Reasons for lack of referral to medical oncology for systemic therapy in stage IV non-small-cell lung cancer: comparison of 2003-2006 with 2010-2011.

INTRODUCTION: Only approximately 25% of stage iv non-small-cell lung cancer (nsclc) patients receive systemic therapy. For such patients, we examined factors affecting referral to a cancer centre (cc) and to medical oncology (mo), and use of systemic therapy. METHODS: Using the Glans-Look Lung Cancer database, we completed a chart review of stage iv nsclc patients diagnosed in Southern Alberta during 2003-2006 and 2010-2011, comparing median overall survival (mos), referral, and treatment in the two cohorts. RESULTS: Of the 922 patients diagnosed in 2003-2006 and the 560 diagnosed in 2010-2011, 94% and 82% respectively were referred to a cc, with 22% and 23% receiving traditional chemotherapy (tctx). Referral to a cc or mo and use of tctx correlated with survival (p < 0.0001): The mos duration was 11.2 months in those receiving tctx and 1.0 months in those not referred to a cc. The overall mos duration was similar in the two cohorts (4.1 months vs. 3.9 months, p = 0.47). Major reasons for lack of referral to mo included poor functional status, rapid decline, and patient wish, which were similar to the reasons for forgoing tctx. In the two cohorts, 87 (9.4%) and 42 (7.5%) patients received epidermal growth factor inhibitors, with a mos duration of 16.2 months. Multivariable analysis showed that male sex [hazard ratio (hr): 1.16; p = 0.008] and pulmonary embolus (hr: 1.2; p = 0.002) correlated with worse survival. In contrast, receipt of chemotherapy (hr: 0.5; p < 0.001) and enrolment in a clinical trial (hr: 0.76; p = 0.049) correlated with better survival. CONCLUSIONS: Our experience confirms that, over time, uptake of systemic therapy, including tctx and targeted therapy, changed little despite their established efficacy. Most of the factors limiting systemic therapy uptake appear to be non-modifiable at the time of referral. Rapid diagnosis and the availability of well-tolerated drugs for all nsclc patients will likely be the most important factors in increasing systemic therapy uptake in this population.

Adenoid cystic carcinoma of head and neck: clinical predictors of outcome from a Canadian centre.

OBJECTIVES: Adenoid cystic carcinoma (acc) is often treated with surgery, with or without adjuvant radiation therapy (rt). We evaluated disease characteristics, treatments, and potentially prognostic variables in patients with acc. METHODS: Our retrospective analysis considered consecutive cases of acc presenting at a tertiary care hospital between 2000 and 2014. Factors predictive of overall survival (os) and disease-free survival (dfs) were identified by univariate analysis. RESULTS: The 60 patients analyzed had a mean age of 58 years (range: 22-88 years), with a 2:1 female:male ratio. Tumour locations included the major salivary glands (40% parotid, 17% submandibular and sublingual), the oro-nasopharyngeal cavity (27%), and other locations (16%). Of the 60 patients, 35 (58%) received surgery with adjuvant rt; 12 (20%), rt only; 13 (22%), surgery only. Of 18 patients (30%) who experienced a recurrence within 5 years, 3 (5%) developed local recurrence only, and the remaining 15 (25%), distant metastasis. The 5-year os and dfs were 64.5% [95% confidence interval (ci): 45.9% to 78.1%] and 46.2% (95% ci: 29.7% to 61.2%) respectively. In patients without recurrence, 5-year os was 77% (95% ci: 52.8% to 89.9%), and in patients with recurrence, it was 42.7% (95% ci: 15.8% to 67.6%). Patients treated with rt only had a 5-year os of 9.2%. Predictors of 5-year dfs were TNM stage, T stage, nodal status, treatment received, and margin status; age, nodal status, treatment received, and margin status predicted 5-year os. CONCLUSIONS: Despite surgery and rt, one third of patients with acc experience distant recurrence. Patients whose tumours are not amenable to surgery have a poor prognosis, indicating a need for alternative approaches to improve outcomes.

