Atopic dermatitis -- topical therapy: do patients apply much too little?

BACKGROUND: The treatment of atopic dermatitis still remains a challenge. Little research has been done on the issue of the extent to which patients correctly use prescribed topical preparations under everyday conditions. AIMS: To investigate what quantity of topical preparations is applied by outpatients in daily routine treatment over a 26-week period and to what extent this consumption is related to the course of the severity of patients' skin conditions. METHODS: Thirty adult outpatients (20 female and 10 male) with atopic dermatitis were examined at four different times during 26 weeks. For treatment and skin care these patients were given a topical glucocorticoid preparation (prednicarbate) and the corresponding emollient. RESULTS: The average severity rating (SCORAD) was 29.6 (before therapy 33.9, after 26 weeks 27.4). The SCORAD indices improved by a mean of 6.5 points (p<0.05). CONCLUSION: Patients who applied the correct amount of the prednicarbate-containing preparations (not less than 90% of 0.5 g/dm(2)) to the areas of affected skin showed a significant improvement in SCORAD indices across the four measuring times.

ADP-ribosylation of Rho proteins inhibits sperm motility.

The highly homologous Rho proteins RhoA, RhoB and RhoC are low-molecular-mass GTP-binding proteins. They are selectively ADP-ribosylated by Clostridium botulinum ADP-ribosyltransferase C3 (C3 exoenzyme). The biological function of the Rho proteins is still unclear; there is evidence that they are involved in the regulation of the filamental network of cells. Here we report that C3 exoenzyme-like toxins ADP-ribosylate small GTP-binding proteins in bovine spermatozoa and inhibit sperm motility. These findings indicate that Rho proteins which reportedly regulate the microfilament system are basically involved in sperm motility.

Rapid serological analysis of bacterial lipopolysaccharides by electrotransfer to nitrocellulose.

Techniques are described for the rapid screening of proteinase K-treated bacterial lysates by electroblot and immunoenzymatic detection to assess O-specificity of antigens and antisera. Conditions are outlined which permit the use of a single polyacrylamide gel for both electrotransfer to nitrocellulose and silver staining. Immunodetection of transferred LPS bands was equally sensitive to silver stain when whole cell or O-specific antisera were used. The techniques were utilized to identify at least 4 O-serotypes among sorbitol fermenting isolates of the fish pathogen, Yersinia ruckeri. Observed variations in the electrophoretic mobilities of lipopolysaccharides from 17 field isolates of Y. ruckeri were used to accurately predict the O-serotype.

Guidelines for drug treatment of male infertility.

The prerequisite for rational therapy of male fertility disorders is an exact diagnosis. While the possibilities of influencing disturbances of spermiogenesis are limited, male adnexal diseases can be successfully treated in many cases. Drugs for the treatment of fertility disorders must be applied with this in mind, and empiric therapy is often performed in addition to causal treatment which, however, may be quite rationally determined. The therapeutic spectrum in andrology includes antibiotic and antiphlogistic agents, mast cell blockers, zinc, vitamins, and immunosuppressive drugs (corticosteroids). These agents are used for the treatment of inflammatory diseases of the testes and the accessory glands or for suppression of antispermatozoal antibodies. Hormonal disturbances are infrequently encountered by the andrologist, but they can be treated, with proven efficacy, with gonadotrophins, gonadotrophin-releasing hormone (GnRH) or androgens. In certain cases that are not hormonally related, the use of antiestrogens (clomifene, tamoxifen) as stimulating agents may be successful. Furthermore, tissue hormone releasing proteases (kallikrein) can be used both therapeutically (especially in motility disturbances that are not due to structural flagellar defects) and diagnostically (in order to distinguish between inflammatory and noninflammatory testicular damage). Anticholinergics and alpha-sympathomimetics are applied to ameliorate ejaculation or emission failure. In addition to a review of these treatment forms, the development of new concepts, e.g. angiotensin converting enzyme (ACE) inhibitors, is discussed.

Treatment of male fertility disturbances. Current concepts.

