A compendium of antibiotic-induced transcription profiles reveals broad regulation of Pasteurella multocida virulence genes.

The transcriptional responses of Pasteurella multocida to eight antibiotics with known mode of actions (MoAs) and one novel antibiotic compound with an unknown MoA were collected to create a compendium of transcriptional profiles for MoA studies. At minimal inhibitory concentration the three bactericidal compounds enrofloxacin, cefquinome and the novel compound had a minor impact on gene regulation with approximately 1% of the P. multocida genome affected, whilst the bacteriostatic compounds florfenicol, tilmicosin, rifampin, trimethoprim and brodimoprim regulated 20% of the genome. Novobiocin was special in that it regulated 40% of all P. multocida genes. Regulation of target genes was observed for novobiocin, rifampin, florfenicol and tilmicosin and signature genes were identified for most antibiotics. The transcriptional profile induced by the novel compound was unrelated to the compendium profiles suggesting a new MoA. The transcription of many P. multocida virulence factors, particularly genes involved in capsule synthesis and export, LPS synthesis, competence, adherence and iron transport were altered in the presence of antibiotics. Virulence gene transcription was mainly negatively affected, however the opposite effect was also observed in the case of rifampin where the up-regulation of the tad locus involved in tight adherence was seen. Novobiocin and trimethoprim caused a marked reduction in the transcription of capsule genes, which correlated with a concomitant reduction of the capsular layer on the surface of P. multocida. The broad negative impact on virulence gene transcription supports the notion that the therapeutic effect of some antibiotics could be a combination of growth and virulence inhibition.

Prostaglandin-induced programmed cell death in Trypanosoma brucei involves oxidative stress.

Recently, we reported the induction of a programmed cell death (PCD) in bloodstream forms of Trypanosoma brucei by prostaglandin D(2) (PGD(2)). As this prostanoid is readily metabolized in the presence of albumin, we were prompted to investigate if PGD(2) metabolites rather than PGD(2) itself are responsible for the observed PCD. In fact, J series metabolites, especially PGJ(2) and Delta(12)PGJ(2), were able to induce PCD more efficiently than PGD(2). However, the stable PGD(2) analog 17phenyl-trinor-PGD(2) led to the same phenotype as the natural PGD(2), indicating that the latter induces PCD as well. Interestingly, the intracellular reactive oxygen species (ROS) level increased significantly under J series metabolites treatment and, incubation with N-acetyl-L-cysteine or glutathione reduced ROS production and cell death significantly. We conclude that PGJ(2) and Delta(12)PGJ(2) formation within the serum represents a mechanism to amplify PGD(2)-induced PCD in trypanosomes via ROS production.

Effects of adrenergic receptor activation and blockade on the systolic preejection period, heart rate, and arterial pressure in man.

We have investigated the possibility that alterations in the duration of the systolic preejection period can be used to estimate adrenergic influences on the human left ventricle. The preejection period was determined from high speed, simultaneous recordings of the phonocardiogram, carotid pulse tracing, and electrocardiogram. The preejection period was shortened by isoproterenol, epinephrine, and moderate doses of norepinephrine-all of which activate beta adrenergic receptors-and by cedilanid-D. It was unaltered by changes in heart rate induced by atropine and right atrial electrical pacing. Beta adrenergic receptor blockade by propranolol abolished the shortening effects of the three catecholamines but did not inhibit that due to cedilanid-D. Vasoconstriction, both alpha adrenergic (epinephrine and norepinephrine after propranolol) and nonadrenergic (angiotensin), prolonged the preejection period. Most of the shortening of the preejection period by beta adrenergic receptor activating agents and cedilanid-D and all of the prolongation accompanying pharmacologic vasoconstriction occurred after the onset of the first heart sound, thereby excluding changes in electrical-mechanical delay as a major factor in the observed preejection period responses. Shortening of the preejection period by beta adrenergic activity induced with isoproterenol was dose-related. Increasing doses of propranolol produced parallel shifts to the right in the isoproterenol dose-response curve. In 37 normal resting subjects intravenous propranolol (10 mg) prolonged the preejection period an average of 10 (SE +/- 1) msec. In six patients with psychogenic sinus tachycardia and a patient with a pheochromocytoma the presence of excessive beta adrenergic influences on the left ventricle was demonstrated by the finding of an initially short preejection period which responded with an abnormally great prolongation to beta adrenergic receptor blockade.

