Immunomagnetic selection of purified monocyte and lymphocyte populations from peripheral blood mononuclear cells following cryopreservation.

Cryopreservation is a method commonly used to store human blood samples. We sought to determine if cryopreserved peripheral blood mononuclear cells (PBMC) could be separated effectively into distinct populations by using monoclonal antibodies and immunomagnetic microspheres. PBMC obtained from healthy blood donors and from human immunodeficiency virus-infected subjects were cryopreserved for as long as 18 months. Recovered cells were separated into CD14+ monocytes and CD4+ T-cell subsets by immunomagnetic selection. Flow cytometry analysis indicated >95% depletion of monocytes from PBMC following immunomagnetic selection with anti-CD14. A highly enriched population of CD4+ T cells was obtained from the CD14-depleted cell fraction by using an anti-CD4 monoclonal antibody and detachable immunomagnetic beads. The CD4+ T cells were subsequently separated into CD4+ CD45RO and CD4+ CD45RA fractions. Each fraction contained >90% enrichment for the respective subpopulation and <5% of the reciprocal subpopulation. No significant differences in cell surface expression of leukocyte markers, in efficiency of selection of PBMC subpopulations, or in mitogen-induced proliferation were detected in freshly isolated or cryopreserved cells. Efficient recovery of cryopreserved specimens means that targeted assays can be performed on selected, prospectively stored samples once clinical endpoints have been achieved.     

Clinical and alimentary history of children attending a pediatric gastroenterology outpatient clinic with functional chronic constipation and its possible complications

In order to better understand the natural history of chronic functional constipation, a questionnaire was applied to 163 children and infants, before beginning standardized treatment. Median age (range) at start was 3 mo(0-108 mo) but age at arrival at the Pediatric Gastroenterology Unit was 53 mo(2-146 mo). In 62.4% of the cases symptoms began before or up to 3 mo after cow's milk introduction and rarely around (-/+ 6 mo) toilet training. Possible complications appeared progressively, often at preschool or school age or as the first noticeable manifestation: recurrent abdominal pain (61.1%), fecal soiling (45.4%), fecal blood (35%), enuresis (23.3%), vomiting (19%), urinary infection (17.9%), urinary retention (8.6%). Abdominal distension was rarely detected on physical examination and was usually discrete. In conclusion, children attended in Botucatu begin their constipation at an early age, frequently associated with weaning,and important complications may ensue along years. This evolution should be avoided by prevention and early treatment of constipation.     

Supernumerary intrathoracic rib,a rare congenital anomaly: Case report and review of the literature

A 17‐year‐old female who presented with cough, chest pain, dyspnea, and hemoptysis was found to have an intrathoracic rib. Patients who are diagnosed with intrathoracic ribs are most often asymptomatic and should undergo limited diagnostic workup. Intrathoracic ribs are rare congenital anomalies incidentally identified after chest radiography performed for another indication, as is the case with this patient. In this case, further evaluation was necessary due to persistent symptoms. Here, we used contrast‐enhanced ultrasound after chest radiography and computed tomography to further evaluate the fatty intrathoracic mass and exclude vascular features suggestive of a tumor.     

Enzyme Immunoassay: Binding of Salmonella Antigens to Activated Microtiter Plates

Abstract

A heat extract prepared from radiolabeled Salmonella cells was used to determine if covalent binding to activated surface of polystyrene plates would improve antigen retention thus contributing to increase sensitivity in an enzyme immunoassay for Salmonella antigen. The effect of treatment with ethylchloroformate on the retention of antigens passively absorbed to polyvinylchloride and polystyrene plates was also investigated. Chemically modified plates retained more radiolabeled antigens after washing than did untreated plates in which the antigens had been physically adsorbed. However, improvement of assay sensitivity depended on the type of plate used for covalent binding of antigen. N-succiniraidyl 3-(2-pyridyldlthio) propionate (SPDP), was found to be potentially useful for mediation of covalent binding of antigens to activated plates.     

Post Sex Affectionate Exchanges Promote Sexual and Relationship Satisfaction

The current research examined the role of post sex affection in promoting sexual and relationship satisfaction in ongoing romantic partnerships. Since romantic partners view the period after engaging in sex as an important time for bonding and intimacy, we sought to determine if and how the duration and quality of post sex affection might promote satisfaction in romantic relationships. In two studies, we tested the link between post sex affectionate behavior (e.g., cuddling, caressing, shared intimacy) and sexual and relationship satisfaction. In Study 1, a cross-sectional survey of individuals in romantic relationships (N = 335), duration of post sex affection was associated with higher sexual satisfaction and, in turn, higher relationship satisfaction. In Study 2, a daily experience study of 101 established couples (N = 202) with a 3-month follow-up, day-to-day changes in post sex affection duration and quality were associated with both partners’ sexual and relationship satisfaction, and engaging in longer and more satisfying post sex affection over the course of the study was associated with higher relationship and sexual satisfaction 3 months later. In general, the pattern of results was consistent for men and women, but the association between the duration of post sex affection and relationship satisfaction was stronger for women than for men (Study 1) and women, but not men, felt more sexually satisfied when their partner reported higher quality post sex affection (Study 2). The findings suggest that the period after sex is a critical time for promoting satisfaction in intimate bonds.     

