Restricted isotype, distinct variable gene usage, and high rate of gp120 specificity of HIV-1 envelope-specific B cells in colostrum compared with those in blood of HIV-1-infected, lactating African women.

A successful HIV-1 vaccine must elicit immune responses that impede mucosal virus transmission, though functional roles of protective HIV-1 Envelope (Env)-specific mucosal antibodies remain unclear. Colostrum is a rich source of readily accessible mucosal B cells that may help define the mucosal antibody response contributing to prevention of postnatal HIV-1 transmission. To examine the HIV-1 Env-specific colostrum B-cell repertoire, single B cells were isolated from 17 chronically HIV-infected, lactating women, producing 51 blood and 39 colostrum HIV-1 Env-specific B-cell antibodies. All HIV-1 Env-specific colostrum-derived antibodies were immunoglobulin (Ig)G1 isotype and had mean heavy chain complementarity-determining region 3 (CDR3) lengths and mutation frequencies similar to those isolated from blood. However, variable heavy chain (VH) gene subfamily 1(∼)69 usage was higher among colostrum than blood HIV-1 Env-reactive antibodies (49% vs. 20%, P=0.006, Fisher's exact test). Additionally, more HIV-1 Env-specific colostrum antibodies were gp120 specific than those isolated from blood (44% vs. 16%, P=0.005, Fisher's exact test). One cross-compartment HIV-1 Env-specific clonal B-cell lineage was identified. These unique characteristics of colostrum B-cell antibodies suggest selective homing of HIV-1-specific IgG1-secreting memory B cells to the mammary gland and have implications for targeting mucosal B-cell populations by vaccination.

Single-cell analysis reveals oligoclonality among 'low-count' monoclonal B-cell lymphocytosis.

Monoclonal B-cell lymphocytosis (MBL) is a preclinical hematologic syndrome characterized by small accumulations of CD5(+) B lymphocytes. Most MBL share phenotypic characteristics with chronic lymphocytic leukemia (CLL). Although some MBL progress to CLL, most MBL have apparently limited potential for progression to CLL, particularly those MBL with normal absolute B-cell counts ('low-count' MBL). Most CLL are monoclonal and it is not known whether MBL are monoclonal or oligoclonal; this is important because it is unclear whether MBL represent indolent CLL or represent a distinct premalignant precursor before the development of CLL. We used flow cytometry analysis and sorting to determine immunophenotypic characteristics, clonality and molecular features of MBL from familial CLL kindreds. Single-cell analysis indicated four of six low-count MBL consisted of two or more unrelated clones; the other two MBL were monoclonal. 87% of low-count MBL clones had mutated immunoglobulin genes, and no immunoglobulin heavy-chain rearrangements of V(H) family 1 were observed. Some MBL were diversified, clonally related populations with evidence of antigen drive. We conclude that although low-count MBL share many phenotypic characteristics with CLL, many MBL are oligoclonal. This supports a model for step-wise development of MBL into CLL.

Hypersensitivity of lymphocytes from chronic lymphocytic leukemia patients to ultraviolet light-C radiation.

Chronic lymphocytic leukemia (CLL) results in the accumulation of mature immunologically defective lymphocytes in GO phase. Lymphocytes from CLL patients were exposed to UVC radiation to determine whether these cells are capable of undergoing apoptosis, as a response to DNA damage. Lymphocytes from CLL patients were found to be readily killed by ultraviolet light-C (UVC) radiation. Cells from healthy donors were minimally affected by doses of UVC ten times higher then those which caused dramatic drops in the metabolism of CLL cells. At four hours after irradiation, the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) had dropped by 50% for CLL cells exposed to a dose of 10 J/m2. In contrast, there was no significant drop for healthy cells exposed to 100 J/m2. Cell death was measured by trypan blue staining, flow cytometry of Annexin V-PI stained cells, and Wright staining. By 24 hours after irradiation, significant amounts of cell death were observed in CLL cells at doses which had no significant effects on viability of healthy lymphocytes. The extreme sensitivity of CLL lymphocytes to UVC indicates that phototherapy should be explored as a potential treatment for this neoplasm.

