Regulation of human basophil adhesion to endothelium under flow conditions: Different very late antigen 4 regulation on umbilical cord blood-derived and peripheral blood basophils

BACKGROUND: Although soluble mediators released by basophils in tissue sites contribute to the chronic injury that occurs in hypersensitivity diseases, only limited information is available about how circulating basophils are recruited to tissues. In particular, the interaction of basophils with endothelium under conditions that mimic physiologic flow has not been explored. OBJECTIVE: We sought to identify adhesion molecules regulating the attachment of human basophils to IL-4-activated human umbilical vein endothelial cells (HUVECs) under flow conditions. METHODS: A parallel-plate flow chamber and blocking mAbs were used to define the adhesion molecules involved in the interactions of peripheral blood basophils (PBBs) and cord blood-derived basophils (CBDBs) with IL-4-activated HUVECs and with Chinese hamster ovary (CHO) cell transfectants expressing specific adhesion molecules. A fluorescent ligand specific for very late antigen 4 (VLA-4) was used to directly examine the VLA-4 affinity state of basophils. RESULTS: Flowing PBBs and CBDBs attached to activated HUVECs and to CHO cells expressing P- or E-selectin. However, only CBDBs attached to vascular cell adhesion molecule 1 (VCAM-1)-transfected CHO cells under flow conditions. The attachment of CBDBs to CHO cells was blocked by mAbs directed against E-selectin, P-selectin, and VCAM-1, whereas attachment of PBBs was blocked by E-selectin and P-selectin mAbs. Activating VLA-4 with Mn(2+) on PBBs resulted in adhesion to the VCAM-1-transfected CHO cells, indicating that VLA-4 activity on PBBs can be regulated, at least in part, through affinity changes. The Mn(2+)-induced upregulation of basophil VLA-4 affinity was demonstrated directly by using a fluorescent ligand for VLA-4 and flow cytometry. CONCLUSIONS: The interaction of human CBDBs and PBBs with endothelium under flow conditions is mediated in part by both P- and E-selectin. VLA-4 additionally contributes to the adhesion of flowing CBDBs. However, the affinity of VLA-4 is too low to support the adhesion under flow conditions of unstimulated PBBs.     

Detection of cytomegalovirus infection during a vaccine clinical trial in healthy young women: seroconversion and viral shedding.

BACKGROUND: An antibody method based on absorption of serum with cytomegalovirus (CMV) glycoprotein B (gB) was developed for detection of infection during clinical trials of CMV gB vaccine. Previous study showed that this method detected the antibody response to infection and was negative with vaccine induced immunity. OBJECTIVES: In an ongoing efficacy trial of CMV gB vaccine the ability of the gB-absorbed CMV IgG assay to detect CMV infection was assessed and compared with viral culture results. STUDY DESIGN: Two hundred and ninety two healthy, seronegative young women in a phase II, double-blind, placebo-controlled, clinical trial of recombinant CMV gB vaccine (sanofi pasteur) with MF59 adjuvant (Chiron) were randomized to receive CMV gB vaccine or placebo (1:1) on a 0, 1 and 6 month schedule. Participants were screened every 3 months for CMV infection using the gB-absorbed CMV IgG assay, and a subgroup was also screened for infection with viral cultures. Viral culture (urine, vaginal swab and saliva) was used to confirm CMV infection in all subjects with a positive gB-absorbed CMV IgG result. RESULTS: Evidence of CMV infection (gB-absorbed CMV IgG levels>or=5.0 AU/ml) was found in 23/292 (7.88%) study participants. The gB-absorbed CMV IgG levels of their first positive serum ranged from 15.7 to 251.0 AU/ml with a mean of 77.0 AU/ml and a median of 44.9 AU/ml. Cytomegalovirus was isolated from all 23 of them from culture specimens collected after their first positive gB-absorbed CMV IgG. The time to first CMV positive culture from first positive gB-absorbed CMV IgG ranged from 0 to 12 weeks with a median of 2 weeks. CONCLUSIONS: The gB-absorbed CMV IgG assay detects CMV infection in CMV gB vaccine clinical trials earlier and more rapidly than virus culture and does not reveal whether subjects received CMV gB vaccine or placebo.     

