Increased chemotaxis and activity of circulatory myeloid progenitor cells may contribute to enhanced osteoclastogenesis and bone loss in the C57BL/6 mouse model of collagen-induced arthritis.

Our study aimed to determine the functional activity of different osteoclast progenitor (OCP) subpopulations and signals important for their migration to bone lesions, causing local and systemic bone resorption during the course of collagen-induced arthritis in C57BL/6 mice. Arthritis was induced with chicken type II collagen (CII), and assessed by clinical scoring and detection of anti-CII antibodies. We observed decreased trabecular bone volume of axial and appendicular skeleton by histomorphometry and micro-computed tomography as well as decreased bone formation and increased bone resorption rate in arthritic mice in vivo. In the affected joints, bone loss was accompanied with severe osteitis and bone marrow hypercellularity, coinciding with the areas of active osteoclasts and bone erosions. Flow cytometry analysis showed increased frequency of putative OCP cells (CD3- B220- NK1.1- CD11b-/lo CD117+ CD115+ for bone marrow and CD3- B220- NK1.1- CD11b+ CD115+ Gr-1+ for peripheral haematopoietic tissues), which exhibited enhanced differentiation potential in vitro. Moreover, the total CD11b+ population was expanded in arthritic mice as well as CD11b+ F4/80+ macrophage, CD11b+ NK1.1+ natural killer cell and CD11b+ CD11c+ myeloid dendritic cell populations in both bone marrow and peripheral blood. In addition, arthritic mice had increased expression of tumour necrosis factor-α, interleukin-6, CC chemokine ligand-2 (Ccl2) and Ccl5, with increased migration and differentiation of circulatory OCPs in response to CCL2 and, particularly, CCL5 signals. Our study characterized the frequency and functional properties of OCPs under inflammatory conditions associated with arthritis, which may help to clarify crucial molecular signals provided by immune cells to mediate systemically enhanced osteoresorption.

Immunohistological and flow cytometric analysis of glycosphingolipid expression in mouse lymphoid tissues.

Expression of neutral glycosphingolipids (GSLs) and gangliosides in normal lymphoid tissues and cells has been studied mostly by biochemical and immunochemical analysis of lipid extracts separated by thin-layer chromatography. GSLs and gangliosides involved in the GM1b biosynthetic pathway were assigned to T-lymphocytes, whereas B-cell gangliosides and GSLs have been poorly characterized in former publications. We used specific polyclonal antibodies in immunohistochemistry and flow cytometry to analyze the distribution of globotriaosylceramide (Gb(3)Cer), globoside (Gb(4)Cer), gangliotriaosylceramide (Gg(3)Cer), gangliotetraosylceramide (Gg(4)Cer), and gangliosides GM3 and GalNAc-GM1b in the mouse thymus, spleen, and lymph node. Immature thymocytes expressed epitopes recognized by all antibodies, except for anti-Gb(4)Cer. Mature thymocytes bound only antibodies to GalNAc-GM1b, Gg(4)Cer, and Gb(4)Cer. In secondary lymphoid organs, antibodies to globo-series GSLs bound to vascular spaces of secondary lymphoid organs, whereas the ganglio-series GSL antibodies recognized lymphocyte-containing regions. In a Western blotting analysis, only GalNAc-GM1b antibody recognized a specific protein band in all three organs. Flow cytometric analysis of spleen and lymph node cells revealed that B-cells carried epitopes recognized by all antibodies, whereas the T-cell GSL repertoire was mostly oriented to ganglio-series-neutral GSLs and GM1b-type gangliosides. The results of immunohistochemistry and flow cytometry were not always identical, possibly because of crossreactivity to glycoprotein-linked oligosaccharides and/or differences between cell surface carbohydrate profiles of isolated cells and cells in a tissue environment.

[Fear of vaccination against hepatitis B--one of the reasons of low vaccination rate among health-care workers]. / Strah od cijepljenja protiv hepatitisa B--jedan od razloga niske procijepljenosti zdravstvenih djelatnika.