Influence of silicone (PDMS) surface texture on human skin fibroblast proliferation as determined by cell cycle analysis.

In vivo biocompatibility of soft-tissue implants is often hampered by development of capsules that eventually might contract and impair implant function. It has been shown that capsule formation can be significantly reduced by using materials with textured surface elements in the micron range. In this study the interaction of human fibroblasts with silicone surfaces was analyzed using cell cycle analysis. Silicone was textured with 2, 5, and 10 microns wide grooves (2MU, 5MU, 10MU, respectively) or kept smooth (SMT). Cell cycle analysis was performed after staining of cells with propidium iodide. Cells proliferated on the fibronectin-preadsorbed silicone, as demonstrated by increased coverage and occurrence of subpopulations in the S and G2/M phase of the cell cycle. Cells on SMT went faster into the S phase than cells on textured silicones. Cells on 10MU showed less proliferation than cells on 2MU and 5MU. Besides the basic percentages of cells in the different cycle phases, DNA profiles were also influenced by incubation time and texture, especially with respect to the presence of hypodiploid populations and asymmetry of the G0/G1 peak. Finally scatter characteristics were influenced. 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay data did not reveal significant differences among the different samples. Fibronectin preadsorption of silicone only resulted in slightly higher MTT conversion. Cell cycle analysis proved to be a sensitive screening method for proliferation on the silicone surfaces and provided information beyond the normal G0/G1, S and G2/M subpopulations.

Effect of surgical and chemical in ovo bursectomy on lymphocyte density scores and histological evaluations of primary and secondary lymphoid organs in hypertensive and hypotensive turkeys.

The effects of surgical and chemical in ovo bursectomy on the histology of primary and secondary lymphoid organs were studied in hypertensive and hypotensive lines of turkeys. The effects of bursectomy were measured by determining the presence of lymphatic nodules and the presence of lymphocytes in secondary lymphoid organs: spleen, cecal tonsil, Meckel's diverticulum, and Peyer's patches; as well as in the thymus and bursa of Fabricius. No treatment effect on splenic nodule formation occurred, but a significantly lower lymphocyte density score was present in the surgically bursectomized group. Both nodule formation and lymphocyte density scores for the cecal tonsil and Peyer's patches were significantly reduced in the surgically bursectomized and testosterone-treated groups as compared with the control and sham groups. The most consistent depression in lymphatic scores in both the surgically and chemically bursectomized groups occurred in the Peyer's patches followed by cecal tonsils and Meckel's diverticulum. As expected, there were no differences in thymocyte density scores. There was no consistent effect on lymphatic scores due to blood pressure selection; however, the hypertensive line showed significantly lower scores than the hypotensive line in the bursa, cecal tonsils, and Peyer's patches. No differences between the sexes were noted in any organ.

Effect of surgical and chemical in ovo bursectomy on hatchability, mortality rate, and antibody response in hypertensive and hypotensive lines of turkeys.

The effect of in ovo bursectomy on hatchability, mortality rate, and antibody response to keyhole limpet hemocyanin (KLH) was studied in hypertensive and hypotensive lines of turkeys. Experiments were conducted to assess the optimal time to perform surgical in ovo bursectomy in turkey embryos for the lowest mortality rate. Factors such as the ease of operation, hatchability, and least deaths were considered. Surgical bursectomies at 25, 24, 23, and 22 day embryonation resulted in 75, 20, 0, and 10% hatchability, respectively. The surgical procedure performed at 25-day embryonation resulted in superior hatchability and ease of operation, and it was less traumatic as evidenced by fewer deaths. The hatchability of embryos injected with testosterone (TE) at 15 days incubation was 55%. Birds subjected to surgery or chemical treatment had increased mortality rates. Cyclophosphamide (CY) treatment at 1 and 2 days posthatch caused a significant increase in deaths over groups not treated with CY. Both primary and secondary KLH responses were significantly lower in bursectomized and TE-injected groups, than in shamoperated or nonoperated control groups.