Preparation of primary monolayer cultures of mouse pancreatic epithelial cells

Summary We describe a technique using normal and diabetic (dbdb) mice to establish primary pancreatic cultures that spread and assume a characteristic epithelial morphology. These cultures contain 4 to 7% beta cells, secrete insulin in response to stimuli for 10 to 14 d, contain few fibroblasts, and have a cell viability that is greater than 95%. The cells attach firmly to glass cover slips and are ideal for the study of insulin secretory granules or contractile proteins using indirect immunofluorescence.     

Evidence for presynaptic 5-hydroxytryptamine3 recognition sites on vagal afferent terminals in the brainstem of the ferret

Antagonists acting at the 5-hydroxytryptamine3 receptor are potent anti-emetic agents in cases of cytotoxic- and radiation-induced vomiting, and binding sites for these compounds have been described in brainstem areas known to be involved in mediation of nausea and vomiting. We have used autoradiography to examine the distribution of one of these antagonists, [3H]granisetron in the caudal brainstem of the ferret, a commonly used animal model for physiological investigations of emesis. The highest density of binding sites was found to be in the dorsomedial region of the nucleus of the solitary tract, the principal terminus for gastric vagal afferent fibres. Lower levels of binding were observed in the area postrema and the dorsal motor nucleus of the vagus. Following unilateral nodose ganglion excision, displaceable binding of [3H]granisetron in the nucleus of the solitary tract was attenuated on the ipsilateral side by 65%. Bilateral subdiaphragmatic vagotomy abolished binding of [3H]granisetron in the entire dorsal vagal complex. These results provide strong circumstantial evidence that 5-hydroxytryptamine3 receptors are located on vagal afferent terminals in the ferret brainstem.     

Modified toluidine blue O stain for Pneumocystis carinii: further evaluation of some technical factors.

A modified toluidine blue O (TBO) stain for Pneumocystis carinii cysts was evaluated with regard to the influence of (i) the age and extent of use of the sulfation reagent, (ii) the source of TBO, (iii) the TBO content of the staining solution, and (iv) the amount of TBO present in the alcohol wash solutions. All TBOs evaluated, except for a new TBO obtained from Roboz Surgical Instrument Co., Inc., Washington, D.C., produced satisfactory results. Each lot of TBO should be quality controlled before use to ensure that the P. carinii cysts stain lavender against a blue background. We have ourselves decided to use only certified TBO with a high dye content. As extensively used sulfation reagent provided less satisfactory results than did either freshly prepared or 1-week-old unused sulfation reagent, we have decided to prepare fresh sulfation reagent at least weekly and to discard used sulfation reagent after 10 slides have been processed.     

Acetoin production by wild-type strains and a lactate dehydrogenase-deficient mutant of Streptococcus mutans.

Eleven different laboratory strains of Streptococcus mutans representing the various serogroups were found to produce an average of 6.0 +/- 4.8 mM acetoin when grown in glucose-containing medium under aerobic conditions. None of the strains produced detectable acetoin when grown anaerobically. A lactate dehydrogenase-deficient mutant produced acetoin both aerobically and anaerobically and in substantially greater amounts than the wild-type strains did. Substitution of mannitol for glucose resulted in decreased acetoin production by wild-type strains and the lactate dehydrogenase-deficient mutant, indicating a role for NADH2 in the regulation of the acetoin pathway. Pyruvate incorporated into the growth medium of a wild-type strain caused acetoin to be produced anaerobically and stimulated acetoin production aerobically. Cell extracts of a wild-type S. mutans strain were capable of producing acetoin from pyruvate and were (partly) dependent on thiamine PPi. Extracts prepared from aerobically grown cells had approximately twice the acetoin-producing activity as did extracts prepared from anaerobically grown cells. The results indicate that acetoin production by S. mutans may represent an auxiliary reaction of pyruvate dehydrogenase in this organism.     

