Differential effects of regulatory T cells on the initiation and regression of atherosclerosis.

OBJECTIVE: Regulatory T cells (Tregs) play an important role in the regulation of T cell-mediated immune responses through suppression of T cell proliferation and cytokine production. In atherosclerosis, a chronic autoimmune-like disease, an imbalance between pro-inflammatory cells (Th1/Th2) and anti-inflammatory cells (Tregs) exists. Therefore, increased Treg numbers may be beneficial for patients suffering from atherosclerosis. In the present study, we determined the effect of a vast expansion of Tregs on the initiation and regression of well-established lesions. METHODS AND RESULTS: For in vivo Treg expansion, LDL receptor deficient (LDLr(-/-)) mice received repeated intraperitoneal injections of a complex of IL-2 and anti-IL-2 mAb. This resulted in a 10-fold increase in CD4(+)CD25(hi)Foxp3(+) T cells, which potently suppressed effector T cells ex vivo. During initial atherosclerosis, IL-2 complex treatment of LDLr(-/-) mice fed a Western-type diet reduced atherosclerotic lesion formation by 39%. The effect on pre-existing lesions was assessed by combining IL-2 complex treatment with a vigorous lowering of blood lipid levels in LDLr(-/-) mice. This did not induce regression of atherosclerosis, but significantly enhanced lesion stability. CONCLUSION: Our data show differential roles for Tregs during atherosclerosis: Tregs suppress inflammatory responses and attenuate initial atherosclerosis development, while during regression Tregs can improve stabilization of the atherosclerotic lesions.

ALT and viral load decline during PEG-IFN alpha-2b treatment for HBeAg-positive chronic hepatitis B.

BACKGROUND: Alanine aminotransferase (ALT) is one of the main indicators for inflammatory activity in chronic hepatitis B. During interferon-based therapy, approximately 25%-40% of patients exhibit an ALT flare. OBJECTIVES AND STUDY DESIGN: To analyze the relation between ALT and HBV-DNA during pegylated interferon alpha-2b (PEG-IFN) treatment and compare different patterns of on-treatment viral load decline with the occurrence of ALT flares. RESULTS: Of the 123 patients included in this study 31 (25%) exhibited an ALT flare during treatment or follow-up. Six out of 8 (75%) host-induced flares, i.e. ALT flares which were followed by a HBV-DNA decrease associated with a favorable treatment outcome, occurred in patients with a delayed HBV-DNA decline pattern (delayed vs. non-delayed decline, p=.022); 5 of these 8 patients exhibited HBeAg loss and 4 even HBsAg loss at the end of follow-up. The prediction of ALT normalization was possible using on-treatment viral load. Based on the difference from baseline, the evolution of viral load and ALT level were strongly interrelated during treatment and follow-up. With a joint model we estimated a correlation coefficient of 0.38 (p<0.001) during the first 4 weeks of the treatment and of 0.72 (p<0.0001) thereafter. CONCLUSION: There was a strong relation between ALT and viral load in HBeAg-positive chronic hepatitis B patients treated with PEG-IFN alpha-2b, especially after 4 weeks of treatment. Patients with a delayed decline in viral load often exhibited a host-induced flare associated with a favorable outcome.

Exacerbation of chronic hepatitis B infection after delivery.

During pregnancy several alterations in the immune status allow mothers to tolerate the genetically different foetal tissues. We investigated the evolution of liver disease during and after pregnancy in chronic hepatitis B patients. Between 1998 and 2006 there were 38 pregnancies in 31 chronic hepatitis B 's' antigen-positive women at our liver unit. Twenty-four subjects (63%) were hepatitis B 'e' antigen (HBeAg)-positive, 14 (37%) HBeAg-negative. In 13 pregnancies (34%), lamivudine therapy was started during the last trimester of pregnancy to lower hepatitis B virus (HBV) DNA levels to reduce the risk of vertical transmission. A significant increase in liver disease activity after pregnancy, defined as a three times increase in alanine aminotransferase (ALT) within 6 months after delivery, occurred in 17 of 38 patients (45%). In those treated with lamivudine during the last trimester of pregnancy, this occurred in even 8/13 patients (62%). Prediction during pregnancy of these exacerbations was not possible using HBV DNA, ALT level, HBeAg status or any other characteristic. The median maximal ALT of these exacerbations was 4.0 x ULN and none led to decompensated liver disease. In conclusion, a significant increase in liver inflammation occurs often after pregnancy. This may be due to a reactivation of the immune system after delivery. Based on our data we recommend monitoring closely and if necessary treating women with chronic HBV shortly after delivery.

