly, during the 6 month baseline period before the start of the study, relapse rates, baseline disability, and the volume of lesions on T2-weighted images were significantly higher in patients who developed NAb during treatment. Because of interpatient variability, no definitive relationship was observed between NAb formation and loss of clinical or magnetic resonance imaging (MRI) response.

Anti-HIV antiviral activity of stavudine in a thymidine kinase-deficient cellular line.

Stavudine (d4T) is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. It is known that stavudine is metabolized in cells to the mono-, di- and triphosphate nucleotides but the enzymes responsible for its phosphorylation are as yet unidentified. In particular, there are conflicting results concerning the role of thymidine kinase 1 (TK1) in stavudine metabolism. To gain new insights into this phenomenon we analysed the antiviral activity of stavudine in a TK1-deficient, resistant cell line. The results indicate that TK1 is responsible for the phosphorylation of stavudine but it is not the only enzyme involved in its activation. The other enzyme(s) that might be involved in the metabolism of stavudine, however, are not able to phosphorylate stavudine with the same efficiency as TK1. Since it has been shown that prolonged treatment with zidovudine may induce an in vivo defect in TK1 activity, it is tempting to speculate that patients treated for a long time with zidovudine could be resistant to further treatment with stavudine.

Interferon antibodies in patients with infectious diseases. Anti-interferon antibodies.

Interferons (IFNs) are generally recognized as the most important therapeutic agent in some infectious diseases such as chronic hepatitis B and C. Since the early clinical trials it was documented that the therapeutic use of IFNs could be complicated by the development of antibodies able to neutralize or to bind to the IFN molecule. After several years of research it is now widely accepted that the presence of circulating anti-IFN antibodies may affect the response to IFN. Here we summarize what is currently know on the clinical significance of antibodies to IFN in IFN-treated viral diseases patients.

Antibodies to interferon (IFN) in hepatitis C patients relapsing while continuing recombinant IFN-alpha2 therapy.

A number of trials have demonstrated that IFN-alpha is effective in chronic hepatitis C virus infection. It is known, however, that a number of chronic hepatitis C patients experience, after an initial response to IFN, disease reactivation or relapse (also called 'breakthrough') while IFN therapy is still ongoing. Since in a number of clinical conditions a significant correlation between development of antibodies to IFN and failure of therapy has been established, we addressed the possibility that the development of antibodies to IFN may take part in the relapse occurring in hepatitis C patients during recombinant IFN-alpha (rIFN-alpha) therapy. The prevalence of neutralizing (NA) and binding antibodies (BA) to rIFN-alpha2 has been evaluated in 45 patients with chronic hepatitis C treated with rIFN-alpha2a who first normalized aminotransferase (ALT) levels, and subsequently showed disease reactivation while on treatment. The presence of NA and BA was tested before therapy, during the response to IFN treatment, and at the time when ALT started to rise again to abnormal levels. The results showed that no patients had detectable antibodies to IFN before therapy and during the period of response to the therapy, while most of them (88.9%) developed NA and/or BA to IFN-alpha2 concomitantly with disease reactivation. In particular, in 29 of the 45 patients (64.4%) ALT normalized on treatment and rose to abnormal levels when NA appeared in their serum, while in 11 of the 16 (68.8%) remaining patients the relapse was associated with BA development. The frequency of seroconversion in these patients is significantly higher than that observed in the control group. These data indicate that antibodies to IFN may be responsible for breakthrough in the majority of patients showing disease reactivation while rIFN-alpha therapy is still ongoing.

Evaluation of a community arthritis program in Australia: dissemination of a developed program.

OBJECTIVE: To evaluate the effectiveness of a community-based arthritis education program conducted in a number of locations throughout the Sydney, Australia, metropolitan area. METHODS: The program, based on earlier work, comprised 6 weekly sessions of 2.5 hours' duration. The study sample included 175 men and women with different types of arthritis, divided into intervention (n = 104) and control (n = 71) groups. Five outcome measures were selected to evaluate effectiveness of the program: pain perception, knowledge level, self-efficacy, disability index, and self-management behavior. RESULTS: The results indicated that the program was effective in increasing knowledge level (F[1,222] = 10.3, P = 0.001 at 6 weeks; F[1,108] = 7.8, P = 0.006 at 6 months), and a statistically significant difference was found in disability satisfaction 6 months after intervention (F[1,98] = 5.9, P = 0.01], but no statistically significant difference was found in pain perception, self-efficacy, and disability index. CONCLUSION: This research supports some of the successful outcomes which follow an arthritis education program: increased knowledge level and increased disability satisfaction.

Zidovudine induces the expression of cellular resistance affecting its antiviral activity.

We have previously shown that multidrug-resistant cells expressing the multidrug transporter P-glycoprotein are less sensitive to the antiviral activity of AZT. Subsequently, we addressed the question whether AZT itself is able to induce cellular resistance to the drug. Indeed, CEM cells propagated in the presence of increasing concentrations of AZT become resistant to the antigrowth and antiviral activity of AZT but do not express detectable level of P-glycoprotein. Sensitivity of these cells to other compounds, such as vinblastine, vincristine, ddI, and ddC remained unchanged, indicating that, in contrast to P-glycoprotein-positive cells, AZT-induced resistance is specific for AZT. Interestingly, in AZT-induced resistant cells the intracellular accumulation of AZT and exogenous deoxythymidine, as well as thymidine kinase activity, are significantly reduced when compared with the parental cell line. Our findings show that AZT itself may directly induce the expression of cellular mechanisms leading to the acquisition of specific cellular resistance that can affect its antiviral activity.

Williams-Beuren syndrome in monozygotic twins with variable expression.

Five sets of monozygotic (MZ) twins with Williams-Beuren syndrome (WBS) have been reported so far. We report on an additional pair of mz twins concordant for WBS but variable expression for the syndrome. Although both faces look different monozygosity of the twins was proven by DNA fingerprint analysis, HLA, and blood group pattern. Both girls had the typical facial appearance with strabismus. Both had developmental delay, mild supravalvular aortic stenosis (SVAS), hypoplasia of both pulmonary arteries and multiple peripheral pulmonary stenoses, and inguinal hernia. Unilateral renal agenesis was seen in one of the twins. In addition the pedigree pointed to a second disorder with probably autosomal dominant inheritance. Both twins had a cleft palate, but their father had cleft lip and the grandfather as well as the greatgrandfather had cleft lip/palate. Findings of linkage analysis in pedigrees with nonsyndromic oral facial cleft were taken to suggest that a major locus for nonsyndromal oral facial cleft is located on the distal portion of chromosome 6. Linkage studies could serve as a starting point to examine a locus associated with WBS. Our observation and reports on the literature support the hypothesis that WBS is a genetic disorder.

C6 reference typing report.

Various C6 protein allotypes were examined using polyacrylamide gel isoelectric focusing followed by immunoblotting or hemolytic overlay. For several 'difficult' allotypes, neuraminidase treatment of samples and long-distance isoelectric focusing gels were applied. Nineteen different allotypes were distinguished besides the two common allotypes C6 A and C6 B. They were designated basically according to the previous statement on C6 nomenclature [Mauff et al., 1980].