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Biomedical subjects

R Huss

Publications and source records attributed to R Huss.

At least 19 recordsLinked to original sources
PubMed2006

Multipotent mesenchymal stem cells reduce interstitial fibrosis but do not delay progression of chronic kidney disease in collagen4A3-deficient mice.

Multipotent mesenchymal stem or stromal cells (MSC) have shown to improve outcome of acute renal injury models, but whether MSC can delay renal failure in chronic kidney disease is not known. We injected primary MSC or saline into mice that lack the alpha3-chain of type IV collagen (COL4A3), a model of chronic kidney disease with close similarities to human Alport disease. Weekly injections of MSC from week 6 to 10 of life prevented the loss of peritubular capillaries and reduced markers of renal fibrosis, that is, interstitial volume, numbers of smooth muscle actin-positive interstitial cells, and interstitial collagen deposits as compared to saline-injected COL4A3-deficient mice. However, renal function, that is, blood urea nitrogen, creatinine levels, proteinuria as well as survival of COL4A3-deficient mice were not affected by MSC injections. Although MSC were found to localize to kidneys of COL4A3-deficient mice after injection, differentiation into renal cells was not detected. However, MSC expressed growth factors, that is, vascular endothelial growth factor (VEGF) and bone morphogenetic protein-7 under basal culture conditions. In fact, VEGF mRNA levels were increased in kidneys of MSC-injected COL4A3-deficient mice and MSC supernatants enhance endothelial cell proliferation in vitro. Thus, weekly injections with MSC prevent loss of peritubular capillaries possibly owing to local production of growth factors rather than by differentiation into renal cells. The maintenance of interstitial vasculature is associated with less interstitial fibrosis but, is insufficient to delay renal failure and survival of COL4A3-deficient mice.

Animals↗
PubMed2005

JAK and STAT proteins are expressed and activated by IFN-gamma in rat pancreatic acinar cells.

The development of acute pancreatitis (AP) is triggered by acinar events, but the subsequent extra-acinar events, particularly a distinct immune response, appear to determine its severity. Cytokines modulate this immune response and are derived not only from immunocytes but also from pancreatic acinar cells. We studied whether pancreatic acinar cells were also capable of responding to cytokines. The JAK/STAT-pathway represents the main effector for many cytokines. Therefore, expression and regulation of JAK and STAT proteins were investigated in rat pancreatic acinar cells. Western blotting showed expression of JAK1, JAK2, Tyk2, and STAT1, STAT2, STAT3, STAT5, STAT6. In addition, STAT1 was reversibly tyrosine-phosphorylated upon the procedure of acinar cell isolation. In contrast, STAT3-phosphorylation occurred spontaneously after pancreas removal and was not reversible within 8 h. STAT1 phosphorylation was also observed upon treatment with IFN-gamma but not upon EGF, TNF-alpha or IL-6, and inhibited by the JAK2-inhibitor AG-490. Immunohistochemistry revealed cytoplasmic expression of unphosphorylated STAT1 in untreated acinar cells and nuclear translocation of phosphorylated STAT1 following IFN-gamma-treatment. Interestingly, although CCK leads to the activation of multiple stress pathways in pancreatic acinar cells, we found no influence of CCK on phosphorylation of STAT1, STAT3, or STAT5 in the pancreas. In conclusion, our data provide further evidence that pancreatic acinar cells are able to interact with immune cells. Besides stimulating immune cells via cytokine secretion, acinar cells are in turn capable of responding to IFN-gamma via JAK2 and STAT1 which may have an impact on the development of AP.

Animals↗
PubMed2004

[Chemokine directed homing of transplanted adult stem cells in wound healing and tissue regeneration].

