Ovarian follicular growth and atresia: the relationship between cell proliferation and survival.

Growth factors and steroids play an important role in the regulation of ovarian follicular development. In cattle, two of the earliest detectable differences between the healthy dominant follicle selected for development to the ovulatory stage and subordinate follicles destined to undergo atresia are the greater availability of IGF and the greater capacity to produce estradiol in the dominant follicle. We have shown that IGF-I and estradiol stimulate the proliferation of bovine granulosa cells in vitro and promote granulosa cell survival by increasing resistance to apoptosis. Furthermore, the ability of IGF-I and estradiol to increase resistance to apoptosis is tied to their ability to promote progression through the cell cycle. Blocking the cell cycle at the transition between the first gap phase and the DNA synthesis phase using a specific inhibitor prevented the protective effects of IGF-I and estradiol against apoptosis. Further experiments showed that the protective effect of IGF-I against apoptosis is mediated by the stimulation of phosphatidylinositol 3-kinase and its downstream target, protein kinase B/Akt. Constitutive activation of Akt by the infection of granulosa cells with a recombinant Akt adenovirus protected against apoptosis, and this effect also depended on cell cycle progression. These experiments show that the protective effect of estradiol and IGF-I against apoptosis depends on unperturbed progression through the cell cycle. Once follicles have developed to the preovulatory stage, the LH surge induces terminal differentiation of granulosa cells and withdrawal from the cell cycle. Bovine granulosa cells withdraw from the cell cycle by 12 h after the LH surge and become resistant to apoptosis, even in the absence of growth factors. Treatment with a progesterone receptor antagonist in vitro caused reentry of granulosa cells into the cell cycle and susceptibility to apoptosis, suggesting that induction of progesterone receptor expression by the LH surge is required for cell cycle withdrawal and resistance to apoptosis. In summary, the susceptibility of granulosa cells to apoptosis depends on the cell cycle. Proliferating granulosa cells in growing follicles depend on growth factors for survival, whereas cells that have terminally differentiated in response to the LH surge are resistant to apoptosis and relatively independent of growth factors for survival.

Susceptibility of ovarian granulosa cells to apoptosis differs in cells isolated before or after the preovulatory LH surge.

The luteinizing hormone (LH) surge initiates the final stages of ovarian follicle development, and induces ovulation and luteinization of preovulatory follicles. To investigate whether exposure to the LH surge alters follicle cell susceptibility to apoptosis, granulosa and theca cells were isolated from bovine preovulatory follicles before and 14 h after injection of GnRH to induce an LH surge. Granulosa cells isolated before the LH surge were susceptible to apoptosis induced by soluble Fas ligand or serum withdrawal, while cells isolated after the LH surge were resistant to apoptosis. Resistance to Fas-mediated apoptosis was not associated with decreased Fas mRNA or protein levels. Pretreatment of granulosa cells isolated after the LH surge with the protein synthesis inhibitor cycloheximide rendered the cells susceptible to Fas-mediated apoptosis, indicating that inhibition of apoptosis was mediated by expression of labile survival factors. Theca cells were sensitive to Fas-mediated apoptosis before and after exposure to the LH surge. Resistance to apoptosis of granulosa cells from preovulatory follicles after the LH surge may be important for normal ovulation and luteinization.

Relationship of Fas ligand expression and atresia during bovine follicle development.

The Fas antigen (Fas) is a cell surface receptor that may be involved in the initiation and progression of follicle cell apoptosis during atresia. Fas initiates apoptosis in sensitive cells after binding Fas ligand (FasL). Other experiments have shown that expression of Fas mRNA and responsiveness to Fas-mediated apoptosis vary in bovine granulosa and theca cells during follicle development. In the present study, FasL mRNA content was measured and Fas and FasL protein expression was examined in bovine granulosa and theca cells of healthy dominant follicles and the two largest atretic subordinate follicles on day 5 of the oestrous cycle (day 0 = oestrus), and of dominant follicles from the first wave of follicle development after they had become atretic and showed no growth for 4 days. FasL mRNA content was higher in granulosa cells from atretic compared with healthy follicles. FasL mRNA content was also higher in theca cells from atretic subordinate compared with healthy dominant follicles on day 5, but did not differ between theca cells from healthy and atretic dominant follicles. Immunohistochemical staining for FasL was more intense in theca compared with granulosa cells and in atretic compared with healthy follicles. Immunohistochemical staining for Fas was more intense in granulosa compared with theca cells and in atretic subordinate compared with healthy dominant follicles on day 5. Immune cells, known to express Fas and FasL, were localized in the theca, but not the granulosa, cell layer of all follicles. Higher concentrations of Fas and FasL in cells from atretic follicles, together with the previous demonstration of increased responsiveness of granulosa cells from subordinate follicles to FasL-induced apoptosis, support a potential role for FasL-mediated apoptosis during ovarian follicle atresia.

