Mesenchymal Stromal Cells Protect Endothelial Cells from Cytotoxic T Lymphocyte-Induced Lysis.

The integrity of the vasculature plays an important role in the success of allogeneic organ and haematopoietic stem cell transplantation. Endothelial cells (EC) have previously been shown to be the target of activated cytotoxic T lymphocytes (CTL) resulting in extensive cell lysis. Mesenchymal stromal cells (MSC) are multipotent cells which can be isolated from multiple sites, each demonstrating immunomodulatory capabilities. They are explored herein for their potential to protect EC from CTL-targeted lysis. CD8(+) T cells isolated from human PBMC were stimulated with mitotically inactive cells of a human microvascular endothelial cell line (CDC/EU.HMEC-1, further referred to as HMEC) for 7 days. Target HMEC were cultured in the presence or absence of MSC for 24 h before exposure to activated allogeneic CTL for 4 h. EC were then analysed for cytotoxic lysis by flow cytometry. Culture of HMEC with MSC in the efferent immune phase (24 h before the assay) led to a decrease in HMEC lysis. This lysis was determined to be MHC Class I restricted linked and further analysis suggested that MSC contact is important in abrogation of lysis, as protection is reduced where MSC are separated in transwell experiments. The efficacy of multiple sources of MSC was also confirmed, and the collaborative effect of MSC and the endothelium protective drug defibrotide were determined, with defibrotide enhancing the protection provided by MSC. These results support the use of MSC as an adjuvant cellular therapeutic in transplant medicine, alone or in conjunction with EC protective agents such as defibrotide.

Can action research strengthen district health management and improve health workforce performance? A research protocol.

INTRODUCTION: The single biggest barrier for countries in sub-Saharan Africa (SSA) to scale up the necessary health services for addressing the three health-related Millennium Development Goals and achieving Universal Health Coverage is the lack of an adequate and well-performing health workforce. This deficit needs to be addressed both by training more new health personnel and by improving the performance of the existing and future health workforce. However, efforts have mostly been focused on training new staff and less on improving the performance of the existing health workforce. The purpose of this paper is to disseminate the protocol for the PERFORM project and reflect on the key challenges encountered during the development of this methodology and how they are being overcome. METHODS: The overall aim of the PERFORM project is to identify ways of strengthening district management in order to address health workforce inadequacies by improving health workforce performance in SSA. The study will take place in three districts each in Ghana, Tanzania and Uganda using an action research approach. With the support of the country research teams, the district health management teams (DHMTs) will lead on planning, implementation, observation, reflection and redefinition of the activities in the study. Taking into account the national and local human resource (HR) and health systems (HS) policies and practices already in place, 'bundles' of HR/HS strategies that are feasible within the context and affordable within the districts' budget will be developed by the DHMTs to strengthen priority areas of health workforce performance. A comparative analysis of the findings from the three districts in each country will add new knowledge on the effects of these HR/HS bundles on DHMT management and workforce performance and the impact of an action research approach on improving the effectiveness of the DHMTs in implementing these interventions. DISCUSSION: Different challenges were faced during the development of the methodology. These include the changing context in the study districts, competing with other projects and duties for the time of district managers, complexity of the study design, maintaining the anonymity and confidentiality of study participants as well as how to record the processes during the study. We also discuss how these challenges are being addressed. The dissemination of this research protocol is intended to generate interest in the PERFORM project and also stimulate discussion on the use of action research in complex studies such as this on strengthening district health management to improve health workforce performance.

The value of HAZWOPER medical surveillance.

Medical surveillance is mandated for workers with potential exposure to hazardous materials. However, little guidance is provided regarding the components of a medical surveillance testing program for these individuals. This article describes the medical surveillance program for a group of 72 employees who respond to hazardous material releases throughout the United States. Conditions related to chemical exposures were not identified in this group. However, several non-occupational health conditions were identified, including a relatively high prevalence of one or more signs of metabolic syndrome. Medical surveillance may provide valuable information regarding an individual's underlying health status and non-occupational health conditions to be addressed at an early stage.

Good governance and corruption in the health sector: lessons from the Karnataka experience.