A major challenge in stem cell biology is to study the underlying mechanisms of tissue specific homing and differentiation. Recent results suggest that bone marrow derived stem cells can give rise to multiple cell types. Because chemokines and chemokine receptors are associated with development, differentiation and homing of immune cells, we undertook efforts to study the chemokine receptor expression profile of human adult stem cells to identify their potential role in tissue specific homing prior to transdifferentiation. Using human bone marrow-derived stem cell lines, we could demonstrate functional chemokine receptor expression of various chemokine receptors. The expression of CXCR5 and CCR7, associated with secondary lymphoid organ homing as well as CXCR4 and CCR10, involved in organ specific homing and CXCR3, CCR5 and CCR1, which are involved in inflammation events, suggested a role of chemokine receptors in tissue specific homing of stem cells. To proof the specific homing of stem cells in vivo, we used murine stem cell lines, stably introduced green fluorescent protein under control of CMV promotor into the cells and injected them intravenously into mice. We demonstrate the homing of these stem cells to lymphnode and thymus as well as mucosal tissue, while stem cells home exclusively to a site of lesion during wound healing and tissue regeneration. Our data suggest that chemokine biology may play a pivotal role in the homing of stem cells to specific tissues and niches prior to (trans)differentiation, while the homing changes during tissue damage and other adequate lesions.

Adult↗
PubMed2003

The p53-dependent effects of macrophage migration inhibitory factor revealed by gene targeting.

Macrophage migration inhibitory factor (MIF) is a mediator of host immunity and functions as a high, upstream activator of cells within the innate and the adaptive immunological systems. Recent studies have suggested a potentially broader role for MIF in growth regulation because of its ability to antagonize p53-mediated gene activation and apoptosis. To better understand MIF's activity in growth control, we generated and characterized a strain of MIF-knockout (MIF-KO) mice in the inbred, C57BL/6 background. Embryonic fibroblasts from MIF-KO mice exhibit p53-dependent growth alterations, increased p53 transcriptional activity, and resistance to ras-mediated transformation. Concurrent deletion of the p53 gene in vivo reversed the observed phenotype of cells deficient in MIF. In vivo studies showed that fibrosarcomas induced by the carcinogen benzo[alpha]pyrene are smaller in size and have a lower mitotic index in MIF-KO mice relative to their WT counterparts. The data provide direct genetic evidence for a functional link between MIF and the p53 tumor suppressor and indicate an important and previously unappreciated role for MIF in carcinogenesis.

Alleles↗
PubMed2001

Asthma education in rural communities.

Asthma is a common disease of airway obstruction in school-aged children. Adequate management of asthma in children leads to fewer missed school days, fewer hospitalizations, fewer emergency room visits, and an increase in quality of life. Most asthma educational programs and evaluations have focused on urban rather than rural populations. The purpose of this study was to identify parental asthma needs, develop rural asthma education materials, and evaluate the effectiveness of these educational materials in improving the knowledge and asthma care effectiveness of parents of children with asthma in a rural community. Seven parents were contacted by telephone and administered a pre- and posttest questionnaire analyzing their level of knowledge about asthma and their quality of life. Asthma educational materials were mailed to all parents in the study before administering the posttest. Results indicated that all parents needed additional education about asthma, especially regarding medications. Pre- and posttest scores showed improvements in three areas of knowledge: long-term asthma medications, controlling roaches in the home, and daily peak flow monitoring. There was a significant improvement between pre- and posttest results from the activity domain of quality of life. Eighty-five percent of the parents reported that they had either initiated changes in their home, or planned to in the future, from reading the educational materials. The parents' response to the educational materials that they received by mail was positive, indicating that they may not have received enough information about how to care for children with asthma before our study. The data suggest that distribution of asthma educational materials in rural communities can increase parental knowledge about asthma and lead to positive changes in behavior that can improve their children's health.

Asthma↗
PubMed2001

Highly efficient retroviral gene transfer into immortalized CD34(-) cells and organ distribution after transplantation into NOD/SCID mice.