Apoptosis of bovine granulosa cells after serum withdrawal is mediated by Fas antigen (CD95) and Fas ligand.

Ovarian follicular atresia occurs by apoptosis of granulosa and theca cells. The Fas antigen (Fas), a cell surface receptor that triggers apoptosis when activated by Fas ligand (FasL), may be involved in this process. A possible role of the Fas pathway in mediating serum withdrawal-induced apoptosis of granulosa cells was examined. Granulosa cells collected from 5- to 10-mm bovine follicles were cultured in DMEM-F12 containing serum for 3 days, deprived of serum, and live cells were counted at various times after serum withdrawal. Cell death increased significantly 6 h after serum withdrawal (21% +/- 7%; P: < 0.05 vs. 0 h) and continued to increase until 24 h (43% +/- 6%). No further increases in cell death were observed through 72 h. Detection of the translocation of phosphatidylserine to the outer surface of the cell membrane by annexin V binding indicated that cells died by apoptosis. Quantitative reverse transcriptase-polymerase chain reaction assays showed no changes in Fas mRNA levels but a 4.7-fold increase in FasL mRNA 3 h after serum withdrawal (P: < 0.05 vs. 0 h). FasL mRNA remained elevated through 24 h and returned to basal levels at 48 h. Immunohistochemical staining showed that both Fas and FasL protein increased on the cell surface within 3 h and remained elevated through 12 h (the last time point tested). Binding of FasL to Fas was blocked with two reagents that bind to the extracellular domain of FasL: an anti-FasL antibody and Fas:Fc, a chimeric protein consisting of the Fc portion of human immunoglobulin G and the extracellular domain of human Fas. Cell death 24 h after serum withdrawal was reduced 55% +/- 10% and 34% +/- 12% by anti-FasL antibody and Fas:Fc, respectively (P: < 0.05 vs. no blocking protein). In conclusion, serum withdrawal-induced apoptosis of bovine granulosa cells is mediated at least partially by Fas/FasL interactions. These results are consistent with a potential role of Fas in an autocrine or paracrine pathway to trigger ovarian follicular atresia.

Treatment of a patient with a Class II malocclusion, impacted canine, and severe malalignment.

A case report of the orthodontic treatment of a male adolescent with a unilateral dental Class II malocclusion, an impacted canine, severe maxillary malalignment, and a canted maxillary anterior occlusal plane. Treatment consisted of full fixed appliances, extraction of the maxillary right first premolar, and surgical exposure of the impacted canine. Treatment vastly improved the patient's facial and dental esthetics. A Class I skeletal and dental relationship was established, along with a functional anterior guidance. The dental arches were coordinated and the dental midlines coincident with the midsagittal plane. This case report was presented to the American Board of Orthodontics in partial fulfillment of the requirements for the certification process conducted by the Board.

Regulation of Fas antigen (Fas, CD95)-mediated apoptosis of bovine granulosa cells by serum and growth factors.