First-, second-, third-line therapy for mRCC: benchmarks for trial design from the IMDC.

BACKGROUND: Limited data exist on outcomes for metastatic renal cell carcinoma (mRCC) patients treated with multiple lines of therapy. Benchmarks for survival are required for patient counselling and clinical trial design. METHODS: Outcomes of mRCC patients from the International mRCC Database Consortium database treated with 1, 2, or 3+ lines of targeted therapy (TT) were compared by proportional hazards regression. Overall survival (OS) and progression-free survival (PFS) were calculated using different population inclusion criteria. RESULTS: In total, 2705 patients were treated with TT of which 57% received only first-line TT, 27% received two lines of TT, and 16% received 3+ lines of TT. Overall survival of patients who received 1, 2, or 3+ lines of TT were 14.9, 21.0, and 39.2 months, respectively, from first-line TT (P<0.0001). On multivariable analysis, 2 lines and 3+ lines of therapy were each associated with better OS (HR=0.738 and 0.626, P<0.0001). Survival outcomes for the subgroups were as follows: for all patients, OS 20.9 months and PFS 7.2 months; for those similar to eligible patients in the first-line ADAPT trial, OS 14.7 months and PFS 5.6 months; for those similar to patients in first-line TIVO-1 trial, OS 24.8 months and PFS 8.2 months; for those similar to patients in second-line INTORSECT trial, OS 13.0 months and PFS 3.9 months; and for those similar to patients in the third-line GOLD trial, OS 18.0 months and PFS 4.4 months. CONCLUSIONS: Patients who are able to receive more lines of TT live longer. Survival benchmarks provide context and perspective when interpreting and designing clinical trials.

Assessment of treatment patterns associated with injectable disease-modifying therapy among relapsing-remitting multiple sclerosis patients.

BACKGROUND: Availability of oral disease-modifying therapy (DMT) for relapsing-remitting multiple sclerosis (RRMS) may affect injectable DMT (iDMT) treatment patterns. OBJECTIVE: The objective of this paper is to evaluate iDMT persistency, reasons for persistency lapses, and outcomes among newly diagnosed RRMS patients. METHODS: Medical records of 300 RRMS patients initiated on iDMT between 2008 and 2013 were abstracted from 18 US-based neurology clinics. Eligible patients had ≥3 visits: pre-iDMT initiation, iDMT initiation (index), and ≥1 visit within 24 months post-index. MS-related symptoms, relapses, iDMT treatment patterns (i.e. persistency, discontinuation, switching, and restart), and reasons for non-persistency were tracked for 24 months. RESULTS: At 24 months, iDMT persistency was 61.0%; 28.0% of patients switched to another DMT, 8.0% discontinued, and 3.0% stopped and restarted the same iDMT. The most commonly identified reasons for non-persistency were perceived lack of efficacy (22.2%), adverse events (18.8%), and fear of needles/self-injecting (9.4%). At 24 months, 38.0% of patients had experienced a relapse and 11.0% had changes in MRI lesion counts. Patients without MS-related symptoms at index reported increases in the incidence of these symptoms at 24 months. CONCLUSIONS: Non-persistency with iDMT remains an issue in the oral DMT age. Many patients still experienced relapses and disease progression, and should consider switching to more effective therapies.

Freezing immature oocytes.

The establishment of a long-term preservation system for mammalian oocytes is important for the development of both biological and medical sciences. A number of efforts have been made to develop this system. In human reproductive medicine, the development of an oocyte cryopreservation system can improve the efficacy of the current assisted reproductive technology (ART) for infertile patients with severe reproductive disorders. In this article, the technical development of cryopreservation programs for human oocytes and its biological background were reviewed. Clinical outcome after the use of this technology was further introduced.

Effect of dietary lipid on gamma-glutamyl transferase-positive foci during hepatocarcinogenesis in rats.