BACKGROUND: CD34(-) stem cells are apparently the earliest progenitors of hematopoiesis and mesenchymal tissues. The majority of those progeny rests in the BM as fibroblast-like cells, but can also circulate the peripheral blood. Nevertheless, CD34(-), fibroblast-like cells can be isolated from BM aspirates and PBMC, mediated by their ability to adhere to the plastic surface of tissue culture flasks. In standard colony assays, CD34(-), fibroblast-like cells produce a significant number of colony-forming-units (CFUs), mainly CFU-F (fibroblast). METHODS: Despite advanced cell-culture techniques and the application of various growth factors, the life span of those multipotent stem cells is limited. Therefore, we immortalized and cloned fibroblast-like, CD34(-) stem cells and used retroviral constructs containing the green-fluorescence protein (GFP) to determine the gene-transfer efficiency and their use for gene marking prior to transplantation into NOD/SCID mice. RESULTS: We could demonstrate a highly efficient retroviral gene transfer into those immortalized CD34(-), fibroblast-like hematopoietic cells (up to 95% transduced cells), maintaining their ability to produce CFUs, as well as a distinct organ distribution after transplantation into the recipient animals, functioning as SCID-repopulating cells (SRC). Transplanted cells could be detected in the BM, as well as other parenchymal organs, such as the lung, liver, skin, small intestine and brain. DISCUSSION: CD34(-), fibroblast-like progenitor cells can give rise to hematopoietic progeny, but also home to mesenchymal organ sites in recipient animals. There is increasing evidence that pluripotent CD34(-) stem cells can be isolated from various sources and still maintain their capabilities to generate progeny of different tissues. This could be a promising approach to using peripheral-blood derived stem cells for cellreplacement therapy and tissue engineering.

Animals↗
PubMed2001

Rural school nurses' asthma education needs.

School nurses play an important role in identifying children with asthma and providing care during school hours. Educational programs designed to improve nurses' asthma knowledge and practices have concentrated on urban settings. The purpose of this investigation was to determine asthma-related practices and educational needs of rural school nurses. A survey about asthma was mailed to school nurses in all counties of the state of Maryland and in Washington, D.C. Responses were compared between rural Maryland counties and counties from the remainder of Maryland and Washington, D.C. The survey addressed attitudes and beliefs, function and roles, medication administration, and educational needs about asthma. We found that rural nurses used peak flow meters less often to assess and monitor asthma, requested fewer referrals for asthma, had fewer interactions with health room assistants, and had reduced access to asthma educational resources. Also, they provided less asthma education in the schools than other school nurses. These results suggest a need for comprehensive asthma educational programs in rural areas that are based on national guidelines, and that address the unique needs of rural school nurses. These programs should also emphasize the need for open communication between rural school nurses, health room assistants, primary care providers, and parents/caregivers.

Adult↗
PubMed2001

Mesenchymal differentiation and organ distribution of established human stromal cell lines in NOD/SCID mice.

Two human stromal cell lines were established previously from bone marrow-derived primary long-term cultures by immortalization using the SV40 large T antigen and cellular cloning. After irradiation, the fibroblast-like cell lines L87/4 and L88/5 support hematopoietic differentiation of allogeneic cord blood cells in vitro. The stromal cells do not express CD34 and CD50, but some adhesion molecules and integrins, such as CD44, CD54 and CD58. Their expression profiles on RNA and protein levels are suggestive of their osteogenic potency. The quality and quantity of osteocalcin and osteopontin protein expression depended on the culture conditions. Expression of the osteogenic markers increased over time in culture, especially in cells growing in clusters. The stromal cells also expressed collagens I and V, but did not show any expression of collagens II and III. The potentially osteoblastic stromal cells were transplanted into NOD/ SCID recipient mice by intravenous injection and were found in various mesenchymal organs up to 10 weeks after transplantation. Osteocalcin-positive human stromal cells could be detected in the bone marrow, thymus, liver, brain and gut of the recipient animals. In summary, there is evidence that human bone-marrow-derived stromal cells have to be considered mesenchymal progenitors, persistently expressing osteogenic markers in vitro and in vivo.

Animals↗
PubMed2000

In vitro-generated stem cell leukaemia showing altered cell cycle progression with distinct signalling of the tyrosine-phosphorylated rasGAP-associated p62(dok) protein.

In an attempt to gain more insight into the events of leukaemic transformation, a cell line overexpressing MHC class II (DR) was generated by transfecting an early CD34-negative haematopoietic progenitor stem cell line with the appropriate constructs. The stable transfection with genes for DR antigens leads to cellular transformation. The DR(+) transformed cell clones express a tyrosine-phosphorylated DR heterodimer and show a significantly different morphology. DR(+) clones present the morphology of an immature myeloid neoplasia expressing alpha-naphthyl-acetate-esterase (ANAE), but neither myeloperoxidase nor CD34. While D064 cells predominately grow adherent as fibroblast-like cells, the DR(+) clones display a decrease in adherent growth. Although both cell lines express similar amounts of the interleukin-6 (IL-6) signal transducer gp130, the DR-transfected cells still show activation of STAT factors by IL-6, whereas D064 cells do not. Although the transformed clones present acceleration of cell-cycle transition and growth, the G(0)/G(1) progression inhibitor p27(kip-1) is up-regulated, while the expression of proteins involved in the S/G(2) phase transition, such as cyclin B and cdc2 (p34), is suppressed. Instead cyclin D3, one of the G(0)/G(1) progression factors, is up-regulated, as well as tyrosine-phosphorylated p62(dok), suggesting dysregulation of cell cycle-controlling proteins. In addition, DR(+) leukaemia-like cells also overexpress Bcl-2, while bax expression is suppressed, compared with the wild-type (wt) parental haematopoietic stem cell line.

