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Brain trauma was clinically associated with increased osteogenesis in the appendicular skeleton. We showed previously in C57BL/6J mice that mild traumatic brain injury (mTBI) transiently induced bone formation in the femur via the cannabinoid-1 (CB1) receptor. Here, we subjected ICR mice to mTBI and examined the bone response in the skull using microCT. We also measured mast cell degranulation (MCD)72 h post-injury. Finally, we measured brain and calvarial endocannabinoids levels post-mTBI. mTBI led to decreased bone porosity on the contralateral (untouched) side. This effect was apparent both in young and mature mice. Administration of rimonabant (CB1 inverse agonist) completely abrogated the effect of mTBI on calvarial porosity and significantly reduced MCD, compared with vehicle-treated controls. We also found that mTBI resulted in elevated levels of anandamide, but not 2-arachidonoylglycerol, in the contralateral calvarial bone, whereas brain levels remained unchanged. In C57BL/6J CB1 knockout mice, mTBI did not reduce porosity but in general the porosity was significantly lower than in WT controls. Our findings suggest that mTBI induces a strain-specific CB1-dependent bone anabolic response in the skull, probably mediated by anandamide, but seemingly unrelated to inflammation. The endocannabinoid system is therefore a plausible target in management of bone response following head trauma.

Brain trauma was clinically associated with increased osteogenesis in the appendicular skeleton. We showed previously in C57BL/6J mice that mild traumatic brain injury (mTBI) transiently induced bone formation in the femur via the cannabinoid-1 (CB1) receptor. Here, we subjected ICR mice to mTBI and examined the bone response in the skull using microCT. We also measured mast cell degranulation (MCD)72 h post-injury. Finally, we measured brain and calvarial endocannabinoids levels post-mTBI. mTBI led to decreased bone porosity on the contralateral (untouched) side. This effect was apparent both in young and mature mice. Administration of rimonabant (CB1 inverse agonist) completely abrogated the effect of mTBI on calvarial porosity and significantly reduced MCD, compared with vehicle-treated controls. We also found that mTBI resulted in elevated levels of anandamide, but not 2-arachidonoylglycerol, in the contralateral calvarial bone, whereas brain levels remained unchanged. In C57BL/6J CB1 knockout mice, mTBI did not reduce porosity but in general the porosity was significantly lower than in WT controls. Our findings suggest that mTBI induces a strain-specific CB1-dependent bone anabolic response in the skull, probably mediated by anandamide, but seemingly unrelated to inflammation. The endocannabinoid system is therefore a plausible target in management of bone response following head trauma.

Small molecules targeting peripheral CB1 receptors have therapeutic potential in a variety of disorders including obesity-related, hormonal, and metabolic abnormalities, while avoiding the psychoactive effects in the central nervous system. We applied our in-house algorithm, iterative stochastic elimination, to produce a ligand-based model that distinguishes between CB1R antagonists and random molecules by physicochemical properties only. We screened ∼2 million commercially available molecules and found that about 500 of them are potential candidates to antagonize the CB1R. We applied a few criteria for peripheral activity and narrowed that set down to 30 molecules, out of which 15 could be purchased. Ten out of those 15 showed good affinity to the CB1R and two of them with nanomolar affinities (Ki of ∼400 nM). The eight molecules with top affinities were tested for activity: two compounds were pure antagonists, and five others were inverse agonists. These molecules are now being examined in vivo for their peripheral versus central distribution and subsequently will be tested for their effects on obesity in small animals.

Among a multitude of hormonal and metabolic complications, individuals with Prader-Willi syndrome (PWS) exhibit significant bone abnormalities, including decreased BMD, osteoporosis, and subsequent increased fracture risk. Here we show in mice that loss of Magel2, a maternally imprinted gene in the PWS critical region, results in reduced bone mass, density, and strength, corresponding to that observed in humans with PWS, as well as in individuals suffering from Schaaf-Yang syndrome (SYS), a genetic disorder caused by a disruption of the MAGEL2 gene. The low bone mass phenotype in Magel2-/- mice was attributed to reduced bone formation rate, increased osteoclastogenesis and osteoclast activity, and enhanced trans-differentiation of osteoblasts to adipocytes. The absence of Magel2 in humans and mice resulted in reduction in the fatty acid amide bone homeostasis regulator, N-oleoyl serine (OS), whose levels were positively linked with BMD in humans and mice as well as osteoblast activity. Attenuating the skeletal abnormalities in Magel2-/- mice was achieved with chronic administration of a novel synthetic derivative of OS. Taken together, Magel2 plays a key role in modulating bone remodeling and mass in PWS by affecting OS levels and activity. The use of potent synthetic analogs of OS should be further tested clinically as bone therapeutics for treating bone loss. © 2018 American Society for Bone and Mineral Research.

