Regulation of platelet lifespan in the presence and absence of thrombopoietin signaling.

UNLABELLED: Essentials We examined platelet survival in models of absent or enhanced thrombopoietin (TPO) signaling. Platelet lifespan is normal in transgenic mice with chronically enhanced TPO signaling. Mpl deficiency does not negatively affect platelet lifespan in the absence of thrombocytopenia. We conclude that TPO and its receptor Mpl are dispensable for platelet survival in adult mice. SUMMARY: Background It is well established that thrombopoietin (TPO), acting via its receptor Mpl, is the major cytokine regulator of platelet biogenesis. The primary mechanism by which TPO signaling stimulates thrombopoiesis is via stimulation of Mpl-expressing hematopoietic progenitors; Mpl on megakaryocytes and platelets acts to control the amount of TPO available. TPO could potentially reduce platelet and/or megakaryocyte apoptosis, and therefore increase the platelet count. However, the effect of TPO receptor signaling on platelet survival is unresolved. Methods and results Here, we investigated platelet survival in mouse models of absent or enhanced TPO signaling. In the absence of thrombocytopenia, Mpl deficiency did not negatively influence platelet lifespan, and nor was platelet survival affected in transgenic mice with chronically increased TPO signaling. Conclusions We conclude that TPO and its receptor Mpl are dispensable for platelet survival in adult mice.

BCL-2 is dispensable for thrombopoiesis and platelet survival.

Navitoclax (ABT-263), an inhibitor of the pro-survival BCL-2 family proteins BCL-2, BCL-XL and BCL-W, has shown clinical efficacy in certain BCL-2-dependent haematological cancers, but causes dose-limiting thrombocytopaenia. The latter effect is caused by Navitoclax directly inducing the apoptotic death of platelets, which are dependent on BCL-XL for survival. Recently, ABT-199, a selective BCL-2 antagonist, was developed. It has shown promising anti-leukaemia activity in patients whilst sparing platelets, suggesting that the megakaryocyte lineage does not require BCL-2. In order to elucidate the role of BCL-2 in megakaryocyte and platelet survival, we generated mice with a lineage-specific deletion of Bcl2, alone or in combination with loss of Mcl1 or Bclx. Platelet production and platelet survival were analysed. Additionally, we made use of BH3 mimetics that selectively inhibit BCL-2 or BCL-XL. We show that the deletion of BCL-2, on its own or in concert with MCL-1, does not affect platelet production or platelet lifespan. Thrombocytopaenia in Bclx-deficient mice was not affected by additional genetic loss or pharmacological inhibition of BCL-2. Thus, BCL-2 is dispensable for thrombopoiesis and platelet survival in mice.

Impacts of enterotoxin gene cluster-encoded superantigens on local and systemic experimental Staphylococcus aureus infections.

Staphylococcus aureus is both a component of the normal skin flora and an important pathogen. It expresses a range of recognized and putative virulence factors, such as enterotoxins with superantigenic properties. Several superantigen genes, i.e., seg, sei, selm, seln, and selo, are encoded by the enterotoxin gene cluster (egc), which is found in the majority of S. aureus isolates. Carriage of egc is associated with fitness of S. aureus in the gut microbiota, but it is not known if it contributes to pathogenicity. We constructed egc+ (functional for the seg, selm, and selo genes) and isogenic egc- S. aureus mutants, and investigated their virulence profiles in murine infection models. No effect of egc was seen in a local skin and soft tissue infection model, but in an invasive infection model, increased weight loss was observed after infection with the egc+ as compared to the egc- mutant. Mortality and arthritis were not affected by egc status. Our data suggest that egc has limited effects on the virulence of S. aureus. It may primarily function as a colonization factor increasing commensal fitness, although it might have some aggravating effects on the infection when the bacteria reach the blood.

Loss of Bak enhances lymphocytosis but does not ameliorate thrombocytopaenia in BCL-2 transgenic mice.