Acute Disease↗
PubMed2000

Quiescence of CD34-negative haematopoietic stem cells is mediated by downregulation of Cyclin B and no stat activation.

The CD34-negative, adherent growing, fibroblast-like canine haematopoietic stem cell line D064 was recently identified as the earliest progenitor population in the bone marrow. D064 cells are predominately quiescent. Quiescence is mediated by the accumulation of the cyclin-dependent kinase inhibitor p27(kip-1)and in parallel, by the downregulation of Cyclin B, leading to an accumulation of quiescent cells in the G(0)/G(1)-phase of the cell cycle. Stem cell factor (SCF), the ligand for the tyrosine kinase receptor c-kit, usually induces differentiation of the CD34-negative stem cells into CD34-positive haematopoietic precursors. SCF also suppresses the expression of c-myc-dependent Cyclin E, which is not transcribed initially, but expression occurs later on. Interleukin 6 (IL-6) instead rather promotes proliferation, but fails to induce proliferation in the majority of CD34-negative stem cells due to no STAT activation in quiescent cells. Nevertheless, the potential of quiescent D064 cells to proliferate eventually, becomes apparent by the low-level expression of IL-6 dependent STAT factors. D064 cells also spontaneously start to express Bax, while Bcl-2 is downregulated in parallel. In summary, CD34-negative haematopoietic stem cells dwell in the marrow or other niches as quiescent cells, until they can respond to autocrine or paracrine growth factor-mediated signals.

Animals↗
PubMed2000

Impairment of genomic DNA binding to a putative dysfunctional receptor on erythrocytes independent of complement and antibodies in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by a variety of autoantibodies and other immune abnormalities indicative of an immunological hyperactivity. Antibodies against native DNA, however, are a disease-specific marker and play a major role in the pathogenesis of systemic or organ-specific disease manifestations. Nevertheless, the mechanisms causing the appearance of autoantibodies and immune complexes in SLE are not yet understood. Here, we report that chromosomal DNA and other forms of nucleic acids are usually cleared from circulation by binding to a yet unidentified receptor-like protein on the surface membrane of erythrocytes, independently from complement or antibodies. The binding kinetics of DNA and other nucleic acids to erythrocytes are significantly altered in SLE patients, showing an overall reduced binding capability and presaturated binding kinetics. Significant amounts of chromosomal DNA can be isolated from erythrocytes of SLE patients but not from normal controls. Electron microscopy shows electron-dense particles on the surface of SLE erythrocytes (approximate size 20-40 nm). Comparative genomic hybridization reveals that the nucleic acid isolated from erythrocytes of SLE patients is of genomic and random origin, leading to an accumulation of "free" nucleic acids in the periphery, which eventually induces a B-cell immune response.

Antibodies↗
PubMed2000

Perspectives on the morphology and biology of CD34-negative stem cells.

