Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility.

Human mitochondrial DNA polymerase, encoded by POLG, contains a polyglutamine tract encoded by a CAG microsatellite repeat. Analysis of POLG genotypes in different populations identified an association between absence of the common, ten-repeat allele and male infertility typified by a range of sperm quality defects but excluding azoospermia.

Morphological study of the effects of the GnRH superagonist deslorelin on the canine testis and prostate gland.

The present study is part of a programme of research designed to evaluate the efficacy of the GnRH superagonist,deslorelin (D-Trp6-Pro9-des-Gly10-LHRH ethylamide), as a contraceptive for male dogs. Adult dogs were assigned to a completely randomized design comprising six groups of four animals. Each dog in the control group received a blank implant (placebo) and each dog in the other five groups received a 6 mg deslorelin implant. One group of deslorelin treated dogs was sacrificed on each of days 16, 26, 41, 101 and 620, and testicular and prostate tissues were collected for study by light and electron microscopy. On days 16 and 26 after implantation, we observed partial disruption of the seminiferous tubules, with early spermatids shed into the lumen. On days 41 and 101 after implantation, 90­100% of the seminiferous tubules were atrophic and aspermatogenic.On day 101 after implantation, 99% of all sections showed atrophy of the epithelium and shrinkage of epithelial height in the ductus epididymides. On days 41 and 101 after implantation, prostate tissue showed complete atrophy of the glandular epithelium (100% of sections) and an apparent increase in the relative proportion of connective tissue. At the electron microscopic level, in dogs treated with deslorelin for 41 and 101 days, the Sertoli cells were smaller and their nucleoli appeared smaller than in the control dogs. The nucleoli of the Leydig cells were atrophied and prostate glandular epithelium showed reduced epithelial height, a trophy of the nucleolus and an absence of secretory granules.Tissues collected during the recovery phase revealed a complete recovery of spermatogenesis. In conclusion, slow release implants containing deslorelin induce a striking a trophy of the testes and prostate gland by 26 days after implantation, explaining the previously reported loss of ejaculate and arrest of sperm output. At histological level,the entire process appears to be completely reversible, in accordance with data on endocrine variables and semen production.

Dose-response studies for pituitary and testicular function in male dogs treated with the GnRH superagonist, deslorelin.

We tested the effect of dose of GnRH superagonist on pituitary and testicular function in a study with four groups of four male dogs. The Controls received blank implants and the other three groups received implants containing 3, 6 or 12 mg deslorelin (D-Trp6-Pro9-des-Gly10-GnRH ethylamide). In all deslorelin-treated groups, there was initially an acute increase in plasma concentrations of LH and testosterone, followed by declines such that both hormones became undetectable after approximately 12 days. There was a dose-response in some of these early aspects of the hormone profiles. With respect to long-term effects of treatment, the 12-mg dose had significantly greater effects than the smaller doses for the duration of minimum testicular volume [366 +/- 77, mean +/- SEM (3 mg), 472 +/- 74 (6 mg), and 634 +/- 59 (12 mg) days], absence of ejaculate [416 +/- 88 (3 mg), 476 +/- 83 (6 mg), and 644 +/- 67 (12 mg) days], undetectable plasma concentrations of LH and testosterone [367 +/- 64 (3 mg), 419 +/- 72 (6 mg), and 607 +/- 69 (12 mg) days], the delay until complete recovery of LH and testosterone secretion [394 +/- 65 (3 mg), 484 +/- 72 (6 mg) and 668 +/- 47 (12 mg) days], and the delay until testes had regrown to normal volume [408 +/- 77 (3 mg), 514 +/- 74 (6 mg), 676 +/- 59 (12 mg) days]. The time taken to restore full ejaculates was also longest for the 12-mg dose: 716 +/- 67 (12 mg) days vs 440 +/- 66 (3 mg) and 538 +/- 83 (6 mg) days after implantation. There was no correlation between delay to recovery of normal ejaculate quality and body mass. We conclude that the dose-response relationship with deslorelin implants is not expressed with respect to the degree of suppression of reproduction, but on the maximum duration of suppression and thus to delay until recovery.