Effect of anti-relaxin antiserum on sperm motility in vitro.

Rabbit anti-relaxin antisera, but not normal rabbit sera, causes a rapid decline of motility of washed human sperm. Preincubation of the antisera with relaxin eliminates this effect. This sperm immobilization effect can serve as a basis of a rapid screening test for anti-relaxin antisera and as a novel adjuvant to barrier contraceptive methods.

Hyalocytes synthesize and secrete inhibitors of retinal pigment epithelial cell proliferation in vitro.

BACKGROUND: Retinal pigment epithelial (RPE) cells that enter the vitreous in pathologic conditions, such as retinal detachment, may proliferate and contribute to the formation of epiretinal membranes. OBJECTIVE: To study whether hyalocytes, endogenous vitreous cells, play a role in modulating the proliferation of RPE cells. METHODS: Cell proliferation was measured by tritiated thymidine incorporation in density-arrested human RPE cells after incubation with media that had been conditioned by cultured bovine hyalocytes. Preliminary characterization of inhibitory activity in hyalocyte-conditioned medium was performed, including blocking experiments with a neutralizing antibody to transforming growth factor-beta 2 (TGF-beta) and proliferation assays that used MV-1-Lu mink lung epithelial cells. Northern blots were done to asses hyalocyte expression of TGF-beta messenger RNA. RESULTS: Hyalocyte-conditioned medium inhibited tritiated thymidine incorporation in RPE cells and MV-1-Lu mink lung epithelial cells in the presence or absence of serum or protease inhibitors. A portion of the inhibitory activity was neutralized by an antibody directed against TGF-beta. Northern blots of hyalocyte RNA demonstrated the presence of messenger RNA for TGF-beta 2. These data suggest that TGF-beta is responsible for a portion of the inhibitory activity secreted by hyalocytes. Additional inhibitory activity is attributable to one or more low-molecular-weight molecules distinct from TGF-beta. CONCLUSIONS: Hyalocyte-conditioned medium inhibits RPE cell proliferation in vitro through TGF-beta and at least one other molecule. Production of these factors by hyalocytes in vivo could provide a deterrent for epiretinal membrane formation that may be perturbed under pathologic conditions.

Upregulation of vascular endothelial growth factor in ischemic and non-ischemic human and experimental retinal disease.

Vascular endothelial growth factor (VEGF) is induced by hypoxia and it has been implicated in the development of iris and retinal neovascularization (NV) in ischemic retinopathies in which it has been suggested that Muller cells are responsible for increased VEGF production. VEGF, however, is also known to be a potent mediator of vascular permeability in other tissues and may perform this function in retina. Immunohistochemical staining for VEGF was performed on a variety of human and experimental ischemic and non-ischemic ocular disorders in which blood retinal barrier (BRB) breakdown is known to occur to determine if there is an upregulation of VEGF in these conditions. We found increased VEGF immunoreactivity in ganglion cells of rats with oxygen-induced ischemic retinopathy and in ganglion cells, the inner plexiform layer, and some cells in the inner nuclear layer of rats with experimental autoimmune uveoretinitis (EAU), in which there was no identifiable ischemia or NV. In rats with EAU, VEGF staining intensity increased from 8 to 11 days after immunization, coincident with BRB failure. These results were confirmed using two distinct anti-VEGF antibodies and by immunoblot and the immunohistochemical staining was eliminated by pre-incubating the antibodies with VEGF peptide. VEGF staining was also increased in the retina and iris of patients with ischemic retinopathies, such as diabetic retinopathy and retinal vascular occlusive disease, and in patients with disorders in which retinal ischemia does not play a major role, such as aphakic/ pseudophakic cystoid macular edema, retinoblastoma, ocular inflammatory disease or infection, and choroidal melanoma. VEGF was primarily localized within retinal neurons and retinal pigmented epithelial cells in these cases. In addition or in association with its role of inducing NV, VEGF may contribute to BRB breakdown in a variety of ocular disorders and blockage of VEGF signaling may help to reduce some types of macular edema.

