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.     

Panniculitis–an unusual cutaneous manifestation of systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune systemic disease characterized by small vessel involvement that leads to tissue ischemia and fibroblast stimulation resulting in accumulation of collagen (fibrosis) in the skin and internal organs. Lipomembranous panniculitis is a peculiar type of fat necrosis and has been reported with clinical conditions, commonly with peripheral vascular diseases. We describe a case of a 43‐year‐old woman with SSc manifestations, who presented with black scaly skin plaques, associated with thickening of the subcutaneous fat tissue, on the lateral surface of her thighs, her calves, gluteal area and lower abdomen. Biopsy revealed lipomembranous panniculitis. Lipomembranous changes have been seen in connective tissue disorders such as lupus profundus, morphea, systemic sclerosis and panniculitis associated with dermatomyositis, but rarely in thighs, calves, gluteal area and lower abdomen. Almeida MSTM, Lima SCB, Carvalho LL, Almeida JVM, Santos LG, Rolim JRA, Rocha TE. Panniculitis–An unusual cutaneous manifestation of systemic sclerosis.     

Single DNA sequence common to all chlamydial species employed for PCR detection of these organisms.

Chlamydial infection is responsible for a wide spectrum of diseases of the eye, genitourinary tract, and lung. This group of organisms is also implicated in the pathogenesis of coronary artery disease as well as arthritis. Since cross-species infection is widely reported (though probably underestimated), it is an advantage to have a rapid and reliable method to detect all forms of chlamydiae in patient samples. We have identified a 160/163-bp DNA fragment in Chlamydia which is highly conserved in all chlamydial species. A polymerase chain reaction method based on this sequence has been developed to detect, in clinical samples, chlamydiae which have been shown to be positive by fluorescent-staining immunoassay; this method can be utilized in combination with restriction endonuclease cleavage to identify individual chlamydial species. Thus we have developed a sensitive and rapid detection method and have used it on samples from patients with respiratory and genital infections.     

Antibody levels correlate with detection of Trypanosoma cruzi DNA by sensitive polymerase chain reaction assays in seropositive blood donors and possible resolution of infection over time

BACKGROUND: The clinical significance of anti‐Trypanosoma cruzi low‐level reactive samples is incompletely understood. Polymerase chain reaction (PCR)‐positive rates and antibody levels among seropositive blood donors in three countries are described. STUDY DESIGN AND METHODS: Follow‐up samples were collected from T. cruzi–seropositive donors from 2008 through 2010 in the United States (n = 195) and Honduras (n = 58). Also 143 samples from Brazil in 1996 to 2002, originally positive by three serologic assays, were available and paired with contemporary follow‐up samples from these donors. All samples were retested with Ortho enzyme‐linked immunosorbent assay (ELISA). PCR assays were performed on coded sample panels by two laboratories (Blood Systems Research Institute [BSRI] and American Red Cross Holland Laboratory [ARC]) that amplified kinetoplast minicircle DNA sequences of T. cruzi. RESULTS: PCR testing at BSRI yielded slightly higher overall sensitivity and specificity (33 and 98%) compared with those at the ARC (28 and 94%). Among seropositive donors, PCR‐positive rates varied by country (p < 0.0001) for the BSRI laboratory: Brazil (57%), Honduras (32%), and the United States (14%). ELISA signal‐to‐cutoff ratios (S/CO) were significantly higher for PCR‐positive compared to PCR‐negative donors (p < 0.05 for all comparisons). Additionally, PCR‐negative Brazilian donors exhibited greater frequencies of antibody decline over time versus PCR‐positive donors (p = 0.003). CONCLUSION: For all three countries, persistent DNA positivity correlated with higher ELISA S/CO values, suggesting that high‐level seroreactivity reflects chronic parasitemia. Significant S/CO declines in 10% of the PCR‐negative Brazilian donors may indicate seroreversion after parasite clearance in the absence of treatment.     

Successful immune tolerance induction by FVIII in hemophilia A patients with inhibitor may occur without deletion of FVIII‐specific T cells

Summary. Background: The development of an inhibitor is the major complication facing patients with hemophilia A treated by administration of factor (F) VIII concentrates. Restoration of tolerance to FVIII can be achieved by prolonged administration of FVIII (immune tolerance induction, ITI). Although ITI has been used for more than 30 years in patients with hemophilia A and inhibitor, its mechanism of action is still poorly understood. Objectives: As administration of high doses of antigen can induce the apoptosis of the T cells recognizing the antigen, a potential mechanism of action of ITI may be the deletion of FVIII‐specific T cells. Patients/Methods: We studied the CD4+ T‐cell response to FVIII in five (one mild, one moderate and three severe) patients successfully desensitized by administration of FVIII and in control subjects. Results: Following repeated stimulation with autologous dendritic cells loaded with FVIII, FVIII‐specific T oligoclonal cell lines were expanded from the blood of one of the successfully desensitized patients. The FVIII‐specific T cells produced IL‐5, IL‐13 and IL‐2. By contrast, FVIII‐specific T‐cell lines could not be derived from three patients with mild hemophilia A without inhibitor or from four normal control subjects. Conclusions: These data represent the first analysis of the cellular mechanisms regulating the induction of tolerance to FVIII. They demonstrate that successful tolerance induction may occur without deletion of FVIII‐specific T cells.     

Absolute configuration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol formed metabolically from 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is an important metabolite of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK). Using the chiral derivatizing agent, (R)- (+)-alpha-methylbenzyl isocyanate [(R)-(+)-MBIC], previous work has shown that the enantiomeric ratio of metabolically formed NNAL and its glucuronide derivative may be species dependent. However, the absolute configuration of such NNAL has not been previously reported. Synthetically prepared racemic NNAL was converted to diastereomeric esters by reaction with (R)-(+)- and (S)-(-)-alpha-methoxy-alpha- (trifluoromethyl)phenylacetic acid (MTPA) chloride (Mosher's reagent) and the products were characterized by 1H-NMR. Based on chemical shift data, the absolute configuration of NNAL in each diastereomeric ester was assigned. Hydrolysis of (R)-NNAL-(R)-MTPA gave (R)-NNAL. This was converted to the corresponding carbamate by reaction with (R)-(+)-alpha- MBIC and the absolute configurations of the diastereomeric carbamates formed by reaction of (R)- and (S)-NNAL with (R)-(+)-MBIC were thereby assigned. Conversion of metabolically produced NNAL to the same carbamates allowed us to assign the NNAL formed from NNK by rat liver microsomes as (R)-NNAL. The major and minor NNAL-glucuronide diastereomers found in the urine of patas monkeys and humans exposed to NNK were similarly assigned; they were formed from (R)-NNAL and (S)- NNAL, respectively.      

Participation of older newly-diagnosed cancer patients in an observational prospective pilot study: an example of recruitment and retention

Background  

There have been few prospective observational studies which recruited older newly-diagnosed cancer patients, and of these only some have reported information on the number needed to screen to recruit their study sample, and the number and reasons for refusal and drop-out. This paper reports on strategies to recruit older newly-diagnosed cancer patients prior to treatment into an observational prospective pilot study and to retain them during a six-month period.