Implications of micro-RNA profiling for cancer diagnosis.

Micro-RNAs (miRNAs) are a large class of small non-coding RNAs that regulate protein expression in eucaryotic cells. Initially believed to be unique to the nematode Caenorhabditis elegans, miRNAs are now recognized to be important gene regulatory elements in multicellular organisms and have been implicated in a variety of disease processes, including cancer. Advances in expression technologies have facilitated the high-throughput analysis of small RNAs, identifying novel miRNAs and showing that these genes may be aberrantly expressed in various human tumors. These studies suggest that miRNA expression profiling can be correlated with disease pathogenesis and prognosis, and may ultimately be useful in the management of human cancer.

Pituitary and testicular endocrine responses to exogenous gonadotrophin-releasing hormone (GnRH) and luteinising hormone in male dogs treated with GnRH agonist implants.

The present study tested whether exogenous gonadotrophin-releasing hormone (GnRH) and luteinising hormone (LH) can stimulate LH and testosterone secretion in dogs chronically treated with a GnRH superagonist. Twenty male adult dogs were assigned to a completely randomised design comprising five groups of four animals. Each dog in the control group received a blank implant (placebo) and each dog in the other four groups received a 6-mg implant containing a slow-release formulation of deslorelin (d-Trp6-Pro9-des-Gly10-LH-releasing hormone ethylamide). The same four control dogs were used for all hormonal challenges, whereas a different deslorelin-implanted group was used for each challenge. Native GnRH (5 microg kg(-1) bodyweight, i.v.) was injected on Days 15, 25, 40 and 100 after implantation, whereas bovine LH (0.5 microg kg(-1) bodyweight, i.v.) was injected on Days 16, 26, 41 and 101. On all occasions after Day 25-26 postimplantation, exogenous GnRH and LH elicited higher plasma concentrations of LH and testosterone in control than deslorelin-treated animals (P < 0.05). It was concluded that, in male dogs, implantation of a GnRH superagonist desensitised the pituitary gonadotrophs to GnRH and also led to a desensitisation of the Leydig cells to LH. This explains, at least in part, the profound reduction in the production of androgen and spermatozoa in deslorelin-treated male dogs.

Differentially expressed DNA sequences following recovery from unilateral testicular torsion in rat.

The molecular response during recovery from torsion-induced stress in the testis is diverse with a variety of mechanisms. In this study, using unilateral testicular torsion in rat as a model, we used subtractive hybridisation to identify differentially expressed DNA sequences in the torsioned and control testes. Three genes were identified as being down regulated in the torsioned testis compared with controls: Control Testis genes 1, 2 and 3 (CT1, CT2 and CT3). Two genes were up regulated in the torsioned testes: Torsioned Testes genes 1 and 2 (TT1 and TT2). Differential expression was confirmed by Reverse Northern blot analysis. An homology search revealed that CT1 had 88% homology with rat metallothionein cDNA; CT2 had 81% homology with rat cell surface antigen in MHC class I, but no homology could be found for CT3. TT1 had 92% identity with rat Rieske iron-sulphur protein mRNA whereas TT2 had 73% identity with a human clone of unknown function (RP 11-252D22). These results indicate that changes in gene expression occur following torsion induced stress, and that identification of differentially expressed genes may provide insights into the mechanisms of cellular tissue damage in this model.

Low-dose oral natural human interferon-alpha in 29 patients with HIV-1 infection: a double-blind, randomized, placebo-controlled trial.

