Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis

Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.     

Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4

Heparin-induced thrombocytopenia (HIT) is an important complication of heparin therapy. Although there is general agreement that platelet activation in vitro by the HIT IgG is mediated by the platelet Fc receptor, the interaction among the antibody, heparin, and platelet membrane components is uncertain and debated. In this report, we describe studies designed to address these interactions. We found, as others have noted, that a variety of other sulfated polysaccharides could substitute for heparin in the reaction. Using polysaccharides selected for both size and charge, we found that reactivity depended on two independent factors: a certain minimum degree of sulfation per saccharide unit and a certain minimum size. Hence, highly sulfated but small (< 1,000 daltons) polysaccharides were not reactive nor were large but poorly sulfated polysaccharides. The ability of HIT IgG to recognize heparin by itself was tested by Ouchterlony gel diffusion, ammonium sulfate and polyethylene glycol precipitation, and equilibrium dialysis. No technique demonstrated reactivity. However, when platelet releasate was added to heparin and HIT IgG, a 50-fold increase in binding of radio-labeled heparin to HIT IgG was observed. The releasate was then depleted of proteins capable of binding to heparin by immunoaffinity chromatography. Only platelet factor 4-immunodepleted releasate lost its reactivity with HIT IgG and heparin. Finally, to determine whether the reaction occurred on the surface of platelets or in the fluid phase, washed platelets were incubated with HIT IgG or heparin and after a wash step, heparin or HIT IgG was added, respectively. Reactivity was only noted when platelets were preincubated with heparin. Consistent with these observations was the demonstration of the presence of PF4 on platelets using flow cytometry. These studies indicate that heparin and other large, highly sulfated polysaccharides bind to PF4 to form a reactive antigen on the platelet surface. HIT IgG then binds to this complex with activation of platelets through the platelet Fc receptors.     

Specificity of autoantibodies in autoimmune thrombocytopenia

In 42 patients with autoimmune thrombocytopenia (AITP) and a positive direct platelet suspension immunofluorescence test (PSIFT), the antigenic specificity of the autoantibodies was studied. Because the autoantibodies were often not detectable in the serum and additional HLA antibodies may disturb the reaction pattern with the platelet panel, we used eluates prepared from the patients' platelets for this study. Thirty-five patients had antibodies equally reactive with normal platelets, irrespective of their antigenic make-up, but not with the platelets from two Glanzmann's disease patients. Absorption and elution experiments in two patients showed that his was probably not due to the presence of a combination of anti-Zwa and anti-Zwb antibodies. Thus, the majority of autoantibodies against platelets seems to be directed against antigenic determinants not present on Glanzmann's disease platelets, but perhaps located on the platelet-membrane glycoproteins IIb and/or IIIa. In ten patients, antibodies of no, or still unknown, specificity were detected. Three of these had additional antibodies not reactive with the platelets of the two Glanzmann patients.     

Silent cerebral infarction,income, and grade retention among students with sickle cell anemia

Children with sickle cell anemia have a higher‐than‐expected prevalence of poor educational attainment. We test two key hypotheses about educational attainment among students with sickle cell anemia, as measured by grade retention and use of special education services: (1) lower household per capita income is associated with lower educational attainment; (2) the presence of a silent cerebral infarct is associated with lower educational attainment. We conducted a multicenter, cross‐sectional study of cases from 22 U.S. sites included in the Silent Infarct Transfusion Trial. During screening, parents completed a questionnaire that included sociodemographic information and details of their child's academic status. Of 835 students, 670 were evaluable; 536 had data on all covariates and were used for analysis. The students' mean age was 9.4 years (range: 5–15) with 52.2% male; 17.5% of students were retained one grade level and 18.3% received special education services. A multiple variable logistic regression model identified that lower household per capita income (odds ratio [OR] of quartile 1 = 6.36, OR of quartile 2 = 4.7, OR of quartile 3 = 3.87; P = 0.001 for linear trend), age (OR = 1.3; P < 0.001), and male gender (OR, 2.2; P = 0.001) were associated with grade retention; silent cerebral infarct (P = 0.31) and painful episodes (P = 0.60) were not. Among students with sickle cell anemia, household per capita income is associated with grade retention, whereas the presence of a silent cerebral infarct is not. Future educational interventions will need to address both the medical and socioeconomic issues that affect students with sickle cell anemia. Am. J. Hematol. 89:E188–E192, 2014. © 2014 Wiley Periodicals, Inc.     