Our previous studies have shown that bovine granulosa cells cultured in basal media supplemented with 5% fetal bovine serum (BM-FBS) are resistant to apoptosis induced by recombinant Fas ligand (FasL) unless pretreated with interferon-gamma (IFN). Experiments were conducted to test the hypothesis that serum and growth factors alter the susceptibility of granulosa cells to FasL-induced apoptosis. Granulosa cells were cultured in BM-FBS, BM containing insulin, transferrin, selenium, and BSA (BM-ITS), and in BM-ITS supplemented with insulin-like growth factor-I (IGF). Cells were susceptible to FasL-induced killing in BM-ITS (27% killing) but were resistant in BM-FBS and in BM-ITS containing IGF (P < 0.05 vs. killing in BM-ITS). Exposure of phosphatidylserine residues on the outer cell membrane, an early marker of apoptosis, was stimulated by FasL and prevented in the presence of IGF. Neutralization of IGF activity in serum with IGF binding protein 3 reduced the protective effect of FBS on FasL-induced killing (P < 0.05), suggesting that IGF is an inhibitory component in FBS. Cotreatment with IFN overcame the inhibitory effects of serum and IGF on FasL-induced killing (31% and 29% killing, respectively, P > 0.05), but IFN did not potentiate killing of cells cultured in BM-ITS. IFN increased expression of Fas antigen (Fas, the receptor for FasL) mRNA five- to sevenfold (P: < 0. 05) and increased immunostaining for Fas protein similarly in all types of media. Addition of the growth factors epidermal growth factor or basic fibroblast growth factor to BM-ITS also inhibited FasL-induced killing (P < 0.05), whereas keratinocyte growth factor, transforming growth factor, platelet-derived growth factor, FSH, and LH had no effect. In summary, FasL-induced killing is inhibited by FBS and certain growth factors. IFN increased expression of Fas similarly in all types of media but was required for FasL-induced killing only in BM containing FBS or IGF. Therefore, modulation of responsiveness to FasL-induced apoptosis by growth factors and IFN is not directly related to the level of Fas expression.

Responsiveness of mouse corpora luteal cells to Fas antigen (CD95)-mediated apoptosis.

Regression of the corpus luteum (CL) occurs by apoptosis. The Fas antigen (Fas) is a cell surface receptor that induces apoptosis in sensitive cells when bound to Fas ligand or agonistic anti-Fas monoclonal antibodies (Fas mAb). A potential role for Fas to induce apoptosis in dispersed CL cell preparations was tested in cells isolated from mice on Days 2-4 of pseudopregnancy. Total CL dispersates, containing steroidogenic luteal cells, fibroblasts, and endothelial cells, were cultured. The effect of pretreatment of cultures with cytokines interferon gamma (IFN) and tumor necrosis factor alpha (TNF) was examined because these cytokines demonstrated effects on Fas-mediated apoptosis in other cell types. Fas mAb had no effect on viability of CL cells cultured in 5% fetal bovine serum (FBS) and pretreated with or without IFN or TNF, but Fas mAb did kill 23% of the cells in cultures pretreated with IFN + TNF. Fas mRNA was detectable in cultured CL cells and was increased 2.1-, 2. 0-, and 11.8-fold by treatment with TNF, IFN, or IFN + TNF, respectively. CL cells treated with the protein synthesis inhibitor cycloheximide (CX) were killed by Fas mAb in the absence of cytokine pretreatment (34%); pretreatment with IFN or IFN + TNF further potentiated killing (62% and 96%, respectively), whereas pretreatment with TNF had no effect (42%). Cells cultured in medium supplemented with insulin, transferrin, and selenium instead of FBS were killed by Fas mAb in the presence of IFN (23%) or IFN + TNF (29%) but not in the presence of TNF. Cells derived from the mouse CL have a functional Fas pathway that is inhibited by FBS and activated by treatment with CX, IFN, and IFN + TNF.

Expression and activity of the Fas antigen in bovine ovarian follicle cells.

The Fas antigen is a cell surface receptor that triggers apoptosis when bound to Fas ligand (FasL). Studies were undertaken to determine whether the cow provides a suitable model to study the role of the Fas pathway in inducing apoptosis of ovarian cells during follicular atresia. Expression of Fas antigen mRNA and responsiveness to FasL-induced killing in vitro were measured. Effects of the cytokines tumor necrosis factor (TNF)-alpha and interferon-gamma (IFN) were studied because of previous demonstrations of their role in Fas-mediated apoptosis in other cell types. Fas antigen mRNA was detectable in cultured granulosa and theca cells, and expression was increased by treatment with IFN but not TNF. Granulosa and theca cells were resistant to FasL-induced killing unless pretreated with IFN. TNF had no effect on FasL-induced killing. Granulosa and theca cell cultures in which killing occurred in response to FasL stained positively for annexin V, an early marker for cells undergoing apoptosis. These results provide a basis for further studies using the bovine ovary to examine the role of the Fas antigen in follicular atresia.

