PADGEM (GMP140) is a component of Weibel-Palade bodies of human endothelial cells

PADGEM protein (PADGEM), also known as GMP140, is a platelet alpha- granule membrane protein that is translocated to the external membrane after platelet activation. Although the biosynthesis of this protein was originally thought to be confined to megakaryocytes, the synthesis of PADGEM in endothelial cells was recently demonstrated (McEver et al: Blood 70:1974a, 1987). We now describe the subcellular localization of this protein in endothelial cells. Immunofluorescence staining of permeabilized human umbilical vein endothelial cells with KC4, a well characterized monoclonal antibody to PADGEM, showed positively stained elongated structures similar in distribution and shape to Weibel-Palade bodies. Their identity as Weibel-Palade bodies was confirmed by double label immunofluorescence using KC4 and a polyclonal antiserum to von Willebrand factor (vWf), a protein known to be specifically stored in these organelles. All Weibel-Palade bodies were found to contain PADGEM. In contrast to strong perinuclear staining produced with anti- vWf antibodies, no significant perinuclear staining was obtained with KC4, indicating that relatively little PADGEM is present in the endoplasmic reticulum and in the Golgi apparatus. In endothelial cells treated with secretagogues that stimulate vWf release the elongated structures positive for PADGEM disappeared, further identifying these structures as Weibel-Palade bodies. This observation extends the parallels between Weibel-Palade bodies and alpha-granules and suggests a possible functional association between vWf and PADGEM.     

缺血性卒中或短暂性脑缺血发作患者的卒中预防指南

这份新声明旨在为缺血性卒中或短暂性脑缺血发作存活者的缺血性卒中预防提供全面和及时的循证推荐，循证推荐包括对危险因素的控制，动脉粥样硬化性疾病的干预措施，心源性栓塞的抗栓治疗以及非心源性卒中抗血小板药的应用。另外，还为其他多种特殊情况下复发性卒中的预防提供了推荐、包括动脉夹层分离、卵圆孔未闭、高同型半胱氨酸血症、高凝状态、镰状细胞病、脑静脉窦血栓形成、女性卒中（特别是与妊娠和绝经后激素替代治疗相关卒中），脑出血后肮凝药的应用，以及该指南在高危人群中执行和应用的特殊措施。     

Borrelia burgdorferi and Interleukin-1 Promote the Transendothelial Migration of Monocytes In Vitro by Different Mechanisms

A prominent feature of Lyme disease is the perivascular accumulation of mononuclear leukocytes. Incubation of human umbilical vein endothelial cells (HUVEC) cultured on amniotic tissue with either interleukin-1 (IL-1) or Borrelia burgdorferi, the spirochetal agent of Lyme disease, increased the rate at which human monocytes migrated across the endothelial monolayers. Very late antigen 4 (VLA-4) and CD11/CD18 integrins mediated migration of monocytes across HUVEC exposed to either B. burgdorferi or IL-1 in similar manners. Neutralizing antibodies to the chemokine monocyte chemoattractant protein 1 (MCP-1) inhibited the migration of monocytes across unstimulated, IL-1-treated, or B. burgdorferi-stimulated HUVEC by 91% ± 3%, 65% ± 2%, or 25% ± 22%, respectively. Stimulation of HUVEC with B. burgdorferi also promoted a 6-fold ± 2-fold increase in the migration of human CD4⁺ T lymphocytes. Although MCP-1 played only a limited role in the migration of monocytes across B. burgdorferi-treated HUVEC, migration of CD4⁺ T lymphocytes across HUVEC exposed to spirochetes was highly dependent on this chemokine. The anti-inflammatory cytokine IL-10 reduced both migration of monocytes and endothelial production of MCP-1 in response to B. burgdorferi by approximately 50%, yet IL-10 inhibited neither migration nor secretion of MCP-1 when HUVEC were stimulated with IL-1. Our results suggest that activation of endothelium by B. burgdorferi may contribute to formation of the chronic inflammatory infiltrates associated with Lyme disease. The transendothelial migration of monocytes that is induced by B. burgdorferi is significantly less dependent on MCP-1 than is migration induced by IL-1. Selective inhibition by IL-10 further indicates that B. burgdorferi and IL-1 employ distinct mechanisms to activate endothelial cells.     

A soluble form of Fc gamma RIII is present in human serum and other body fluids and is elevated at sites of inflammation.

We have developed a highly sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA) to measure the concentration of Fc gamma RIII in serum and other body fluids. This ELISA is based on the use of monoclonal antibody (MoAb) (3G8) to Fc gamma RIII and a rabbit antiserum against Fc gamma RIII. The lower limit of detection of this ELISA was 1.5 nmol/L. The concentration of soluble Fc gamma RIII in normal serum ranged from 7.3 to 75.9 nmol/L. Soluble Fc gamma RIII was also present in other normal biologic fluids such as saliva, urine, and seminal fluid, but at much lower concentrations than that found in serum. Rabbit anti-Fc gamma RIII immunoblotted polypeptides immunoprecipitated with MoAb 3G8. Fc gamma RIII immunoprecipitated from a neutrophil lysate migrated from 40 to 76 Kd, whereas Fc gamma RIII immunoprecipitated from serum from the same donor migrated from 40 to 66 Kd. The soluble form of Fc gamma RIII apparently was bound to serum IgG, because immunoprecipitation of soluble Fc gamma RIII by MoAb 3G8 coprecipitated polypeptides that were identified by goat antihuman IgG. Incubation of neutrophils in vitro at 4 degrees C and 37 degrees C showed that Fc gamma RIII was released after 30 minutes of incubation at 37 degrees C. To determine whether there was a correlation between the concentration of soluble Fc gamma RIII in biologic fluids and inflammatory diseases, we measured the concentration of Fc gamma RIII in the bronchoalveolar lavage fluid from patients with adult respiratory distress syndrome (ARDS) and in the synovial fluid from patients with various forms of arthritis. In ARDS, we found concentrations of soluble Fc gamma RIII that were five to seven times higher than that found in the bronchoalveolar lavage fluids from healthy adults. The concentration of soluble Fc gamma RIII in the synovial fluid from patients with rheumatoid arthritis ranged from 10 nmol/L to 28 mumol/L. These results suggest that activated neutrophils, such as those at sites of inflammation, may release Fc gamma RIII.     

