Decline of Bone Marrow–Derived Hematopoietic Progenitor Cell Quality During Aging in the Rat

Several studies have shown that aging is associated with quantitative and qualitative alterations of the stem and progenitor cell compartment. The current results indicate that there is a significant age-associated decline in the proliferative capacity of rat myeloid progenitor cells. In contrast, no difference was found in the frequency of myeloid progenitor cells in the bone marrow of young versus old rats. Furthermore, a significant shift towards higher proliferative capacity of myeloid progenitors was observed after lifelong voluntary exercise. These data emphasize that aging is accompanied by a loss of proliferative capacity and that voluntary exercise could retard this process.     

Rasip1 mediates Rap1 regulation of Rho in endothelial barrier function through ArhGAP29

Rap1 is a small GTPase regulating cell–cell adhesion, cell–matrix adhesion, and actin rearrangements, all processes dynamically coordinated during cell spreading and endothelial barrier function. Here, we identify the adaptor protein ras-interacting protein 1 (Rasip1) as a Rap1-effector involved in cell spreading and endothelial barrier function. Using Förster resonance energy transfer, we show that Rasip1 interacts with active Rap1 in a cellular context. Rasip1 mediates Rap1-induced cell spreading through its interaction partner Rho GTPase-activating protein 29 (ArhGAP29), a GTPase activating protein for Rho proteins. Accordingly, the Rap1–Rasip1 complex induces cell spreading by inhibiting Rho signaling. The Rasip1–ArhGAP29 pathway also functions in Rap1-mediated regulation of endothelial junctions, which controls endothelial barrier function. In this process, Rasip1 cooperates with its close relative ras-association and dilute domain-containing protein (Radil) to inhibit Rho-mediated stress fiber formation and induces junctional tightening. These results reveal an effector pathway for Rap1 in the modulation of Rho signaling and actin dynamics, through which Rap1 modulates endothelial barrier function.The small GTPase Rap1 regulates both integrin-mediated and cadherin-mediated adhesions. Rap1 can increase cell adhesion by inducing the allosteric activation and clustering of integrins, thereby increasing cell–extracellular matrix (ECM) adhesion (1–3). Upon cell–ECM engagement, Rap1 induces cell spreading, due to increased cell protrusion and decreased cell contraction, indicating changes in actin dynamics (4, 5). In addition, Rap1 regulates both epithelial and endothelial cell–cell adhesion (6–11). Particularly the role of Rap1 in controlling endothelial cell junctions is important, as weakening of the endothelial barrier can result in pathologies such as chronic inflammation, atherosclerosis, and vascular leakage (12–14). Activation of Rap1 in endothelial cells results in stabilization of junctions and consequently increased barrier function through the recruitment of β-catenin, resulting in stabilization of vascular endothelial (VE)–cadherin at cell–cell junctions (15–18) and rearrangements of the actin cytoskeleton (6, 7, 19–21). These rearrangements of the actin cytoskeleton include the disruption of radial stress fibers and the induction of cortical actin bundles, and consequently a switch from discontinuous, motile junctions into linear, stable junctions (6–8, 20). Rap1 achieves this at least in part by regulating Rho-signaling (6, 7, 10, 19, 20). The molecular mechanism of how Rap1 regulates Rho, however, remains largely elusive, although the Rap1-effector Krev interaction trapped protein 1 (Krit-1)/cerebral cavernous malformations 1 protein (CCM1) has been proposed to be involved (15, 16, 22).In this study, we identified a Rap1-signaling cascade, comprising ras-interacting protein 1 (Rasip1), ras-association and dilute domain-containing protein (Radil), and Rho GTPase-activating protein 29 (ArhGAP29), affecting both cell spreading and endothelial barrier function by regulating the Rho-signaling cascade.     

Immunohistochemical analysis as a means to predict responsiveness to rituximab treatment

OBJECTIVE: Anti-CD20-mediated B cell depletion with rituximab is a new and effective therapy for rheumatoid arthritis (RA). Although B cells in peripheral blood (PB) are consistently depleted in all patients, the clinical effects are more heterogeneous, possibly related to differences in the depleting effects of lymphoid or solid tissues. The aim of this study was to investigate B cell depletion in different compartments (PB, bone marrow, and synovium) and determine predictive variables for responsiveness to rituximab therapy. METHODS: Before and 12 weeks after rituximab treatment, samples of PB, bone marrow, and synovium were collected from 25 patients with RA refractory to disease-modifying antirheumatic drugs and tumor necrosis factor-blocking agents. CD19+ and CD20+ B cells in PB and bone marrow were measured by flow cytometric analysis, whereas CD79a+ and cytoplasmic CD20+ B cells in the synovium were stained by immunohistochemistry. The effects of rituximab on serum Ig and autoantibodies were measured by enzyme-linked immunosorbent assay. RESULTS: Rituximab effectively depleted the CD20+ subset of B cells in the PB, bone marrow, and synovium of RA patients. Rituximab significantly reduced autoantibody production (anti-citrullinated protein antibodies [ACPAs] and rheumatoid factor [RF]), in part due to a nonspecific decrease in total Ig production. Importantly, positivity for circulating ACPA IgM, in combination with a high infiltration of CD79a+ B cells in the synovium, but not of CD138+ plasma cells, was a predictor of clinical outcome after rituximab treatment. ACPA IgM titers were independently associated with synovial infiltration of CD20-,CD79a+ B cells, but not with CD138+ plasma cells. CONCLUSION: These data provide novel insights into the mechanisms of CD20-mediated B cell depletion in the lymphoid and solid tissues of RA patients and suggest a pivotal role for ACPA IgM-producing plasmablasts in RA.     