Expression and function of Fas antigen vary in bovine granulosa and theca cells during ovarian follicular development and atresia.

Fas antigen is a receptor that triggers apoptosis when bound by Fas ligand (FasL). A role for Fas antigen in follicular atresia was studied in follicles obtained during the first wave of follicular development during the bovine estrous cycle (estrus is Day 0). Granulosa and theca cells were isolated from healthy dominant follicles and the two largest atretic subordinate follicles on Day 5, atretic dominant follicles on Days 10-12, and preovulatory follicles on Day 1. Fas antigen mRNA levels were highest in granulosa cells from subordinate as compared to other follicles, and lowest in theca cells from healthy Day 5 dominant as compared to other follicles. FasL alone had no effect on viability of granulosa or theca cells but became cytotoxic in the presence of interferon-gamma (IFN). IFN has been shown to induce responsiveness to Fas antigen-mediated apoptosis in other cell types. In the presence of IFN, killing of granulosa cells by FasL was greater in subordinate compared to healthy dominant follicles on Day 5, did not differ between healthy and atretic dominant follicles, and was similar in theca among all follicles. Granulosa cells from preovulatory follicles, which had been exposed to the LH surge in vivo, were completely resistant to FasL-induced killing. In summary, Fas antigen expression, and responsiveness to Fas antigen-mediated apoptosis, vary during follicular development.

Potentiation of Fas-mediated apoptosis of murine granulosa cells by interferon-gamma, tumor necrosis factor-alpha, and cycloheximide.

The Fas antigen is a transmembrane receptor belonging to the tumor necrosis factor-alpha (TNF) receptor family that, when activated by Fas ligand or agonistic antibodies, induces death by apoptosis. Although the presence of Fas antigen in ovarian tissues has been demonstrated, little is known about whether Fas antigen is functional in the ovary. This report shows that murine granulosa cells are initially resistant to antibody-induced Fas-mediated apoptosis, but will undergo apoptosis when cotreated with TNF and interferon-gamma (IFN) or cycloheximide (CX). Granulosa cells were obtained from follicles of 23-day-old mice 2 days after injection of PMSG. Twenty-four hours after plating, cells were pretreated with either 0 or 200 U/ml IFN, which has been shown to induce Fas antigen expression and is required for Fas-mediated killing in many cell types. At 48 h, cells were treated with 2 microg/ml control IgG, 2 microg/ml anti-Fas antigen antibody (Fas mAb), 10 ng/ml TNF, or Fas mAb and TNF. Cytotoxicity (percent killing) relative to control IgG was determined at 72 h by counting granulosa cells after trypsinization. In the absence of IFN, no cytotoxicity was observed. In the presence of IFN, neither TNF or Fas mAb alone was cytotoxic, but the combination of Fas mAb and TNF resulted in 25% killing (P < 0.05). Fas antigen messenger RNA (mRNA) was detectable in cultures not treated with cytokines and was increased 5-fold by TNF, 2-fold by IFN, and 17-fold by the combination of IFN and TNF. To test whether the presence of a labile inhibitor(s) of Fas-mediated killing in granulosa cells is the cause of resistance to Fas mAb, the protein synthesis inhibitor CX was used. Experiments were performed as described above, except that cells were treated with 0.5 microg/ml CX in conjunction with other treatments at 48 h. Fas mAb treatment in the presence of CX induced 25% cell death without IFN pretreatment and 38% with IFN (P < 0.05). TNF treatment in the presence of CX had no effect alone, but potentiated the effects of Fas mAb, resulting in 56% killing in the absence of IFN and 86% killing in the presence of IFN (P < 0.05). Cells stained positively for DNA fragmentation and annexin V binding, features characteristic of apoptosis. Because initial experiments showed that treatment with TNF alone increased Fas mRNA levels, the effect of pretreating cells for 24 h with TNF before treatment with Fas mAb was tested. Pretreatment with TNF or IFN alone did not promote Fas mAb-mediated killing, but combined pretreatment with TNF and IFN resulted in 25% killing in response to Fas mAb. Treatment of cells with the combination of IFN and TNF induced a 19-fold increase in Fas antigen mRNA levels. Corresponding increases in Fas antigen protein expression on the surface of cells in response to cytokine treatments were detected by immunocytochemistry. Human TNF did not duplicate the effects of mouse TNF in inducing Fas antigen mRNA expression and Fas mAb-induced killing. As human TNF interacts exclusively with the type I, but not the type II, TNF receptor in the mouse, potentiating effects of mouse TNF on the Fas pathway are probably mediated via the type II TNF receptor. The effects of cytokine treatments on levels of mRNA for FAP-1, an inhibitor of Fas-mediated apoptosis, were determined. FAP-1 mRNA was detectable in untreated granulosa cells, and levels were not altered by treatment with TNF and/or IFN. In summary, the Fas-mediated pathway of apoptosis is functional in mouse granulosa cells that are stimulated with IFN and TNF. These cytokines may function at least partially by increasing Fas antigen expression. Granulosa cells appear to have inhibitors of the Fas antigen pathway, as treatment with CX potentiates Fas-mediated death. TNF promotes Fas-mediated killing in the presence and absence of CX. Therefore, TNF is not likely to act simply by increasing Fas antigen expression or decreasing protein inhibitors of the Fas pathway, because TNF remains effec

