Localization of type I and III collagen and fibronectin production in injured gastrocnemius muscle.

The healing of muscle rupture consists of two simultaneous processes, regeneration of disrupted muscle fibers and production of connective tissue scar. These two processes are at the same time supportive to and competitive with each other. Their balanced progression is necessary for optimal healing. Synthesis of three connective tissue proteins, collagen types I and III and fibronectin, was analyzed during the regeneration process from 2 days to 3 weeks by Northern blot and in situ hybridization and immunohistochemistry. For this purpose a partial standard rupture of the gastrocnemius muscle was induced in 56 rats by a strike with a blunt spring-loaded hammer. Northern blot analysis of the specific mRNAs during the healing process revealed distinctly different expression patterns for fibronection and type I and III collagens. During the early stages (days 2 and 3) fibronectin, derived mainly from plasma, was abundant in the traumatized area, but local production of fibronectin mRNA by fibroblasts had also already started by day 2, closely followed by that of type III collagen. This early active synthesis of type III collagen and fibronectin was followed by a decrease after 1 week. The production of type I collagen mRNAs was activated somewhat later and remained elevated for at least 3 weeks. The muscle cells did not contain procollagen mRNAs. The observed sequence of connective tissue proteins reflects the particular function that each carries out during muscle wound healing (e.g., fibronectin in fibroblast trapping, type III collagen in plasticity/flexibility, and type I collagen in tensile strength).     

Interleukin-6 -174G/C polymorphism and longevity: a follow-up study

Increased rate of inflammation has been observed to be associated with aging. This is manifested, e.g. as increased blood levels of proinflammatory cytokines, such as interleukin-6 (IL-6). The production of IL-6 is, at least partially, genetically determined the single nucleotide polymorphism (SNP) at the promoter (-174G/C) being decisive. Consequently, some studies have demonstrated that the -174G/C genotype frequencies are different in very old persons as compared to younger ones. However, the results published this far have been conflicting. One of the main confounding factors in these kind of case/control association studies is the undetected difference in the population structure. To avoid this, we now have collected the mortality data of our cohort of 285 nonagenarians (representing mortality between 90 and 95 years of age) and correlated these to the IL-6 genotype. The frequency of -174 allele G was clearly higher in the survivors (n = 114) than in the non-survivors (n = 171).     

Helicobacter pylori-induced indoleamine 2,3-dioxygenase activity in vivo is regulated by TGFB1 and CTLA4 polymorphisms

The chronic gastric infection caused by Helicobacter pylori is known to be associated with several, probably interrelated, immunomodulatory effects, such as protection from atopic diseases, induction of CD4+ CD25+ T regulatory (T(reg)) cells and increase in indoleamine-pyrrole 2,3-dioxygenase (IDO) -dependent suppressive mechanisms. As these mechanisms, as well as the strength of the infection, are very probably genetically controlled, we selected candidate genes (TGFB1, CTLA4) known to be involved in the activation of T(reg) cells. We examined the association of their polymorphisms (TGFB1 C-509T, CTLA4 A+49G) with blood IDO activity in H. pylori seropositive individuals. Genotypes were determined from 391 healthy adults. H. pylori infection was verified by detecting H. pylori IgG antibodies in sera. Concentrations of tryptophan (trp) and kynurenine (kyn), the main metabolite, were determined by reverse-phase high-performance liquid chromatography, and kyn/trp ratio was used as an indicator of IDO activity. The activity was higher in H. pylori seropositive individuals, but this increase was only detected in individuals with CTLA4+49 AA genotype or in carriers of TGFB1-509 allele T. This suggests that H. pylori induced IDO activity is regulated by TGFB1 and CTLA4, and that IDO is a mediator of the T cell suppressive effects of these genes.     

Lipidomics: A Tool for Studies of Atherosclerosis

Lipids, abundant constituents of both the vascular plaque and lipoproteins, play a pivotal role in atherosclerosis. Mass spectrometry-based analysis of lipids, called lipidomics, presents a number of opportunities not only for understanding the cellular processes in health and disease but also in enabling personalized medicine. Lipidomics in its most advanced form is able to quantify hundreds of different molecular lipid species with various structural and functional roles. Unraveling this complexity will improve our understanding of diseases such as atherosclerosis at a level of detail not attainable with classical analytical methods. Improved patient selection, biomarkers for gauging treatment efficacy and safety, and translational models will be facilitated by the lipidomic deliverables. Importantly, lipid-based biomarkers and targets should lead the way as we progress toward more specialized therapeutics.     

Dendritic cells in human peripheral blood: effective enrichment from the nonadherent cells

Dendritic cells (DC) from human peripheral blood were enriched using a method including adherence on plastic, depletion of phagocytic cells, flotation on density gradient column and panning on antibody-coated surfaces. The course of cell separation was evaluated by characterizing the morphological, antigenic and functional features of the different cell fractions. Using the population depleted of strongly adherent cells as the source, we were able to achieve a cell fraction containing 40-65% of DC (the main contaminants being monocytes and natural killer cells). Functionally these cells were highly stimulatory in autologous mixed leukocyte reaction. On the other hand, cells which primarily adhered well and were detached after overnight culture contained less than 5% of DC (the main contaminants being monocytes) after the same purification protocol. The calculated yields of DC were 1-2 X 10(6) and less than 0.5 X 10(6) from the nonadherent and adherent populations, respectively. Thus we concluded that the adhesiveness of DC from human blood is so weak that they can more efficiently and in a more reproducible way be enriched from the primarily nonadherent cell fraction.     

Circulating cell-free DNA is associated with mortality and inflammatory markers in nonagenarians: the Vitality 90+ Study

Aging is characteristically accompanied by a low-grade inflammatory state and increased cellular death. In this study, we analyzed the associations between the plasma cell-free DNA (cf-DNA) level and immunoinflammatory factors and 4-year mortality. The distribution of qualitative cf-DNA patterns in relation to the total cf-DNA concentration and the antinuclear antibody (ANA) serostatus was also evaluated. The study population consisted of n=258 nonagenarians who were participants in the Vitality 90+ Study. Cf-DNA levels were positively correlated with the C-reactive protein (CRP) level, the interleukin-1 receptor antagonist (IL-1ra) level, the serum amyloid A (SAA) level and the indoleamine 2,3-dioxygenase enzyme activity (IDO), weight and chair-stand test time and inversely correlated with the HDL cholesterol level. The total cf-DNA concentration also remained as an independent predictor of 4-year all-cause mortality. A predominance of low-molecular-weight cf-DNA was observed in ANA-seropositive subjects but this cf-DNA pattern was not associated with mortality. The cf-DNA concentration could thus represent a novel biomarker for systemic inflammation and mortality in the elderly.