We examined 120 health care workers from two Zagreb hospitals with regard to hepatitis B vaccination, and the reasons for not accepting the vaccination. The majority of the examinees (83, 88%) were often exposed to HBV infection and many experienced needlestick exposure to blood (63, 67%). Despite all this, only 55 (67%) examinees routinely use some protective devices at work, but 26 (33%) do not. 57 (60%) examinees believe they can be infected with hepatitis B through vaccination and 57 (72%) believe other "dangerous" complications are also possible. Only 33 (35%) examinees received complete vaccination while others received only one (30, 37%) or two doses of vaccine (30, 35%). 33 (66%) examinees did not accept the vaccination. Only 8.3 (10%) examinees know the procedure after the needlestick exposure to blood. A total of 38 (60%) examinees attended educative lectures on HBV infection before the vaccination. Therefore, poor knowledge about vaccination against hepatitis B among our health care workers demands continuous education and permanent vaccination to avoid occupational hepatitis B.

Activated T lymphocytes suppress osteoclastogenesis by diverting early monocyte/macrophage progenitor lineage commitment towards dendritic cell differentiation through down-regulation of receptor activator of nuclear factor-kappaB and c-Fos.

Activated T lymphocytes either stimulate or inhibit osteoclastogenesis from haematopoietic progenitors in different experimental models. To address this controversy, we used several modes of T lymphocyte activation in osteoclast differentiation--mitogen-pulse, anti-CD3/CD28 stimulation and in vivo and in vitro alloactivation. Osteoclast-like cells were generated from non-adherent immature haematopoietic monocyte/macrophage progenitors in murine bone-marrow in the presence of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) and monocyte-macrophage colony-stimulating factor (M-CSF). All modes of in vivo and in vitro T lymphocyte activation and both CD4(+) and CD8(+) subpopulations produced similar inhibitory effects on osteoclastogenesis paralleled by enhanced dendritic cell (DC) differentiation. Osteoclast-inhibitory effect was associated with T lymphocyte activation and not proliferation, and could be replaced by their culture supernatants. The stage of osteoclast differentiation was crucial for the inhibitory action of activated T lymphocytes on osteoclastogenesis, because the suppressive effect was visible only on early osteoclast progenitors but not on committed osteoclasts. Inhibition was associated specifically with increased granulocyte-macrophage colony-stimulating factor (GM-CSF) expression by the mechanism of progenitor commitment toward lineages other than osteoclast because activated T lymphocytes down-regulated RANK, CD115, c-Fos and calcitonin receptor expression, and increased differentiation towards CD11c-positive DC. An activated T lymphocyte inhibitory role in osteoclastogenesis, confirmed in vitro and in vivo, mediated through GM-CSF release, may be used to counteract activated bone resorption mediated by T lymphocyte-derived cytokines in inflammatory and immune disorders. We also demonstrated the importance of alloactivation in osteoclast differentiation and the ability of cyclosporin A to abrogate T lymphocyte inhibition of osteoclastogenesis, thereby confirming the functional link between alloreaction and bone metabolism.

Alteration of newly induced endochondral bone formation in adult mice without tumour necrosis factor receptor 1.

Tumour necrosis factor (TNF)-alpha, a major proinflammatory cytokine, exerts its role on bone cells through two receptors (TNFR1 and TNFR2). TNFR1, but not TNFR2, is expressed by osteoblasts and its function in bone formation in vivo is not fully understood. We compared in vivo new bone formation in TNFR1-deficient (TNFR1(-/-)) mice and wild-type mice, using two models of bone formation: intramembranous ossification following tibial marrow ablation and endochondral ossification induced by bone morphogenetic protein (BMP)-2. Intramembranous osteogenesis in TNFR1(-/-) mice did not differ from the wild-type mice either in histomorphometric parameters or mRNA expression of bone-related markers and inflammatory cytokines. During endochondral osteogenesis, TNFR1(-/-) mice formed more cartilage (at post-implantation day 9), followed by more bone and bone marrow (at day 12). mRNAs for BMP-2, -4 and -7 were increased during the endochondral differentiation sequence in TNFR1(-/-) mice. The expression of receptor activator of NF-kappa B ligand (RANKL) and receptor activator of NF-kappa B (RANK), as assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR), was also increased significantly during endochondral ossification in TNFR1(-/-) mice. In conclusion, signalling through the TNFR1 seems to be a negative regulator of new tissue formation during endochondral but not intramembranous osteogenesis in an adult organism. BMPs and RANKL and its receptor RANK may be involved in the change of local environment in the absence of TNFR1 signalling.

[Aortic valve insufficiency due to rupture of the cusp in a patient with multiple trauma]. / Insuffizienz der Aortenklappe aufgrund einer Ruptur des Klappensegels bei einem polytraumatisierten Patienten.