Strengthening good governance and preventing corruption in health care are universal challenges. The Karnataka Lokayukta (KLA), a public complaints agency in Karnataka state (India), was created in 1986 but played a prominent role controlling systemic corruption only after a change of leadership in 2001 with a new Lokayukta (ombudsman) and Vigilance Director for Health (VDH). This case study of the KLA (2001-06) analysed the:Scope and level of poor governance in the health sector; KLA objectives and its strategy; Factors which affected public health sector governance and the operation of the KLA. We used a participatory and opportunistic evaluation design, examined documents about KLA activities, conducted three site visits, two key informant and 44 semi-structured interviews and used a force field model to analyse the governance findings. The Lokayukta and his VDH were both proactive and economically independent with an extended social network, technical expertise in both jurisdiction and health care, and were widely perceived to be acting for the common good. They mobilized media and the public about governance issues which were affected by factors at the individual, organizational and societal levels. Their investigations revealed systemic corruption within the public health sector at all levels as well as in public/private collaborations and the political and justice systems. However, wider contextual issues limited their effectiveness in intervening. The departure of the Lokayukta, upon completing his term, was due to a lack of continued political support for controlling corruption. Governance in the health sector is affected by positive and negative forces. A key positive factor was the combined social, cultural and symbolic capital of the two leaders which empowered them to challenge corrupt behaviour and promote good governance. Although change was possible, it was precarious and requires continuous political support to be sustained.

[Cell-based strategies for salivary gland regeneration]. / Zellbasierte Strategien für die Speicheldrüsenregeneration.

Xerostomia as a side effect of radiotherapy or due to Sjögren's disease leads to considerable impairment of the quality of life of the affected patients. Preventive treatment approaches such as intensity-modulated radiotherapy, surgical transfer of a submandibular gland to a site outside the radiation field or administration of amifostin during radiation treatment are not yet completely established in clinical practice and are not applicable for all patients. Symptomatic treatment with pilocarpin or synthetic saliva leads to an improvement of the symptoms only in some patients, and in the case of pilocarpin significant systemic anticholinergic side-effects might occur. Because large numbers of patients are affected and current treatment options are not satisfactory, it is essential to develop new treatment options. In parallel with the in vitro production of functional salivary gland constructs by means of tissue engineering techniques, attempts are currently under way to experimentally restore salivary gland function by genetic treatment approaches such as transfection of the affected salivary glands with aquaporins or pro-angiogenic factors. In addition, the in vivo application of stem cells is under investigation. In the present paper, we discuss the clinical and radiobiological background of xerostomia and highlight possible innovative future treatment options.

Natural history and surgical treatment of brown tumor lesions at various sites in refractory primary hyperparathyroidism.

OBJECTIVE: Nowadays, the occurrence of brown tumor lesions or osteitis fibrosa cystica caused by long-lasting primary hyperparathyroidism are very rare, since measuring serum calcium became available routinely in the mid-1970s. It is a tumor-like lesion that may affect the entire skeleton, often presenting with diffuse focal bone pain or by pathological fracture. METHODS: We describe our experience of brown tumor lesions at different skeletal sites that were treated at our trauma centre within the last two years. This included surgical therapy for the indications (i) pain at the pelvis, (ii) increased risk for pathological fracture at the tibia and (iii) acute radicular symptoms at the lumbar spine. The literature was reviewed for the current understanding of the pathophysiology as well as therapy of brown tumor lesions in primary hyperparathyroidism. RESULTS: Curettage of a left-sided iliac crest brown tumor terminated focal pain. A less invasive stabilisation system and bone cement decreased both patient pain and the fracture risk of brown tumor lesion sites of the shinbone; and internal fixator including laminectomy at the lumbar spine ended radicular symptoms. CONCLUSION: Patients with refractory primary hyperparathyroidism should be monitored closely by endocrinologists and the patient's serum calcium level should be adjusted as far as possible. Radiography is required only if focal bone pain or pathological fractures or radicular symptoms occur. Surgery should be considered if large bone defects with spontaneous fracture risk or increasing pain are present. Tumor curettage, Palacos plombage and less invasive stabilisation systems have proved to be acceptable surgical options.

Inhibition of the mammalian target of rapamycin impedes lymphangiogenesis.

Lymphatic complications are common side effects of mammalian target of rapamycin (mTOR) inhibitor-based immunosuppression in kidney transplantation. Therefore, we investigated whether the mTOR inhibitor rapamycin, besides its known antihemangiogenic effect, also impedes regenerative lymphangiogenesis. In a murine skin flap model, rapamycin impaired recovery of lymphatic flow across surgical incisions resulting in prolonged wound edema in these animals. Importantly, the antilymphangiogenic effect of rapamycin was not related to a general inhibition of wound healing as demonstrated an in vivo Matrigeltrade mark lymphangiogenesis assay and a model of lymphangioma. Rapamycin concentrations as low as 1 ng/ml potently inhibited vascular endothelial growth factor (VEGF)-C driven proliferation and migration, respectively, of isolated human lymphatic endothelial cells (LECs) in vitro. Mechanistically, mTOR inhibition impairs downstream signaling of VEGF-A as well as VEGF-C via mTOR to the p70S6 kinase in LECs. In conclusion, we provide extensive experimental evidence for an antilymphangiogenic activity of mTOR inhibition suggesting that the early use of mTOR inhibitor following tissue injury should be avoided. Conversely, the antilymphangiogenic properties of rapamycin and its derivates may provide therapeutic value for the prevention and treatment of malignancies, respectively.