The effect of dietary lipid on gamma-glutamyl transferase-positive (GGT-positive) foci was investigated. Female Sprague-Dawley rats were dosed with diethylnitrosamine (15 mg/kg) at 24 h of age. After weaning, they were fed nutritionally complete semipurified diets for 3 months. Rats fed 15% corn oil had significantly lower hepatic phospholipid eicosapentaenoate and docosahexaenoate than rats fed 7.5% corn oil plus 7.5% fish oil, 5% corn oil plus 10% fish oil (P < 0.05). However, rats fed 15% corn oil had significantly greater hepatic phospholipid arachidonate than rats fed the other two diets (P < 0.05), suggesting that n-3 polyunsaturated fatty acids were incorporated into hepatic phospholipid at the expense of n-6 polyunsaturated fatty acids. Hepatic PGF2alpha content was significantly greater in rats fed 15% corn oil than in rats fed the other two diets (P < 0.05). Rats fed fish oil had significantly lower hepatic vitamin E content than rats fed corn oil (P < 0.05). Hepatic lipid peroxidation (TBARS) tended to increase with increased dietary fish oil (P < 0.05). Dietary lipid did not influence GGT-positive foci area or number. In conclusion, dietary lipid affected hepatic PGF2alpha production, however, showed no effect on GGT-positive foci area and number. This may suggest that PGF2alpha is not the underlying mechanism for GGT-positive foci during hepatocarcinogenesis.

Regulatory effects of dietary n-3 and n-6 lipids on plasma and hepatic lipid levels, liver cell number and microsomal protein content in spontaneously hypertensive rats.

Weanling male spontaneously hypertensive rats were fed semipurified diets containing either corn or fish oil for 8 weeks. Rats fed on fish oil diet had significantly lower plasma triglyceride, total cholesterol and HDL-cholesterol levels than rats fed on corn oil diet (P < 0.05). Moreover, rats fed on fish oil diet had significantly lower liver total lipid and triglyceride concentrations than rats fed on corn oil diet (P < 0.05). Dietary lipids were reflected in plasma fatty acid composition. Rats fed on fish oil diet had significantly greater plasma eicosapentaenoate (EPA) and docosahexaenoate (DHA) (n-3 PUFAs) with an accompanying decrease in plasma linoleate (LA) and arachidonate (AA) (n-6 PUFAs), in comparison with the rats fed corn oil (P < 0.05). Those results would suggest that the n-3 PUFAs were incorporated into plasma lipids at the expense of the n-6 PUFAs. Rats fed on corn oil diet had significantly greater liver DNA content than rats fed on fish oil diet (P < 0.05), thereby implying that the n-3 PUFAs in fish oil had an inhibitory effect on liver cell proliferation. Furthermore, rats fed on fish oil diet had significantly greater hepatic microsomal protein content than rats fed on corn oil diet (P < 0.05), indicating that fish oil exerted a stimulatory effect on hepatic microsomal enzymes.

Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program.

This study describes the results with immature human follicular oocytes harvested from unstimulated ovaries, matured in vitro, fertilized, and transferred to an agonadal recipient. Two hundred seventy immature oocytes were aspirated from 23 ovaries removed for various gynecological indications from August 1988 to October 1989. The numbers of follicular oocytes collected from ovaries were compared by patients' ages and the stages of menstrual cycle. Immature oocytes in vitro were incubated with either mature follicular fluid (FF) or fetal cord serum (FCS). The maturation rate in the mature FF group was 55.8%, significantly higher than the 35.9% in the FCS group. In addition, mature FF group was shown to provide a significantly higher fertilization rate than the FCS group (81.0% versus 31.6%). More fertilized eggs developed into normal embryos in the nonstimulated cycle group than in stimulated cycles with routine treatment. Finally, five embryos were transferred to a woman with premature ovarian failure on day 18 of a steroid replacement cycle. She subsequently delivered healthy triplet girls. These results suggest that in vitro maturation of immature oocytes from unstimulated ovaries with mature follicular fluid could be used successfully in a donor oocyte program after in vitro fertilization.

