Characterization of Virus-Responsive Plasmacytoid Dendritic Cells in the Rhesus Macaque

Plasmacytoid dendritic cells (PDC) are potent producers of alpha interferon (IFN-α) in response to enveloped viruses and provide a critical link between the innate and adaptive immune responses. Although the loss of peripheral blood PDC function and numbers has been linked to human immunodeficiency virus (HIV) progression in humans, a suitable animal model is needed to study the effects of immunodeficiency virus infection on PDC function. The rhesus macaque SIV model closely mimics human HIV infection, and recent studies have identified macaque PDC, potentially making the macaque a good model to study PDC regulation. In this study, we demonstrate that peripheral blood PDC from healthy macaques are both phenotypically and functionally similar to human PDC and that reagents used for human studies can be used to study macaque PDC. Both human and macaque PBMC expressed IFN-α in response to herpes simplex virus (HSV), the prototypical activator of PDC, as measured by using an IFN bioassay and IFN-α-specific enzyme-linked immunospot assays. Similar to human PDC, macaque PDC were identified by using flow cytometry as CD123⁺ HLA-DR⁺ lineage⁻ cells. In addition, like human PDC, macaque PDC expressed intracellular IFN-α, tumor necrosis factor alpha, macrophage inflammatory protein 1β/CCL4, and IFN-inducible protein 10/CXCL10 upon stimulation with HSV, all as determined by intracellular flow cytometry. We found that IFN regulatory factor 7, which is required for the expression of IFN-α genes, was, similar to human PDC, expressed at high levels in macaque PDC compared to monocytes and CD8⁺ T cells. These findings establish the phenotypic and functional similarity of human and macaque PDC and confirm the utility of tools developed for studying human PDC in this animal model.     

Rheumatic heart disease: proinflammatory cytokines play a role in the progression and maintenance of valvular lesions

Heart lesions of rheumatic heart disease (RHD) patients contain T-cell clones that recognize heart proteins and streptococcal M peptides. To functionally characterize heart-infiltrating T lymphocytes, we evaluated their cytokine profile, both directly in situ and in T-cell lines derived from the heart (HIL). Interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, and IL-10 expressions were characterized in 20 heart tissue infiltrates from 14 RHD patients by immunohistochemistry. IFN-gamma-, TNF-alpha-, and IL-10-positive cells were consistently predominant, whereas IL-4 was scarce in the valves. In agreement with these data, the in vitro experiments, in which 13 HILs derived from heart samples of eight patients were stimulated with M5 protein and the immunodominant M5 (81-96) peptide, IL-4 was detected in HIL derived from the atrium (three of six) but not from the valve (zero of seven). IFN-gamma and IL-10 production were detected in culture supernatants in 11 of 13 and 6 of 12 HILs, respectively. The predominant IFN-gamma and TNF-alpha expression in the heart suggests that Th1-type cytokines could mediate RHD. Unlike in reversible myocardium inflammation, the significantly lower IL-4 expression in the valvular tissue (P = 0.02) may contribute to the progression of the RHD leading to permanent valvular damage (relative risk, 4.3; odds ratio, 15.8). The lack of IL-4 in vitro production by valve-derived HIL also emphasizes the more severe tissue destruction in valves observed in RHD.     

Expression of blood group antigens in urinary tract tumours: prospective fluorescence study using cryostat sections of fresh frozen tissues.

Cryostat sections of fresh frozen tissues were used in a prospective study of blood group H and A antigen fluorescence in 73 transitional cell carcinomas of the bladder. The aim was to evaluate antigen expression without subjecting the tumour tissues to organic solvents that extract blood group active glycolipids. Deletion of the genetically predicted antigen was twice as common in tumours of pT1 or greater stage than those of pTa stage and also twice as common in poorly differentiated than in moderately well differentiated tumours. The considerable heterogeneity and overlap, however, in patterns of reactivity in tumours of various histopathological stages and grades and the effect of secretor status on antigenicity meant that there was no obvious antigenic feature that correlated precisely with invasive stage or differentiation grade. It remains to be determined whether the antigen positive and antigen negative tumours represent different disease entities with differing clinical courses. Our results indicate, however, that studies of the blood group antigens in urinary tract tumours are more likely to be of value in research into biochemical disorders in the neoplastic process than in routine clinical assessment as a guide to treatment.     

Influence of selected wound dressings on PMN elastase in chronic wound fluid and their antioxidative potential in vitro

Exudates from non-healing wounds contain elevated levels of proteolytic enzymes, like elastase from polymorphonuclear granulocytes (PMN elastase), reactive oxygen species (ROS) and reactive nitrogen species (RNS). The overproduction of proteolytic enzymes leads to reduced concentrations of growth factors and proteinase inhibitors, resulting in an imbalance between degradation and remodelling processes. Thus, the reduction of protein-degrading enzymes and scavenging of ROS and RNS seem to be suitable ways to support the healing process of chronic stagnating wounds. The aim of this study was to test selected wound dressings from different biomaterials (collagen, oxidized regenerated cellulose (ORC) and ORC/collagen mixture), regarding their antioxidative potential in vitro and their influence on the concentration and activity of PMN elastase in chronic wound fluid. Antioxidant capacity of the investigated wound dressing was determined by a pholasin-based chemiluminescent assay. PMN elastase concentration was determined by means of ELISA. Enzyme activities could be measured by a fluorescence assay. As the presented data demonstrates, all tested materials showed antioxidant capacity. In addition, the investigated materials were able to reduce the concentration and activity of PMN elastase. Beside other aspects, such as biocompatibility, biodegradability, fluid absorption and clinical effects (e.g. angiogenesis and microcirculation), the understanding of these properties may help to support the further refinement of wound dressings for improved wound healing.     