The CD34 antigen is the classical indicator molecule of pluripotent hematopoietic stem cells. But there is more and more evidence that progenitors of a yet uncommitted stem cell population do not express this surrogate marker. The bone marrow and other sites of hematopoiesis consist also of fibroblast-like stromal cells, quiescent hematopoietic stem cells, and mesenchymal stem cells. Depending on their stage of differentiation, CD34- stem cells cannot only generate hematopoietic progenitors, but also more specified mesenchymal precursors, such as osteoblasts, chondrocytes, myocytes, adipocyts, and others. The stromal cell compartment produces not only matrix proteins, such as collagens, fibronectin and others, but also the essential growth factors, which initiate and support the differentiation of primary quiescent, but eventually activated CD34- stem cells into CD34+ hematopoietic progenitors. In vivo studies have shown that long-term hematopoietic and mesenchymal reconstitution can be achieved with CD34- stem cell lines, isolated from various sources, although the frequency of CD34- stem cells seams to be quite low among the progenitor population. Some authors deny the reconstitution ability of CD34- cells. The majority of CD34- stem cells are quiescent fibroblast-like cells, which can be identified in the bone marrow biopsy as "bone lining cells". Some of those bone lining cells show protein synthesis and contain secretory vesicles. Recent studies have demonstrated that there is a surprising plasticity of the earliest stem cell population, consisting of cells with stromal cell function as well as hematopoietic and mesenchymal progenitors. The new insights into the biology of totipotent stem cells give us novel perspectives for cell- and gene therapy of various malignant and nonmalignant diseases and the possibility to replace defective organ functions with autologous CD34- stem cells.

Animals↗
PubMed2000

Isolation of primary and immortalized CD34-hematopoietic and mesenchymal stem cells from various sources.

Based on historical radiation experiments in rodents, the hematopoietic stem cell was defined by its biological properties and later by the expression of certain surface antigens (e.g., CD34), as well as the absence of lineage-specific markers (e.g., DR). Quite recently it was shown that hematopoietic reconstitution can also be achieved by CD34- stem cells, which can be isolated from the bone marrow, peripheral blood and cord blood cells. CD34-stem cells are considered to be predominately part of the quiescent stem cell pool of hematopoietic and mesenchymal stem cells. Due to novel techniques, CD34-stem cells can be expanded on the level of a true stem cell but also directed towards their differentiation into specified tissues or organ systems. This requires the establishment of primary fibroblast-like CD34- stem cells in vitro and their possible reversible and transient immortalization with optimized vector systems.

Animals↗
PubMed2000

Evidence of peripheral blood-derived, plastic-adherent CD34(-/low) hematopoietic stem cell clones with mesenchymal stem cell characteristics.

The hematopoietic system of vertebrates can be completely reconstituted with hematopoietic stem cells derived from the bone marrow, fetal liver, or cord blood, or even from peripheral-blood-derived cells. A cellular marker to identify those cells is the proteoglycan CD34, although we have shown that the earliest identifiable hematopoietic stem cell is a CD34(-) fibroblast-like cell which can differentiate into CD34(+) hematopoietic precursors. Peripheral blood mononuclear cells were isolated from the heparinized blood of a dog and incubated in tissue culture in the presence of interleukin 6. After 10-14 days, an adherent layer of fibroblast-like cells had developed and cells were immortalized using the SV-40 large T antigen. Cells were cloned and subcloned by measures of limiting dilution, and various fibroblast-like clones were established. These fibroblast-like cells either do not express the CD34 antigen or express CD34 on a low level, although transcribing CD34. The CD34(-/low) cells express osteocalcin as a mesenchymal cell marker. The fibroblast-like cells eventually differentiate spontaneously in vitro into CD34(+) precursors and show colony formation. Prior to autologous stem cell transplantation, one clone of choice (IIIG7) was transfected with a retroviral construct containing the green-fluorescence protein (GFP). The recipient dog was totally irradiated with 300 cGy and received a stem cell transplant with GFP-containing, immortalized, fibroblast-like monoclonal autologous stem cells (0.5 x 10(8)/kg dog). No additional growth factors were applied. The peripheral blood counts recovered after 23 days (WBC >500; platelets >10,000). A peripheral blood smear showed some dim but definite, although timely, limited expression of the GFP protein in nucleated peripheral blood cells just five weeks after transplantation. A bone marrow biopsy showed GFP-positive cells in the marrow cavity predominantly as "bone-lining cells."

Animals↗
PubMed1999

CDK-inhibitor independent cell cycle progression in an experimental haematopoietic stem cell leukaemia despite unaltered Rb-phosphorylation.