Effects of 1,2-propanediol and freezing-thawing on the in vitro developmental capacity of human immature oocytes.

OBJECTIVE: To investigate effects of 1,2-propanediol and freezing-thawing treatment on the maturation and developmental capacity of the human immature oocytes obtained from unstimulated ovaries. DESIGN: Intact cumulus-enclosed immature oocytes collected from unstimulated ovaries were divided into three groups, such as no treatment as control (group 1), only 1,2-propanediol-treated (group 2), and cryopreserved group (group 3). Oocytes in group 1, group 2, and survived oocytes from cryopreservation in group 3 were cultured for 48 hours. A random selection of matured oocytes was inseminated with normal donor sperm to evaluate the fertilization and developmental capacity. SETTING: Infertility Medical Center at the CHA General Hospital, Seoul, Korea. PATIENT(S): Oocytes were obtained from patients undergoing gynecological surgery. MAIN OUTCOME MEASURE(S): Rates of survival, maturation to metaphase II, fertilization, and cleavage. RESULT(S): Survival rate after freezing-thawing in group 3 was 55.1% (54/98). Oocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum, 10 IU/mL pregnant mare serum gonadotropin, and 10 IU/mL hCG. Maturation rates were 76.8% (63/82), 67.1% (47/70), and 59.3% (32/54) in the groups 1, 2, and 3, respectively. Maturation rate in group 3 was significantly lower than that of group 1. Fertilization rates were 90.5% (19/21), 81.0% (17/21), and 42.9% (6/14), and cleavage rates were 94.7% (18/19), 88.2% (15/17), and 16.7% (1/6) in groups 1, 2, and 3, respectively. Fertilization and cleavage rates of survived oocytes in group 3 also were significantly lower than those of groups 1 and 2. CONCLUSION(S): Results suggest that the pretreatment with 1.5 M 1,2-propanediol itself before the freezing has no inhibitory effect on the maturation, fertilization, and cleavage of human immature oocytes in vitro. However, the freezing-thawing procedure used had detrimental effects on the maturation and developmental capacity.

Pregnancies and deliveries after in vitro maturation culture followed by in vitro fertilization and embryo transfer without stimulation in women with polycystic ovary syndrome.

OBJECTIVE: To improve the efficacy of an IVF-ET program for unstimulated patients with polycystic ovary syndrome (PCOS) with the use of culture for oocyte maturation. DESIGN: Prospective studies with the comparison of different ET procedures from March 1995 through February 1998. SETTING: University-affiliated hospital. PATIENT(S): Ninety-four cycles in 64 consenting patients with PCOS. INTERVENTION(S): Immature oocytes were retrieved from unstimulated patients with PCOS and subsequently cultured and fertilized in vitro. Zygote intrafallopian transfer (ZIFT), uterine ET, or a combined approach of ZIFT + uterine ET was subsequently performed. MAIN OUTCOME MEASURE(S): Laboratory and clinical data. RESULT(S): Among 1, 280 immature oocytes (13.6 +/- 7.5 oocytes per patient) retrieved, 89% (1,139) were morphologically normal, and 62.2% (708/1,139) of the normal oocytes matured in vitro after culture for 48 hours. When intracytoplasmic sperm injection was performed, 68% (481/708) developed to the normal pronuclear stage, and 88.1% of the embryos cocultured with Vero cells (266/302) cleaved. Eighty-five ET cycles were conducted and pregnancy was established in 23 cycles (27.1%), which consisted of 8 after uterine ET and 15 after a combined approach. Seventeen patients delivered 20 normal infants. CONCLUSION(S): The IVF-ET method using no ovarian stimulation followed by in vitro maturation culture can be a feasible assisted reproductive technology for treatment of PCOS with various complications.

Embryotropic role of hemoglobin and ethylenediaminetetraacetic acid in preimplantation development of ICR mouse 1-cell embryos.