Pharmacokinetics of single-dose intravenous or intramuscular administration of gentamicin in roosters.

Healthy mature roosters (n = 10) were given gentamicin (5 mg/kg of body weight, IV) and, 30 days later, another dose IM. Serum concentrations of gentamicin were determined over 60 hours after each drug dosing, using a radioimmunoassay. Using nonlinear least-square regression methods, the combined data of IV and IM treatments were best fitted by a 2-compartment open model. The mean distribution phase half-life was 0.203 +/- 0.075 hours (mean +/- SD) and the terminal half-life was 3.38 +/- 0.62 hours. The volume of the central compartment was 0.0993 +/- 0.0097 L/kg, volume of distribution at steady state was 0.209 +/- 0.013 L/kg, and the total body clearance was 46.5 +/- 7.9 ml/h/kg. Intramuscular absorption was rapid, with a half-life for absorption of 0.281 +/- 0.081 hours. The extent of IM absorption was 95 +/- 18%. Maximal serum concentration of 20.68 +/- 2.10 micrograms/ml was detected at 0.62 +/- 0.18 hours after the dose. Kinetic calculations predicted that IM injection of gentamicin at a dosage of 4 mg/kg, q 12 h, and 1.5 mg/kg, q 8 h, would provide average steady-state serum concentrations of 6.82 and 3.83 micrograms/ml, with minimal steady-state serum concentrations of 1.54 and 1.50 micrograms/ml and maximal steady-state serum concentrations of 18.34 and 7.70 micrograms/ml, respectively.

Pharmacokinetics of single doses of gentamicin given intravenously and intramuscularly to turkeys.

The disposition and absorption kinetics of gentamicin were studied in healthy, mature male and female turkeys (n = 10). Single doses of gentamicin (5 mg/kg) were injected either i.v. or i.m. with a 30-day rest period between each treatment. Baseline and serial venous blood samples (n = 17) were collected from each turkey. Serum concentrations of gentamicin were determined in duplicate for 24 h after each treatment, using radio-immunoassay. Using nonlinear least-square regression methods, the combined data of the i.v. and i.m. treatments were best described by a two-compartment open model. Kinetic analysis of the data after a single i.v. dose provided the following mean values: t1/2 alpha = 0.170 +/- 0.093 h, t1/2 beta = 2.57 +/- 0.79 h, MRT = 3.62 +/- 0.96 h, Vc = 0.090 +/- 0.017 l/kg, Vd(ss) = 0.172 +/- 0.024 l/kg, Vd(area) = 0.190 +/- 0.030 l/kg, and Clt = 49.8 +/- 9.8 ml/h/kg. After a single i.m. dose, the following mean values were determined: MRT = 5.10 +/- 1.73 h, t1/2abs = 0.74 +/- 0.66 h, tlag = 0.07 +/- 0.19 h, Clt/F = 50.7 +/- 12.5 ml/h/kg, Vd(area)/F = 0.193 +/- 0.044 l/kg, and F = 102 +/- 21%. Kinetic calculations made with the single i.m. data predicted that an i.m. injection of gentamicin at the dosage rate of 3 mg/kg q. every 12 h would provide average steady state serum concentrations of 4.93 micrograms/ml.

Effects of fenbendazole administration on hematology, clinical chemistries and selected hormones in the white Pekin duck.