OBJECTIVE: To evaluate clinical efficacy and toxicity of low-dose oral natural human interferon-alpha (nHuIFN alpha) on CD4+ lymphocyte counts and clinical symptoms in patients with HIV-1 infection. DESIGN: Double-blind, randomized, placebo-controlled trial with crossover. SETTING: Private practice specializing in the treatment of patients with AIDS. PATIENTS, PARTICIPANTS: Only patients with HIV-1 infection and CD4+ lymphocyte counts between 200 and 500 x 10(6)/l were included for study. Thirty out of thirty-one patients at study entry completed treatment with placebo, and 29 completed nHuIFN alpha treatment. Mean patient age was 36 years (range, 25-58 years). The 30 patients included 26 men, of whom 22 were homosexual, and four women; five were drug users and none were currently on zidovudine therapy, although three had been previously. INTERVENTIONS: Patients were randomly assigned to cohorts of 10 to receive either 200 IU nHuIFN alpha once daily orally absorbed or placebo with crossover after 6 weeks. MAIN OUTCOME MEASURES: Every 2 weeks, a detailed history, physical examination, and laboratory tests, including CD4+ and CD8+ lymphocyte counts, were conducted. RESULTS: There was only a slight, transient increase in mean CD4+ lymphocyte counts after 4 weeks of treatment with nHuIFN alpha, compared with a slight decline when placebo was administered. This effect reached statistical significance in a subgroup of patients only and was not sustained after 6 weeks. There were no significant changes in weight and clinical symptoms. All patients remained HIV-1-antibody-positive. Treatment-related adverse reactions were not observed. CONCLUSIONS: Our double-blind, randomized, placebo-controlled clinical trial did not confirm a previous report of efficiency of oral nHuIFN alpha. Although non-toxic, our data do not justify the widespread use of low-dose oral nHuIFN alpha in HIV-infected patients outside controlled clinical trials.

Oral use of interferon.

Interferon-alpha (IFN-alpha) given orally has biological activity in humans and other animals. The dose providing the most benefit delivers IFN-alpha to the oral mucosa in a concentration (10(2)-10(3) IU), similar to that naturally produced in the nasal secretions during respiratory infections. In contrast, conventional IFN therapy employs parenteral doses of > 10(6) IU and, for this reason, orally administered IFN therapies have been called low-dose treatments. Efficacy in both animal disease models and human studies has been reported, and the mechanisms whereby oral administration has a systemic effect are under active study in a number of laboratories.

Natural human interferon-alpha administered orally as a treatment of bovine respiratory disease complex.

Natural human interferon-alpha (nHuIFN-alpha) from three sources was given orally to 368 calves experiencing a natural outbreak of bovine respiratory disease complex (BRDC). In one study, 200 calves were given one treatment daily for 3 days of placebo or 20, 200, or 2,000 IU of nHuIFN-alpha before shipment. Calves treated with 20 or 200 IU had a significant (p < 0.05) weight gain benefit for the first 21 days in the feedlot, if they had rectal temperatures <40 degrees C when treated with nHuIFN-alpha. In a second trial, 168 mixed-breed calves (five groups randomized to 31-36 calves/group) were treated with one dose of placebo or 200 or 400 IU of nHuIFN-alpha after shipment to the feedlot. Using this regimen, a dose of 200 IU per calf significantly (p < 0.08) decreased the number of sick calves per group and delayed development of BRDC. Results of these studies demonstrate that oral administration of nHuIFN-alpha, which may partially mimic the nasally secreted IFN response reported during BRDC, may be beneficial in cattle.

Single-step method for the determination of the amount of the common deletion in mitochondrial DNA.

A fluorescent-based quantitative PCR assay to measure the percentage of the common deletion in rat mitochondria is reported. The amount of the common deletion is measured relative to the amount of total mitochondrial (mt)DNA. The use of a heterologous competitor construct allows the reaction to be monitored to ensure that exponential accumulation of products occurs. The use of fluorescence-labeled primers provides a safe and sensitive means to quantify products without any adjustment for size. This assay will allow the quantitative determination of the common deletion with one dilution range.