OBJECTIVE: To evaluate embryotropic action of hemoglobin (Hb) and ethylenediaminetetraacetic acid (EDTA) on preimplantation embryo development. DESIGN: In vitro model study using mouse embryos. SETTING: University affiliated hospital, Pochon CHA University. ANIMALS: Four-week-old block strain ICR mice naturally mated after superovulation. INTERVENTION(S): One-cell embryos were cultured in serum-free, modified preimplantation-1 medium, to which 1 microg/ml Hb and/or 0.1 mM EDTA were added. MAIN OUTCOME MEASURE(S): Preimplantation development and blastomere number. RESULT(S): More (P<.05) 1-cell embryos developed to the 4-cell (52% vs. 67%-84%), 8-cell (48% vs. 65%-81%), and blastocyst (40% vs. 61%-79%) stages after the addition of hemoglobin (Hb) and/or EDTA than after no addition. Highest proportion of embryos developed to each stage after the combined addition of Hb+EDTA. EDTA specifically stimulated the development before the 8-cell stage, which was as similar as Hb+EDTA. On the contrary, higher ratio of morula to blastocyst transformation was obtained after the addition of Hb or Hb+EDTA than after no addition (0.76 vs. 0.96-0.98). Significant increases in the cell number of blastocysts (46.5-47.2 vs. 53.2 cells), inner cell mass (ICM) cells (16.7-17.5 vs. 21 cells), and the ratio of ICM cells to trophoblasts (0.3-0.37 to 0.39) were found after the combined addition of Hb+EDTA, compared with no addition or with the addition of EDTA or Hb alone. CONCLUSIONS: Hb and EDTA have stage-specific effects on supporting preimplantation embryo development; Hb promotes both the development before the 8-cell stage and the morula to blastocyst transformation, whereas EDTA mainly promotes the development to the 8-cell stage. The combined exposure of embryos to Hb and EDTA improves not only preimplantation development but also the growth and quality of blastocysts.

Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage.

OBJECTIVE: To investigate effects of cryoprotectant and cryopreservation on the chromosome and microtubule configuration of human immature oocytes. DESIGN: Intact cumulus-enclosed immature oocytes were collected from unstimulated ovaries and divided into three groups: group 1, no treatment (control); group 2, only 1,2-propanediol treatment, and group 3, cryopreserved oocytes. Oocytes in groups 1 and 2, and oocytes that survived after cryopreservation in group 3 were cultured for 48 hours. SETTING: Infertility Medical Center at the CHA General Hospital, Seoul, Korea. PATIENT(S): Oocytes were obtained from patients undergoing gynecologic surgery. MAIN OUTCOME MEASURE(S): Maturation rate and abnormality in chromosomes by fluorescence in situ hybridization and in the spindle by immunostaining for tubulin. RESULT(S): There was no effect of propanediol-only treatment on the chromosomal (41.4%) and spindle abnormalities (35.3%) in group 2 compared with control oocytes (31.8% and 22.2%, respectively), whereas a statistically significant increase in abnormalities in chromosomes (77.8%) and spindles (70%) was found in group 3. CONCLUSION(S): Human oocytes matured in vitro after cryopreservation at the germinal vesicle stage showed increased incidence of chromosomal and spindle abnormalities. These abnormalities may impair the capacity for further development of the embryos derived from frozen-thawed oocytes.

Improved human oocyte development after vitrification: a comparison of thawing methods.