Medical therapy of male infertility aims to improve or normalise the fertility status of a subfertile patient. However, this can be a frustrating task due to limited knowledge about the pathophysiology of male reproductive functions, and the fact that pharmacological therapy is mainly empirical and less often specific. Nevertheless, the spectrum of treatment approaches has increased within the last decade and comprises hormonal and non-hormonal compounds. Hormonal therapy is performed with antioestrogens (clomiphene, tamoxifen), gonadotrophin-releasing hormone (GnRH), prolactin inhibitors (bromocriptine), gonadotrophins (hMG, hCG), androgens (testosterone, mesterolone), and testosterone aromatase inhibitors (testolactone). Tissue hormone-releasing proteases (kallikrein) can also be applied, liberating kinins as mediator substances with different effects at the cellular level. Non-hormonal therapy includes improvement of testicular microcirculation by oxpentifylline, antimicrobial and anti-inflammatory agents, drugs to improve or allow emission and ejaculation, and psychotropic and antispasmodic drugs to diminish functional disturbances induced by emotional stress. Treatment schedules are either specifically or empirically based. If treatment is based on a pathophysiological concept which implies strong patient selection, success of treatment is excellent. In contrast, despite an increased number of compounds, empirically based therapies remain unpredictable and the results are moderate and often not reproducible. However, when different drugs are compared with a placebo group in selected, well-controlled patients with idiopathic normogonadotrophic oligozoospermia, pregnancy rates will be in the range of 30 to 40% within an observation period of 1 year, as compared with the spontaneous conception rate of between 10 and 20%.

Production and characterization of monoclonal antibodies to the major protein of boar outer dense fibers.

Outer dense fibers (ODF) are structural elements in the mammalian sperm tail which surround the axoneme in the midpiece and principal piece and probably may help to maintain the elastic structures and elastic recoil of the sperm tail. In the present study, we have generated and characterized and describe a series of monoclonal antibodies (mAbs) against the 30 kDa major protein from boar ODF. For antibody screening an ELISA was developed using a newly developed method to fix the ODF proteins to the solid phase. A total of seven mAbs were selected and characterized by ELISA, Western blot and immunofluorescence. The mAbs recognize the major protein component of boar ODF on preparative Western blot and mark the mid- and principal piece of demembranated flagella. These mAbs also recognize the mid- and principal piece of demembranated human spermatozoa from normozoospermic patients, but not from those with asthenozoospermia. For the first time, we succeeded in obtaining hybridoma cell lines that secrete mAbs of class IgM, which react with the 30 kDa protein of boar ODF.

Acrosin activity of cryo-preserved human spermatozoa.

The acrosin activity of human spermatozoa was determined in fresh, glycerolated, and cryo-preserved semen specimens. In 119 semen specimens, mean increases in acrosin activity of 62% in glycerolated and 56% in cryo-preserved samples were found. Thus, no statistically significant differences in mean acrosin activity were found between glycerolated and cryo-preserved spermatozoa. However, 23.5% of the cryo-preserved samples showed a significant decrease in extractable acrosin activity when compared with untreated controls. In oligospermic specimens (less than 40 million spermatozoa/ml), a statistically significant decrease in acrosin activity due to cryo-injury was detectable. Differentiation of specimens responding to glycerol pretreatment with an increase in extractable acrosin activity from those responding with a decrease showed a different freezing behavior, indicating two ejaculate types with respect to acrosin extraction. Samples frozen without glycerol protection showed the same amounts of extractable acrosin activity as did glycerol-protected specimens. The stability of acrosin in acidic acrosomal extracts during liquid nitrogen freeze treatment was confirmed.

Antisperm antibodies in infertile and homosexual men: relationship to serologic and clinical findings.

The incidence and significance of antisperm antibodies in different groups of men were evaluated by a modified enzyme-linked immunosorbent assay. In serum, 4.0% of dermatologic patients (n = 223), 9.6% of andrologic patients (n = 178), and 28.6% of homosexual men (n = 42) were positive for IgG and/or IgM antibodies. In seminal fluids, 7.3% of the andrologic patients had IgA (and IgG) antibodies to spermatozoa. Only 1 of 29 positive men had antibodies both in serum and in seminal fluid. No correlation between antisperm antibodies and IgG/IgM concentrations was found in serum, whereas in seminal plasma men with antisperm antibodies showed higher IgG/IgA concentrations than men without (IgA, 3.2 versus 1.7 mg/dl; IgG, 9.8 versus 6.3 mg/dl). It is concluded that there is a high incidence of antisperm antibodies among homosexual men, probably because of contact of spermatozoa with the immune system by passive anal intercourse. There is little correlation between antisperm antibodies in serum and seminal plasma of infertile men because of a lack of relevant antibody transfer from the serum and the formation of local antibodies in seminal plasma. Antisperm antibodies in seminal fluid are associated with elevated local IgG and IgA concentrations.