Industrial Pseudomonas folliculitis.

INTRODUCTION: Complaints of poor water quality and skin rashes among workers at a US cardboard manufacturing facility were investigated to determine potential causes. METHODS: Employees were interviewed regarding work duties and health symptoms. Areas of dermatitis in affected employees were visually examined. Collected water samples were tested for potential chemical and microbial contaminants. RESULTS: A total of 27 employees were identified with complaints of recent skin rashes affecting primarily the upper and lower extremities. Dermatitis complaints were associated with water contact and work in areas with poor water quality. Water testing showed high levels of Pseudomonas aeruginosa. Other tested substances were not at levels of concern. CONCLUSIONS: Overgrowth of P. aeruginosa occurred in the water system shortly after the facility switched to a closed-loop water recycling system and was the most likely cause of the observed dermatitis. To our knowledge, this is the first reported outbreak of Pseudomonas folliculitis in an industrial setting.

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.

[Adult stem cells regenerate the endocrine pankreas and normalize hyperglycaemia and insulin production in diabetic mice]. / Adulte Stammzellen regenerieren das endokrine Pankreas und normalisieren Hyperglykämie und Insulinproduktion in einem Diabetesmodell der Maus.

AIMS: The potential role of adult stem cells in the regeneration of beta cells in diabetes is still controversial. Although islet cell transplantation is currently the most pursued field of research, we have investigated the capacity of multipotent adult stem cells to correct hyperglycaemia in an experimental murine diabetes model. METHODS: Cloned stem cells were labelled with eGFP or transfected with a pTie2-RFP construct to show endothelial differentiation in vivo. The beta cell toxin alloxan was injected intravenously and all mice became hyperglycaemic (> 400 mg/dl) within two days and lost more than 90 % of their beta cell mass. Stem cells were then injected either directly into the pancreas or given systemically. RESULTS: Mice that received stem cell transplantation reached normal blood glucose levels within 14 days and the beta cell mass fully recovered within one month after treatment, regaining normal body weight soon after stem cell infusion. The host pancreas then dissociated and further analysed. The eGFP+ donor cells did not express insulin and other endocrine markers, but showed a red fluorescence (RFP+) and CD31 expression instead, characteristics of endothelial cells after pTie2 activation. It was further shown that remaining (eGFP-) beta cells showed increased cell cycle activity. CONCLUSIONS: Endothelial differentiation from transplanted stem cells, induced by the environment of an injured pancreas, allows the regeneration of insulin production either through proliferation of still existing and residual beta cells in the islet or the recruitment and differentiation of beta cell progenitors mostly from the duct region via enhanced vasculogenesis and microcirculation.

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.

[Chemokine directed homing of transplanted adult stem cells in wound healing and tissue regeneration]. / Chemokine-vermitteltes Homing von transplantierten adulten Stammzellen während der Wundheilung und Geweberegeneration.

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.

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.

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.

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.

Asthma severity, atopic status, allergen exposure and quality of life in elderly persons.

BACKGROUND: Although asthma can be associated with significant airflow obstruction in those over the age of 65, it is often underdiagnosed and undertreated. OBJECTIVE: To describe severity of asthma, allergy skin test sensitivities, indoor allergen exposures, and the impact on quality of life (QOL) and health status in elderly persons with asthma. METHODS: A cross-sectional data analysis with 80 elderly persons with asthma recruited from medical, geriatric, and allergy/immunology tertiary care centers. Asthma severity was determined by symptoms and measurements of lung function. House dust specimens were collected from mattresses and bedroom carpets and analyzed separately for the major allergens of house dust, using monoclonal antibody-based immunoenzymetric assays. QOL was measured using Juniper's Asthma Quality of Life Questionnaire. Health status was measured using the Short Form Health Survey Medical Outcome Questionnaire which included Ferrans and Powers' Quality of Life Index subscales. RESULTS: Two-thirds of participants had either moderate or severe persistent asthma. Skin tests to a battery of common airborne allergens were positive to at least one allergen in 56 of the 75 participants tested (74.7%). Reservoir dust allergen levels were often high enough to place participants at risk of symptoms or at risk of developing sensitization. Increased asthma severity was associated with significantly lower QOL and a trend toward decreased health status. CONCLUSIONS: Asthma is a significant chronic problem in the elderly. Atopy was common. Asthma severity impacts on these participants' QOL and health status. Results support interventions aimed at identifying allergens precipitating attacks and reducing them in the home.

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.

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.

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.

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.