Fas antigen-mediated apoptosis of ovarian surface epithelial cells.

The Fas antigen is a cell surface receptor that, when engaged by Fas ligand or specific agonistic antibodies, triggers apoptosis. The effect of an agonistic monoclonal antibody to mouse Fas antigen (Fas mAb, clone J02) on the viability of cells from dispersed mouse corpora lutea (CL cultures) was tested. Cultures were prepared by enzymatic digestion of CL from day 4-7 pseudopregnant mice. Cultures were pretreated with 0, 1, 10, 100, or 1000 U/ml murine interferon-gamma (IFN) at 72 h of culture. IFN has been shown to increase Fas antigen expression in a number of cell types. At 96 h (time zero), cultures were treated with Fas mAb or IgG. By 4 h after Fas mAb treatment, discrete homogeneous patches of cells within the cultures showed characteristic signs of apoptosis, including blebbing of cell membranes, detachment, and disappearance from the culture. CL cultures contain luteal, stromal, and endothelial cells; fibroblasts; and surface epithelial cells (OSE). Cells dying in response to Fas mAb were identified as OSE. Affected cells had the cobblestone appearance and distinct nuclei typical of epithelial cells. Unlike luteal cells, OSE did not stain with the lipophilic dye, Nile red. The cells did not stain with acetylated low density lipoprotein conjugated to the fluorescent marker octadecyl indocarbocyanine, a marker for endothelial cells and monocytes. Cells in patches stained positively for cytokeratin, a marker for epithelial cells. Fas-mediated cytotoxicity was quantified by counting the number of cells present in discrete patches of OSE 0 and 8 h after Fas mAb treatment. Fas mAb treatment had no effect in cultures pretreated with 0 or 1 U/ml IFN, but induced significant death of OSE in cultures pretreated with 10, 100, and 1000 U/ml IFN (37 +/- 11%, 54 +/- 18%, and 60 +/- 11%, respectively). There was no apparent effect of Fas mAb on other cell types within the CL cultures. To confirm that cells dying in response to Fas mAb were OSE, experiments were also performed on enriched cultures of OSE prepared by enzymatic digestion of the outer surface of the ovary. In enriched OSE cultures pretreated with 200 U/ml IFN, there was 44% killing in response to Fas mAb, whereas in cells not pretreated with IFN, there was no effect. In situ fluorescent end labeling of DNA in CL cultures indicated that treatment with IFN and Fas mAb induced DNA fragmentation in OSE typical of apoptosis. Immunocytochemistry of CL cultures indicated that Fas antigen was expressed in OSE pretreated with IFN. Quantitative reverse transcriptase-PCR showed that IFN pretreatment increased Fas antigen messenger RNA levels 2.3-fold in enriched cultures of OSE. In summary, OSE in CL cultures and enriched cultures of OSE undergo apoptosis in response to Fas mAb when pretreated with IFN. In vivo, OSE undergo programmed cell death before ovulation and rapidly proliferate to repair the surface of the ovulatory follicle after ovulation. Most ovarian cancers are derived from the OSE. The results have implications for both normal ovarian function and oncogenesis in the ovary.

