CTLA4-Ig Directly Inhibits Osteoclastogenesis by Interfering With Intracellular Calcium Oscillations in Bone Marrow Macrophages

CTLA4-Ig (cytotoxic T-lymphocyte antigen 4-immunoglobulin; Abatacept) is a biologic drug for rheumatoid arthritis. CTLA4 binds to the CD80/86 complex of antigen-presenting cells and blocks the activation of T cells. Although previous reports showed that CTLA4-Ig directly inhibited osteoclast differentiation, the whole inhibitory mechanism of CTLA4-Ig for osteoclast differentiation is unclear. Bone marrow macrophages (BMMs) from WT mice were cultured with M-CSF and RANKL with or without the recombinant mouse chimera CTLA4-Ig. Intracellular calcium oscillations of BMMs with RANKL were detected by staining with calcium indicator fura-2 immediately after administration of CTLA4-Ig or after one day of treatment. Calcium oscillations were analyzed using Fc receptor gamma- (FcRγ-) deficient BMMs. CTLA4-Ig inhibited osteoclast differentiation and reduced the expression of the nuclear factor of activated T cells NFATc1 in BMMs in vitro. Calcium oscillations in BMMs were suppressed by CTLA4-Ig both immediately after administration and after one day of treatment. CTLA4-Ig did not affect osteoclastogenesis and did not cause remarkable changes in calcium oscillations in FcRγ-deficient BMMs. Finally, to analyze the effect of CTLA4-Ig in vivo, we used an LPS-induced osteolysis model. CTLA4-Ig suppressed LPS-induced bone resorption in WT mice, not in FcRγ-deficient mice. In conclusion, CTLA4-Ig inhibits intracellular calcium oscillations depending on FcRγ and downregulates NFATc1 expression in BMMs. © 2019 American Society for Bone and Mineral Research.     

Neglect‐induced pseudo‐thrombotic thrombocytopenic purpura due to vitamin B12 deficiency

Although thrombotic thrombocytopenic purpura (TTP) is rare, early diagnosis and treatment are important for decreasing the mortality rate. Acquired vitamin B12 deficiency is frequently overlooked because of its rarity in developed countries, particularly in children and adolescents. The hematological changes in vitamin B12 deficiency present as megaloblastic anemia, increased lactate dehydrogenase, vasoconstriction, increased platelet aggregation, and abnormal activation of the coagulation followed by microangiopathy as well as neutropenia and thrombocytopenia. We report herein the case of a 15‐year‐old girl who had been neglected, which might have caused pseudo‐TTP through malnutrition, particularly vitamin B12 deficiency. When we encounter cases of TTP in children, clinicians must be aware of the possibility of malnutrition, particularly with vitamin B12 deficiency, even in developed countries, and investigate the cause of malnutrition including neglect.     

Action of Isoflurane on the Substantia Gelatinosa Neurons of the Adult Rat Spinal Cord

Background: Although isoflurane, a volatile anesthetic, can block the motor response to noxious stimulation (immobility and analgesia) and suppress autonomic responsiveness, how it exerts these effects at the neuronal level in the spinal cord is not fully understood.

Methods: The effects of a clinically relevant concentration (1 rat minimum alveolar concentration [MAC]) of isoflurane on electrically evoked and spontaneous excitatory/inhibitory transmission and on the response to exogenous administration of the [gamma]-aminobutyric acid type A receptor agonist muscimol were examined in lamina II neurons of adult rat spinal cord slices using the whole cell patch clamp technique. The effect of isoflurane on the action potential-generating membrane property was also examined.

Results: Bath-applied isoflurane (1.5%, 1 rat MAC) diminished dorsal root-evoked polysynaptic but not monosynaptic excitatory postsynaptic currents. Glutamatergic miniature excitatory postsynaptic currents were also unaffected by isoflurane. In contrast, isoflurane prolonged the decay phase of evoked and miniature [gamma]-aminobutyric acid type A receptor-mediated inhibitory postsynaptic currents and increased the amplitude of the muscimol-induced current. Isoflurane had little effect on action potential discharge activity.     

Amiloride and urinary trypsin inhibitor inhibit urothelial cancer invasion

OBJECTIVE: To determine the contribution of urokinase-type plasminogen activator (uPA) and plasmin in the invasion of highly invasive urothelial cancer cells. METHODS: We compared expression levels of mRNA and protease activity of uPA and plasmin formation in primary cultures of the noninvasive transitional cell carcinoma, UCT-1, and in the highly invasive type, UCT-2. By using in vitro cell invasion assay system, we evaluated the effects of amiloride and urinary trypsin inhibitor (UTI), which inhibit uPA and plasmin, respectively, on invasion by both cell lines. RESULTS: Expression levels of mRNA, protein, and activities of uPA were significantly higher (p<0.005) and resulted in more plasminogen activation in UCT-2 than in UCT-1. Amiloride and UTI significantly inhibited plasmin formation and the invasion of both cell lines (p<0.001). CONCLUSIONS: High expression levels of mRNA, activities of uPA and high plasmin formation significantly potentiated the invasiveness of urothelial cancer cells. Thus, inhibitors of uPA and plasmin, such as amiloride and UTI, respectively, could be useful therapeutic tools with which to treat urothelial cancer.     

