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BACKGROUND: Clinical indications for lymphocyte subset enumeration by flow cytometry include monitoring of disease progression and timing of therapeutic intervention in infection with human immunodeficiency virus. Until recently international standardisation has not been possible due to a lack of suitable stable reference material. METHODS: This study consisted of two trials of a stabilised whole blood preparation. Eleven participants were sent two standard protocols for staining plus gating strategy and asked to report absolute counts for lymphocyte subsets. RESULTS: No significant difference was detected between the two methods when results from the two assays and all partners were pooled. Significant differences in results from the different partners were observed. However, representative mean counts were obtained for geometric means, geometric coefficient of variation, and 95% confidence interval for CD3 910 cells/mul, 9%, and 888 to 933, respectively), CD4 (495 cells/mul, 12%, and 483 to 507), and CD8 (408 cells/mul, 13%, and 393 to 422). CONCLUSION: We have introduced a stabilised blood preparation and a well-characterized biological standard. The availability of this reference material greatly simplifies the validation of new techniques for CD4(+) T-cell enumeration and the expansion of external quality assurance programmes for clinical laboratories, including those that operate in resource-restricted environments. (c) 2005 Wiley-Liss, Inc.

Objective To determine the indications for distal pancreatectomy for chronic pancreatitis and to evaluate the risks, functional loss, and outcome of the procedure. Summary Background Data Chronic pancreatitis is generally associated with continued pain, parenchymal and ductal hypertension. and progressive pancreatic dysfunction, and it is a cause of premature death in patients who receive conservative treatment. Good results have recently been reported by the authors and others for resection of the pancreatic head in this disease, but distal pancreatectomy is a less popular option attended by variable success rates. It remains a logical approach for patients with predominantly left-sided pancreatic disease, however. Methods A personal series of 90 patients undergoing distal pancreatectomy for chronic pancreatitis over the last 20 years has been reviewed, with a mean postoperative follow-up of 34 months (range 1–247). Pancreatic function was measured before and after operation in many patients. Results Forty-eight of 84 patients available for follow-up had a successful outcome in terms of zero or minimal, intermittent pain. There was one perioperative death, but complications developed in 29 patients, with six early reexplorations. Morbidity was unaffected by associated splenectomy or right-to-left dissection. Late mortality rate over the follow-up period was 10%; most of these late deaths occurred because of failure to abstain from alcohol. Preoperative exocrine function was abnormal in two thirds of those tested and was unchanged at follow-up. Diabetic curves were seen in 10% of patients preoperatively, while there was an additional diabetic morbidity rate of 23% related to the procedure and late onset of diabetes (median duration 27 months) in another 23%. Diabetic onset was related to percentage parenchymal resection as well as splenectomy. Outcome was not clearly dependent on the etiology of pancreatitis or on disease characteristics as assessed by preoperative imaging. However, patients with pseudocyst disease alone did better than other groups. Twenty-one of 36 patients who failed to respond to distal pancreatectomy required further intervention, including completion pancreatectomy, neurolysis, and sphincteroplasty. Thirteen of these 21 patients achieved long-term pain relief after their second procedure. Conclusions Distal pancreatectomy for chronic pancreatitis from any etiology can be performed with low mortality and a good outcome in terms of pain relief and return to work in approximately 60% of patients. Little effect is seen on exocrine function of the pancreas, but there is a diabetic risk of 46% over 2 years. Pseudocyst disease is associated with the best outcome, but other manifestations of this disease, including strictures, calcification, and limited concomitant disease in the head of the pancreas, can still be associated with a good outcome.

The flow cytometers that are currently supported by industry provide accurate CD4 + -T-cell counts for monitoring human immunodeficiency virus disease but remain unaffordable for routine service work under resource-poor conditions. We therefore combined volumetric flow cytometry (measuring absolute lymphocyte counts in unit volumes of blood) and simpler protocols with generic monoclonal antibodies (MAbs) to increase cost efficiency. Volumetric absolute counts were generated using CD45/CD4 and CD45/CD8 MAb combinations in two parallel tubes. The percentage values for the various subsets were also determined within the leukocyte and lymphocyte populations utilizing a fully automated protocol. The levels of agreement between the newly developed method and the present industry standards, including both volumetric and bead-based systems using a full MAb panel for subset analysis, were tested by Bland-Altman analyses. The limits of agreement for CD4 counts generated by the volumetric methods using either CD45/CD4 (in a single tube) or the full Trio MAb panel (in three tubes) on the CytoronAbsolute flow cytometer were between −29 and +46 cells/mm 3 with very little bias for CD4 counts (in favor of the Trio method: +8 CD4 + lymphocytes/mm 3 ; 0.38% of lymphocytes). The limits of agreement for absolute CD4 counts yielded by the volumetric CD45/CD4 method and the bead-based method were between −118 and +98 cells/mm 3 , again with a negligible bias (−10 CD4 + lymphocytes/mm 3 ). In the volumetric method using CD45/CD8, the strongly CD8 + cells were gated and the levels of agreement with the full Trio showed a minor bias (in favor of the Trio; +40 CD8 + cells/mm 3 ; 5.2% of lymphocytes) without a significant influence on CD4/CD8 ratios. One trained flow cytometrist was able to process 300 to 400 stained tubes per day. This workload extrapolates to a throughput of >30,000 samples per year if both CD45/CD4 and CD45/CD8 stainings are performed for each patient or a throughput of >60,000 samples if only CD45/CD4 counts are tested in a single tube. Thus, on the basis of the high efficiency and excellent agreement with the present industry standards, volumetric flow cytometers with automated gating protocols and autobiosamplers, complemented by generic CD45, CD4, and CD8 MAbs used in two-color immunofluorescence, represent the most suitable arrangements for large regional laboratories in resource-poor settings.