Bax and Bak are critical effectors of apoptosis. Although both are widely expressed and usually functionally redundant, recent studies suggest that Bak has particular importance in certain cell types. Genetic and biochemical studies indicate that Bak activation is prevented primarily by Mcl-1 and Bcl-xL, whereas Bax is held in check by all pro-survival Bcl-2 homologues, including Bcl-2 itself. In this study, we have investigated whether loss of Bak or elevated Mcl-1 modulates haemopoietic abnormalities provoked by overexpression of Bcl-2. The Mcl-1 transgene had little impact, probably because the expression level was insufficient to effectively reduce Bak activation. However, loss of Bak enhanced lymphocytosis in vavP-BCL-2 transgenic mice and increased resistance of their thymocytes to some cytotoxic agents, implying that Bak-specific signals can be triggered in certain lymphoid populations. Nevertheless, lack of Bak had no significant impact on thymic abnormalities in vavP-BCL-2tg mice, which kinetic analysis suggested was due to accumulation of self-reactive thymocytes that resist deletion. Intriguingly, although Bak(-/-) mice have elevated platelet counts, Bak(-/-)vavP-BCL-2 mice, like vavP-BCL-2 littermates, were thrombocytopaenic. To clarify why, the vavP-BCL-2 platelet phenotype was scrutinised more closely. Platelet life span was found to be elevated in vavP-BCL-2 mice, which should have provoked thrombocytosis, as in Bak(-/-) mice. Analysis of bone marrow chimaeric mice suggested the low platelet phenotype was due principally to extrinsic factors. Following splenectomy, blood platelets remained lower in vavP-BCL-2 than wild-type mice. However, in Rag1(-/-) BCL-2tg mice, platelet levels were normal, implying that elevated lymphocytes are primarily responsible for BCL-2tg-induced thrombocytopaenia.

Transgenic, inducible RNAi in megakaryocytes and platelets in mice.

BACKGROUND: RNA interference (RNAi) is a powerful tool for suppressing gene function. The tetracycline (tet)-regulated expression system has recently been adapted to allow inducible RNAi in mice, however its efficiency in a particular cell type in vivo depends on a transgenic tet transactivator expression pattern and is often highly variable. OBJECTIVE: We aimed to establish a transgenic strategy that allows efficient and inducible gene knockdown in particular hematopoietic lineages in mice. METHODS AND RESULTS: Using a tet-regulated reporter gene strategy, we found that transgenic mice expressing the rtTA (tet-on) transactivator under control of the cytomegalovirus (CMV) promoter (CMV-rtTA mice) display inducible reporter gene expression with unusual and near-complete efficiency in megakaryocytes and platelets. To test whether the CMV-rtTA transgene can drive inducible and efficient gene knockdown within this lineage, we generated a novel mouse strain harboring a tet-regulated short hairpin RNA (shRNA) targeting Bcl-x(L) , a pro-survival Bcl-2 family member known to be essential for maintaining platelet survival. Doxycycline treatment of adult mice carrying both transgenes induces shRNA expression, depletes Bcl-x(L) in megakaryocytes and triggers severe thrombocytopenia, whereas doxycycline withdrawal shuts off shRNA expression, normalizes Bcl-x(L) levels and restores platelet numbers. These effects are akin to those observed with drugs that target Bcl-x(L) , clearly demonstrating that this transgenic system allows efficient and inducible inhibition of genes in megakaryocytes and platelets. CONCLUSIONS: We have established a novel transgenic strategy for inducible gene knockdown in megakaryocytes and platelets that will be useful for characterizing genes involved in platelet production and function in adult mice.

Protection against experimental Staphylococcus aureus arthritis by vaccination with clumping factor A, a novel virulence determinant.

The importance of the fibrinogen-binding adhesin clumping factor A (ClfA) in the pathogenesis of Staphylococcus aureus septic arthritis was examined in an animal model. The protective effect of active and passive immunization with ClfA also was investigated in S. aureus infection models. The severity of arthritis was markedly reduced in mice challenged intravenously with a clfA mutant, compared with mice infected with the wild-type strain. Mice immunized with recombinant ClfA and challenged with S. aureus developed less-severe arthritis than did mice immunized with a control antigen. Passive immunization of mice with rat and rabbit anti-ClfA antibodies protected against S. aureus arthritis and sepsis-induced death, indicating that the protection by active immunization is antibody mediated. Taken together, these data strongly suggest that ClfA is a crucial virulence determinant for septic arthritis and an excellent target for the generation of immune therapies directed against S. aureus.