A splice variant of trkB and brain-derived neurotrophic factor are co-expressed in retinal pigmented epithelial cells and promote differentiated characteristics.

There is evidence suggesting reciprocal trophic interactions between photoreceptors and the retinal pigmented epithelium (RPE), but the factors involved have not been identified. In this study, we investigated the hypothesis that one or more known neurotrophic factors act upon the RPE. Cultured human and freshly isolated bovine RPE cells demonstrated saturable specific binding for [125I]labeled BDNF, NT-4/5 and NT-3 with little specific binding for CNTF and none for NGF. Cross-competition experiments showed that BDNF is the preferred ligand and cross-linking of [125I]BDNF resulted in a doublet at 160 kd that was increased in RPE cells incubated in all-trans retinoic acid. There was basal phosphorylation of a 145 kd protein recognized by an anti-trk antibody that was increased in RPE cells pulsed with BDNF. RT-PCR with primers spanning the transmembrane domain demonstrated that RPE cells express trkB mRNA lacking a region homologous to exon 9 of chicken trkB, a splice variant that has been demonstrated to preferentially interact with BDNF. Northern blots demonstrated that cultured RPE cells also express mRNA for BDNF. BDNF did not stimulate proliferation or increase survival of RPE cells in serum-free medium, but promoted a differentiated morphology and increased the expression of cellular retinaldehyde binding protein, a marker of the differentiated state in RPE cells. An RPE cell line that spontaneously shows differentiated features showed a high level of BDNF mRNA. These data demonstrate that RPE cells express a short splice variant of trkB whose activation correlates with expression of differentiated characteristics and the cells themselves are capable of producing a ligand for the receptors. Signaling through trkB could play a role in differentiation of RPE cells during development and maintenance of the differentiated state in adult RPE.

Sperm motility.

PIP: Current literature dealing with sperm motility is reviewed. Laboratory observation has led to the hypothesis that the sperm tail moves when the microtubules, powered by adenosine triphosphate (ATP), slide past one another, moving by means of their dynein arms, which are actual molecules of ATP activity. This sliding microtubule hypothesis is considered to explain the motility of the 9+2 sperm tails and all cilia and flagella. The clinical usefulness of determinations of various glycosidases in semen has been assessed in recent years. The neutral alpha-glycosiderase specific activity of sperm has been correlated with the percentage motility. It is postulated that an abnormal function of the epididymis may be related to decreased production of alpha-glucosidases. The subjective nature of visual assessment of motility is a persistent problem for researchers. Objective appraisal is hampered by variation in the appraisals of different researchers and striking, unexplained differences in motility in successive visits. Quantitative methods that can provide reproducible information on overall motility and percentage of motile sperm and can distinguish the speed of progression from ineffective vibrating and circling motions are not yet available to clinicians. Factual knowledge of the process of sperm maturation in the epididymis and the special quality of the environment in the epididymis that supports maturation or sperm storage is lacking. Various groups of compounds have been used to stimulate the in vitro motility of sperm, including methyl xanthines, nucleotides, carnitine and acetylcarnitine, arginine, and kallikrein. Sperm washing will occasionally enhance motility. There is agreement that sperm lose their motility within 15 days after vasectomy. The split-ejaculate method has demonstrated that the distribution of sperm cells is not uniform throughout the ejaculatory process. Insemination with the 1st portion of the split ejaculate has been effective in achieving pregnancy in infertile couples. Therapy for increasing sperm motility includes low dosage androgen and human chorionic gonadotropin. Research has been directed at improving the survival of motile sperm during the process of freezing, storing, and thawing human semen. However, storage of fertile semen does not guarantee future fertility.^ieng

Clinical experience with sperm antibody testing.