Optimal control of FES-assisted standing up in paraplegia using genetic algorithms

A practical system for Functional Electrical Stimulation (FES) assisted standing up in paraplegia should involve only a minimum of manual set up and tuning. An improved tuning method, using a genetic algorithm (GA) is proposed and demonstrated using computer simulation. Specifically, the GA adjusts the parameters of fuzzy logic (FL) and gain-scheduling proportional integral derivative (GS-PID) controllers that electrically stimulate the hip and knee musculature during the sit-stand maneuver. These new GA designed controllers were found to be effective in coordinating volitional and FES control according to formulated criteria. The latter was based on the deviations from a desired trajectory of the knee and hip joints and the magnitude of the voluntary upper body forces. The magnitude of the average arm forces were slightly higher when compared with the open-loop maximal stimulation of the hip and knee musculature; however, the terminal knee velocities were significantly reduced to less than 10 degrees /s. For practical implementation, the number of trials required to optimize the FL and GS-PID controllers can be reduced by a proposed pre-training procedure using a computer model scaled to the individual. The GA designed controllers remain near optimal provided the model-subject mismatch is small.     

Routine antenatal screening for hepatitis B using pooled sera: validation and review of 10 years experience.

AIMS: To validate the sensitivity of universal antenatal screening for hepatitis B surface antigen (HBsAg) by testing pools of 10 sera, and to review 10 years' experience using this method. METHODS: 66,945 antenatal patients were tested between 1986 and 1996 using the pooled method. All sera from 1996 (n = 6050) were retrieved and retrospectively tested individually. An in vitro determination of the effect of pooling on sensitivity was performed by checkerboard neutralisation assay. RESULTS: 26 HBsAg positive women were detected by universal screening over 10 years; 12 had non-European surnames and five had known risk factors for hepatitis B infection. High titre anti-HBs sera in the pool reduced the sensitivity of the HBsAg assay, though the effect was only significant at low levels of HBsAg carriage. CONCLUSIONS: The prevalence of hepatitis B is extremely low in the antenatal population served by Plymouth PHL. Pooling is unlikely to reduce sensitivity enough to lead to significant preventable vertical transmission, and is a cost-effective and valid strategy in areas of low seroprevalence.     

Filamentous actin bundles in the kidney

The distribution of filamentous actin bundles in the rat kidney was studied using a fluorescent phallotoxin label and transmission electron microscopy. The microvillous brush border lining proximal tubules, smooth muscle in renal vessels, and renal corpuscles were the structures most intensely labeled with rhodamine phalloidin. Closer evaluation of renal corpuscles revealed intense labeling of filamentous actin within podocyte foot processes enveloping the glomerular capillary loops. Rhodamine phalloidin also labeled basal bands of filamentous actin in the parietal epithelium and basal bands of actin in proximal and distal tubules. Finally, a band of filamentous actin was evident along the innermost aspect of the kidney capsule, within cells which often joined to form sinus-like compartments.     

Induction of experimental autoimmune thyroiditis in B cell-depleted mice

The effect of B cell depletion on the induction and severity of murine experimental autoimmune thyroiditis was investigated. Thirteen CBA mice were given repeated intraperitoneal doses of 700 micrograms purified rabbit anti-mouse Ig antibody from 24 hours to 8 weeks after birth. Controls were given normal rabbit IgG (14 mice) or were left uninjected (10 mice). At six weeks all mice received two doses of 70 micrograms murine thyroid extract in complete Freund's adjuvant. Only 2/13 of the anti-Ig treated mice were fully B cell-deficient as determined by serum IgM, spleen cell immunofluorescence and responsiveness to LPS; however, the levels of anti-thyroglobulin autoantibodies were very low in 7/13 mice. The results demonstrate that thyroiditis can be actively induced in the absence of B cells and autoantibodies but that B cells may play a role in increasing disease severity.