Prospective Evaluation of Diagnostic Accuracy of Dried Blood Spots from Finger Prick Samples for Determination of HIV-1 Load with the NucliSENS Easy-Q HIV-1 Version 2.0 Assay in Malawi

HIV-1 viral load (VL) testing is not widely available in resource-limited settings. The use of finger prick dried blood spot (FP-DBS) samples could remove barriers related to sample collection and transport. Measurement of VL using DBS from EDTA venous blood (VB-DBS) in place of plasma has previously been validated using the NucliSENS Easy-Q HIV-1 v2.0 assay, but information on the accuracy of FP-DBS samples for measuring VL is limited. This prospective study, conducted at Thyolo District Hospital in southern Malawi, compared VL levels measured on FP-DBS samples and plasma using the NucliSENS Easy-Q HIV-1 v2.0 assay. Comparability was assessed by means of agreement and correlation (131 patients with VLs of ≥100 copies/ml), sensitivity, and specificity (612 patients on antiretroviral treatment [ART]). Samples of EDTA venous blood and FP-DBS from 1,009 HIV-infected individuals were collected and prepared in the laboratory. Bland-Altman analysis found good agreement between plasma and FP-DBS VL levels, with a mean difference of −0.35 log₁₀, and 95% limits of agreement from −1.26 to 0.55 log₁₀. FP-DBS had a sensitivity of 88.7% (95% confidence interval [CI], 81.1 to 94.4%) and a specificity of 97.8% (95% CI, 96.1 to 98.9%) using a 1,000-copies/ml cut point and a sensitivity of 83.0% (95% CI, 73.4 to 90.1%) and a specificity of 100% (95% CI, 99.3 to 100%) using a 5,000-copies/ml cut point. This study shows that FP-DBS is an acceptable alternative to plasma for measuring VL using the NucliSENS Easy-Q HIV-1 v2.0. We are conducting a second study to assess the proficiency of health workers at preparing FP-DBS in primary health care clinics.     

Expression at mRNA level of cytokines and A238L gene in porcine blood-derived macrophages infected <Emphasis Type="Italic">in vitro</Emphasis> with <Emphasis Type="Italic">African swine fever virus</Emphasis> (ASFV) isolates of different virulence

Summary. Porcine macrophage cultures were infected with two ASFV isolates of variable virulence and mRNA levels of several relevant macrophage-derived cytokines were quantified by real time PCR. At six hours post infection, a clear enhancement of mRNA expression of TNF, IL6, IL12 and IL15 was observed in macrophages infected with the low virulent ASFV/NH/P68 (NHV) when compared to those infected with the highly virulent ASFV/L60 (L60). The sequence of the A238L gene homologue to the cellular IB was found identical in both viral isolates and its expression at mRNA level was higher in macrophages infected with NHV when compared to macrophages infected with L60. Furthermore our results suggest a negative correlation between the mRNA expression of A238L gene and the mRNA expression of the above mentioned cytokines (with the exception of IL10) in L60 infected macrophages in opposition to the positive correlation (with exception of the IL1) suggested in NHV infection. Overall, our data strongly emphasize that virulence of ASFV isolates may depend on their capacity to regulate the expression of macrophage-derived cytokines relevant for the development of host protective responses by yet unknown mechanisms triggered by the virus at early stages of the cellular infection.Received March 25, 2003; accepted July 3, 2003 Published online August 18, 2003     

Acute myocardial infarction in the puerperium.

The authors describe the case of a forty-two-year-old patient with acute myocardial infarction (MI) on the third day of puerperium, who underwent direct angioplasty. The authors review the main physiological cardiovascular and hemostatic changes in pregnancy, which are usually well tolerated but which increase the probability of cardiovascular events when risk factors or previous cardiac disease are associated. Blood volume and cardiac output increase by around 50% during pregnancy, hemoglobin concentration falls, and reduced peripheral vascular resistance leads to a fall in arterial blood pressure during the first half of pregnancy. End-diastolic pressure, stroke volume and cardiac output increase in the immediate puerperium, and cardiovascular risk is highest in the perinatal period. The incidence of MI during pregnancy and puerperium is low; however, it is on the increase due to the growing number of pregnancies in women close to the end of their reproductive age.