A functional polymorphism of TIR-domain-containing adaptor protein is not associated with axial spondyloarthritis.

OBJECTIVE: A genetic variant of the toll-like receptor (TLR)2/4 adaptor protein TIRAP (single nucleotide polymorphism (SNP) C539T) was identified in a UK and in several African populations. The heterozygous genotype of this SNP has been associated with protection from severe infections. This allele results in an attenuated response to bacterial pathogens. As an exaggerated innate immune response to pathogens has been implicated in spondyloarthritis (SpA) pathogenesis, we analysed if the heterozygous C/T genotype was underrepresented in axial SpA compared with healthy controls. METHODS: 204 patients with axial SpA and 175 population-matched controls were included. SNP C539T was determined with a sequence-specific polymerase chain reaction and direct sequencing. RESULTS: The frequency of the haplotypes was similar in cases and controls (87% for C and 13% for T in both groups). The C/T genotype, which attenuates TLR signalling, was not underrepresented in cases versus controls (19% in controls vs 24% in cases, p = 0.44). The T/T genotype, was slightly lower in cases than in controls, although this was not significant (3.4% in controls vs 1% in cases, p = 0.15). Within the cases, there were no differences in disease phenotype or activity between patients with the C/C or C/T genotype. CONCLUSIONS: This study did not show significant associations of SNP S180L of the TLR2/4 adaptor protein TIRAP with axial SpA.

Review article: Success and failure of nucleoside and nucleotide analogues in chronic hepatitis B.

BACKGROUND: Strong suppression of viral replication and normalization of alanine aminotransferase is feasible with nucleos(t)ide analogues. It is estimated viral replication and liver inflammation can be controlled in 90% of patients with chronic hepatitis B with the current available treatments. AIM: To review the studies currently available on the management of chronic hepatitis B with nucleos(t)ide analogues. RESULTS: Although very potent, nucleos(t)ide analogues are not effective in every patient. Some factors are known to influence treatment outcome, but many host and viral factors are still unknown. Stopping rules have to be defined to assess treatment efficacy in an early stage and change the regimen. Discontinuation of nucleos(t)ide analogues is often followed by reactivation of HBV. Data on the risk factors for relapse are necessary in order to decide if treatment can be safely discontinued. Another major drawback of nucleos(t)ide analogues is the emergence of resistance. The efficacy of compounds for the treatment of mutant virus and the impact of cross-resistance is largely unknown. The use of combination therapy to prevent resistance looks promising, but has to be proven. CONCLUSIONS: HBV has become a treatable disease, however much research is needed to optimize treatment for individual patients and treatment failures.

A new method for the rapid and long term growth of human neural precursor cells.

A reliable source of human neural tissue would be of immense practical value to both neuroscientists and clinical neural transplantation trials. In this study, human precursor cells were isolated from the developing human cortex and, in the presence of both epidermal and fibroblast growth factor-2, grew in culture as sphere shaped clusters. Using traditional passaging techniques and culture mediums the rate of growth was extremely slow, and only a 12-fold expansion in total cell number could be achieved. However, when intact spheres were sectioned into quarters, rather than mechanically dissociated, cell cell contacts were maintained and cellular trauma minimised which permitted the rapid and continual growth of each individual quarter. Using this method we have achieved a 1.5 million-fold increase in precursor cell number over a period of less than 200 days. Upon differentiation by exposure to a substrate, cells migrated out from the spheres and formed a monolayer of astrocytes and neurons. No oligodendrocytes were found to develop from these human neural precursor cells at late passages when whole spheres were differentiated. This simple and novel culture method allows the rapid expansion of large numbers of non-transformed human neural precursor cells which may be of use in drug discovery, ex vivo gene therapy and clinical neural transplantation.