Of the 724 patients examined for circulating spermagglutinating antibodies by the Kibrick spermagglutination test, 95 (13.1%) were found to be positive. Almost 20% of the 406 male patients demonstrated autoantibodies. This high incidence may be explained by the selected population studied. Of 50 males tested who demonstrated more than 10% agglutination in their semen, but otherwise were normospermic, 42% demonstrated autoantibodies. Of the 318 females examined, 14 (4.4%) demonstrated sperm-agglutinating antibodies. Of the 315 male and female patients examined for circulating sperm-immobilizing antibodies by the Isojima sperm-immobilization test, 14 (4.5%) were found to have antibodies. Of the 162 males treated, 10 (6%) were positive for antibodies by this technique, as were 4 (3%) of the 153 women tested.

Stimulation of ejaculated human spermatozoa by caffeine.

Caffeine (1, 3, 7-trimethyl-2, 6-dioxypurine), a cyclic nucleotide phosphodiesterase inhibitor, increased the motility of ejaculated human spermatozoa. Both the percent motility and the grade of forward progression were significantly increased (P smaller than 0.001) and maintained in the presence of 6 mM of caffeine for up to five hours at 37 C. Increases in percent motility greater than two-fold occurred among the groups having low initial motility. Increases in samples with a high initial percent motility (greater than 60%) were not as dramatic, but they were still significant. In addition, the grade of motility or forward progression was increased in all groups up to one full unit. The longevity of the spermatozoa, judged by activity, was also increased for up to five hours.

Circulating sperm-agglutinating antibodies in azoospermic men with congenital bilateral absence of the vasa deferentia.

Congenital bilateral absence of the vasa deferentia has been found to be the cause for azoospermia in 101 patients who have been examined in our urologic practice since 1956. This represents approximately 2% of the 5112 patients seen during that time because of problems relating to infertility. Of these 101 men, 29 have had their sera tested for the presence of circulating sperm-agglutinating antibodies. The Shulman15 modification of the macroscopic gelatin sperm-agglutination test of Kibrick et al6 was used. None of these patients had had testicular biopsy. Significantly high titers (1:32 or greater) of antibodies were found in 18 (62.1%) men, low titers (1:16 or less) in five (17.2%), and no antibodies in six (20.7%).

Evaluation of a new silastic seminal fluid collection device.

A new Silastic seminal fluid collection device has been tested and compared in vitro with glass jars, latex condoms, and the Milex polyethylene sheath. In a series of tests utilizing 50 different semen samples and observing the effects on motility over a period of 4 hours, the Silastic seminal fluid collection device demonstrated sperm survival slightly better than that with glass jars, superior to that with the polyethylene Milex sheath, and far greater survival (by several hours) than with the latex condom. The Silastic collection device was found to be more comfortable and more pleasurable to the patients than was the Milex sheath. The accuracy of seminal volume measurements was also found to be better in the Silastic collection device.

Prolactin, fructose, and zinc levels found in human seminal plasma.

Fructose, zinc, and prolactin determinations were performed on the seminal plasma of normospermic (n = 30), oligospermic (n = 30), and azoospermic (n = 30) men to determine whether there was a correlation between any of these substances and seminal quality. None of these azoospermic men had congenital bilateral absence of the vasa, but in a separate group of such men (n = 10) these same studies were performed. Fructose was determined photometrically, zinc by atomic absorption, and prolactin by radioimmunoassay. Zinc and fructose levels in all three groups showed no statistical differences. Prolactin was found to be significantly higher in the seminal plasma of normospermic men than was found in the seminal plasma of oligospermic or azoospermic men. In men with congenital bilateral absence of the vasa and seminal vesicles, zinc was elevated, fructose was absent, and prolactin was markedly depressed.

Successful management of infertility due to polyzoospermia.

Polyzoospermia is a rare cause of male infertility. In our clinical practice, patients with exceedingly high sperm counts (ranging from 650 million/ml to 1.75 billion/ml) have invariably been found to have associated poor sperm motility and poor sperm migration in postcoital tests, although the sperm appear morphologically normal. Cases of two infertile couples are presented in which each husband had sperm counts greater than 1 billion/ml. Two methods are reported for the successful management of their infertility problems: (1) precoital dilutional douching and (2) in vitro seminal fluid dilution with 5% dextrose-Ringer's lactate solution and artificial insemination homologous. Each method resulted in improved sperm motility, postcoital tests, and pregnancy.