Opening the flood gates: interferon-alpha treatment for Sjögren's syndrome.

Interferon (IFN)-alpha is the main IFN produced in response to viral infection. Low levels of IFNalpha can be detected in nasal secretions after exposure to viruses in vivo. Radioimmunoassay has shown that endogenous IFNalpha is low in children, reaches a peak in young adults, and gradually declines with aging. Importantly, this endogenous IFNalpha is significantly decreased in patients with Sjögren's syndrome (SS). IFNalpha has been tested as a therapeutic agent in patients with SS. Intramuscular human leucocyte IFNalpha increases saliva production significantly in patients with SS. Improvements have been noted in lacrimal function and in dryness symptoms. Since IFNalpha infrequently induces autoimmune phenomena and high dose IFNalpha treatment sometimes has a serious adverse event profile, treatment focus has shifted to use of low dose orally-administered IFNalpha. In a single-masked controlled trial, 60 patients with SS randomly received natural human IFNalpha 150IU 3 times a day in an oral lozenge formulation or sucralfate as control for 6 months. At study end, 15 (50%) of the 30 IFNalpha-treated patients had saliva production increases at least 100% above baseline. IFNalpha treatment was well tolerated and no patients withdrew. Labial minor salivary gland biopsies indicated significant decreases in lymphocytic infiltration accompanied by a significant increase in intact salivary gland tissue after 6 months of treatment. In another 12-week double-masked, randomised, placebo-controlled trial, stimulated saliva production in patients with SS receiving IFNalpha lozenges 150IU 3 times daily was significantly increased. This dosage was also suggestive of benefit for 5 of 7 subjective measures of oral and ocular comfort. The tolerability profile of these low dose oral IFNalpha lozenges is excellent; no serious adverse events have been recorded. Adverse effects were generally mild and there were no clinically significant changes in laboratory or clinical safety measures. Low oral doses of natural human IFNalpha thus appear to improve secretory function and relieve dryness in patients with SS without causing significant adverse events. Endogenous or orally administered IFNalpha may activate oropharyngeal lymphoid and epithelial cells and induce production of potent soluble factors which could mediate immunological reactivity. It has been suggested that IFNalpha/beta potentiates clonal expansion and survival of CD8 T cells. Stimulating effects have also been demonstrated on natural killer cell activity, which has been shown to be depressed in patients with SS. It is likely that some combination of these immunological effects results in anti-inflammatory activity and ameliorates signs and symptoms of SS.

Evaluation of luteal support therapy in a randomized controlled study within a gamete intrafallopian transfer program.

A randomized controlled study of luteal support therapy (using intramuscular injections of progesterone and/or human chorionic gonadotropin) was conducted in a trial designed to minimize variables that might adversely affect the change of pregnancy. After applying rigid selection criteria, 207 women were recruited into one of four groups. Mathematical modeling was applied to the results to determine if there were degrees of improvement in uterine receptivity relative to various grades of embryo quality ("E" factor). Although the trial size was insufficient to enable the detection of significant improvements in the pregnancy rates that ranged from 27.5% for non-treatment to 41.2% for those receiving combined treatment, the birth rates were significantly better with luteal support (11.8% versus 29.4%). Similarly, the overall implantation rate just failed to reach statistical significance for luteal support, but the ongoing implantations were significantly better (3.6% versus 9.0%). Data modeling indicated that luteal support, particularly with the combined regimen, could improve the ongoing implantation rate by up to 2.5-fold when the E factor was poorest.

Influence of pentoxifylline in severe male factor infertility.

Two in vitro fertilization sperm preparation protocols using pentoxifylline (long and short exposure before insemination) were studied in 57 couples (61 cycles) with male factor infertility. For each cycle, oocytes were divided into two groups for insemination using either pentoxifylline-treated or control semen. Fertilization rates improved over controls in the short protocol (P less than 0.001) and fewer couples experienced fertilization failure (P = 0.02). Sixteen pregnancies ensued (30% per collection with the short protocol), and 4 were from cases with less than 1.0 X 10(6) progressively motile sperm count per milliliter, 1 being as low as 0.2 X 10(6) progressively motile count per milliliter. Seventeen healthy infants have now delivered and pregnancy wastage is not increased. Pentoxifylline is thus a useful sperm treatment for cases of male factor infertility.