OBJECTIVE: To evaluate the developmental competence of vitrified human oocytes thawed using two different methods to establish an effective cryopreservation protocol. DESIGN: In vitro model study. SETTING: University-affiliated hospital. PATIENT(S): Patients who underwent a long protocol of ovarian stimulation with GnRH and gonadotropins. INTERVENTION(S): Vitrified oocytes from the patients were thawed using either a four-step method with 2.5-minute intervals or a four-step method with 5-minute intervals. MAIN OUTCOME MEASURE(S): Morphologic normality, maturation, fertilization, and development of the oocytes to the blastocyst stage. RESULT(S): The two thawing methods did not significantly affect the morphologic normality (84%-100%), maturation (75%-100%), fertilization (38%-71%), polyspermy (more than three pronuclei; 0%-20%), or parthenogenetic activation (only female pronucleus; 0%-8%) of the vitrified oocytes. However, more of the vitrified oocytes developed to the two-cell (71%-100% versus 50%-67%), four-cell (71%-93% versus 0%-50%), eight-cell (46%-71% versus 0%), and blastocyst (23%-36% versus 0%) stages after thawing using the four-step method with 2.5-minute intervals than using the four-step method with 5-minute intervals. CONCLUSION(S): Vitrified human oocytes developed to the blastocyst stage with IVF. A four-step thawing method with 2.5-minute intervals was more effective in supporting preimplantation embryo development than a four-step thawing method with 5-minute intervals.

In vitro blastocyst formation of human oocytes obtained from unstimulated and stimulated cycles after vitrification at various maturational stages.

OBJECTIVE: To evaluate the developmental competence and chromosomal normality of oocytes vitrified at various times after maturation culture. DESIGN: In vitro model study. SETTING: A university-affiliated hospital. PATIENT(S): Unstimulated women who underwent cesarean section or oophorectomy and infertile women who underwent a long protocol of GnRH stimulation. INTERVENTION(S): Retrieved oocytes were vitrified at 0 or 48 hours after culture in unstimulated cycles and at 0, 8-15, or 24-28 hours after culture in stimulated cycles. MAIN OUTCOME MEASURE(S): Postthaw morphologic normality, maturation, fertilization, cleavage, blastocyst formation, and chromosome number. RESULT(S): In the 53 oocytes that were obtained from unstimulated cycles, no statistically significant differences were found in rates of morphologic normality (range, 56%-63%) or fertilization (range, 31%-37%) according to the time of vitrification. In the 50 oocytes that were obtained from stimulated cycles, more of those that were vitrified at 24-28 hours were morphologically normal than those that were vitrified at 0 or 8-15 hours. Regardless of these differences, high cleavage rates (83%-100%) were obtained that did not differ significantly among the treatment groups. In both cycles, 20%-43% of cleaved oocytes developed to the blastocyst stage by 6 days after IVF. All the karyotyped blastocysts, three from unstimulated cycles and four from stimulated cycles, had a normal number of chromosomes. CONCLUSION(S): Vitrified and thawed oocytes from unstimulated or stimulated cycles developed to the blastocyst stage, regardless of when vitrification occurred; the number of chromosomes in the blastocysts was normal.

Increased expression and localization of a serine protease inhibitor, protease nexin-1 (PN-1), in the ovary and uterus during implantation in rat.

Protease nexin-1 (PN-1) is a serine protease inhibitor (serpin) that inactivates several proteases, including thrombin, urokinase, plasminogen activators (PA), and plasmin. It also plays a role in regulating proteolytic activity generated by PA system. PN-1 is known to be involved in tissue remodeling, cellular invasiveness, matrix degradation, and tumor growth. However, the role of PN-1 in female reproductive tracts, such as the uterus, ovary, and oviduct, during pregnancy is not known. The present study was designed to investigate the changes of PN-1 mRNA level and localization in the tracts during implantation and early pregnancy by using reverse transcription (RT)-polymerase chain reaction (PCR) and in situ hybridization. We found that PN-1 mRNA levels were coordinately regulated during early pregnancy in a stage- and tissue-specific manner, such that an increased expression of PN-1 gene appeared at the time of the implantation period in the uterus and ovary. Both the uterus and ovary synthesized PN-1 mRNA and their maximal PN-1 expression occurred on Day 6.5 postcoitum (p.c.). On 13.5 days of pregnancy, PN-1 level was low in the uterus and ovary. On the other hand, PN-1 mRNA in the oviduct did not show after 6.5 days of pregnancy. It appears that PN-1 mRNA in the uterus and ovary was highly regulated during early pregnancy, which might have an important role in implantation of rat blastocysts. PN-1 was localized in endometrial stromal cells of the uterus and in granulosa cells of the unstimulated primary follicles in the ovary during periimplantation period. Also, PN-1 mRNA expression was higher at implantation period than that at nonimplantation period of pregnancy. In conclusion, PN-1 is expressed in female reproductive tracts and highly regulated during implantation and early pregnancy.