Stimulation of human sperm motility by relaxin.

Relaxin has recently been identified in human seminal plasma (SP). This investigation was undertaken to determine the effect of relaxin on sperm motility. Washed ejaculated human sperm were incubated with or without porcine relaxin. In a series of 37 normal specimens (motility greater than or equal to 60%) relaxin attenuated the decline in grade of forward progression and percentage of motile sperm. Relaxin had a similar effect on a series of 47 subfertile specimens (motility 30% to 40%). Endogenous relaxin may play a role in delaying declines of motility and grade of forward progress in ejaculated sperm.

Follicle-stimulating hormone, luteinizing hormone, and testosterone levels found in human seminal plasma.

Radioimmunoassays were performed on the seminal plasma of normospermic, oligospermic, and azoospermic men to determine the levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone. FSH levels in the seminal plasma of all three groups were found to be similar to the levels normally found in blood serum. LH levels in the seminal plasma of azoospermic and oligospermic men were within the normal range found in blood serum but were elevated in the normospermic samples. Testosterone levels in the seminal plasma of all three groups tested were considerably lower than the normal range found in blood serum.

The effect of an anti-insulin antiserum on human sperm motility.

Inhibition of sperm motility by anti-relaxin antiserum has been previously reported. This effect may be secondary to a nonspecific effect of immunoglobulins binding to sperm or to a specific inhibition of relaxin activity at the sperm surface. The current investigation demonstrates that an anti-insulin antiserum does not inhibit sperm motility in the same system in which an anti-relaxin antiserum results in inhibition. This supports the hypothesis that the anti-relaxin antiserum effect is specific.

Stimulation of human sperm cervical mucus penetration in vitro by relaxin.

The effect of relaxin, a peptide hormone present in seminal plasma, on the penetration of cervical mucus by washed human sperm in vitro was studied. Penetration was analyzed by two methods. The total number of sperm per 100 X field at the 10-mm, 20-mm, and 30-mm points along a mucus-filled capillary tube was counted and the farthest distance 100 sperm and 5 sperm/100 X field penetrated the capillary tube was measured. These measurements were made at 30, 60, and 90 minutes. Treating washed sperm with physiologic concentrations of relaxin resulted in significantly increased sperm penetration as compared with sperm treated with albumin or buffer. Replacing seminal plasma to washed sperm resulted in equivalently increased penetration. The findings strongly suggest that relaxin is necessary for proper penetration of cervical mucus by human sperm.

The effect of relaxin and prostaglandin E2 on the motility of human spermatozoa.

Relaxin and prostaglandin E2 (PGE2) are present in human semen and have been shown to affect sperm motility. The authors further examined the effects of porcine relaxin and PGE2 on the motility of human spermatozoa. A dose-response study revealed that PGE2 at a concentration of 25 micrograms/ml is most effective in improving the motility of washed human sperm. Relaxin (100 ng/ml), PGE2 (25 micrograms/ml), or the two combined have no effect on the motility of spermatozoa in fresh, normal semen, suggesting that the constituents of fresh semen are optimal for motility. Relaxin and PGE2 individually improve the motility of washed spermatozoa. However, relaxin, but not PGE2, improves the motility of sperm in semen incubated at 37 degrees C for 5 hours (aged). In contrast to the individual substances, a combination of relaxin + PGE2 has no effect on the motility of washed spermatozoa or aged spermatozoa, suggesting that these two substances antagonize each other's actions on sperm motility. The presence of both relaxin and PGE2 in seminal plasma with normal motility spermatozoa suggests that other factors in seminal plasma regulate the effects of these substances on sperm motility.

Human semen relaxin and its correlation with the parameters of semen analysis.

The relaxin content of 92 normal semen samples and 85 semen samples selected at random was correlated to the parameters measured on routine semen analysis in those samples. The concentration of immunoactive relaxin in a sample of semen plasma did not correlate with any of the parameters commonly used for semen analysis: count, percent motility, grade of forward progression, volume, and morphology.