A CD34-negative haematopoietic progenitor cell line, D064, derived from canine bone marrow stromal cells is able to differentiate into haematopoietic progenitors under the influence of growth factor-mediated signalling. While differentiating, these cells eventually start to express MHC class II molecules (DR homologues) on their surface. The stable transfection of the fibroblast-like wild-type cells with retroviral constructs containing the cDNA for the canine MHC class II DR-genes (DRA and DRB) induces a change in morphology, accelerates cell cycle progression and leads to a loss of anchorage-dependent growth. Transfected cells show features of an immature stem cell leukaemia, such as giant cell formation. In wild-type D064 cells the accumulation of the cyclin-dependent kinase inhibitor (cdki) p27kip-1 induces differentiation, which is dependent upon signalling via the ligand for the tyrosine kinase receptor c-kit (stem cell factor). DR-transfected cells instead apparently grow independently of any growth factor-mediated signals and express high levels of the cdkis p27kip-1 and especially p21(waf-1/cip-1), concurrently with accelated cell cycle progression. In contrast to the overexpression of cdkis and despite accelerated cell cycle progression, the expression of the G2/M phase transition kinase p34cdc2 is significantly reduced in DR-transfected and transformed cells as compared to the haematopoietic wild-type cell line D064. This might suggest a possible alternative cell cycle progression pathway in this experimental stem cell leukaemia by by-passing the G0/G1 phase arrest, although retinoblastoma (Rb)-phosphorylation remains unaltered. These results provide evidence that mechanisms normally controlling the cell cycle and early haematopoietic differentiation are disrupted by the constitutive transcription and expression of MHC class II genes (DR) leading to a progression and growth of this experimental stem cell leukaemia independent from cell cycle controlling regulators such as p27 and p21.

Animals↗
PubMed1999

Hematopoietic reconstitution of syngeneic mice with a peripheral blood-derived, monoclonal CD34-, Sca-1+, Thy-1(low), c-kit+ stem cell line.

Previous work had revealed that a CD34- fibroblast-like cell is the earliest hematopoietic progenitor population. This cell type is able to differentiate into hematopoietic progeny of all lineages and circulates in the peripheral blood, from where it can be isolated by IL-6-mediated plastic adherence. We isolated peripheral blood-derived mononuclear cells (MNC) from male CBA mice and established in vitro a fibroblast-like, adherent growing cell layer. Cells were immortalized by SV-40 transfection for cellular cloning. Monoclonal fibroblast-like cell clones were established, and the surface expression of early stem cell markers was determined by flow cytometry. Clones were CD34-, Sca-1+, Thy-1(low), and c-kit+. Lethally irradiated female CBA mice were successfully transplanted with a fibroblast-like cell clone, R-M26/2-1. After syngeneic transplantation, peripheral blood counts were back to normal in transplanted mice on days 15-20, and fluorescence in situ hybridization (FISH) revealed the sole presence of male hematopoietic cells in the BM of female recipients at weeks 7, 9, 11, and 16 after transplantation. Immunohistochemistry for the expression of CD34, Sca-1, Thy-1, and c-kit showed the presence of the phenotype of the transplanted stem cell clone along the bone spicules in the marrow cavity, giving rise to HPC of all lineages. In summary, we have shown that a CD34-, Sca-1+, Thy-1(low), and c-kit+ fibroblast-like cell is consistent with the phenotype of the earliest hematopoietic and repopulating stem cell and can be isolated from peripheral blood cells.

Animals↗
PubMed1998

A method to determine the coverage of ivermectin distribution in onchocerciasis-control programmes.

Onchocerciasis is a human filarial infection responsible for an estimated 750,000 blind and severely visually disabled people. In some African countries, this disease represents the main cause of blindness, with considerable socio-economic impact on the affected communities. Since the introduction of ivermectin as a microfilaricidal agent, there is hope that visual loss from onchocerciasis can be eliminated through community-based ivermectin-distribution programmes. The African Programme for Onchocerciasis Control (APOC) has now been initiated, by the World Health Organization and World Bank, to distribute ivermectin in 19 African countries where onchocerciasis is endemic. Estimates of ivermectin coverage in distribution programmes for onchocerciasis control have so far been based on the number of treatments given, in any one year, to a target population. There is a need for a standardized method to evaluate, measure and monitor coverage over time. In April 1996, a cluster random-sampling method was used to calculate coverage with ivermectin in an urban population of 30,000 people in the Central African Republic. The method was adapted from that used to determine coverage by the WHO Expanded Programme on Immunization. It proved to be inexpensive and easy to perform, requiring only local equipment and personnel. The method used and results obtained in Bossangoa and the potential use of the technique in other distribution programmes are discussed.

Adult↗