Factors associated with fertility of nulliparous dairy heifers following a 10-day fixed-time artificial insemination program with sex-sorted and conventional semen.

OBJECTIVE: To examine factors associated with fertility on dairy farms that used a common fixed-time artificial insemination (FTAI) program in yearling heifers. METHODS: Records were analysed from 954 yearling heifers on 10 south-west Victorian dairy farms that used a common FTAI program, involving the insertion of a 1.9-g progesterone-releasing device for 10 days; 2 mg oestradiol benzoate at insertion; 500 µg cloprostenol on day 7; and FTAI 48 h after device removal. Weight, age, expression of oestrus, sire, semen type (frozen sex-sorted or frozen conventional) and timing of insemination were examined for their relationship with first-service conception rates. RESULTS: Heifers over 300 kg body weight were 1.18-fold more likely to express oestrus during the FTAI program. For every extra 1 kg, there was a 1.5% increase in the likelihood of expressing oestrus. First-service conception rates were 40.3% and 56.0% for sex-sorted and conventional semen, respectively, and were significantly higher when oestrus was expressed. The difference was greater for sex-sorted semen (3.4-fold) compared with conventional semen (1.5-fold). The interval from device removal to insemination varied between 47 and 51.4 h and had no significant effect on conception rates. However, there was a trend towards a higher conception rate for sex-sorted semen when inseminations were performed >50 h after device removal. CONCLUSIONS: Increased fertility was associated with larger heifers and heifers that expressed oestrus, particularly when sexed-sorted semen was used. Variation in the timing of AI with respect to device removal between 47 and 51.4 h did not adversely affect conception rates.

The immunoglobulin heavy chain gene 3' enhancers induce Bcl2 deregulation and lymphomagenesis in murine B cells.

Human follicular B-cell lymphoma is associated with the t(14;18) chromosomal translocation that juxtaposes the Bcl2 proto-oncogene with the immunoglobulin heavy chain (Igh) locus, resulting in the deregulated expression of Bcl2. Our previous studies have shown that the Igh 3' enhancers deregulate the Bcl2 expression in vitro. However, the effects of the Igh 3' enhancer elements on Bcl2 expression in vivo are not known. To investigate the role of the Igh 3' enhancers in Bcl2 deregulation, we used gene targeting to generate knock-in mice in which four DNase I-hypersensitive regions from the murine Igh 3' region were integrated 3' of the Bcl2 locus. Increased levels of Bcl2 mRNA and protein were observed in the B cells of Igh-3'E-bcl2 mice. B cells from Igh-3'E-bcl2 mice showed an extended survival in vitro compared with B cells from wild-type (Wt) mice. The Bcl2 promoter shift from P1 (the 5' promoter) to P2 (the 3' promoter) was observed in B cells from Igh-3'E-bcl2 mice, similar to human t(14;18) lymphomas. The IgH-3'E-bcl2 mice developed monoclonal B-cell follicular lymphomas, which were slowly progressive. These studies show that the Igh 3' enhancers have an important role in the deregulation of Bcl2 and B-cell lymphomagenesis in vivo.

miR-449a targets HDAC-1 and induces growth arrest in prostate cancer.

Histone deacetylases (HDACs) are frequently overexpressed in broad range of cancer types, where they alter cellular epigenetic programming to promote cell proliferation and survival. However, the mechanism by which HDACs become overexpressed in human cancers remains somewhat of a mystery. In this study, we investigated the expression and functional significance of miR-449a in prostate cancer cells. Using real-time PCR, we found that miR-449a is downregulated in prostate cancer tissues relative to patient-matched control tissue. Introduction of miR-449a into PC-3 prostate cancer cells resulted in cell-cycle arrest, apoptosis and a senescent-like phenotype. In silico analysis of 3'-UTR regions identified a number of genes involved in cell-cycle regulation as putative targets of miR-449a. Using a luciferase 3'-UTR reporter system, we established that HDAC-1 (histone deacetylase 1), a gene that is frequently overexpressed in many types of cancer, is a direct target of miR-449a. Further, our data indicate that miR-449a regulates cell growth and viability in part by repressing the expression of HDAC-1 in prostate cancer cells. Our findings provide new insight into the function of miRNA in regulating HDAC expression in normal versus cancerous tissue.

