Effect of Shipment, Storage, Anticoagulant, and Cell Separation on Lymphocyte Proliferation Assays for Human Immunodeficiency Virus-Infected Patients

Lymphocyte proliferation assays (LPA), which can provide important information regarding the immune reconstitution of human immunodeficiency virus (HIV)-infected patients on highly active antiretroviral therapy, frequently involve shipment of specimens to central laboratories. In this study, we examine the effect of stimulant, anticoagulant, cell separation, storage, and transportation on LPA results. LPA responses of whole blood and separated peripheral blood mononuclear cells (PBMC) to different stimulants (cytomegalovirus, varicella-zoster virus, candida and tetanus toxoid antigens, and phytohemagglutinin) were measured using fresh specimens shipped overnight and frozen specimens collected in heparin, acid citrate dextrose (ACD), and citrate cell preparation tubes (CPT) from 12 HIV-infected patients and uninfected controls. Odds ratios for positive LPA responses were significantly higher in separated PBMC than in whole blood from ACD- and heparin-anticoagulated samples obtained from HIV-infected patients and from ACD-anticoagulated samples from uninfected controls. On separated PBMC, positive responses were significantly more frequent in fresh samples compared with overnight transportation for all antigens and compared with cryopreservation for the candida and tetanus antigens. In addition, viral antigen LPA responses were better preserved in frozen PBMC compared with specimens shipped overnight. CPT tubes yielded significantly more positive LPA results for all antigens, irrespective of the HIV patient status compared with ACD, but only for the candida and tetanus antigens and only in HIV-negative controls compared with heparin. Although HIV-infected patients had a significantly lower number of positive antigen-driven LPA responses compared with uninfected controls, most of the specimen processing variables had similar effects on HIV-positive and -negative samples. We conclude that LPA should be performed on site, whenever feasible, by using separated PBMC from fresh blood samples collected in either heparin or ACD. However, if on-site testing is not available, optimal transportation conditions should be established for specific antigens.     

Expression of sialyl-Lewis X, an E-selectin ligand, in inflammation, immune processes, and lymphoid tissues.

The carbohydrate structure sialyl-Lewis X (SLex) can function as a ligand for E-selectin, formerly known as endothelial leukocyte adhesion molecule-1 (ELAM-1). This study was performed to analyze the expression of SLex by leukocytes and other cell types in the context of inflammatory and immune processes. Human peripheral blood cells were examined by flow cytometry using monoclonal antibody CSLEX1 directed against SLex. Cell surface SLex was found in abundance on nearly all isolated polymorphonuclear leukocytes (PMN) and monocytes, and at low levels on a substantial portion (up to 40%) of natural killer cells. This moiety was expressed also on approximately 10% of peripheral blood T cells. Immunohistochemistry was performed on various human tissues involved in inflammatory or immune processes and on secondary lymphoid tissues. In acute appendicitis, endothelial cells of postcapillary venules expressed E-selectin, and most PMN, both within vessels and extravasated, expressed SLex. A substantial number of monocytes/macrophages in inflamed appendiceal, synovial, and dermal tissues also reacted with antibody CSLEX1; however, only rare tissue macrophages in uninflamed nonlymphoid sites showed expression of SLex. These observations are consistent with the concept that SLex on circulating PMN and monocytes functions as a ligand for endothelial E-selectin in the development of inflammatory reactions. SLex-positive lymphocytes also were seen, notably, T lymphocytes in inflamed skin. An unexpected finding was that the CSLEX1 antibody also reacted with venular endothelium in certain lymphoid tissues and in inflamed appendix, but not with endothelium in normal appendix. Whether the SLex antigen identified on endothelium represents de novo expression or passive adsorption remains to be determined.     

Nodular regenerative hyperplasia of the liver in a case of myelofibrosis with extramedullary hematopoiesis and secondary portal venous hypertension.

Nodular regenerative hyperplasia of the liver was identified at autopsy in a patient with myelofibrosis with extramedulary hematopoiesis, an association not previously reported. Portal venous hypertension, documented during the patient's terminal hospitalization, was ascribed, in part, to a high rate of blood flow through the enlarged spleen. Possible mechanisms accounting for the development of nodular regenerative hyperplasia of the liver, and evidence provided by this case pertinent to these mechanisms, are discussed.     

Reliability of CD4 quantitation in human immunodeficiency virus-positive children: implications for definition of immunologic response to highly active antiretroviral therapy

Our objective was to develop data-based algorithms for definition of immunologic response to AIDS therapies in pediatric patients, taking account of T-cell subset measurement errors. The study design involved cross-protocol analysis of 2,148 enrollees in six completed Pediatric AIDS Clinical Trials Group trials. We used standard quantitation of T-cell subsets; linear modeling with mean-dependent measurement error variance was used to develop 95% tolerance limits for change in CD4%. For individuals with a CD4% of approximately 25%, the measurement error-based 95% tolerance interval ranges from 15% to 35%, whereas for individuals with a CD4% of approximately 5%, the tolerance interval ranges from 3% to 7%. When pairs of CD4% measures taken within a time interval of less than 30 days are averaged to estimate steady-state CD4%, tolerance interval width decreases by approximately 30%. A simple graphical tool that provides a data-based criterion for immunologic response over and above variation ascribable to T-cell measurement error is provided. Variability in CD4% due to measurement error is substantial, increases with level of CD4%, and complicates assessment of immunologic response to therapy. Replicates of CD4% measures could be used to improve precision of interpretation of CD4% measures.     