Obesity-related structural and functional changes in the kidney develop early in the course of obesity and occur independently of hypertension, diabetes, and dyslipidemia. Activating the renal cannabinoid-1 receptor (CB1R) induces nephropathy, whereas CB1R blockade improves kidney function. Whether these effects are mediatedviaa specific cell type within the kidney remains unknown. Here, we show that specific deletion of CB1R in the renal proximal tubule cells did not protect the mice from obesity, but markedly attenuated the obesity-induced lipid accumulation in the kidney and renal dysfunction, injury, inflammation, and fibrosis. These effects associated with increased activation of liver kinase B1 and the energy sensor AMP-activated protein kinase, as well as enhanced fatty acidβ-oxidation. Collectively, these findings indicate that renal proximal tubule cell CB1R contributes to the pathogenesis of obesity-induced renal lipotoxicity and nephropathy by regulating the liver kinase B1/AMP-activated protein kinase signaling pathway.

B lymphocyte induced maturation protein-1 (Blimp-1) is a transcription repressor of the Krueppel-like family. Blimp-1 plays important roles in developmental processes, such as of germ cells and hair follicle stem cells. In B lymphocytes Blimp-1 orchestrates the terminal differentiation into plasma cells. We discovered that Blimp-1 undergoes SUMOylation by SUMO-1. This SUMOylation is modulated by the SUMO protease SENP1. While Blimp-1 is relatively stable in 293T cells, a fusion with SUMO1 rendered it to rapid proteasomal degradation. Increase in SENP1 activity stabilized Blimp-1, while a decrease promoted its degradation. Our data indicate that SUMOylation of Blimp-1 regulates its intracellular stability. Structured summary of protein interactions Blimp1 physically interacts with SUMO1 by anti tag coimmunoprecipitation (View Interaction 1 , 2 ). SUMO1 physically interacts with Blimp1 by anti tag coimmunoprecipitation (View interaction) .

Matrix metalloproteinases (MMPs) are a family of over twenty zinc-dependent enzymes that hydrolyze connective tissue and are involved in a variety of diseases, which are associated with undesired tissue breakdown. Previously we described the synthesis of a series of achiral alkyl and cycloalkylcarbamoylphosphonic acids and their biological evaluation. Herein we report the effect of chirality and geometrical isomerism on the potency and selectivity of inhibition. The inhibitory potencies of pairs of enantiomeric and stereoisomeric alkyl and cycloalkylcarbamoylphosphonic acids were evaluated on recombinant MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 enzymes. The results show that the enantiomers and stereoisomers studied differ considerably in their inhibitory potencies and selectivities on the enzyme subtypes studied. Such a result is consistent with the assumption that the carbamoylphosphonates interact with a chiral environment such as an enzyme.

Matrix metalloproteinases (MMPs) are a family of over 20 zinc-dependent enzymes that hydrolyze connective tissue and are involved in a variety of diseases, which are associated with undesired tissue breakdown. This paper reports the synthesis, characterization, and biological evaluation of a novel class of MMP inhibitors based on the carbamoylphosphonic acid function. We report a series of 10 open chain N-alkylcarbamoylphosphonic acids (ranging from R = C(1) to C(6) groups), eight N-cycloalkylcarbamoylphosphonic acids (ranging from cyclopropyl to cyclooctyl rings), and four N,N-dialkylcarbamoylphosphonic acids. The compounds were evaluated in three in vitro models, which consisted of (a) the in vitro invasion across a reconstituted basement membrane, (b) determination of the IC(50) values on recombinant MMP-1, MMP-2 MMP-3, MMP-8, and MMP-9 enzymes, and (c) an in vitro capillary formation model, which is a model of angiogenesis. Several of the compounds were also tested in an in vivo murine melanoma model. The following general conclusions have been reached: Most compounds show selectivity for MMP-2 over the other MMP subtypes examined. Cycloalkylcarbamoylphosphonic acids are more potent than comparable open-chain alkyl compounds. Optimal activity against MMP-2 among the cycloalkyl derivatives was shown by N-cyclopentylcarbamoylphosphonic acid (3m). N,N-Dialkylcarbamoylphosphonic acids that were examined showed weak or no activity. The compounds examined showed toxic effects neither in vitro nor in vivo in the concentrations used. Carbamoylphosphonic acids are water soluble at physiological pH and are stable indefinitely.