The nitration of protein tyrosine residues by peroxynitrous acid has been associated with pathological conditions. Here it is shown, using a sensitive competitive enzyme-linked immunosorbent assay and immunoblotting for nitrotyrosine, that spontaneous nitration of specific proteins occurs during a physiological process, the activation of platelets by collagen. One of the main proteins nitrated is vasodilator-stimulated phosphoprotein. Endogenous synthesis of nitric oxide and activity of cyclo-oxygenase were required for the nitration of tyrosine. The nitration was mimicked by addition of peroxynitrite to unstimulated platelets, although the level of nitrotyrosine formation was greater and its distribution among the proteins was less specific.

Atherosclerotic plaques develop in the arterial wall from complex multicellular processes following the early recruitment of circulating monocytes. Infiltration of monocytes is mediated by cell adhesion molecules (CAMs), including vascular cell adhesion molecule-1 (VCAM-1) which is rapidly induced in endothelial cells in response to cytokines. Apolipoprotein E (apo E), a 34-kDa polypeptide, helps protect against atherosclerosis, in part, because apo E phospholipid particles secreted by macrophages may have local protective effects within lesions. Here we have investigated whether purified plasma apo E, complexed with dimyristoyl phosphatidylcholine (DMPC) vesicles, can inhibit cytokine-induced vascular cell adhesion molecule-1 (VCAM-1) expression in human umbilical vein endothelial cells (HUVECs). Expression of VCAM-1 in endothelial cells after exposure to tumour necrosis factor-alpha (TNF-alpha) or interleukin 1beta (IL-1beta) was quantified by ELISA and shown to be partially inhibited by 17beta-estradiol (40-60% inhibition) or by S-nitroso-L-glutathione, a nitric oxide donor (20-25%). However, preincubations with physiological concentrations (10-100 microg protein/ml) of apo E DMPC did not downregulate VCAM-1 expression, even with extended preincubation times. These findings were confirmed using a fluorescence-activated cell sorter (FACS) for analysis which indicated additionally that apo E-DMPC had no effect on sub-populations within the HUVEC cultures. Finally, apo E-DMPC vesicles were also unable to suppress TNF-alpha-induced upregulation of E-selectin or intercellular adhesion molecule-1 (ICAM-1). We conclude that plasma apo E is unlikely to be important in limiting endothelial activation.

Peroxynitrite, a reactive oxidising species resulting from a reaction between nitric oxide and the superoxide anion, modifies proteins by nitration of certain amino acids such as tyrosine. Tissue factor (TF), a transmembrane protein, is expressed on cells under inflammatory conditions and initiates the coagulation cascade. The extracellular domain of TF is rich in tyrosine. Exposure of recombinant TF and cellular TF to peroxynitrite was associated with a reduction in procoagulant activity. This was accompanied by an elevated level of nitrotyrosine residues. Peroxynitrite may have a protective role by attenuation of the thrombotic properties of TF.

Mouse bladder epithelium has been successfully transplanted to the bladders of syngeneic mice and has survived for at least twenty weeks. The fate of the transplanted tissue was followed using a fluorescein label. The recipient bladders were prepared by stripping the urothelium either by a surgical or a chemical method. The possibility of adopting a comparable technique for the treatment of early bladder cancer in man is discussed.

The temporal and spatial regeneration of denuded mouse bladders was characterized using antibody markers to mucosal and submucosal elements in bladder tissue. Mechanical stripping of bladder mucosa resulted in a plane of cleavage in the submucosa leaving the muscle layers intact. Fully regenerated bladders were observed after 14 to 21 days although submucosal elements showed abnormal vacuolation. Implantation of immortalized epithelial cell line BBN3 into denuded bladders resulted in solid tumor formation while, in contrast, transplantation of MB331 showed cystic structures with no indication of invasion. Confirmation of the epithelial and implanted origin of cells lining the lumina of implanted bladders were shown using different keratin antibodies. The benign behavior of MB331 in vivo is suggestive of a cell line representing a preneoplastic stage in carcinogenesis and demonstrates an approach to assess the in vivo phenotype of cell parameters established in vitro.