Spatial expression of a DNA repair gene, N-methylpurine-DNA glycosylase (MPG) during development in mice.

BACKGROUND: DNA repair is a crucial phenomenon that maintains the chromosome integrity of genome which are continuously damaged by endogenous and exogenous alkylating agents. If the damaged DNA is not repaired, it may lead to mutation, chromosomal aberration, aging and cancer. N-methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, removes N-methylpurine and other damaged purines in DNA. MATERIALS AND METHODS: MPG mRNA expression was revealed at various stages of mouse development from day 7.5 p.c. (post coitum) embryo to day 400 mature adult by Northern blot hybridization or RT-PCR. RESULTS: MPG transcripts were abundant in the mouse embryo during pregnancy and in adult testis and ovary. The MPG mRNA level in the testis was low in 1-week-old mice, but the level showed its maximum among the organs tested in 4-week-old young adults. In placenta, the level of MPG mRNA continuously decreased from day 7.5 p.c. to day 17.5 p.c. CONCLUSIONS: The spatial expression of MPG gene is highly regulated. Transcription of MPG is maximum in rapidly dividing and growing tissues during development. These data suggest that an elevated rate of MPG transcription is required for DNA replication.

Gene amplification and expression of the DNA repair enzyme, N-methylpurine-DNA glycosylase (MPG) in HPV-infected cervical neoplasias.

BACKGROUND: Lethal and mutagenic damages of DNA is caused by a variety of agents including viruses. It is known that HPV is one of the major causes of cervical carcinogenesis and that cells eliminate DNA lesions with DNA repair enzymes. However, the role of N-methylpurine-DNA glycosylase (MPG) is not known in the development of cervical cancer. MATERIALS AND METHODS: Multiplex polymerase chain reaction (PCR) was used for the detection and typing of HPV in the biopsy. Gene amplification of MPG was measured by a PCR-based assay. The mRNA levels of MPG were determined by reverse transcription-PCR using hypoxanthine-guanine phosphoribosyl transferase as the reference gene. An immunohistochemical technique was used to examine the distribution of MPG in the tissues. RESULTS: Of 68 Korean cervical neoplasia patients, 86.8% showed HPV infection. High-risk HPV 16/18 were the most prevalent but positive only in 47.3% of the invasive cancer patients. Gene amplification of MPG was significantly increased in high-risk HPV-infected tissues as compared to low-risk HPV-infected and normal tissues (p < 0.05). The mRNA levels of MPG were higher in HPV-infected invasive carcinoma than normal cervical tissues. Immunohistochemical staining revealed that the intracellular expression and distribution (localization) of MPG altered in the cervical neoplasia. Interestingly, MPG expression in CIN III and invasive carcinoma (IC) was much higher than normal and CIN I. Granular positivity of MPG was notable in the perinuclear regions of the cytoplasm in HPV-infected invasive cancer. CONCLUSION: This is the first report on MPG expression in cervical neoplasia. Our results indicate that the gene amplification and expression of MPG were increased in high-risk HPV-infected cervical neoplasias and the intracellular distribution of MPG protein was altered, suggesting a role of MPG in carcinogenesis.

In vitro maturation and in vitro fertilization of bovine oocytes cultured in a chemically defined, protein-free medium: effects of carbohydrates and amino acids.