Preliminary results using pentoxifylline in a pronuclear stage tubal transfer (PROST) program for severe male factor infertility.

In vitro trials with washed spermatozoa incubated in medium containing 1 mg/ml of the methyl xanthine phosphodiesterase inhibitor PF showed improved counts of total motile and total progressively motile spermatozoa in cases of oligospermia/asthenospermia. Application of this agent in a PROST program for a series of nine couples presenting for treatment with histories of failed fertilization in vitro resulted in five pregnancies (four singleton, one triplet) and the subsequent delivery of normal infants. The results warrant further evaluation of this sperm treatment for cases of severe male factor infertility.

Fertilization of human oocytes in relation to varying delay before insemination.

Fertilization and pregnancy rates in an in vitro fertilization and embryo transfer program were studied after a range of insemination times of between 1 and 26 hours after oocyte recovery. There was no significant variation in fertilization rate across this range. The pregnancy rate showed no significant variation for insemination between 3 and 16 hours after aspiration. However, it was disappointing at 2 hours (3%), and no pregnancies were achieved from the nine patients whose ova were inseminated 20 or more hours after aspiration. It is concluded that mature oocytes can be inseminated in vitro at any time between 3 and 16 hours after aspiration and still retain the same potential to produce a pregnancy.

A test of the human sperm acrosome reaction following ionophore challenge. Relationship to fertility and other seminal parameters.

Acrosome reaction capacity was tested on semen samples from 53 fertile and 26 subfertile men. Preparations were divided into two aliquots after 3 or 24 hours of culture. One aliquot received 10 mumol/L calcium ionophore A23187 in dimethyl sulfoxide (DMSO) and the other received DMSO alone. Acrosome reactions were scored on ethanol-permeabilized smears using fluorescein isothiocyanate (FITC)-conjugated Pisum sativum lectin. The following factors were analyzed: the spontaneous reaction rates (control); induced reaction rates (ionophore-challenged); and the difference between the two, being the proportion of spermatozoa in the population capable of reacting in response to calcium influx (acrosome reaction to ionophore challenge [ARIC]). While spontaneous reactions bore no relation to fertility, induced reactions and ARICs were significantly reduced or absent in subfertile men, indicating acrosomal dysfunction as a likely cause of fertilization failure. The test was shown to have a predictive value for fertility comparable to that of the hamster ovum sperm penetration assay and to be a simple and cost-effective addition to existing semenology.

Oral therapy with human interferon alpha in calves experimentally injected with infectious bovine rhinotracheitis virus.

Fifty-six calves, seronegative for infectious bovine rhinotracheitis (IBR) virus, were randomly divided into 7 equal groups (n = 8) and given 0.0, 0.05, 0.50, or 5.00 international units (IU) of natural or recombinant human interferon alpha per kg body weight (nHuIFN-alpha or rHuIFN-alpha, respectively) orally once daily for 4 consecutive days, starting 2 days before intranasal inoculation with virulent IBR virus. Calves given 0.05 IU nHuIFN-alpha/kg bwt had significantly greater weight gain at days 15 (P < 0.10) and 25 (P < 0.05) than the placebo-treated (0.0 IU) control group. The treatment groups given 0.05 and 0.5 IU nHuIFN-alpha/kg bwt nHuIFN-alpha had fewer days with temperature > 40 degrees C (P < 0.05 and P = 0.10, respectively), and lower mean rectal temperatures on days 8 and 11 (0.05 IU/kg bwt; P < 0.10) or on day 11 (0.5 IU/kg bwt; P < 0.10). None of the calves given 0.05 IU nHuIFN-alpha/kg bwt required antibiotic therapy. Calves given 0.50 IU/kg bwt of nHuIFN-alpha, or 0.05 IU/kg bwt of rHuIFN-alpha had fewer (P < 0.05) total days of antibiotic therapy compared to controls. These data indicate that low dose oral IFN-alpha treatment significantly reduced the clinical effects of IBR virus infection in feedlot cattle in an interferon dose-dependent fashion.