Fas antigen-mediated apoptosis in human granulosa/luteal cells.

The Fas antigen is a transmembrane receptor that can trigger apoptosis in a variety of tumor and hematopoietic cells. Ovarian follicular atresia and luteolysis are thought to occur by apoptosis. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry, we demonstrated that human granulosa/luteal cells express the Fas antigen. An anti-human Fas antigen monoclonal antibody (Fas mAb; clone CH-11), which induces apoptosis in other cell types by binding to the Fas antigen, induced significant cell death (30%) in cultures pretreated with interferon gamma (IFN gamma). This agrees with studies on tumor cell lines showing that IFN gamma enhances cytotoxic effects of Fas mAb. Granulosa/luteal cells exhibited morphological characteristics typical of apoptosis, including membrane blebbing and condensed chromatin. DNA fragmentation into oligonucleosomal units of approximately 180 bp, typical of apoptosis, was detected at elevated levels in Fas mAb-treated cultures via 3' end-labeling and gel electrophoresis. Examination of cultured cells in situ for apoptotic DNA cleavage by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) indicated that more apoptotic death occurred in Fas mAb-treated cultures than in control cultures. Effects of hCG-induced luteinization of cultures on Fas mAb-induced cytotoxicity was examined: combined pretreatment with IFN gamma and hCG induced a synergistic increase in Fas mAb-induced cytotoxicity (40%) over that obtained with IFN gamma-pretreatment alone (15%). In summary, granulosa/luteal cells express the Fas antigen and are sensitive to Fas mAb-induced apoptosis. Human CG synergized with IFN gamma to increase Fas mAb-induced death.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Pseudomonas aeruginosa IgG subclass titers in patients with cystic fibrosis: correlations with pulmonary function, neutrophil chemotaxis, and phagocytosis.

To explore possible mechanisms for the association between elevated immunoglobulin levels and lower pulmonary function in cystic fibrosis patients, we measured serum IgG subclass levels and anti-P. aeruginosa IgG subclass titers and correlated levels with neutrophil phagocytosis and chemotaxis. Serum was obtained from 13 cystic fibrosis patients colonized with the same serotype of P. aeruginosa, 12 noncolonized patients, and 12 normal volunteers. All anti-P. aeruginosa IgG subclass titers were elevated in serum from colonized patients. IgG3 level and anti-P. aeruginosa IgG3 titer were inversely correlated with pulmonary function. Phagocytosis of P. aeruginosa by neutrophils correlated with serum IgG3 level and was increased by opsonization with serum from colonized patients. Chemotactic index was increased in serum from colonized patients and inversely correlated with pulmonary function chest roentgenogram score. Chemotactic index directly correlated with anti-P. aeruginosa IgG3 titer and serum IgG3. These data demonstrate that cystic fibrosis patients with increased IgG3 levels are in poorer clinical condition and that their serum enhances neutrophil function. Such patients may have increased pulmonary inflammation with subsequent lung damage.

Association of Epstein-Barr virus infection and pulmonary exacerbations in patients with cystic fibrosis.

We observed severe pulmonary exacerbations during primary Epstein-Barr virus (EBV) infection in adolescent patients with cystic fibrosis. Since EBV is not a known respiratory tract pathogen in cystic fibrosis, we studied retrospectively all EBV-susceptible patients ages 6 to 18 years with chronic Pseudomonas respiratory tract colonization hospitalized for a pulmonary exacerbation during an 18-month period. Patients with serologic evidence of primary EBV infection (n = 5) were compared to control patients without EBV (n = 7). Before admission the groups had similar pulmonary function tests, clinical scores and frequency of hospitalization. On admission patients with EBV had significant weight loss, lower pulmonary function tests and lower clinical scores compared with controls. All remained significantly different 6 months after admission. Frequency of exacerbations requiring hospitalization increased after EBV infection but remained unchanged in controls. Primary EBV infection can be associated with severe pulmonary exacerbations and subsequent deterioration in clinical course in cystic fibrosis patients.