Influence of economic restraints and reduced specialist resources on delivery and quality of orthodontic care.

In 1993 and 1994, economic restrictions were introduced in the County of Ostergötland. The aim of this study was to investigate the influence on delivery and quality of orthodontic care, i.e. any subsequent change in number of patients receiving orthodontic treatment both by General Public Dental Service (GPDS) and by specialist clinic, the choice of appliance, and treatment outcomes, and also any changes in the total number of appliance treatments by general practitioners. Records were examined for 236 and 213 patients registered in 1994 and 1997, respectively, at an orthodontic clinic in the western district of Ostergötland. The total number of appliance treatments by general practitioners was estimated. The number of patients receiving initial treatment by a general practitioner and subsequently by an orthodontist, was relatively unchanged during the period. Quad helix predominated in both 1994 and 1997. The best treatment outcomes were achieved by quad helix and maxillary removable appliances, and the poorest by activators and headgear. In conclusion the total number of appliance treatments by general practitioners decreased as well as treatments requiring patient compliance over an extended period, findings which might be a consequence of the coincident economic restriction.

Treatment of lower second premolar agenesis by autotransplantation: four-year evaluation of eighty patients.

The frequency of agenesis of the second lower premolar is 2.5-4%. In growing patients, early extraction of the deciduous molar and subsequent closure of the space is a common therapy, but in some cases space closure is deemed unlikely and autotransplantation is an alternative. The aim of the study was to analyze the outcome of autotransplantation in replacing missing lower second premolars and to evaluate the associated presurgical orthodontic treatment. The material consisted of records of all patients with teeth transplanted to the lower second premolar region during the period 1988-89 at the Department of Oral Surgery, Eastman Institute, Stockholm. The following variables were registered: sex, age, number of transplanted teeth, donor tooth, root development, recipient site, orthodontic treatment, persisting temporary molar, total number of congenitally missing teeth, the surgeon responsible, and clinical and radiological follow-up variables. Of 110 transplanted teeth, 99 had not completed root formation, and in 11 teeth the root formation was completed. The success rates after 4 years were 92% and 82%, respectively. Both premolars and molars served as donor teeth, but the main donor tooth was the upper second premolar. Fourteen percent had been orthodontically treated only because of the transplantation, i.e. to open the space for the donor tooth. Treatment of agenesis of the second lower premolar by autotransplantation has a good prognosis. In growing individuals the transplant not only maintains growth and development of the alveolar ridge but also provides a permanent solution to the agenesis.

Staphylococcus aureus-induced inflammation and bone destruction in experimental models of septic arthritis.

Staphylococcus aureus is the most common cause of septic arthritis. This disease often leads to severe joint destruction and high mortality. An experimental model of S. aureus arthritis has been developed to study the course of inflammation and joint destruction, to elucidate the role of bacterial and host factors for joint pathology and mortality, and to develop therapeutical and preventive devices against septic arthritis and sepsis. Results show that the innate immune system is crucial in defending the host against staphylococcal infection while components of the specific immune system, T and B lymphocytes and their products, are detrimental to the host, mediating joint destruction and increasing mortality rates. Staphylococcal capsule polysaccharides, toxins, cell wall-attached adhesins and possibly also the chromosomal DNA are virulence determinants in S. aureus arthritis. Several vaccine candidates have recently been described which protects against staphylococcal infections, e.g. staphylococcal surface polysaccharides, enterotoxins devoid of their superantigenic properties and collagen adhesin. There are also new approaches suggested for treatment of ongoing infections, such as the combined use of antibiotics and corticosteroids.

The binding of calcium to the B-repeat segment of SdrD, a cell surface protein of Staphylococcus aureus.