The alloantigen-specific immunosuppressive activity of estradiol-chlorambucil conjugate (KM2210) and its beneficial effect on allogeneic bone marrow transplantation in mice.

KM2210, a conjugate of estradiol and chlorambucil (CBL), which was originally developed as an anti-breast cancer agent, inhibits proliferative response of human mononuclear cells to alloantigens in mixed lymphocyte culture in a dose-dependent manner, but has no effect on their response to phytohemagglutinin. Neither estradiol benzoate nor CBL alone showed these unique actions. The suppressive effect of KM2210 on MLC was abrogated by adding of anti-transforming growth factor-beta (TGF-beta) antibody to the culture, but was not affected by the addition of interleukin-2, suggesting that KM2210, unlike CBL, displays its actions via TGF-beta. In experimental allogeneic bone marrow transplantation using mice, daily oral administration of KM2210 (2 mg/kg/day) for 30 days posttransplant significantly inhibited the alloantigen-specific immune reactions. Furthermore, the survival rate of the KM2210-treated mice was significantly higher than that of the cyclosporine-treated (2 mg/kg/day, p.o.) mice, and no adverse effect of KM2210 on hematopoietic recovery was found. These results strongly suggest possible clinical benefits of KM2210 as a new immunosuppressive agent for the prevention and treatment of graft-versus-host disease and other allospecific immune reactions.     

Association of adiponectin mutation with type 2 diabetes: a candidate gene for the insulin resistance syndrome

Adiponectin, also referred to as AdipoQ or ACRP30, is a plasma protein produced and secreted exclusively from adipose tissue. The protein contains a collagen-like domain and a C1q-like globular domain. A protease-generated globular segment enhances fatty acid oxidation in muscles, thereby modulating lipid and glucose metabolism. Plasma adiponectin levels are inversely correlated with the severity of insulin resistance. A recent genome-wide scan study mapped a susceptibility locus for type 2 diabetes and the metabolic syndrome to chromosome 3q27, where the adiponectin gene is located. Here, we screened Japanese patients with type 2 diabetes and age- and BMI-matched nondiabetic control subjects for mutations in adiponectin gene. We identified four missense mutations (R112C, I164T, R221S, and H241P) in the globular domain. Among these mutations, the frequency of I164T mutation was significantly higher in type 2 diabetic patients than in age- and BMI- matched control subjects (P < 0.01). Furthermore, plasma adiponectin concentrations of subjects carrying I164T mutation were lower than those of subjects without the mutation. All the subjects carrying I164T mutation showed some feature of metabolic syndrome, including hypertension, hyperlipidemia, diabetes, and atherosclerosis. Our findings suggest that I164T mutation is associated with low plasma adiponectin concentration and type 2 diabetes.     

T-786C polymorphism in endothelial NO synthase gene affects cerebral circulation in smokers: possible gene-environmental interaction

Effects of smoking on white matter lesions, such as lacunar infarction and leukoaraiosis, are still controversial. We hypothesized that the endothelial NO synthase (eNOS) genotype was a modulating factor for the effect of smoking on cerebral circulation. We took a cross-sectional population from the participants of a health examination to study the effects of smoking and a single-nucleotide polymorphism in the eNOS gene, T-786C. Smokers and nonsmokers were defined as having a smoking index (cigarettes per day times years) of >/=200 and 0, respectively. One hundred sixty-six male nonsmokers and 344 male smokers were recruited. Cerebral blood flow was measured by the (133)Xe inhalation method. Genotyping of T-786C was performed by using a newly developed allele-specific polymerase chain reaction. Smokers were exposed to greater oxidative stress, as estimated by urinary F(2)-isoprostane excretion. In smokers, CC homozygotes of T-786C showed a significant decrease of cerebral blood flow (56.6+/-13.3, 57.6+/-11.5, and 44.0+/-7.2 mL/min per 100 g tissue for TT, TC, and CC, respectively; P=0.03 by ANOVA) and a significant increase of cerebrovascular resistance, whereas the eNOS genotype did not affect these parameters in nonsmokers. This result indicated that the eNOS genotype could modify cerebrovascular circulation in a general population by potentiating the adverse effect of smoking.