Effects of hemolysis and storage on quantification of hormones in blood samples from dogs, cattle, and horses.

Veterinary diagnostic endocrinology laboratories frequently receive hemolyzed plasma, serum, or blood samples for hormone analyses. However, except for the previously reported harm done by hemolysis to canine insulin, effects of hemolysis on quantification of other clinically important hormones are unknown. Therefore, these studies were designed to evaluate effects of hemolysis on radioimmunoassay of thyroxine, 3,5,3'-triiodothyronine, progesterone, testosterone, estradiol, cortisol, and insulin in equine, bovine, and canine plasma. In the first experiment, hormones were measured in plasma obtained from hemolyzed blood that had been stored for 18 hours. Blood samples were drawn from pregnant cows, male and diestrous female dogs, and male and pregnant female horses. Each sample was divided into 2 equal portions. One portion was ejected 4 times with a syringe through a 20-gauge (dogs, horses) or 22-gauge (cows) hypodermic needle to induce variable degrees of hemolysis. Two subsamples of the blood were taken before the first and after the first, second, and fourth ejections. One subsample of each pair was stored at 2 to 4 C and the other was stored at 20 to 22 C for 18 to 22 hours before plasma was recovered and stored at -20 C. The second portion of blood from each animal was centrifuged after collection; plasma was recovered and treated similarly as was blood. Concentrations of thyroxine in equine plasma, of 3,5,3'-triiodothyronine, estradiol, and testosterone in equine and canine plasma, and of cortisol in equine plasma were not affected by hemolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of 6 and 8 hourly tobramycin dosing intervals in treatment of pulmonary exacerbations in cystic fibrosis patients.

The efficacy and toxicity of a shortened tobramycin dosing interval in the treatment of exacerbations of Pseudomonas aeruginosa pulmonary infection in cystic fibrosis patients were evaluated prospectively. Patients ages 13 to 30 years received 34 treatment courses and were randomized by pairs to receive tobramycin administered either every 6 or 8 hours. Peak serum concentrations were adjusted to 8 to 10 micrograms/ml; thus a larger total daily dosage was administered to patients receiving tobramycin every 6 hours. The shorter dosing interval was associated with better pulmonary function at follow-up and significantly longer time before next hospital admission for a pulmonary exacerbation. During the study hospitalization there were no differences in pulmonary function tests, clinical score, sputum carriage of P. aeruginosa, toxicity or necessary length of hospitalization. A 6-hour tobramycin dosing interval was more efficacious than an 8-hour dosing interval in the treatment of cystic fibrosis patients.

Respiratory tract colonization with Pseudomonas aeruginosa in cystic fibrosis: correlations between anti-Pseudomonas aeruginosa antibody levels and pulmonary function.

Chronic Pseudomonas aeruginosa respiratory tract colonization in patients with cystic fibrosis is associated with development of antibodies to the organism. In contrast to the protection usually afforded by humoral immunity to a bacterial pathogen, the immune response to P. aeruginosa may help perpetuate infection and contribute to pulmonary damage in cystic fibrosis. To determine if specific anti-P. aeruginosa antibody levels correlated with pulmonary dysfunction, we measured antibodies to seven P. aeruginosa serotypes, and correlated the geometric mean titer with pulmonary function tests. Patients were divided into groups without P. aeruginosa colonization (n = 20), with recent colonization (n = 20), and with chronic colonization (n = 60). Noncolonized patients had normal pulmonary function or mild obstructive lung disease, and low anti-P. aeruginosa titers. Pulmonary function tests in recently colonized patients were not different from those of noncolonized patients, but antibody titers were higher. Following colonization FEV1 declined and titers increased rapidly. Patients with chronic colonization had worse pulmonary function and higher titers, but while the former were stable the latter gradually increased. An inverse correlation was found between anti-P. aeruginosa titer and FVC, FEV1, and FEF25-75 (P less than 0.001) in these patients; age was not a factor. The strong correlation between severity of lung disease and anti-P. aeruginosa titer demonstrates that an exaggerated immune response to P. aeruginosa is associated with pulmonary damage in patients with cystic fibrosis.

