c-Src links a RANK/αvβ3 integrin complex to the osteoclast cytoskeleton

RANK ligand (RANKL), by mechanisms unknown, directly activates osteoclasts to resorb bone. Because c-Src is key to organizing the cell's cytoskeleton, we asked if the tyrosine kinase also mediates RANKL-stimulated osteoclast activity. RANKL induces c-Src to associate with RANK(369–373) in an αvβ3-dependent manner. Furthermore, RANK(369–373) is the only one of six putative TRAF binding motifs sufficient to generate actin rings and activate the same cytoskeleton-organizing proteins as the integrin. While c-Src organizes the cell's cytoskeleton in response to the cytokine, it does not participate in RANKL-stimulated osteoclast formation. Attesting to their collaboration, αvβ3 and activated RANK coprecipitate, but only in the presence of c-Src. c-Src binds activated RANK via its Src homology 2 (SH2) domain and αvβ3 via its SH3 domain, suggesting the kinase links the two receptors. Supporting this hypothesis, deletion or inactivating point mutation of either the c-Src SH2 or SH3 domain obviates the RANK/αvβ3 association. Thus, activated RANK prompts two distinct signaling pathways; one promotes osteoclast formation, and the other, in collaboration with c-Src-mediated linkage to αvβ3, organizes the cell's cytoskeleton

M-CSF regulates the cytoskeleton via recruitment of a multimeric signaling complex to c-Fms Tyr-559/697/721.

M-CSF is known to induce cytoskeletal reorganization in macrophages and osteoclasts by activation of phosphatidylinositol 3-kinase (PI3K) and c-Src, but the detailed mechanisms remain unclear. We find, unexpectedly, that tyrosine (Tyr) to phenylalanine (Phe) mutation of Tyr-721, the PI3K binding site in the M-CSF receptor c-Fms, fails to suppress cytoskeletal remodeling or actin ring formation. In contrast, mutation of c-Fms Tyr-559 to Phe blocks M-CSF-induced cytoskeletal reorganization by inhibiting formation of a Src Family Kinase SFK·c-Cbl·PI3K complex and the downstream activation of Vav3 and Rac, two key mediators of actin remodeling. Using an add-back approach in which specific Tyr residues are reinserted into c-Fms inactivated by the absence of all seven functionally important Tyr residues, we find that Tyr-559 is necessary but not sufficient to transduce M-CSF-dependent cytoskeletal reorganization. Furthermore, this same add-back approach identifies important roles for Tyr-697 and Tyr-721 in collaborating with Tyr-559 to recruit a multimeric signaling complex that can transduce signals from c-Fms to the actin cytoskeleton

Cdc42 regulates bone modeling and remodeling in mice by modulating RANKL/M-CSF signaling and osteoclast polarization

The modeling and remodeling of bone requires activation and polarization of osteoclasts, achieved by reorganization of the cytoskeleton. Members of the Rho subfamily of small GTPases, including Cdc42, are known regulators of cytoskeletal components, but the role of these proteins in bone physiology and pathophysiology remains unclear. Here, we examined loss-of-function mice in which Cdc42 was selectively ablated in differentiated osteoclasts and gain-of-function animals wherein Cdc42Gap, a protein that inactivates the small GTPase, was deleted globally. Cdc42 loss-of-function mice were osteopetrotic and resistant to ovariectomy-induced bone loss, while gain-of-function animals were osteoporotic. Isolated Cdc42-deficient osteoclasts displayed suppressed bone resorption, while osteoclasts with increased Cdc42 activity had enhanced resorptive capacity. We further demonstrated that Cdc42 modulated M-CSF–stimulated cyclin D expression and phosphorylation of Rb and induced caspase 3 and Bim, thus contributing to osteoclast proliferation and apoptosis rates. Furthermore, Cdc42 was required for multiple M-CSF– and RANKL-induced osteoclastogenic signals including activation and expression of the differentiation factors MITF and NFATc1 and was a component of the Par3/Par6/atypical PKC polarization complex in osteoclasts. These data suggest that Cdc42 regulates osteoclast formation and function and may represent a promising therapeutic target for prevention of pathological bone loss