The effect of low dose oral human interferon alpha therapy on diarrhea in veal calves.

Low doses of recombinant human interferon alfa 2a (rHuIFN-alpha 2a), were given orally in milk replacer formula to veal calves to determine the efficacy of rHuIFN-alpha 2a for protection against diarrhea, ear and/or respiratory tract infections common in vealing operations. Calves given rHuIFN-alpha 2a had fewer days and a lower incidence of diarrhea, compared to placebo-treated calves. Calves treated with rHuIFN-alpha 2a had significantly (P < 0.05) fewer ear infections and fewer total days of ear infection than did placebo-treated calves. The mortality rate was lower in the rHuIFN-alpha 2a treatment group (1.6%) than in the placebo treatment group (2.9%) and calves given rHuIFN-alpha 2a had a greater average weight gain (13.1 lbs. more per calf) than calves given placebo. These data demonstrate that orally administered rHuIFN-alpha 2a exhibited a protective effect against clinically significant signs of disease in veal calves, reduced the mortality rate in this population, and enhanced average weight gain.

The use of interferon modulates the negative effects of heat stress on poultry production.

A trial was conducted with broilers reared in two temperature environments; one was thermoneutral and the other had cycling ambient temperatures. Human interferon alpha (HuIFN-alpha) was added to the drinking water daily at four dose levels (0.0, 0.01, 0.1 and 1.0 international units (IU) per ml of drinking water). The trial began with 21 day old chicks housed either in a thermoneutral (24 degrees C) or a cycling ambient temperature (24-36 degrees C) environment. Interferon added to water at the highest concentration (1.0 IU/ml) improved surviability of birds in the cycling ambient temperature (24-36 degrees C) environment (P < 0.05). Birds housed in the cycling (24-36 degrees C) environment, drinking the lowest concentration of IFN-alpha 0.01 IU/ml, had a significantly improved weight gain-to-feed ratio. Oral IFN-alpha reduced the cost of production for birds reared in a cycling ambient temperature environment.

Comparison of the long-term effects of treatment with oral and parenteral interferon alpha in chronic viral hepatitis patients.

This report presents the interferon alpha (IFN-alpha) treatment results for 75 patients with chronic hepatitis B virus (HBV) (51 cases) and hepatitis C virus (HCV) (24 cases) induced hepatitis in maximal 61 months follow-up. Among the group of 51 patients with chronic HBV hepatitis, 35 were treated orally with IFN-alpha in the form of lozenges in low daily doses (37.5-150 U). The treatment was completed in 32 cases. The remaining 16 patients with chronic HBV hepatitis completed the treatment with parenteral IFN-alpha (3 x 10(6) U, 3 times a week). Positive results measured by the use of seroconversion in the HBe-antigen system were obtained for 68.7% (5-61 months follow-up) and 56.2% (7-44 months follow-up) of the patients treated with oral and parenteral IFN-alpha, respectively. Among the group of 24 patients with chronic HCV hepatitis, the first 6 patients were initially treated with IFN-alpha in the form of lozenges, in low daily doses. Biochemical remission was not achieved in these patients; genotype 1b was documented in 4 of them. Both, the first 6 patients (after a break) and the remaining 18 were treated with IFN-alpha parenterally, as in HBV patients. Temporary clinical and biochemical remission was achieved in 62.5% of the cases during the treatment, however the durable remission observed during 6-29 months of follow-up was achieved in 20.4 of the cases only.