Lesions of the heart valve caused by blunt chest trauma is rare, but when it does occur it can significantly injure the patient. On the basis of autopsy studies, research shows that heart valves are injured in less than 5% of patients who have died due to impact thoracic trauma. Among the heart valves, the aortic valve is the most often lacerated, which has been proved by relevant autopsy and clinical studies. Aortic valve lesions can be the only injury, but it is possible that additional heart or large vessel injuries are also present (myocardial contusion, rupture of the atrial septum, aortic rupture, rupture of the left common carotid artery). The force that causes such an injury is often great and often causes injuries to other organs and organ systems. In a multiple trauma patient, it is very important to specifically look for heart-related injuries because it is possible that they may be overlooked or missed by the surgeon, because of other obvious injuries. We describe the case of a 41-year-old man with multiple trauma who was diagnosed with aortic valve insufficiency due to rupture of the left coronary cusp 6 weeks after a road accident. Valvuloplasty was performed. Seven years later the patient is free of symptoms and is in good physical condition. Echocardiography showed normal dimensions of the heart chambers, a normal thickness of the heart walls, and normal systolic and diastolic function of the left ventricle. Heart valves are morphologically normal, and only an unimportant aortic insufficiency was noticed by echocardiography.

Cellular and molecular interactions between immune system and bone.

Functional interdependence between immune and bone systems is reflected in a number of regulatory molecules acting on the cells of both systems and common precursors for bone and immune cells. Therefore, the disturbances of the immune system may affect bone metabolism, and vice versa. This review addresses the roles of two major immune cell populations, T and B lymphocytes, in the regulation of bone metabolism. Experimental models and human diseases demonstrated that T lymphocytes may produce many bone cell regulatory cytokines, including two essential stimulators of osteoclastogenesis: receptor for activation of nuclear factor kappa b (NF-kappa B) (RANK) ligand (RANKL) and macrophage colony-stimulating factor. The effect of T lymphocytes on osteoclastogenesis may be both stimulatory and inhibitory, and depends on the activation stage and pattern of cytokine production. We showed that acute removal of T lymphocytes stimulated osteoclast differentiation in vitro and enhanced new cartilage and bone formation at non-osseous sites in vivo. B lymphocytes may be even more closely related to bone cells, as B lymphopoiesis requires an intimate contact with osteoblastic/stromal cells, and estrogens, powerful regulators of bone mass, are also involved in the differentiation of the B lymphocyte lineage. Also, B lymphocyte progenitors may give rise to functional osteoclasts. Both B and T lymphocytes may act through the RANKL/RANK/osteoprotegerin cytokine system, which has been independently discovered within immune and bone systems. These cytokines have crucial roles in the development and function of osteoclasts, dendritic cells, and T and B lymphocytes, as well as in the thymus and lymph node organogenesis. The cytokines produced by immune cells may affect bone cell function and vice versa, but the full complexity of these interactions awaits further investigation.

Role of B lymphocytes in new bone formation.

Although there may be a close relationship between B lymphocytes and osteoclasts, or bone resorbing cells, little is known about the role of B lymphocytes in bone formation. We compared in vivo new bone induction in mice homozygous for the B-cell deficient (microMT) gene knockout, which lack functional B lymphocytes, with bone induction in control wild-type (C57BL/6) mice. Our comparison used two models of new bone induction in vivo: endochondral osteoinduction by subcutaneous implantation of recombinant human bone morphogenetic protein (rhBMP-2) and osteogenic regeneration after tibial bone marrow ablation. The expression of bone-specific proteins (bone sialoprotein, osteopontin, and osteocalcin) and inflammatory/immunomodulatory cytokines (interleukin-1alpha and -1beta, interleukin-6, and tumor necrosis factor-alpha) was assessed by Northern blot analysis or reverse transcription-polymerase chain reaction, respectively. Ossicles induced by rhBMP-2 were larger in volume and mass in microMT knockout mice, but relative volumes of the newly induced bone, cartilage, and bone marrow were similar in the two groups. Six days after tibial bone marrow ablation, microMT knockout mice resorbed the initial blood clot faster and formed more trabecular bone, paralleled by greater levels of bone sialoprotein mRNA than in the wild-type mice. microMT knockout and wild-type mice also differed in the expression pattern of inflammatory/immunomodulatory cytokines during the development of the newly induced bone, suggesting that a genetic lack of B lymphocytes may create a change in the immunological milieu at the site of new bone induction, which stimulates the initial accumulation and proliferation of mesenchymal progenitor.