Immunolocalization of beta 1 integrins in platelets and platelet- derived microvesicles

Human platelets contain several adhesion receptors belonging to the integrin superfamily. At least three beta 1 integrins are present on platelets and have been shown to mediate platelet adhesion to collagen, fibronectin, and laminin. To study the cellular localization of the beta 1 integrins in platelets, we produced a polyclonal antibody by immunization of goat 172 with purified beta 1 subunit from HPB-ALL cells. Antibody 172 (Ab172) specifically immunoblotted a 135-Kd protein in a lysate of whole platelets. The reactivity of Ab172 with platelet membrane proteins was further determined by immunoprecipitation of lysates of surface-radioiodinated platelets. Ab172 immunoprecipitates, resolved by nonreducing/reducing two-dimensional sodium dodecyl sulfate- polyacrylamide gel electrophoresis consisted of three labeled proteins with migrational properties of platelet glycoprotein (GP)Ia, GPIc and GPIIa. Neither GPIIb/IIIa nor the vitronectin receptor were immunoprecipitated by Ab172, confirming a lack of cross-reactivity with the beta 3 integrins in platelets. Immunofluorescence studies using Ab172 were performed to investigate the cellular distribution of beta 1 integrins in platelets. Fluorescent labeling of intact cells demonstrated the presence of beta 1 antigen on the surface of resting cells. Permeabilization of platelets with Triton X-100 showed the presence of an intracellular pool of beta 1 antigen. Double-label experiments using Ab172 and AP-2 (anti-GPIIb/IIIa) showed identical labeling patterns, suggesting a similar subcellular distribution for these integrins. Following thrombin stimulation, permeabilized cells showed a centralized clearing of both beta 1 antigen and GPIIb/IIIa as well as an intensification of surface labeling for beta 1 antigen. These findings suggest the translocation of intracellular beta 1 antigen to the platelet surface as a result of thrombin stimulation. Because platelet-derived microvesicles have been reported to contain GPIIb/IIIa, we investigated the possible distribution of beta 1 integrins in these structures. Microvesicles, produced as a result of platelet activation, were labeled with Ab172, suggesting the distribution of beta 1 integrins in these structures as well as in intact cells.     

Cystic optic nerve sheath meningioma.

A 58-year-old woman with a 35-year history of left proptosis underwent neuroimaging that revealed a large cystic lesion. Surgery revealed an optic nerve sheath meningioma associated with cyst formation. The cyst was part of the tumor, a phenomenon that is well described in intracranial meningiomas but not in optic nerve sheath meningiomas.     

Immunohistochemical study of apoptosis markers and involvement of chemokine CXCR4 in skin Merkel cell carcinoma

BACKGROUND: Merkel cell carcinoma (MCC) is a rare, aggressive cancer of the skin that mainly affects elderly patients. Because of its rarity, there is no established treatment or proven markers to guide therapy or prognosis. Immunohistochemical expression of apoptosis proteins is considered a useful marker of both malignancy and tumour progression. Apoptosis plays a fundamental role in skin homeostasis, and apoptotic cells have been detected in normal and diseased skin. Chemokines possess a wide range of biological activities and CXCR4 is expressed in some cancer cells, where it plays an efficient role in metastasis formation. OBJECTIVE: To identify immunohistochemical parameters that can help clinicians select the most suitable therapy for skin MCC. DESIGN: Antibodies against ki67, bcl-2, p53, survivin, p16 and CXCR4 were tested to assess the usefulness of these antigens as indices of proliferation potential and predictors of prognosis. METHODS: Immunohistochemical detection of apoptosis inhibitors and CXCR4 was performed on tissue from 12 patients with primary MCC. After excision of the primary lesion, five survived and had no metastases, and seven experienced local recurrence or lymph node metastases. RESULTS: Expression of ki67 and survivin was increased in patients with local recurrence or metastasis (retrospectively classified as 'poor prognosis') compared with those with a 'good prognosis', and bcl-2 expression was significantly greater (P=0.003). P53 and p16 immunostaining was moderate in both groups. A positive correlation was observed between survivin and mutant p53 in the poor prognosis group (r=0.593, P=0.033; regression coefficient). High values of p53 were measured in patients with high levels of survivin and vice versa. CXCR4 was not detected at all. CONCLUSIONS: Our results show strong MCC cell apoptosis inhibition and a high cell proliferation capacity. The positive correlation between survivin and p53 may be a predictor of MCC spread via the lymphatic network. Absent CXCR4 expression may reflect a less aggressive form, with less efficient development of distant and non-organ-selective metastasis formation.     