A human antibody binds to alpha-galactose receptors and mimics the effects of Clostridium difficile toxin A in rat colon

BACKGROUND & AIMS: Nearly all human sera contain an immunoglobulin G antibody (antigalactose) that binds the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc expressed on cells from most mammals but not humans. Because the Clostridium difficile toxin A receptor in rodents contains this trisaccharide, the aim of this study was to examine whether antigalactose could mimic the enterotoxic effects of toxin A and bind to receptors containing this trisaccharide. METHODS: Fluid secretion, [3H]-mannitol permeability, and release of rat mast cell protease II and prostaglandin E2 were measured after luminal exposure of rat colon to either purified human anti-galactose, control immunoglobulin G, toxin A, or buffer. RESULTS: Toxin A (5 micrograms) and antigalactose (250 micrograms) but not control immunoglobulin (250 micrograms) stimulated colonic fluid secretion and caused increased mannitol permeability and rat mast cell protease II release. Antigalactose and toxin A and, to a lesser degree, control immunoglobulin G also stimulated release of prostaglandin E2, but only toxin A produced acute inflammation of rat colonic mucosa. Antigalactose and toxin A bound specifically to a single class of colonic brush border receptors with dissociation constants of 10(-6) mol/L and 5.4 x 10(-8) mol/L, respectively. CONCLUSIONS: Fluid secretion, increased permeability, and mast cell activation occur in rat colon when toxin A or human antigalactose immunoglobulin G bind to receptors bearing the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc. (Gastroenterology 1996 Jun;110(6):1704-12)     

Pharmacogenetics of morphine: Potential implications in sickle cell disease

Morphine is frequently used to treat painful episodes associated with sickle cell disease (SCD) but may fail to provide adequate analgesia in many patients. This concise review focuses on unique disease related changes in physiologic variables associated with SCD that impacts pharmacokinetics and pharmacodynamics of morphine and may contribute to the variability in analgesia. Emerging evidence suggests that the allelic variants in the genes involving the opioid (UGT2B7, OPRM1, and ABCB1 genes) and nonopioid system (COMT gene) can alter the efficacy of morphine.     

Bone-marrow microinvolvement in non-small cell lung cancer is not a reliable indicator of tumour recurrence and prognosis.

AIMS: This study aimed to examine the incidence of bone-marrow microinvolvement in non-small cell lung cancer (NSCLC) patients and its correlation with tumour recurrence and prognosis. METHODS: Between March 1997 and August 1998, we analysed 96 bone-marrow specimens (from the posterior iliac crest) of NSCLC patients before surgery. Tumour differentiation showed well differentiated carcinoma in six, moderately differentiated carcinoma in 69, and poorly differentiated carcinoma in 21. p-TNM staging showed stage Ia in five, stage Ib in 33, stage IIb in 19, stage IIIa in 26, stage IIIb in eight, and stage IV in five. The specimens were examined by immunohistochemical staining with anti-human cytokeratin AE1/AE3 and clone MNF116 mixed solution (Ab1, n=96) and/or Ber-EP4 (Ab2, n=80) to detect the presence of malignant epithelial cells in the bone marrow. RESULTS: Positive results were observed in 21 patients (21. 9%). The occurrence of bone-marrow microinvolvement was not related to patient age, sex, cell type, or TNM status. The 30-month disease-free survival rates were 50.2% and 53.9% in bone-marrow negative and bone-marrow positive patients, respectively (P=0.5670); the 30-month cumulative survival rates were 66.7% and 67.6% in bone-marrow negative and bone-marrow positive patients, respectively (P=0.9351). Multivariate analysis failed to demonstrate bone-marrow microinvolvement as an independent prognostic factor. CONCLUSIONS: Our results show that bone-marrow microinvolvement is not unusual, and its occurrence cannot be translated into early tumour recurrence or poor outcome during an intermediate-term follow-up, which means bone-marrow microinvolvement may be an epiphenomenon rather than true metastasis in NSCLC.     

N-(1-乙氧羰基-3-苯氨甲酰基丙基)甘氨酰甘氨酸衍生物的合成

报道4个N-(1-[1-乙氧羰基-3-(对甲)苯氨甲酰基]丙基甘氨酰｝-N-取代甘氨酸(XI_1～4)和5个1-[1-乙(或甲)氧羰基-3-(对甲)苯氨甲酰基]丙基-4-取代-1,4-哌嗪-2,5-二酮(XII_1～5)共9个估计有血管紧张素转化酶抑制活性化合物的合成和鉴定。所有这些化合物及9个相应的酯(X_1～9)均未见文献报道。药理初试结果,化合物XII₂,XII₅,XI₄和XII₁均有较强降压活性。