The aim of the present study was to determine the effect of nickel-containing alloys on lymphocyte subsets in an experimental setting. Plates of alloys containing nickel (Ceramalloy, Talladium, Cerillium, Rexillium) or gold (Orion) were implanted subcutaneously into mice. The levels of CD4+ and CD8+ T-lymphocyte subpopulations and of Smig+ B lymphocytes were determined at various intervals following implantation, using monoclonal antibodies and flow cytometry. No changes were detected in the proportion of the lymphocyte subsets tested. One month after implantation, the mean fluorescence intensity of CD4, CD8 or Smig, in the peripheral blood lymphocytes (PBL) of the nickel alloy-implanted animals, was significantly higher than that prior to this procedure. Only a mild increase in CD4 and CD8 was noted after implantation of the gold alloy. The observed effects are most likely attributable to the surgical trauma, and do not indicate that nickel-containing dental alloys influence T cell subsets in this murine model.

Stimulation of peripheral blood lymphocytes (PBL) with phytohemagglutinin (PHA) strikingly increased the proportion of CD4+CD8+ cells. Highly purified CD4+ and CD8+ lymphocyte populations cultured in the presence of PHA consistently failed to coexpress the CD8 and CD4 markers. Similarly, exposure of highly purified CD4+ cells to PHA and recombinant interleukin-2 resulted in augmented expression of CD25 but failed to induce the expression of CD8. When purified preparations of either CD4+ or CD8+ cells were activated separately for 3 days and incubated together for an additional 5 h, a considerable proportion of CD4+CD8+ cells was found in the mixture. Cycloheximide treatment did not prevent the appearance of the CD8 marker on CD4 cells. CD4+CD8+ cells isolated from PBL exposed for 3 days to PHA lost their CD8 antigenicity within 24–48 h in the absence of PHA. Increased levels of soluble CD4 and CD8 antigens were found in supernatant fluids of PHA-stimulated cells. T cells failed, however, to bind soluble markers even after prolonged incubation in the presence of supernatant fluids. Our studies show that activation of CD4+ cells per se does not elicit the CD4+CD8+ phenotype and that soluble T cell markers do not bind to T cells. Rather, it seems that direct cell–cell contact is required for the transfer of CD8 molecules from CD8+ cells to the membrane of CD4+ cells.

The aim of the present study was to determine whether activation of human T-cells in vitro results in the expression of markers characteristic for T-cells on the surface of B-lymphocytes and to correlate antigenic changes with the release of soluble T-cell antigens. Peripheral blood lymphocytes (PBL) were exposed in vitro to phytohemagglutinin (PHA) for 3 days. Changes in the phenotype of the cells were determined by flow cytometry and the level of soluble T-cell antigens was assessed by ELISA. PHA-activated PBL released elevated quantities of soluble CD2, CD4, and CD8 compared with control cultures. Following PHA stimulation the proportion of CD4+CD8+ and HLA-DR+ cells increased. In addition, after 3 days of activation with PHA about 80% of the CD19+ cells (B-cells) reacted with F(ab)2 fragments of CD2 monoclonal antibodies (MoAbs). A high proportion of B-cells in activated cultures also reacted with F(ab)2 fragments of anti-CD8 MoAb and anti-CD4 MoAb. The removal of either CD4+ or CD8+ T-cells from the cultures prior to stimulation with PHA drastically reduced the proportion of B-cells expressing CD4 or CD8, respectively. The attachment of T-cell markers to the surface B-lymphocytes may constitute a new mechanism for B-cell regulation by T-cells.