The effects of single i.v. and p.o. doses (5 mg/kg) of fenbendazole, were evaluated on thyroxine, tri-iodothyronine, corticosterone, hematology, clinical chemistries, and serum proteins in 10 white Pekin ducks. Fenbendazole was administered i.v. (n = 5) as a 3% dimethyl sulfoxide solution and p.o. (n = 5) as a 10% commercial suspension. Serum enzyme concentrations, total protein and protein fractions, glucose, cholesterol, uric acid, sodium, and potassium were unchanged from baseline values. Serum triglycerides decreased consistently in the i.v.-treated group but remained unchanged in the p.o.-treated group. Serum chloride was consistently elevated above baseline values for both i.v.- and p.o.-treated ducks, while inorganic phosphate was consistently decreased only in the i.v.-treated group. Hemoglobin and hematocrit values generally were below baseline values. Leukocyte values varied considerably and were not significantly different from baseline values. Serum thyroxine and tri-iodothyronine values in both the i.v.- and p.o.-treated groups were not changed significantly from baseline values. Serum corticosterone values were not changed in the i.v.-treated groups but they were decreased at various times in the p.o.-treated group. Although there were some sporadic significant changes in the parameters measured versus baseline values all values remained within the physiologic limits for ducks. The safety of fenbendazole has been previously demonstrated for several species.

Comparative albumin determinations in ducks, chickens, and turkeys by electrophoretic and dye-binding methods.

Serum total protein, albumin, and globulin concentrations in male ducks, turkeys, and chickens were compared, using electrophoretic and dye-binding methods, as well as using a bovine and chicken albumin standard. When a chicken standard was used for determination of albumin and globulin concentrations by automated methods, results were more comparable with results of electrophoresis than were those when a bovine standard was used.

Histological evaluation of the vascular system for the severity of atherosclerosis in hyper and hypotensive male and female turkeys: comparison between young and aged turkeys.

Relationships among age, blood pressure, and atherosclerosis were studied in turkeys genetically selected for high and low systolic blood pressure ranging in age from 2 days to 104 weeks. Relationships between sex and atherosclerosis were studied only at 104 weeks of age. Abdominal aortas were examined in all birds in each age group. The left and right internal, external, and common carotid arteries, cranial and caudal thoracic aortas, area of coeliac orifice, sciatic junction, left and right sciatic arteries, left and right coronary arteries, and left and right deep branches of the coronary arteries were examined in the 104-week-old group. Mean abdominal aortic plaque scores for the different age groups increased linearly to the age of 12 weeks; after this time scores reached a plateau. Except for the first and last time period, the hypertensive group of males showed a significantly greater abdominal aortic plaque score than the hypotensive group. The right sciatic artery was the only other blood vessel in the 104-week-old group that had a significantly greater plaque score in the hypertensive line when compared with the hypotensive line. In the 104-week period, only the left and right sciatic arteries showed significantly higher plaque scores in the males than in the females. Also, only the male turkeys showed an increase in plaque scores with a concomitant rise in systolic pressure. In 104-week-old females there was no evidence of intimal change in either the left and right internal carotids, left and right common carotids, cranial thoracic aorta, or the right coronary artery. The hypertensive line had significantly greater blood pressure and greater ventricular weight, length, and sagittal width than the hypotensive line. The 104-week-old turkeys had more lipid staining material within the plaque than the younger birds, and calcification was evident in two of the older turkeys.

Effect of deprival of rete testis fluid on the morphology of efferent ductules.

One week after unilateral cannulation of the rete testis and ligation of the efferent ductules, samples of the proximal, middle and distal segments of the efferent ductules of 6 goats were examined by light and electron microscopy and compared with normal contralateral efferent ductules. The pseudostratified columnar epithelium consisted of ciliated, nonciliated and basal cells. The number of clear vacuoles decreased markedly in the proximal and middle segments following deprivation of androgen-rich rete testis fluid. The epithelium of the distal segment of the cannulated side had few large clear vacuoles compared to the normal side, which had a high concentration of large vacuoles. Since the large vacuoles decreased in all three segments following ligation, they were thought to be absorptive. Some cells of the distal segment of the cannulated side contained a single, huge, basal vacuole. Electron-dense, membrane-bound granules were abundant in the proximal segment of normal ductules. After cannulation these granules were still present. It was concluded that the electron-dense granules were insensitive to rete testis fluid and that they did not arise from the fluid leaving the rete testis.