Human Infection Caused by Leptospira fainei

We report a human case of leptospirosis in which the spirochete was detected by dark-field microscopy examination of cerebrospinal fluid (CSF) and isolated from both CSF and blood. Leptospira fainei was identified by sequencing the 16S rDNA gene, which had been amplified by polymerase chain reaction. This case confirms the role of L. fainei as a human pathogen and extends its distribution to southern Europe

Talin1 and Rap1 are critical for osteoclast function

To determine talin1's role in osteoclasts, we mated TLN1(fl/fl) mice with those expressing cathepsin K-Cre (CtsK-TLN1) to delete the gene in mature osteoclasts or with lysozyme M-Cre (LysM-TLN1) mice to delete TLN1 in all osteoclast lineage cells. Absence of TLN1 impairs macrophage colony-stimulating factor (M-CSF)-stimulated inside-out integrin activation and cytoskeleton organization in mature osteoclasts. Talin1-deficient precursors normally express osteoclast differentiation markers when exposed to M-CSF and receptor activator of nuclear factor κB (RANK) ligand but attach to substrate and migrate poorly, arresting their development into mature resorptive cells. In keeping with inhibited resorption, CtsK-TLN1 mice exhibit an ∼5-fold increase in bone mass. Osteoclast-specific deletion of Rap1 (CtsK-Rap1), which promotes talin/β integrin recognition, yields similar osteopetrotic mice. The fact that the osteopetrosis of CtsK-TLN1 and CtsK-Rap1 mice is substantially more severe than that of those lacking αvβ3 is likely due to added failed activation of β1 integrins. In keeping with osteoclast dysfunction, mice in whom talin is deleted late in the course of osteoclastogenesis are substantially protected from ovariectomy-induced osteoporosis and the periarticular osteolysis attending inflammatory arthritis. Thus, talin1 and Rap1 are critical for resorptive function, and their selective inhibition in mature osteoclasts retards pathological bone loss

Regulated maturation of malaria merozoite surface protein-1 is essential for parasite growth

The malaria parasite Plasmodium falciparum invades erythrocytes where it replicates to produce invasive merozoites, which eventually egress to repeat the cycle. Merozoite surface protein-1 (MSP1), a prime malaria vaccine candidate and one of the most abundant components of the merozoite surface, is implicated in the ligand–receptor interactions leading to invasion. MSP1 is extensively proteolytically modified, first just before egress and then during invasion. These primary and secondary processing events are mediated respectively, by two parasite subtilisin-like proteases, PfSUB1 and PfSUB2, but the function and biological importance of the processing is unknown. Here, we examine the regulation and significance of MSP1 processing. We show that primary processing is ordered, with the primary processing site closest to the C-terminal end of MSP1 being cleaved last, irrespective of polymorphisms throughout the rest of the molecule. Replacement of the secondary processing site, normally refractory to PfSUB1, with a PfSUB1-sensitive site, is deleterious to parasite growth. Our findings show that correct spatiotemporal regulation of MSP1 maturation is crucial for the function of the protein and for maintenance of the parasite asexual blood-stage life cycle

Long-Term Clinical Protection from Falciparum Malaria Is Strongly Associated with IgG3 Antibodies to Merozoite Surface Protein 3

Using data from malaria cases in Senegal, Pierre Druilhe and colleagues found that antibodies of the IgG3 isotype directed to merozoite surface protein 3 (MSP3) were strongly predictive of clinical outcome

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

The Relationship Between Spirituality and Depression in Family Caregivers of the Elderly

The purpose of this study was to examine the relationship between spirituality and depression in family caregivers of the elderly. This study sought to test the following hypothesis: The level of spirituality will be negatively correlated with the level of depression for family caregivers of the elderly. A descriptive, correlational design utilizing Neuman\u27s wholistic system theory was used with a convenience sample of 44 family caregivers aged between 32 and 88 years. Self-reporting questionnaires were mailed to clients (identified as caregivers) of a home care agency, a caregiver respite program, and a Parkinson\u27s support group all providing services in Northern Michigan. The Spiritual Perspective Scale (SPS) was utilized to measure spirituality, and the short form of the Geriatric Depression Scale (GDS) was utilized to measure depression.; A Pearson correlation revealed no significant relationship existed between the total SPS scores and the total GDS scores (r = {dollar}-{dollar}.106, df = 32, p {dollar}\u3e{dollar}.05). Even though this study\u27s results were not statistically significant, the data indicated a trend toward a negative relationship between the variables. Implications for nursing are discussed