Farage, a new cell line established from a lymph node biopsy of a patient with non-Hodgkin's lymphoma (NHL), constitutes a clonal expansion of cells at a distinct stage of B-cell differentiation. The cells lack both T and myeloid surface markers, express B cell surface antigens including CD19, CD20, CD22, HLA-DR, were positive for C3 receptors and EBNA and expressed BCL-2. No immunoglobulin determinants could be demonstrated on the cell surface. Intracellular IgM and kappa chains were detected, in an unusual but distinct localization and appeared to be localized to the nucleus or to the perinuclear area without any spread to the cytoplasm, as seen in the early B cells. Southern blot DNA analysis showed rearrangement of one of the IgJH alleles. The Farage cells were negative for B cell activation antigens including CD25, CD11b, HC2 and Bly-7. The cells were negative for two anti CD-10 (CALLA) reagents but weakly positive with one. Interestingly they were strongly positive for both nuclear and cytoplasmic T...

The spread of malignant tumor cells from a primary neoplasm to distant organs where they multiply and form new foci is the major cause of death from cancer. Despite the different modalities of cancer treatment, no effective curative therapy of metastatic lesions is available. To possess metastatic potential, a cell has to be able to invade the surrounding tissue, spread via lymphatics and/or the bloodstream, extravasate, and multiply at secondary sites. There is increasing evidence for a positive correlation between matrix metalloproteinase-2 (MMP-2) activity and tumor cell invasion. Agents blocking MMP-2 have been shown to prevent tumor cell invasion in vitro and in vivo. Inhibition of MMPs has, therefore, become the focus of considerable interest in connection with a variety of potential therapeutic applications. We have discovered a nontoxic MMP-2–selective inhibitor effective at nanomolar range on recombinant MMP. This compound, cyclopentylcarbamoylphosphonic acid, significantly inhibited cellular invasion and capillary formation in vitro. Further, i.p. or oral administration of the compound significantly reduced lung metastasis formation and s.c. tumor growth in a murine melanoma model. The effect of this novel compound on lung colonization, capillary formation, and s.c. tumor growth indicates that the compound might also be effective in treatment of primary tumor growth in reduction, or at least in prevention, of further tumor growth, thereby reducing the tumor burden of the patient by a nontoxic approach.

The CD4 molecule functions as a receptor for the binding and infectivity of the human immunodeficiency virus (HIV). It is of interest, therefore, to develop procedures for its down-regulation. In the present study, the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the expression of cell surface antigens of the HL-60 promyelocytic leukemia cell line was analyzed. Exposure of HL-60 cells to 1,25(OH)2D3 resulted in down-regulation of CD4 as assessed by their staining with the Leu-3a monoclonal antibody (MoAb). This treatment increased the staining of HL-60 cells with the monocyte-specific 63D3 MoAb. In contrast to the rapid elimination of cell surface CD4 by exposure of HL-60 to phorbol myristate acetate (PMA), the maximal reduction of CD4 by 1,25(OH)2D3 was attained within 48 h after the beginning of the exposure.

A new T-cell line (Amsalem) was established from the peripheral blood of a patient with pre-T leukemia. Amsalem cells are unique in that they possess antigenic determinants associated with the E-receptor, yet fail to form rosettes with sheep red blood cells (SRBC). Amsalem cells were found to possess morphological and cytochemical features characteristic of T-lymphocytes, and were sensitive to the cytotoxic effect of rabbit antisera specific for T-cell antigens. In immunofluorescent tests with monoclonal antibodies, Amsalem cells showed a strong reactivity with the OKT-11A and A-22 antibodies, specific for the E-receptor. The cells were reactive with OKT-4 and showed a very weak reactivity with OKT-6 and OKT-8. No reactivity was found with the OKT-3, Leu 7, Leu 11, and OKM1 antibodies. Amsalem cells failed to form rosettes with SRBC; however, mouse anti-Amsalem serum inhibited the formation of E-rosettes. It is concluded that the Amsalem cell line is a line of pre-T leukemia cells characterized by a dissociation between its inability to form E-rosettes and the presence of antigenic constituents of the E-receptor.