In the Sdr family of Staphylococcus aureus cell surface proteins, three recently cloned members (Josefsson, E., McCrea, K., Ni Eidhin, D., O'Connell, D., Cox, J. A., Hook, M., and Foster, T. (1998) Microbiology, in press) display variable numbers of B-repeats, i.e. segments of 110-113 residues that probably make up one folding unit. Each B-repeat contains one conserved EF-hand motif and two acidic stretches. Equilibrium dialysis revealed that segment B1-B5 of SrdD contains 14 Ca2+-binding sites with high affinity ([Ca2+]0.5, 4 microM), whereas flow dialysis yielded 5 sites of high affinity (class I) and 10 of low affinity (class II). The discrepancy could be explained by the slow induction of high affinity in the class II sites. Kinetic experiments using fluorescent Ca2+ indicators corroborated slow binding of Ca2+ at the latter sites. Circular dichroism and Trp fluorescence showed that, whereas the Ca2+ form is well folded, the metal-free form seems strongly disorganized. The Ca2+-induced conformational changes comprise both fast and slow steps, giving thus a structural support for the induction of class II Ca2+-binding sites. The B-repeats may act as rulers or springs that modulate the distance between the interactive A region and the bacterial cell surface.

Suppression of type II collagen-induced arthritis by the endogenous estrogen metabolite 2-methoxyestradiol.

OBJECTIVE: To evaluate the antiarthritic properties of 2-methoxyestradiol, an endogenous metabolite of estradiol, on type II collagen-induced arthritis (CIA) in DBA/1 mice. METHODS: The effects of treatment with 2-methoxyestradiol on the development of CIA were evaluated clinically and histologically. The in vitro effects of 2-methoxyestradiol on lymphocyte and endothelial cell proliferation and differentiation were analyzed by standard methods. RESULTS: The development of CIA was significantly suppressed by 2-methoxyestradiol. Incubation with 2-methoxyestradiol suppressed the in vitro proliferation of endothelial cells, indicating that this compound down-regulates angiogenesis. Endothelial cell production of nitric oxide (NO) was also down-regulated by 2-methoxyestradiol. In contrast to estradiol, 2-methoxyestradiol exerted neither detectable feminizing effects on the sex organs nor inhibition of leukocyte development in hematopoietic organs. CONCLUSION: The development of CIA is suppressed by 2-methoxyestradiol, possibly via inhibition of angiogenesis. Diminished NO production could be of importance in vivo because it is a potent proinflammatory mediator. Since 2-methoxyestradiol exerts only mild side effects compared with estradiol, it is an interesting candidate for therapeutic use in inflammatory diseases.

Measurements of catecholamine-mediated apoptosis of immunocompetent cells by capillary electrophoresis.

Single cell analysis with capillary electrophoresis, a technique capable of detecting zeptomole quantities (10(-21) mole) of neurochemical species, has been used to demonstrate that lymphocytes are capable of active synthesis of dopamine and norepinephrine. Exposure of lymphocytes to catecholamines at concentrations as low as 10 nM leads to decreased proliferation and differentiation, e.g. interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and immunoglobulin (Ig). In addition, both inhibition of dopamine uptake with nomifensine and inhibition of packing of catecholamines into vesicles with tetrabenazine, results in significantly lower levels of dopamine and norepinephrine (p < 0.01 and p < 0.05, respectively). The catecholamine-dependent inhibition of T- and B-lymphocyte activity is mediated via an induction of a Bcl-2/Bax and Fas/FasL involved apoptosis. These findings indicate a novel mechanism for regulation of lymphocyte activity in the central nervous system, whereby elevated regional levels of catecholamines might lead to the immunoprivilege of the brain.

Catecholamines are synthesized by mouse lymphocytes and regulate function of these cells by induction of apoptosis.

The immune and the nervous systems are anatomically closely related and interact with each other by molecules common to both systems, such as cytokines and neurotransmitters. The purpose of this study was to investigate the participation of catecholamines in the neuroimmunological network. The ability of immune cells to produce catecholamines was examined by a highly sensitive capillary electrophoresis assay, which permits detection of easily oxidized catecholamines in the zeptomole (10(-21)) range. In addition, the effects of catecholamines on in vitro proliferation, differentiation and apoptosis of lymphocytes were assessed. Mouse spleen cells and macrophages contained on average 7 x 10(-17) and 2 x 10(-17) mole dopamine per cell, respectively. In the former cell population also norepinephrine was found. Several mouse B- and T-cell hybridomas were also shown to contain endogenously produced dopamine in levels ranging from 7 x 10(-20) to 2 x 10(-18) mole dopamine per cell. In addition, one of the T-cell hybridomas proved to synthesize norepinephrine. The dopamine production of lymphocytes was blocked by the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine, whereas incubation with the precursor L-DOPA increased the dopamine content. Incubation with L-DOPA, dopamine and norepinephrine dose-dependently suppressed mitogen induced proliferation and differentiation of mouse lymphocytes. Even short-time pretreatment of lymphocytes with L-DOPA and dopamine strongly suppressed lymphocyte proliferation and cytokine production. Incubation of lymphoid cells with L-DOPA, dopamine and norepinephrine dose-dependently induced apoptosis which, at least partly, explains the suppressive effects of catecholamines on lymphocyte function. Our results demonstrate that catecholamines: (i) are actively produced by lymphocytes and (ii) have the capacity to act as auto- and/or paracrine regulators of lymphocyte activity through induction of apoptosis.