Impairment and recovery of visual functions after bilateral lesions of superior colliculus

Adult, male hooded rats were tested on a six-choice jumping stand apparatus designed to study their ability to perform visually guided orientation movements with a brightness discrimination task. Upon reaching criterion, the rats suffered either one- or two-stage, bilateral electrolytic lesions of the superior colliculi and then, after a brief recovery period, were retested for their ability to retain the preoperatively learned task and, in addition, perform a more difficult task. Although both brain damaged groups evidenced an impairment in comparison to sham-operated controls, the rats with two-stage lesions were less disabled than their simultaneously operated counterparts.     

Quantitative immunohistochemical detection of oxidized low density lipoprotein in the rabbit arterial wall

Quantitative immunohistochemical techniques were developed for mapping low density lipoprotein (LDL) oxidation within arterial tissue. Antibodies were raised by immunizing rabbits with Cu(2+)-oxidized rabbit LDL. ELISAs showed that they reacted strongly with oxidized rabbit LDL, weakly with other oxidized lipoproteins, and not at all with native LDL. Using optimized histological procedures, the antibodies were applied to sections of calibration gels containing LDL at various concentrations and levels of oxidation, and to sections of aortas from normal and heritable hyperlipidemic rabbits. Binding was measured with a rhodamine-labeled secondary antibody and carefully calibrated techniques of digital imaging fluorescence microscopy. Values obtained using a nonspecific primary antibody were subtracted. Specific binding to calibration sections increased linearly with respect to the concentration of oxidized LDL and the duration of its exposure to Cu2+, approximately linearly with respect to its modified lysine content, and nonlinearly with respect to its relative electrophoretic mobility. Specific staining was detected in sections of aortas from heritable hyperlipidemic but not normal rabbits. In the former, it was higher in the intima than in the media and was greater downstream than upstream of intercostal branch ostia; the average level was lower in those branches with the least intimal thickening but the difference between upstream and downstream regions was larger. These results correlate with the known pattern of lipid deposition in hyperlipidemic rabbit aortas. A small but significant amount of specific staining was observed in sections which were devoid of intimal thickening, which is consistent with LDL oxidation occurring prior to disease or during its earliest stages.     

Isolation and characterization of human and rabbit sperm tail fibrous sheath

Using mechanical and chemical dissection methods, fibrous sheath was isolated both from normal ejaculated human spermatozoa and from rabbit cauda epididymal spermatozoa. The same techniques did not produce a pure preparation of fibrous sheath from ejaculated rabbit spermatozoa, suggesting that further cross-linking and stabilization of sperm structures occurs in response to components of the seminal plasma. The isolation procedures were monitored by phase contrast microscopy and the purity of the fibrous sheath was verified by electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human fibrous sheath revealed at least 14 protein bands of which the most intensely stained were of molecular weight 84, 72, 66.2, 57, 32 and 28.5 kDa. The rabbit fibrous sheath revealed at least 10 protein bands, of which the most intensely stained were 35.2, 32.7 and 28.5 kDa. The amino acid composition of the purified fibrous sheath from human and rabbit spermatozoa was similar, being high in aspartic acid and/or asparagine and glutamic acid and/or glutamine, serine, alanine, leucine, lysine and glycine, but low in histidine, tyrosine and isoleucine. This composition is similar to that reported for the rat and suggests that mammalian sperm tail fibrous sheaths are composed of similar types of proteins, although there are apparent differences in protein components between species.      

Acute and Subacute Toxicity of Aspilia Africana Leaves

This study was designed to evaluate the toxicity of the aqueous extract of Aspilia africana leaves. Oral doses of 500 mg/kg and 1000 mg/kg were administered for 28 days to rats after every 2 days for sub-acute toxicity. For acute toxicity, 5 doses of 2, 4, 8, 12 and 16g/Kg body weight were investigated in mice. The control groups consisted of mice or rats administered with distilled water. The signs of toxicity fluctuated lightly from one mammal to another throughout the experiment. The liver, kidneys and heart weight of rats revealed no significant differences between the test groups and the control. The results indicated that the medium lethal dose (LD₅₀) was found to be greater in females than males with an average of 6.6g/Kg body weight for both sexes. Regardless of the significant differences observed at certain points in some biochemical parameters (ALT, AST, ALP, Creatinine and Glutathione); none showed any linear dose responsiveness. On the other hand, most of the parameters investigated were found to be gender dependent. These results suggested that A Africana can be classified among substances with low toxicity.     

Spontaneous reanastomosis between lymphatic vessels following syngeneic transplantation of the small intestine in the rat

Abstract Spontaneous lymphvascular reanastomosis (SLR) following small bowel transplantation in rats is of clinical relevance for the resorption of long chain fatty acids. Detailed morphological and molecular data concerning the process of lymphvascular reanastomosis are not available in the literature. In this study SLR was investigated using microradiology and scanning electron microscopy. Between the 8^th and 21^st postoperative days following transplantation SLR does not occur between the intestinal trunk of the transplant and the thoracic duct of the recipient. Instead, an indirect connection was observed between the inserted advential lymphatic vessels of the mesenteric artery and lymphatic vessels of the aorta or ductus deferens, which are connected with the thoracic duct.