Evaluation of linkage disequilibrium between chromosome 22q11 single nucleotide polymorphisms in a large outbred population

To assess the utility of linkage disequilibrium (LD) as a tool for fine-mapping disease genes in non-isolated populations, we have assessed the linkage disequilibrium strength among a series of single nucleotide polymorphisms (SNPs) in an approximate 1 Mb region of human chromosome 22q11. Nineteen random SNPs were discovered and tested across this region with an average spacing of 57 kb (range=1.4-289 kb). These 19 SNPs were genotyped in a population consisting of 444 unrelated pedigrees that were largely collected in the U.S. and U.K. Haplotypes for all pedigrees were derived from pedigree data and over 1,400 haplotypes from unrelated individuals were evaluated for linkage disequilibrium between marker alleles. In addition, linkage disequilibrium between marker alleles was also evaluated using estimated haplotypes without genealogical information (i.e., without parental genotype information). Every marker pair combination was tested for a total of 171 tests and 2x2 contingency tables were constructed to measure LD strength. In general the haplotypes derived from pedigree data provided a more conservative estimate of LD strength. Using genealogical information for estimates of D', 59% (10/17) of marker pairs less than 50 kb apart had D' values >0.30. Finally, we observed a 60 kb region with non-significant LD, which could reflect increased recombination in this region.     

Genetic epidemiology in the study of susceptibility/resistance to malaria in the human population

The development of genetic epidemiology methods using recent human genetic map together with the growing availability of candidate genes have led to substantial advances in the identification of host genes in human malaria. Investigation of these genes has progressed along two complementary ways: 1) The search for genes influencing the severe malaria clinical phenotype by means of population based case-control studies which showed the protective role of several red cell genetic defects (sickle cell anemia, a-thalassaemia ...) and that some polymorphisms of the TNF-alpha promoter region could predispose to cerebral malaria; 2) The investigation of the genetic regulation of malaria-related biological phenotypes (infection levels, immune response) by means of familial studies which underlined the influence of the 5q31-q33 chromosomal region in the control of Plasmodium falciparum blood parasitemia and the role of major histocompatibility complex (MHC) and non-MHC genes in the regulation of humoral and cellular response to various malarial antigens. Ongoing studies will precise the role of these genes and probably reveal the existence of other genes not identified yet. The impact of these findings on the understanding of malaria pathogenesis and on the design of future preventive and therapeutic strategies should be considerable.     

Stimulation of human monocyte beta-glucan receptors by glucan particles induces production of TNF-alpha and IL-1 beta.

beta-glucans are pharmacologic agents that rapidly enhance host resistance to a variety of biologic insults through mechanisms involving macrophage activation. To determine whether stimulation of the beta-glucan receptors on human monocytes resulted in cytokine production, monolayers of monocytes were incubated with purified yeast glucan particles and measured for tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) mRNA and protein. By Northern blot analysis, TNF-alpha mRNA was detected within 30 min of incubation with glucan particles, peaked at 2 h, and remained elevated for at least 8 h. Glucan induction of IL-1 beta mRNA followed a similar time-course of initiation and accumulation. By enzyme-linked immunosorbent assays (ELISAs), significant levels of TNF-alpha and IL-1 beta were present in supernatants of glucan-treated cells within 1 h and plateau levels of both cytokines were approached within 4 h. At particle-to-cell ratios of from 0.4 to 18, glucan particles induced dose-dependent increases in TNF-alpha and IL-1 beta mRNA and corresponding increases in TNF-alpha and IL-1 beta proteins. Exposure of monocytes to glucan particles for 0-30 min and washing before continued incubation for 4 h in particle-free buffer induced production and secretion of TNF-alpha and IL-1 beta in a time-dependent fashion compatible with phagocytosis. The pretreatment of monocyte monolayers with trypsin reduced glucan-induced production of TNF-alpha and IL-1 beta in a dose-dependent manner with 5 micrograms/ml of trypsin effecting reductions of greater than 50%. Thus, glucan particles induce human monocyte production of TNF-alpha and IL-1 beta by a mechanism that is dependent on trypsin-sensitive beta-glucan receptors.     

Induction of intervertebral disc-like cells from adult mesenchymal stem cells

The potential of adult mesenchymal stem cells (MSCs) to differentiate towards cartilage, bone, adipose tissue, or muscle is well established. However, the capacity of MSCs to differentiate towards intervertebral disc (IVD)-like cells is unknown. The aim of this study was to compare the molecular phenotype of human IVD cells and articular chondrocytes and to analyze whether mesenchymal stem cells can differentiate towards both cell types after transforming growth factor beta (TGF beta)-mediated induction in vitro. Bone marrow-derived MSCs were differentiated in spheroid culture towards the chondrogenic lineage in the presence of TGF beta(3) dexamethasone, and ascorbate. A customized cDNA-array comprising 45 cartilage-, bone-, and stem cell-relevant genes was used to quantify gene expression profiles. After TGF beta-mediated differentiation, MSC spheroids turned positive for collagen type II protein and expressed a large panel of genes characteristic for chondrocytes, including aggrecan, decorin, fibromodulin, and cartilage oligomeric matrix protein, although at levels closer to IVD tissue than to hyaline articular cartilage. Like IVD tissue, the spheroids were strongly positive for collagen type I and osteopontin. MSC spheroids expressed more differentiation markers at higher levels than culture-expanded IVD cells and chondrocytes, which both dedifferentiated in monolayer culture. In conclusion, mesenchymal stem cells adopted a gene expression profile that resembled native IVD tissue more closely than native joint cartilage. Thus, these cells may represent an attractive source from which to obtain IVD-like cells, whereas modification of culture conditions is required to approach the molecular phenotype of chondrocytes in hyaline cartilage.