Surreptitious ingestion of a long-acting vitamin K antagonist/rodenticide, brodifacoum: clinical and metabolic studies of three cases

The vitamin K metabolism of three patients with factitious purpura due to brodifacoum ingestion was studied. These patients, who presented with bleeding disorders due to deficiency of the vitamin K-dependent blood clotting proteins, were refractory to vitamin K1 at standard doses and required fresh frozen plasma to control bleeding until large doses of vitamin K1 were used. Metabolic studies demonstrated a blockade in vitamin K utilization, consistent with the presence of a vitamin K antagonist, but the patients denied use of anticoagulants. Warfarin assays were negative. We show that the factitious purpura in each patient was due to the surreptitious ingestion of brodifacoum, a potent second generation long-acting vitamin K antagonist used as a rodenticide. The coagulopathies responded to long-term therapy with large doses of vitamin K1. The serum elimination half-time for brodifacoum ranged from 16 to 36 days in these patients. The anticoagulant effect is of long duration, requiring chronic vitamin K treatment. With increasing availability of new rodenticides, factitious purpura due to surreptitious ingestion of these potent vitamin K antagonists is emerging as a new problem, previously associated with warfarin, with important implications for diagnosis and treatment.     

Comparison of the native prothrombin antigen and the prothrombin time for monitoring oral anticoagulant therapy

We have measured the fully carboxylated (native) prothrombin antigen and the undercarboxylated (abnormal) prothrombin antigen in patients treated with sodium warfarin using specific immunoassays to evaluate a new approach for monitoring oral anticoagulant therapy. Plasma and serum samples (391) were assayed for the prothrombin time, native prothrombin antigen, and abnormal prothrombin antigen. The results were correlated with the presence of bleeding or thromboembolic complications at the time of phlebotomy. The native prothrombin antigen correlated with the occurrence of complications in 95% of samples. Of 13 samples from patients with bleeding complications, 13/13 (100%) had a native prothrombin of 12 micrograms/mL or lower. Of seven samples from patients with thromboembolic complications, 6/7 (86%) had a native prothrombin of 24 micrograms/mL or greater. By comparison, a prothrombin time index of 1.5 to 2.5, 1.5 to 2.2, 1.5 to 2.0, or 1.3 to 1.8 identified 6/20 (30%), 9/20 (45%), 11/20 (55%), or 12/20 (60%) patients at risk, respectively. Although the prothrombin time index did correlate with the presence of bleeding complications, the native prothrombin antigen correlated closely with the presence of bleeding and thromboembolic complications. According to these results, the native prothrombin antigen, maintained in a range of 12 to 24 micrograms/mL by regular adjustment of the warfarin dosage, may be associated with a reduced risk of complications due to excessive or insufficient warfarin therapy. On the basis of these preliminary data, we recommend that the native prothrombin antigen be considered to monitor warfarin therapy.     

Improvement in patient‐reported outcomes in a rituximab trial in patients with severe rheumatoid arthritis refractory to anti–tumor necrosis factor therapy

Objective

To assess the effects of treatment with rituximab plus methotrexate on patient‐reported outcomes in patients with active rheumatoid arthritis (RA) who experienced inadequate response to anti–tumor necrosis factor therapy.

Methods

Patients with active RA were randomly assigned to rituximab (1,000 mg on days 1 and 15) or placebo. The primary end point was the proportion of patients with an American College of Rheumatology 20% response at week 24. Additional goals were to assess treatment effects on pain, fatigue, functional disability, health‐related quality of life, and disease activity by comparing mean changes between groups. The analysis was conducted in the intent‐to‐treat population. The proportion of patients who achieved the minimum clinically important difference on the Health Assessment Questionnaire (HAQ) disability index (DI), Functional Assessment of Chronic Illness Therapy–Fatigue (FACIT‐F), and Short Form 36 (SF‐36) was determined.

Results

Rituximab patients had statistically significantly greater pain relief. The FACIT‐F showed significantly greater improvement in rituximab patients than placebo patients from weeks 12 through 24. Mean improvement from baseline in functional disability (measured by the HAQ DI) was significantly greater in rituximab patients from weeks 8 to 24. The mean ± SD change from baseline for the SF‐36 Physical Component Score was 6.64 ± 8.74 for rituximab patients and 1.48 ± 7.32 for placebo patients (P < 0.0001). The mean change from baseline for the SF‐36 Mental Component Score was 5.32 ± 12.41 for rituximab patients and 2.25 ± 12.23 for placebo patients (P = 0.0269).

Conclusion

Rituximab produced rapid, clinically meaningful, and statistically significant improvements in patient‐reported pain, fatigue, functional disability, health‐related quality of life, and disease activity. These effects were sustained throughout the study.