Immunomodulating and anti-inflammatory properties of the sympatholytic compound 6-hydroxydopamine.

The objective of this study was to analyse the anti-inflammatory and immunosuppressive properties of 6-hydroxydopamine (6-OHDA), a well known sympatholytic compound. Collagen type II arthritis, a T cell-dependent autoimmune disease, was significantly suppressed by a short-term administration of 6-OHDA at the time of the disease onset. Similar outcome was observed when in vivo models of T cell-dependent and independent inflammatory reactions were applied. In contrast, long-term pretreatment with 6-OHDA and hence efficient sympatholysis did neither affect the course of arthritis nor the outcome of T cell-dependent and independent inflammatory reactions. These findings, together with evidence of dose-dependent in vitro inhibitory effects of 6-OHDA on lymphocyte proliferation and differentiation, indicate that the anti-inflammatory features of this compound are mediated through a direct action on effector cells rather than by sympatholysis.

Traumatic injuries to permanent teeth among Swedish school children living in a rural area.

Traumatic dental injuries in children and adolescents are a common problem, and several studies have shown that the prevalence of these injuries is increasing. The aim of this study was to make a cross-sectional investigation of all traumatized permanent teeth in children living in a rural area and to estimate the time required for each treatment. Patient records from all children aged between 7 and 17 years living in the district of Boxholm were examined. Patients with documentation concerning any traumatic injury were invited to an examination and interview. The following variables were examined: age, sex, etiology, organized sport activity, type of injury, treatment, number of dental visits, and time utilized for dental care. Dental injuries were recorded in 11.7% (88) of children (63% boys, 37% girls) and prevalence was highest in the 8-12 year-old age group for boys (75%) and the 7-9 year-old age group for girls (63%). The most frequent etiological factors were collision during play, and falling over. In boys, 10% of all dental injuries were caused by violence. Simple enamel fractures of maxillary central incisors were the most common type of injury. The mean treatment time was 74 minutes for each trauma incident, and in 35% of cases, only one dental visit was required. It was concluded that the frequency of traumatic dental injuries in a rural area is lower than in urban areas.

Neutrophil mediated inflammatory response in murine lupus.

We have studied the ability of the lupus prone MRL lpr/lpr (MRL/lpr) and (NZBxNZW)F1 (NZB/W) female mice to raise granulocyte mediated inflammatory responses. These autoimmune strains, known to exhibit severe anergy as concerns T cell dependent immune function, are not well analysed with respect to neutrophil-mediated inflammatory responses. An in vivo model of granulocyte mediated inflammation has been developed in our laboratory. A single intradermal injection of olive oil into mouse footpad induces massive infiltration of polymorphonuclear cells (PMNC) within 24 h. This extravasation of PMNC gives rise to a localized footpad swelling, which can be easily and reproducibly measured and relates to severity of the inflammatory process. T cell independence of this inflammatory model was ascertained by in vivo T cell depletion using monoclonal antibodies to CD4 and CD8 molecules. Olive oil triggered inflammation was inducible in both young and aged lupus mice. The intensity of footpad swelling upon olive oil injection was similar in lupus mice and in healthy control strains. In contrast, aged MRL/lpr and NZB/W mice showed severely depressed T cell dependent inflammatory responses as assessed by delayed type hypersensitivity reaction to sheep red blood cells. We conclude that the PMNC mediated inflammatory potential is not affected in severely diseased lupus mice. The increased numbers of circulating PMNC together with intact PMNC function may explain why severely immune deficient lupus mice seldom show clinical signs of bacterial infection.