Differences in matrix composition between calvaria and long bone in mice suggest differences in biomechanical properties and resorption: Special emphasis on collagen

The mammalian skeleton consists of bones that are formed in two different ways: long bones via endochondral ossification and flat bones via intramembranous ossification. These different formation modes may result in differences in the composition of the two bone types. Using the 2D-difference in gel electrophoresis technique and mass spectrometry, we analyzed the composition of murine mineral-associated proteins of calvaria and long bone. Considerable differences in protein composition were observed. Flat bones (calvariae) contained more soluble collagen (8x), pigment epithelium derived factor (3x) and osteoglycin (4x); whereas long bones expressed more chondrocalcin (3x), thrombospondin- 1 (4x), fetuin (4x), secreted phosphoprotein 24 (3x), and thrombin (7x). Although cystatin motifs containing proteins, such as secreted phosphoprotein 24 and fetuin are highly expressed in long bone, they did not inhibit the activity of the cysteine proteinases cathepsin B and K. The solubility of collagen differed which coincided with differences in collagen crosslinking, long bone containing 3x more (hydroxylysine)-pyridinoline. The degradation of long bone collagen by MMP2 (but not by cathepsin K) was impaired. These differences in collagen crosslinking may explain the differences in the proteolytic pathways osteoclasts use to degrade bone. Our data demonstrate considerable differences in protein composition of flat and long bones and strongly suggest functional differences in formation, resorption, and mechanical properties of these bone types.     

Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney

The chemokine CXCL16 plays an important role in the recruitment of leukocytes to sites of inflammation influencing the course of experimental glomerulonephritis. Here we show that human kidneys highly express CXCL16 in the distal tubule, connecting tubule and principal cells of the collecting duct with weak expression in the thick ascending limb of Henle. Beside the membrane localization, a soluble form of CXCL16 can be proteolytically released which acts as a chemotactic factor. In human renal tissue the expression pattern of the disintegrin-like metalloproteinase ADAM10 is similar to that of CXCL16, suggesting ADAM10 can potentially cleave CXCL16 in vivo. When we tested this in primary tubular cells we found that blockade of ADAM10 activity inhibited the IFN-gamma induced release of soluble CXCL16. Acute tubular damage in renal allografts was associated with elevated urinary CXCL16 and this correlated with focally increased apical CXCL16 expression in the distal tubules and collecting ducts. Renal allograft biopsies, with a histopathological diagnosis of acute interstitial rejection, showed increased basolateral ADAM10 expression together with high numbers of infiltrating T cells. Our results suggest that CXCL16 and ADAM10 are involved in the recruitment of T cells to the kidney and play an important role in inflammatory kidney diseases.     

Differential localization of coatomer complex isoforms within the Golgi apparatus

Coatomer, the coat protein of coat protein complex (COP)I-vesicles, is a soluble protein complex made up of seven subunits, alpha-, beta-, beta'-, gamma-, delta-, epsilon-, and zeta-COP. Higher eukaryotes have two paralogous versions of the gamma- and zeta- subunits, termed gamma1- and gamma2-COP and zeta1- and zeta2-COP. Different combinations of these subunits are known to exist within coatomer complexes, and gamma1/zeta1-, gamma1/zeta2-, and gamma2/zeta1-COP represent the major coatomer populations in mammals. The role of COPI vesicles in the early secretory pathway is the subject of considerable debate. To help to resolve this discussion, we used quantitative immunoelectron microscopy and found that significant localization differences for COPI-isoforms do exist, with a preference for gamma1zeta1- and gamma1zeta2-coatomer in the early Golgi apparatus and gamma2zeta1-coatomer in the late Golgi apparatus. These differences suggest distinct functions for coatomer isoforms in a manner similar to clathrin/adaptor vesicles, where different adaptor proteins serve particular transport routes.     

Chances of renal recovery for dialysis-dependent ANCA-associated glomerulonephritis

In patients who have anti-neutrophil cytoplasm autoantibody (ANCA)-associated glomerulonephritis and are on dialysis at time of diagnosis, renal function is sometimes insufficiently restored by immunosuppressive treatment, which often coincides with potentially lethal adverse effects. This study investigated the clinical and histologic variables that determine the chances of dialysis independence, dialysis dependence, or death after 12 mo in these patients. Sixty-nine patients who had ANCA-associated glomerulonephritis and were dialysis dependent at diagnosis received uniform, standard immunosuppressive therapy plus either intravenous methylprednisolone or plasma exchange. Eleven clinical and histologic variables were assessed. Univariate and binary logistic regression analyses were performed. Predictive parameters were entered into a two-step binary logistic regression analysis to differentiate among the outcomes of dialysis independence, dialysis dependence, or death. The point at which the chance of therapy-related death exceeded the chance of dialysis independence was determined. The chance of recovery exceeded the chance of dying in most cases. Intravenous methylprednisolone as adjunctive therapy plus <18% normal glomeruli and severe tubular atrophy increased the chance of therapy-related death over the chance of dialysis independence. Plasma exchange treatment plus severe tubular atrophy and <2% normal glomeruli increased the chance of therapy-related death over that of dialysis independence. Even with ominous histologic findings, the chance of renal recovery exceeds the chance of therapy-related death when these patients are treated with plasma exchange as adjunctive therapy.     

Radiographic hip disorders and associated complications in severe cerebral palsy

We performed a cross-sectional study of 160 adult patients with severe cerebral palsy to study the relationship between radiographic hip disorders (migration and deformity of the femoral head), on the one hand, and complications such as handling problems, seating problems, decubitus ulcers, fractures and contractures, on the other hand. Both migration and deformity were positively related to the need for a special seat in the wheelchair and adduction contractures of the hip. We conclude that migration and deformity of the femoral head, if possible, should be prevented in patients with severe cerebral palsy.