Excellent safety and tolerability of the human immunodeficiency virus type 1 pGA2/JS2 plasmid DNA priming vector vaccine in HIV type 1 uninfected adults.

A vaccine consisting of DNA priming followed by recombinant modified vaccinia Ankara (rMVA) boosting has achieved long-term control of a pathogenic challenge with a chimera of simian and human immunodeficiency viruses (SHIV-89.6P) in rhesus macaques. Based on these results, clade B HIV-1 DNA and rMVA immunogens have been developed for trials in humans. We conducted a first-time in humans phase I safety trial using the pGA2/JS2 (JS2) HIV-1 DNA priming vector expressing Gag, Pol, Env, Tat, Rev, and Vpu. Thirty HIV-uninfected adults were vaccinated with 0.3 or 3 mg of JS2 DNA, or a saline placebo, by intramuscular injection at months 0 and 2. Both doses of DNA were safe and well-tolerated with no differences between the control, 0.3 mg, or 3 mg groups (n = 6, 12, and 12, respectively) through 12 months of postvaccination follow- up. A chromium-release assay using fresh peripheral blood mononuclear cells (PBMCs) and a validated IFN-gamma ELISpot assay with frozen PBMCs failed to detect CD4(+) or CD8(+) HIV-1-specific T cell responses. HIV-specific neutralizing antibodies were also not detected. The vaccine is being further developed as a priming vector for a combined DNA plus rMVA prime/boost HIV vaccination regimen.

Use of fish liver PLHC-1 cells and zebrafish embryos in cytotoxicity assays.

Heat shock proteins (HSPs) indicate exposure to cellular stress and adverse cellular effects, thus serving as biomarkers of these effects. The highly conserved Hsp70 proteins are expressed under proteotoxic conditions, whereas small HSPs are expressed in response to stressors acting on the cytoskeleton and cell signaling pathways. Poeciliopsis lucida hepatocellular carcinoma line 1 (PLHC-1) cells have been used extensively for studying effects of cytotoxicity. A number of assays have been developed to examine DNA levels, protein levels, growth rate, morphological changes, and viability. The boundary between sub-lethal and lethal effects of particular stressors has been determined. The methodology and analytical framework for these techniques along with sample assays using cadmium stressed PLHC-1 cells are described. A range of methodologies have been developed in the past decade that allow the analysis and interpretation of gene expression and function in vivo in zebrafish embryos, and many of these are now being applied to the development of embryotoxicity assays. Here we provide the theoretical background and methodology for utilizing Hsp70 expression as an indicator of toxicity in the zebrafish embryo. Hsp70 expression is activated in a tissue-specific manner in zebrafish larvae following exposure to a number of different toxicants, including cadmium. This has allowed the development of an hsp70/eGFP reporter gene system in stable transgenic zebrafish that serves as a reliable yet extremely quick indicator of cell-specific toxicity in the context of the multicellular, living embryo.

Highly efficient blue-light generation from a compact, diode-pumped femtosecond laser by use of a periodically poled KTP waveguide crystal.

We present a simplified, potentially portable, and highly efficient blue-light source from a periodically poled KTP waveguide crystal with a compact femtosecond Cr:LiSAF laser. This light source generates 5.6 mW of blue average output power at 424 nm with 27 mW of incident fundamental in a single-pass extracavity arrangement at room temperature. The overall system efficiency of electrical power to blue light is 0.5%, and the internal second-harmonic generation conversion efficiency is as high as 37%. The slope efficiency of 5.5% pJ(-1) at low pulse energies is, to our knowledge, the highest slope efficiency yet reported for frequency conversion into the blue spectral region.

Polymorphic repeats in the androgen receptor gene in high-risk sibships.