This study was conducted to examine the effects of carbohydrates and amino acids on the maturation and fertilization of bovine oocytes. To evaluate the effect of each treatment without any unpredictable interference, oocytes were cultured in a simply defined medium (modified Tyrode's medium; mT) without the addition of hormones and proteins. In Experiment 1, oocyte maturation to the metaphase-II stage was significantly (P<0.0001) enhanced after the addition of glucose (5.6 mM), lactate (10 mM) and/or pyruvate (0.5 mM) to mT (37-74%) than after no addition (0%). In mT supplemented with glucose, the addition of 19 essential and non-essential amino acids (aa; 0, 0.01, 0.1, 1, 5 or 10%) did not further improve in vitro maturation (Experiment 2) or in vitro fertilization (Experiment 3) of oocytes. However, more (P<0.05) pronuclear formation after in vitro-insemination was found in oocytes matured in mT with 1% aa and glucose than in oocytes matured in mT with glucose alone (56% vs. 35%). Penetration of spermatozoa into the ooplasm was initiated at 3 h after insemination and pronuclear formation from 8 h (Experiment 4). When cultured inseminated oocytes were examined up to 192 h post insemination, a significant (P<0.05) increase in the number of 2-cell (18 v. 38%) and 8-cell embryos, (7 v. 20%) and morulae (0 v. 8%) was found after the addition of 1% aa to mT with glucose than after no addition (Experiment 5). A limited number of oocytes matured in mT with aa and glucose developed to the blastocyst stage (6%). These results indicate that exogenous carbohydrates and amino acids are prerequisites for the maturation and fertilization of bovine oocytes in vitro. Glucose alone promotes the nuclear maturation of oocytes, whereas amino acids aid the pronuclear formation of fertilized oocytes.

Development of in vitro matured bovine oocytes after cryopreservation with different cryoprotectants.

In vitro matured bovine oocytes at the metaphase-II stage were slowly frozen in phosphate buffered saline (PBS) containing 1.0 M glycerol, 1.0 M dimethylsulfoxide (DMSO) or 1.0 M propylene glycol (PROH). When thawed rapidly, more (P<0.05) oocytes were morphologically normal after being frozen with DMSO (86%) or PROH (83%) than with glycerol (62%). When inseminated in vitro with frozen-thawed bull spermatozoa, higher (P<0.05) penetration rates were observed in DMSO (79%) or PROH (76%) than in glycerol (48%). The percentages of oocytes developing to the 2-cell stage at 48 h postinsemination were also significantly (P<0.05) higher in DMSO (51%) and PROH (54%) than in glycerol (33%). However, a significant increase in the proportions of 8-cell embryos (46 vs 21 to 26%; P<0.05) at 72 h postinsemination and morulae (14 vs. 6 to 8%; P<0.05) was derived from oocytes frozen with PROH than with DMSO or glycerol. In conclusion, the type of cryoprotectant used is one of the critical factors affecting developmental competence of bovine oocytes frozen at the metaphase-II stage. For this stage of oocytes, PROH was the most effective, yielding a large number of 8-cell embryos and morulae than either glycerol or DMSO in a slow freezing method combined with a 3-step thawing protocol.

Soluble adhesion molecules and cytokines in tumor-associated tissue eosinophilia of nasopharyngeal carcinoma.

The phenomenon of tumor-associated tissue eosinophilia (TATE) is seen in some cases of nasopharyngeal carcinoma (NPC) and is characterized by the eosinophils breaking through the vascular wall and pervading the tumor stroma. The margination and trans-endothelial migration of eosinophils in a typical inflammatory reaction depend on the activating effects of certain cytokines and the expression of adhesion molecules on the eosinophils and endothelial cells. In order to investigate whether the adhesion molecules and activating cytokines play a role in eosinophil tumor infiltration, we measured the serum levels of 3 adhesion molecules, intercellular adhesion molecule-1, E-selectin and vascular cell adhesion molecule-1, and 2 cytokines, IL-3 and IL-5, in 60 NPC patients and 40 normal healthy subjects. We found that the NPC patients had higher serum levels of all three soluble adhesion molecules than the normal subjects but the levels of adhesion molecules failed to correlate with the TATE phenomenon. The levels of IL-3 and IL-5 appeared not to differ between the NPC and control groups. We postulate that the three soluble adhesion molecules do not play a major role in TATE and that their elevation in serum may be due to local and/or systemic immune responses.