Intravenous immune globulin treatment of pulmonary exacerbations in cystic fibrosis.

The effect of intravenously administered immune globulin (IVIG) on patients with cystic fibrosis with an acute exacerbation of pulmonary infection was evaluated in a double-blind study. Patients at least 12 years of age, with chronic respiratory tract colonization with Pseudomonas aeruginosa and hospitalized with a reduction in pulmonary function, were randomly assigned to receive 20% dextrose (control subjects: n = 8) or 100 mg/kg IVIG (Gamimune) (experimental subjects: n = 8) on days 1, 2, and 3; all patients received intravenous antibiotics and chest physiotherapy. There were no differences between groups on admission; patients had moderate to severe disease as measured by Shwachman-Kulczycki scores and pulmonary function tests. Both groups improved clinically. The IVIG treatment was associated with significant increases in forced vital capacity and forced expiratory volume in 1 second (p less than 0.01) and with greater percent improvement in forced expiratory volume and forced expiratory flow (25% to 75%) (p less than 0.05). There was no effect on length of hospitalization (18.3 +/- 11.9 days control vs 17.6 +/- 6.5 experimental). The C3 level was decreased at discharge in IVIG-treated patients; circulating immune complex levels were unchanged. One patient in each group experienced side effects. There were no differences on follow-up at 6 weeks. We conclude that IVIG infusion early in treatment for pulmonary exacerbations in cystic fibrosis patients with moderate to severe disease may be associated with greater improvement in pulmonary function than standard treatment alone.

Calcium-dependent translocation of calbindin-D28k from intestine to blood.

Calbindin-D (vitamin D-induced calcium-binding protein; CaBP) is known to be present in blood at concentrations which vary directly with levels in the intestinal mucosa. Employing a sensitive radioimmunoassay and sampling mesentery venous blood, the present experiments demonstrated a direct relationship between intestinal calcium absorption and serum CaBP. Solutions containing 150 mM NaCl and 45Ca-labeled calcium chloride (5 or 20 mM) were placed in the lumen of ligated duodenal preparations in situ and mesentery venous blood sampled with time. The concentration of absorbed 45Ca in serum was maximal at 5 min, followed by a significant increase in mesentery CaBP maximizing at 15-20 min. Elevation of serum CaBP was not observed when calcium in the dosing solution was omitted or replaced by either glucose or glycine. The possible transfer of absorbed calcium from the enterocyte to the circulation as a CaBP complex was ruled out by calculations revealing that considerably more calcium was transferred than could be accounted for by the low and high affinity binding sites on the protein. It is proposed that vitamin D-dependent enhanced transcellular calcium transport constitutes a stimulus for the increased release of intestinal CaBP into the circulation.

Circannual changes in serum testosterone concentrations of adult and yearling woodchucks (Marmota monax).

Testicular volumes and serum testosterone concentrations were determined biweekly in 5 adult and 4 yearling woodchucks maintained indoors from December through August. Food and water were provided ad libitum except for 2 mo beginning at the winter solstice when feeding and lighting (12L:12D) supplemental to available natural light were discontinued. Temperatures fluctuated with outdoor temperatures greater than 4 degrees C. No significant hibernation occurred. Testes in adults were small in December (0.3-1.8 cm3), largest in February and March (3.5-5.6 cm3), and smallest in late June (0.1-0.5 cm3). Testosterone was basal in December (less than 0.6 ng/ml), maximal (3.4-6.6 ng/ml) between early January and late March, and minimal from April through August (less than 0.8 ng/ml). In yearlings, maximum testes volumes (1.6 cm3) and serum testosterone (0.9 +/- 0.2 ng/ml) were less, and occurred later, than in adults. Testosterone levels and testis volumes measured in newly captured woodchucks in March and April and again 2-3 mo later were generally similar to those of their laboratory counterparts. Thus, in woodchucks: annual cycles of testosterone production and of testes recrudescence and regression parallel each other with maxima during the short, late-winter breeding season; those cycles are not altered significantly by the absence of hibernation or the present conditions of captivity; and yearling males apparently are not an important part of the breeding population.