BACKGROUND: Genetic susceptibility may explain some familial clusters of prostate cancer. The polymorphic androgen receptor (AR) gene, which mediates androgen activity in the prostate, is a candidate gene that may influence predisposition to the disease. METHODS: We analyzed the polymorphic (CAG)n and (GGN)n repeats within the AR gene in men from 51 high-risk prostate cancer sibships, which included at least one affected and one unaffected man (n = 210). We compared repeat lengths of men with prostate cancer (n = 140) to their brothers (n = 70) without disease, stratified by median age at diagnosis of affected men within each sibship. Conditional logistic regression was used to compute odds ratios (OR) and 95% confidence intervals to evaluate associations between prostate cancer and repeat length. RESULTS: The OR for prostate cancer associated with short (CAG)n repeats (< 22) compared to longer repeats (> or =22) was 1.13 (95% CI 0.5-2.4) overall, but was higher in sibships with a median age of <66 years at diagnosis (OR = 1.72, 95% CI 0.5-6.0). The (GGN)n array also was not associated with prostate cancer in general. However, in older men (> or = 66 years), there was a modest elevation in risk (OR = 1.56, 95% CI 0.6-4.1) among those with short repeats (GGN of < or =16). Men with both a short (CAG)n (< 22) and a short (GGN)n (< or =16) array were not at higher risk (OR = 1.06) compared to men with two long repeats [(CAG)n > or =22 and (GGN)n >16)]. CONCLUSIONS: These results suggest that the (CAG)n and (GGN)n repeats in the AR gene do not play a major role in familial prostate cancer.

Clinical evaluation of the therapeutic effectiveness of ethinyl oestradiol and oestrone sulphate on prolonged bleeding in women using depot medroxyprogesterone acetate for contraception. World Health Organization, Special Programme of Research, Development and Research Training in Human Reproduction, Task Force on Long-acting Systemic Agents for Fertility Regulation.

A placebo-controlled randomized clinical trial was conducted in six centres to compare the effects of a 14 day treatment with either 50 micrograms ethinyl oestradiol daily or 2.5 mg oestrone sulphate daily, on depot medroxyprogesterone acetate (DMPA)-induced prolonged bleeding. Out of 1035 women admitted to the study, 278 requested treatment and were given ethinyl oestradiol (n = 90), oestrone sulphate (n = 91) or placebo (n = 97). Ethinyl oestradiol was successful in stopping the bleeding episode in 93% of cases, compared with oestrone sulphate and placebo which had success rates of 76 and 74% respectively. However, the relative advantage of ethinyl oestradiol was marginal, with an average reduction of 1 bleeding day and 3 spotting days compared with the other two groups. Immediately after treatment, women given ethinyl oestradiol had less bleeding but a more unpredictable pattern than the other two groups. In the long term, there were no differences between the bleeding patterns or the discontinuation rates for any reason in the three groups, and the most important single reason for discontinuation in those groups remained 'menstrual problems'. In summary, the study showed that treatment of DMPA-induced prolonged bleeding with ethinyl oestradiol had a limited short-term effect but no beneficial effect on the acceptability of DMPA as a contraceptive method. Treatment with oestrone sulphate was no different from placebo.

PIP: The findings of a multicenter clinical trial challenge the practice of estrogen treatment of the prolonged or irregular vaginal bleeding associated with depot medroxyprogesterone acetate (DMPA) contraceptive use. Included in the study were 1035 DMPA users (mean age, 27 years) from Alexandria, Egypt; Bangkok, Thailand; Chiang Mai, Thailand; Jakarta, Indonesia; Karachi, Pakistan; and Manila, Philippines. 456 (44%) of these women experienced a bleeding episode lasting more than 7 days during their first 6 months of DMPA use. Of these, only 278 (61%) requested treatment. These 278 women were randomly allocated to receive 50 mcg of ethinyl estradiol (n = 90), 2.5 mg of estrone sulfate (n = 91), or placebo (n = 97) daily for 14 days. The treatment stopped the bleeding episode for 93% of women in the ethinyl estradiol group, 76% of those in the estrone sulfate group, and 74% of women receiving a placebo. The ethinyl estradiol advantage was marginal, however. On average, women treated with ethinyl estradiol had their bleeding episode shortened by 1 bleeding day and 3 spotting days. Immediately after treatment, women given ethinyl estradiol had less bleeding and spotting days than their counterparts in the 2 other groups, but demonstrated a more unpredictable pattern, including a greater range of lengths of bleeding/spotting-free intervals. Three months after treatment, there were no differences between the 3 groups in vaginal bleeding patterns.