Anti-inflammatory properties of estrogen. I. In vivo suppression of leukocyte production in bone marrow and redistribution of peripheral blood neutrophils.

We have recently demonstrated that estrogen has differential immunomodulatory properties in mice because it stimulates antibody responses but inhibits T cell-mediated inflammation. In the present study we have examined the influence of estrogen on T cell-independent inflammatory responses. A local inflammation was induced by intradermal injection of olive oil and cholera toxin. Treatment of castrated mice with pharmacological and even physiological doses of estradiol significantly suppressed the inflammatory response, as measured by footpad swelling and documented by histologic examination. Even a single injection of a low dose of estradiol (3.2 micrograms/mouse) given up to 4 days prior to the administration of the phlogistic compound reduced the inflammatory response. Our results demonstrate that the mechanisms whereby estrogen exerts its anti-inflammatory property are probably not mediated by modulation of corticosteroid production. Instead, it is evidenced that estrogen significantly suppresses the bone marrow production of leukocytes and affects the distribution of polymorphonuclear cells in peripheral blood. The possible implications of our results are discussed in the context of the modulation of inflammatory rheumatic diseases.

Peripheral denervation suppresses the late phase of delayed-type hypersensitivity.

The impact of peripheral denervation on antigen-specific immune responses was analysed on the B and T cell levels. Delayed-type hypersensitivity (DTH) response to sheep red blood cells (SRBC) or oxazolone was used in mice as a model system of in vivo T cell reactivity. Serum antibody levels to SRBC, measured by an enzyme-linked immunosorbent assay (ELISA), were evaluated to analyse the B cell response. The effect of peripheral denervation on immune responsiveness was studied both at the point of sensitization and elicitation. The results show that peripheral denervation significantly suppresses the local DTH response. The late, inflammatory phase but not the early phase of the DTH reaction was reduced after denervation (p less than 0.01). The DTH suppression by denervation was similar irrespective of whether the neurectomy was performed 1 or 4 weeks prior to sensitisation. Even in the contralateral limb, with intact innervation, the inflammatory DTH reactivity was decreased. We believe that this phenomenon might be due to abrogation of the reflex arch since the denervation procedure did not give rise to a significant systemic downregulation of DTH. Also, peripheral denervation significantly suppressed footpad swelling induced by local administration of cholera toxin, a potent phlogistic compound. The antibody response against the same immunogen was not influenced by denervation. Our results suggest that denervation of a mixed peripheral sensory/motor nerve abrogates the formation of both T cell-dependent and independent inflammatory responses.

Frequency and phenotypic feature of autoantibody-producing cell precursors in the preclinical stage of murine lupus.

The present study addresses the question of whether there is a difference in the frequencies of autoantibody-producing B-cell precursors in healthy compared with lupus-prone mouse strains. Spleen mononuclear cells (MNC) from 4-week-old (i.e. at the preclinical stage of lupus) mice were activated in vitro for 3 and 6 days with lipopolysaccharides (LPS) and the numbers of IgG, IgA and IgM autoantibody-producing cells were analysed by the ELISPOT assay. The results indicate a high frequency of IgM autoantibody-secreting cells after both 3 and 6 days in vitro stimulation. In spite of high frequencies of IgG-producing cells appearing late during the course of LPS stimulation, no IgG or IgA autoantibody producing cells were detected. No significant differences in the autoantibody repertoire were noted between healthy and lupus-prone mice, indicating that independent of the genetic background the immune system has the capacity to react with autoantibody production. Phenotypic analysis of LPS-induced, IgM-secreting B cells showed clearly that the majority of them were surface IgM+, CD5+ but Thy-1-.

Boston thoracic brace in the treatment of idiopathic scoliosis. Initial correction.

The Boston thoracic brace, i.e., a Boston brace with axillary support, was used to treat thoracic scoliosis in 44 patients. The mean initial correction for curves with the apex at T8-T9 was 15.9 degrees +/- 6.1 degrees (54%). This was a great and significant improvement over results obtained with both the Boston brace and the Boston Milwaukee brace. This degree of correction confirms earlier observations that the increased efficiency of the brace outweighs the disadvantages of the axillary component and encourages further efforts in the direction of nonsurgical treatment of the early stages of scoliosis.