Long-term survival of human central nervous system progenitor cells transplanted into a rat model of Parkinson's disease.

Progenitor cells were isolated from the developing human central nervous system (CNS), induced to divide using a combination of epidermal growth factor and fibroblast growth factor-2, and then transplanted into the striatum of adult rats with unilateral dopaminergic lesions. Large grafts were found at 2 weeks survival which contained many undifferentiated cells, some of which were migrating into the host striatum. However, by 20 weeks survival, only a thin strip of cells remained at the graft core while a large number of migrating astrocytes labeled with a human-specific antibody could be seen throughout the striatum. Fully differentiated graft-derived neurons, also labeled with a human-specific antibody, were seen close to the transplant site in some animals. A number of these neurons expressed tyrosine hydroxylase and were sufficient to partially ameliorate lesion-induced behavioral deficits in two animals. These results show that expanded populations of human CNS progenitor cells maintained in a proliferative state in culture can migrate and differentiate into both neurons and astrocytes following intracerebral grafting. As such these cells may have potential for development as an alternative source of tissue for neural transplantation in degenerative diseases.

Restricted growth potential of rat neural precursors as compared to mouse.

Epidermal growth factor (EGF) responsive precursors isolated from the developing mouse striatum could be continually expanded in culture as free-floating spheres of cells for over 50 days. Under identical conditions, EGF-responsive precursors from the developing rat striatum could only be expanded for between 21 and 28 days, after which crisis ensued and there was a reduction in cell number at each passage. The outer regions of 28-day-old rat spheres contained a heterogeneous population of both dividing and dying cells while the cores were full of dying cells, many of which showed features consistent with apoptosis. Fibroblast growth factor-2 (FGF-2) alone did not lead to an expansion in rat striatal precursor cell number under the conditions used here. EGF combined with FGF-2 acted synergistically on cell growth, but did not prevent the final senescence and death of the rat precursors.

Co-expression of MAP-2 and GFAP in cells developing from rat EGF responsive precursor cells.

In this study we have performed a detailed analysis of EGF-responsive precursors as they develop into neurons and astrocytes using antibodies to nestin, microtubule-associated protein 2 (MAP-2c and MAP-2ab) and glial fibriallary acidic protein (GFAP). Surprisingly, at early time points, most GFAP-positive cells also stained for MAP-2c, and we postulate that this may be a normal stage of astroglial development. At 7 days most of the cells had developed into astrocytes and MAP-2ab-positive cells only represented 5% of the total neuronal population. This study shows that (i) MAP-2c is a marker for early precursors, (ii) the majority of cells developing from. EGF-responsive precursors develop into glia and (iii) only a small population of cells arising from expanded populations of EGF-responsive precursors develop into neurons expressing MAP-2ab. Thus, certain critical signals important for full neuronal differentiation may be missing from this system.

Mutational analysis of the cucumber necrosis virus coat protein gene.

A series of frameshift, deletion, and inversion mutations were made in the coat protein (CP) gene of the icosahedral cucumber necrosis tombusvirus (CNV) to investigate the role of the CP protruding (P) domain in the production of virus particles and, also, to investigate the basis for the accumulation of CP deletion derivatives previously reported in plants inoculated with PD(-), a P-domainless CNV CP mutant. P-domainless coat protein subunit could be detected in extracts of CP mutant-infected plants; however, virus-like particles could not, suggesting that the P domain is essential for tombusvirus particle assembly and/or stability. In addition, each of the P-domain mutants analyzed invariably produced coat protein deletion derivatives in infected plants whereas shell domain mutants rarely produced deletion derivatives. Finally, P-domain inversion and deletion mutants accumulated deletion derivatives very rapidly in comparison to P-domain frameshift mutants. Protoplast studies show that PD(-) RNA inoculum does not undergo further deletion in infected protoplasts, suggesting that PD(-) CP deletion derivatives preferentially accumulate in plants because they have a greater capacity for cell-to-cell movement.