Annual patterns of luteinizing hormone, follicle stimulating hormone, testosterone and inhibin in normal men.

Reproductive functions in most animals demonstrate seasonal fluctuations that allow young to be born at a time of the year favourable for their survival. Whether there is a seasonal change in the human reproductive system is unclear. In the present study, we measured serum concentrations of luteinizing hormone, follicle stimulating hormone, testosterone and inhibin in the same 16 normal men sampled monthly for 1 year. A statistically significant increase in all four measured hormones was found in June, with a nadir in August. Our findings suggest that a circannual rhythm of gonadotrophins and testicular hormones exists in normal men. The mechanism leading to this rhythm and the importance of the rhythm in human biology are unknown.

Depot medroxyprogesterone acetate and breast cancer. A pooled analysis of the World Health Organization and New Zealand studies.

BACKGROUND: Although depot medroxyprogesterone acetate (DMPA) (Depo-Provera) has now been approved for marketing as a contraceptive in the United States, there are still unresolved issues about the relation between DMPA and risk of breast cancer. The two substantial case-control studies of this association yielded similar but inconclusive results. Because their designs were compatible, these studies were pooled to obtain more adequate data for analysis. DESIGN: Pooled results from two case-control studies. SETTING: New Zealand (entire country), Thailand (three centers), Mexico (one center), and Kenya (one center). PARTICIPANTS: A total of 1768 women with breast cancer and 13,905 controls, most of whom were younger than 55 years. MAIN OUTCOME MEASURE: Relative risk (RR) of breast cancer in women who had used DMPA. RESULTS: The RR of breast cancer for women who had ever used DMPA was 1.1 (95% confidence interval [CI], 0.97 to 1.4). There was no increase in risk with increasing duration of use of DMPA, but RR estimates were higher in certain subgroups of women. Further analyses suggested that recent (or current) use was the key factor, with women who had started using DMPA within the previous 5 years estimated to have an RR of 2.0 (95% CI, 1.5 to 2.8). CONCLUSIONS: The increased risk of breast cancer observed in recent (or current) users could be due to enhanced detection of breast tumors in women using DMPA or to acceleration of the growth of preexisting tumors. Women who had used DMPA more than 5 years previously had no increase in risk of breast cancer, regardless of their duration of use.

Breast cancer and prolonged lactation. The WHO Collaborative Study of Neoplasia and Steroid Contraceptives.

The possible relationship between breastfeeding and risk of breast cancer was assessed in multinational hospital-based case-control study that was conducted from 1979 to 1986 in 10 countries, including some in which many women lactated for long periods of time. Data were collected by personal interviews of 2336 cases and 14,900 controls. No statistically significant trends in relative risk estimates with duration of lactation were observed in all women combined, in women who were pre-or postmenopausal, in women of various ages at diagnosis, in women with different numbers of livebirths, or in women characterized by the presence or absence of other risk factors for breast cancer. No trends in risk were observed with mean number of months that a woman lactated per breastfed child, or with number of pregnancies followed by treatment with oral or injectable medication to suppress lactation. In premenopausal women, and in those with two or more livebirths, most risk estimates for women who lactated for various lengths of time > 6 months, relative to the risk in women who breastfed for < or = 3 months, were less than unity, but the 95% confidence intervals of all estimates included one. Long-term lactation may reduce slightly the risk of breast cancer, but the evidence for this from the present and prior investigations is not strong.

High-dose and low-dose combined oral contraceptives: protection against epithelial ovarian cancer and the length of the protective effect. The WHO Collaborative Study of Neoplasia and Steroid Contraceptives.

The relations between use of high-dose and low-dose combined oral contraceptives and epithelial ovarian cancer were compared in an international hospital-based case-control study. 393 cases from seven countries were compared with 2561 matched controls. The odds ratio (OR) was somewhat lower for women who used high-dose oestrogen oral contraceptives (OR = 0.68) than for women who used low-dose preparations (OR = 0.81) although the difference could have occurred by chance. After controlling for time since last use, risk was slightly lower for long-term users of high-dose preparations than for long-term users of low-dose pills. Both high-dose and low-dose oral contraceptives protect against ovarian cancer, but the degree of protection may be slightly weaker for the newer, low-dose products.