Temperature-dependent effects of the components of kallikrein-kinin system on sperm motility in vitro.

The effect of kallikrein and bradykinin on sperm motility was studied. For investigation of whether the effect is not temperature dependent, sperm motility and velocity were measured after incubation with kallikrein or bradykinin at different temperatures. The effect of kallikrein on sperm motility and velocity was demonstrated significantly at 22 degrees C. At 37 degrees C, this effect was absent, but with captopril the effect of kallikrein on sperm velocity was observed. Bradykinin stimulated sperm motility and velocity at 22 degrees C and at 33 degrees C. However, it did not stimulate sperm motility at 37 degrees C. With 1.10 phenantroline, the effect of bradykinin on sperm motility was detected. These results indicate that kallikrein and bradykinin stimulate sperm motility and velocity and that their effects are strongly temperature dependent.

Inhibition of angiotensin-converting enzyme--a new concept of medical treatment of male infertility?

OBJECTIVES: To evaluate the effectiveness of systemic captopril therapy. DESIGN: Randomized double-blind study. SETTING: Andrology Unit at the Department of Dermatology, University of Munich, Munich, Germany. PATIENTS: Infertile men suffering from oligozoospermia (5-20 x 10(6) spermatozoa/mL) and/or asthenozoospermia. INTERVENTIONS: Captopril was given orally; samples of seminal plasma were collected twice before treatment and 4 and 12 weeks after captopril administration. MAIN OUTCOME MEASURE: Semen parameters, pregnancy rate, ACE activity, and kinin levels. RESULTS: After 4 weeks of therapy, significant differences between verum group and placebo group were found concerning ACE activity and kinin levels. Sperm density improved significantly after 12 weeks of captopril therapy. All other semen parameters remained unchanged. The pregnancy rate was not improved. CONCLUSIONS: The suitability of captopril in the therapy of oligozoospermia and/or asthenozoospermia for improvement of male infertility seems to be limited.

Analysis of antigen expression on human spermatozoa by means of monoclonal antibodies.

Hybridoma cell lines were obtained by fusing NS-1 mouse myeloma cells with splenocytes from female mice immunized with whole washed human spermatozoa. Nine clonally derived cell lines were selected which secrete monoclonal antibodies against integral spermatozoan antigens. Immunofluorescence studies showed specific binding of the individual monoclonal antibodies to localized regions of the sperm cell: acrosomal cap, equatorial segment, and midpiece. Five of these antibodies also recognized antigenic determinants of isolated acrosin. The monoclonal antibodies provide probes for the immunochemical characterization of sperm antigens and for the elucidation of the role of these antigens in spermatozoan function.

Lymphadenopathy associated virus/human T-cell lymphotropic virus III antibodies in homosexual men with and without sperm antibodies.

For the evaluation of a possible relationship between antisperm antibodies and LAV/HTLV-III antibodies, both markers were determined in the sera of 89 homosexual men. Thirty-one of 89 men (35%) had sperm antibodies in their sera, and 21 of 89 men (24%) had LAV/HTLV-III antibodies. There was no significant relationship between the occurrence of both kinds of antibodies, so that infection with the LAV/HTLV-III retrovirus in homosexual men seems not be influenced by the presence of antisperm antibodies.

Use of a specific zona pellucida (ZP) protein 3 antiserum as a clinical marker for human ZP integrity and function.