Microwave dissociation of antigen-antibody complexes: a new elution technique to permit phenotyping of antibody-coated red cells

To evaluate the effectiveness of microwave irradiation in dissociating IgG from red cells (RBCs), the use of chloroquine diphosphate (CDP) was compared to that of microwaves. Fifteen paired samples of RBCs from 15 patients with positive direct antiglobulin tests (DATs) were treated with both CDP and microwave radiation. Total microwave exposure times ranged from 20 to 100 seconds. Posttreatment DATs were performed, and the reaction grades of the posttreatment DATs were compared. RBC phenotyping was also performed on repeatedly microwaved RBCs to demonstrate possible effects on RBC antigen expression. Microwaves successfully reduced the reaction grade of the DAT in 14 of 15 samples; CDP reduced the reaction grade in 12 of 15 samples. In samples with a DAT of 2+ or greater (n = 13), the microwave method yielded a greater reduction in DAT strength in six cases (results in the other 7 cases were identical with both methods) (p = 0.01). Five of eight cases with a DAT of 3+ showed a greater reduction in the DAT with microwave treatment than with CDP treatment; results in the remaining three cases were identical (p = 0.03). RBC antigenicity remained unchanged after exposure to microwave radiation (A, B, C, c, D, E, e, Fya, Fyb, Jka, Jkb, K, k, S, and s). Microwave treatment required less than 10 minutes per sample, while CDP treatment required 30 to 120 minutes per sample (mean, 88 min). The microwave technique of antigen-antibody dissociation from RBCs provides a rapid and accurate method of facilitating the phenotyping of RBCs coated with warm autoantibodies and is superior to other methods, which destroy RBC antigens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fecal incontinence among morbid obese women seeking for weight loss surgery: an underappreciated association with adverse impact on quality of life

Purpose Morbid obesity is associated with urinary incontinence (UI). The study purpose was to determine the prevalence of fecal incontinence (FI), its associated risk factors, and its impact on quality of life (QOL) in morbidly obese women. Materials and methods A questionnaire-based study on morbidly obese women [body mass index (BMI) ≥ 35 m/kg²], attending a bariatric surgery seminar, was conducted. Data included demographics, past medical, surgical and obstetric history, and obesity-related co-morbidities. Patients who reported of FI, completed the Cleveland Clinic Foundation Fecal Incontinence scale (CCF-FI) and the Fecal Incontinence Quality of Life scale (FIQL). Results Participants included 256 women [median age 45 years (19–70)] and mean BMI of 49.3 ± 9.4 m/kg². FI was reported in 63%. History of obstetric injury (OR: 2.4, 95% CI: 1.33–4.3; p < 0.001) and UI (OR: 1.2, 95% CI: 1.1–1.4; p < 0.001) were significantly associated with FI. There was no association with age, BMI, parity, and presence of diabetes or hypertension. Median CCF-FI score was 7 (1–20); 34.5% scored ≥10. Incontinence for gas was the most frequent type (87%) of FI, followed by incontinence for liquids (80%), which also had the highest impact on QOL (p < 0.01). Mean FIQL scores were >3 for all four domains studied. CCF-FI scores were significantly correlated with FIQL scores in all domains (p = 0.02). Comment The prevalence of FI among morbidly obese women may be much higher than the rates reported in the general population. FI has adverse effects on QOL. Its correlation with UI suggests that morbid obesity may pose a risk of global pelvic floor dysfunction.     

Increased Ca++, Mg++, and Na+ + K+ ATPase activities in erythrocytes of sickle cell anemia

To determine whether diminished activity of the Ca++ extrusion pump could account for the high levels of red blood cell (RBC) Ca++ in sickle cell anemia (SS), we measured calmodulin-sensitive Ca++ ATPase activity in normal and SS RBC. Hemolysates prepared with saponin were compared, since such preparations expressed maximum ATPase activities, exceeding isolated membranes or reconstituted systems of membranes plus cytosol, SS RBC hemolysates had greater Ca++ ATPase activity than normal hemolysates; they exhibited higher Mg++ and Na+ + K+ ATPase activities as well. Assays on density (age) fractions of SS and normal red cells demonstrated that all ATPase activities were highest in low density (young) cells, and activities in SS red cells exceeded those in normals in all fractions studied. Thus, when studied under conditions that maximize enzyme activity, Ca++ ATPase activity, like Mg++ and Na+ + K+ ATPase, is actually increased in SS RBC, probably due to the young red cell population present. The elevated Ca++ levels in these cells are more likely due to an increased Ca++ leak or abnormal calcium binding than to defective extrusion by the ATPase pump.     

Modulation of murine pluripotential stem cell proliferation in vivo by lithium carbonate

The effect of administering lithium in vivo on murine pluripotential stem cell proliferation was studied. Lithium was injected i.p. at various meq/liter concentrations (0.5-5.0 meq/liter). Data was obtained establishing that lithium increased the pluripotential stem cell population from normal mice as measured by the Till and McCulloch CFUs assay. Significant increases were demonstrated in: (1) bone marrow CFUs; (2) bone marrow organ cellularity, and (3) peripheral blood WBC. Marrow CFUs increase was maximum at 4 days post-lithium injection and greatest in the 1 meq/liter group (p < 0.001). Further increases in lithium concentration, i.e., 5 meq/liter, effectively reduced CFUs levels below normal, suggesting toxicity of the drug was apparent at this dose level. We conclude lithium may modulate granulopoiesis by increasing the CFUs stem cell compartment, thereby directly channeling differentiation into the granulopoietic pathway. This increases the committed progenitor stem cell (CFUc) population and ultimately peripheral blood end-stage cells.