Breast cancer and specific types of combined oral contraceptives. The WHO Collaborative Study of Neoplasia and Steroid Contraceptives.

Data on 2,754 cases and 18,565 controls from a multinational hospital-based, case-control study were analysed to determine whether observed associations between combined oral contraceptives and breast cancer are similar for oral contraceptives with varying types and doses of oestrogens and progestins. After stratifying on duration of use, risk was found to be increased in current and recent users, and to decline with time since last use. These associations, of similar strength, were observed for users of products that contain mestranol and ethinyl estradiol, for women who used preparations with progestins derived from 19-nortestosterone and 17-alpha-hydroxyprogesterone, and for those who took preparations with relatively higher and lower doses of oestrogen. When products with equal doses of the same oestrogen or progestin and varying doses of the other hormonal constituent were considered, slightly higher relative risks per year of use were estimated for users of products with relatively higher than lower doses of either the constituent oestrogen or progestin, but the differences in relative risk could readily have occurred by chance. This study provides no evidence that risk of breast cancer in users of oral contraceptives varies by the type of oestrogen or progestin consumed.

Risk of breast cancer in relation to use of combined oral contraceptives near the age of menopause. WHO Collaborative Study of Neoplasia and Steroid Contraceptives.

Data from a multinational, hospital-based, case-control study were analyzed to determine whether use of combined oral contraceptives (OC) around the time of menopause preferentially increases risk of breast cancer. Results show that the relative risk (RR) of breast cancer was increased in women of all ages who had used oral contraceptives within the past year, but not to a greater extent in women near the age of menopause than in younger women. RRs did not increase with duration of OC use after age 45 in either pre- or postmenopausal women. RRs also were not found to be higher in women who were using OCs near the time of either a natural or artificial menopause than in women who used them at other times. This study thus provides no support for the hypothesis that OCs enhance risk of breast cancer by a greater amount when taken around the time of menopause than when taken at other times.

Histologic types of breast carcinoma in relation to international variation and breast cancer risk factors. WHO Collaborative Study of Neoplasia and Steroid Contraceptives.

Associations between breast cancer risk factors and histologic types of invasive breast carcinoma were studied in 2,728 patients. Lobular and tubular carcinomas occurred with increased relative frequency in most high-risk groups. The proportion of these types increased with age to a maximum at 45-49 years and decreased in the following decade. Significantly increased proportions of lobular and tubular carcinomas were also associated with high-risk countries, prior benign breast biopsy, bilateral breast cancer, concurrent mammary dysplasia, high age at first live birth, never-pregnant patients compared to those with a first live birth before age 20, private pay status, and length of education. Nonsignificant increases were associated with family history of breast cancer, less than 5 live births, less than 25 months total of breast feeding, use of oral contraceptives or IUD, and high occupational class. As a general trend, the higher the overall relative risk, the higher the proportion of lobular and tubular carcinomas. The occurrence of other histologic types also increased with increased breast cancer risk, but to a smaller degree than for lobular/tubular carcinomas. It is suggested that all hormonally related, socio-economic and geographic risk factors exert their effect by selectively increasing number of lobular cells at risk. Family history of breast carcinoma and age over 49 years did not follow the general trend parallel increases in the proportion of lobular/tubular carcinomas and breast cancer risk, and may operate through other mechanisms.

Efficient selection of controls for multi-centered collaborative studies of rare diseases.

An increasing number of epidemiologic studies of rare diseases are being conducted as collaborative ventures among several institutions in different geographic areas. Geographic area must be accounted for in the analysis, along with age and other potential confounders. This paper shows that it may not be possible to accurately predict the distribution of persons with rare diseases by age and geographic area. Therefore, selecting controls by matching on age group and geographic area is more efficient than selecting controls in all age and geographic area strata based on prior estimates of expected numbers of cases.