OBJECTIVE: To evaluate binding characteristics of a specific zona pellucida (ZP) protein 3 (ZP3) antiserum to human oocytes in order to determine its usefulness as a clinical marker for human ZP integrity and function and its correlation with IVF outcome. DESIGN: Prospectively designed, blinded, internally controlled study. SETTING: Tertiary care academic center. PATIENTS: Patients undergoing IVF therapy who had either total failed fertilization or partial fertilization were studied. INTERVENTIONS: Metaphase II oocytes showing absence of pronuclear formation were salt stored 48 hours after insemination and bisected into matching hemizonae using micromanipulation. One hemizona was incubated with AS ZP3-6 (an antiserum generated against a synthetic ZP3 peptide derived from an amino acid sequence that is highly conserved in the structure of ZP3), whereas the matching hemizona was incubated with AS ZP3-7, an antiserum detecting exclusively mouse ZP3 (internal, negative control). Antibody binding was visualized using the peroxidase-antiperoxidase method and diaminobenzidine as color reagent. RESULTS: A total of 104 unfertilized oocytes were evaluated. Analysis of variance showed a significant interaction between gamete factor groups (sperm and oocyte) and antiserum factor. Patients with oocyte factor had significantly lower mean staining scores for the AS ZP3-6-treated hemizonae than patients with sperm factor. CONCLUSIONS: These results demonstrate that anomalies of human ZP3 can be identified with AS ZP3-6 and that these ZP abnormalities correlate with fertilization failure during IVF treatment. Thus, this newly developed biomarker may be of clinical significance in the identification of oocyte defects that are associated with fertilization disorders and may help in the decision-making process in the IVF-assisted fertilization setting.

Function of human epididymal spermatozoa.

Human epididymal spermatozoa taken from caput, corpus, and cauda were investigated to determine their fertilizing capacity (22 epididymides from 11 patients who had undergone orchidectomy because of prostatic cancer). The following functions, which have been reported to correlate positively with the fertilization rate, were determined: motility and progressive motility, chromatin condensation (assessed by aniline blue staining), acrosin activity, and induction of acrosome reaction by low temperature. In addition, stimulation of motility by pentoxifylline and phosphatidylcholine was examined. The results showed that motility, progressive motility, normal chromatin condensation, and inducible acrosome reaction increased from the caput to the cauda epididymidis, whereas acrosin activity was normal in all sections. Stimulation of progressive motility, especially that of caput spermatozoa, could be achieved by both pentoxifylline and phosphatidylcholine, the latter being definitely superior. In conclusion, our study confirmed that human spermatozoa in physiological status undergo several steps of maturation during the epididymal transit. Stimulation of sperm motility by phosphatidylcholine may be helpful for patients in whom epididymal spermatozoa are used for assisted reproduction.

Sperm motility under conditions of weightlessness.

The aim of this study was to determine the differences in motility of frozen and thawed bull spermatozoa under conditions of weightlessness compared with ground conditions. The tests were performed within a series of scientific and technologic experiments under microgravity using sounding rockets in the Technologische Experimente unter Schwerelosigkeit (TEXUS) program launched in Kiruna, North Sweden. Using a computerized sperm motility analyzer, significant differences were found in sperm motility under microgravity compared with sperm under gravitational conditions on earth. Computer analysis showed alterations in straight line and curvilinear velocity, as well as in linearity values. The amount of progressively motile spermatozoa, including all spermatozoa with a velocity > 20 microns/second, increased significantly from 24% +/- 9.5% in the reference test to 49% +/- 7.6% in the microgravity test. In conclusion, there is strong evidence that gravity influences sperm motility.

Enzymatic digestion of bradykinin by rat Sertoli cell cultures.

Sertoli cells play a key role in spermatogenesis. To study the involvement of the kallikrein-kinin system in the testis, the pattern of bradykinin-inactivating kininases in rat Sertoli cells was investigated. Exogenous bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) is cleaved at Pro7-Phe8, Phe5-Ser6, and Gly4-Phe5, as demonstrated by high performance liquid chromatography analysis. Degradation of bradykinin was strongly inhibited by phosphoramidon and thiorphan, which are specific inhibitors of neutral metalloendopeptidase-24.11. The kininase type II-specific inhibitors, captopril and enalapril, were only partially effective in preventing peptidolysis. This indicates that the main kininases responsible for rapid bradykinin inactivation are neutral metalloendopeptidase and, to a lesser extent, kininase type II. Neutral metalloendopeptidase and kininase type II were shown to be located on Sertoli cell membranes. A low degree of bradykinin degradation was detected by simultaneous inhibition of neutral metalloendopeptidase-24.11 and kininase II, pointing out the involvement of further peptidases with minor activities. This remaining activity is probably not due to the action of kininase type I or cysteine proteases, as shown by specific inhibitors. The data presented indicate the occurrence of membrane-bound kininases, which are an important part of the kallikrein-kinin system, in rat Sertoli cell cultures.

Release of angiotensin-converting enzyme (ACE) from human spermatozoa during capacitation and acrosome reaction.

The present study examines the release of angiotensin-converting enzyme (ACE) from human spermatozoa during capacitation conditions and in correlation to acrosome reaction and cell death. The ACE content of spermatozoa was measured by treating the cells with different detergents. Glass wool-filtered and washed human spermatozoa were incubated for 3 hours at 37 degrees C. Percentages of acrosome-reacted and dead spermatozoa did not change significantly, but the ACE release increased from 0 to 2.93 +/- 0.44 mU/100 x 10(6) spermatozoa after 180 minutes (P < 0.001). In order to study the influence of acrosome reaction on ACE release, the acrosome reaction of noncapacitated spermatozoa was induced by 10 microM calcium ionophore A23187. The percentages of acrosome reaction and viability in noncapacitated spermatozoa as well as the ACE release were compared to corresponding data from experiments using capacitated spermatozoa (3 hours, 37 degrees C) from the same donors. Although the number of living acrosome-reacted spermatozoa after ionophore treatment (30.5 +/- 4.0%) was significantly higher than after capacitation (13.3 +/- 2.8%, P < 0.001), ACE release from ionophore-treated, noncapacitated spermatozoa was lower (P < 0.05). The data indicate that ACE release from human spermatozoa during capacitation is independent of acrosome reaction. Measurement of ACE release may be a clinically useful assay for human sperm capacitation.

Acrosin activity of human spermatozoa by means of a simple gelatinolytic technique: a method useful for IVF.

Acrosin activity was determined using a gelatinolysis technique in 100-microliter semen aliquots of 114 patients (normozoospermia, n = 90; asthenozoospermia, n = 12; oligozoospermia, n = 10; polyzoospermia, n = 2) attending an in vitro fertilization (IVF) program. Halo diameter, halo formation rate, and a calculated acrosin activity index correlated significantly with the IVF rates (P = 0.0054, r = 0.396; P = 0.0009, r = 0.401; and P = 0.0003, r = 0.428, respectively). In cases where the halo diameter was < 10 microns and halo formation rate was < 60%, all patients were subfertile or infertile, that is, they showed poor or no fertilization in vitro, respectively. The assay demonstrated a relatively low sensitivity: 25.7% for halo diameter, 37.1% for halo formation rate, and 25.7% for acrosin activity index, respectively. This might be attributed to other sperm functional aspects, such as disturbed acrosome reaction or impaired zona binding.

Influence of gossypol on the secretory function of cultured rat sertoli cells.

Cottonseed gossypol is a potent male contraceptive in several mammalian species including man. Sertoli cells play a crucial role in spermatogenesis. Therefore, the antifertility competence of gossypol may reflect a change in Sertoli cell function. Rat primary cultures were used to examine the effect of gossypol on cell viability, mitochondrial dehydrogenase function, lactate production and secretion of the Sertoli cell-specific protein inhibin. Exposure for 24 h to gossypol (3-6 microM) significantly enhance secretion of lactate but reduce secretion of inhibin without affecting cell viability. At 9-15 microM, the observed decrease of both lactate and inhibin accumulation apparently resulted from Sertoli cell degeneration and death, because viability and mitochondrial function were also reduced. The results suggest that mitochondria of Sertoli cells are a possible target for gossypol-induced infertility.

Determination of active, non-zymogen acrosin, proacrosin and total acrosin in different andrological patients.

A spectrophotometric assay is described which, due to improved extraction conditions, allows quantitative determination of enzymatically active, non-zymogen acrosin, proacrosin and total acrosin activity from human sperm acrosomes. Acrosomal proteinase activity is assessed by acid extraction of the sperm pellet and the suspension medium before and after snap-freezing, followed by zymogen autoactivation. Release of acrosin from the acrosome can be used as a sensitive biochemical marker to characterize acrosomal membrane stability, severe disturbance of which may be the cause of impaired male fertility. Acrosin activities in different populations of semen specimens are reported and compared to data available in the literature. Different degrees of acrosomal membrane alterations are observed in men with oligozoospermia, tetratozoospermia and polyzoospermia. Particularly in oligozoospermia, a significant increase of active, non-zymogen acrosin points to severe acrosomal membrane alterations and, in addition, to a premature activation of proacrosin, which may impair fertilization in certain individuals. Finally, acrosin activity is shown to be significantly influenced by the time of sexual abstinence. It is concluded that determination of acrosin may be a useful indicator of the fertility potential in men.