Effect of exercise intensity on albuminuria in adolescents with Type 1 diabetes mellitus.

AIMS: Exercise may be useful to detect patients with diabetes prone to develop persistent microalbuminuria. We studied the relationship between exercise intensity, measured as maximal oxygen consumption (VO(2)max), and microalbuminuria in patients with Type 1 diabetes mellitus patients. METHODS: We studied 10 patients, age range 10-18 years, with Type 1 diabetes who were normotensive and normoalbuminuric, with less than 10 years since diagnosis. Patients had normal renal function, without infections or clinical evidence of complications. Metabolic control was intensively adjusted in all patients. They underwent three consecutive physical exercise tests, reaching 100, 80 and 60% of the maximal cardiac frequency response. RESULTS: Eight patients had adequate to regular metabolic control. All patients had lower than predicted VO(2)max values. At 60%, only three patients showed microalbuminuria in excess of 20 µg/min, two of them had inadequate metabolic control. Post-exercise microalbuminuria exceeded normal values in nine, seven and three patients when submitted to 100, 80 and 60% of exercise intensity, respectively. CONCLUSIONS: Microalbuminuria increased with exercise intensity. Sex, body composition and VO(2)max were the main factors associated with microalbuminuria. The prognostic significance of albuminuria induced by intense exercise in these subjects with Type 1 diabetes is not yet known.

Symptoms at menopause and care of grandchildren.

OBJECTIVE: To study the possible interaction of care of grandchildren with women's symptoms at postmenopause. METHODS: A cross-sectional, community-based study was carried out recruiting a total of 386 postmenopausal women at public places in the city of León, Mexico. For each woman, we collected data about their interaction with children and grandchildren, and scores of physical and emotional symptoms frequently found at menopause. Levels of follicle stimulating hormone and estradiol in serum were measured. RESULTS: Women who met their children more frequently had decreased sexual interest. Women without children or grandchildren had lower scores on the empty nest syndrome (ENS) and non-specific symptoms of depression (NSSD). The care of grandchildren was associated with loss of sexual interest, depression, NSSD and ENS. Age was inversely associated with hot flushes and anxiety. Schooling was strongly associated with lower scores for hot flushes, depression, anxiety and ENS. CONCLUSIONS: Among other factors associated with symptoms at postmenopause, such as hormone conditions, age and schooling, maternal investment in children and grandchildren may impose a physical and emotional cost that favors the onset of symptoms at postmenopause.

Expression of estrogen receptor alpha and beta in breast cancers of pre- and post-menopausal women.

Expression of estrogen receptors (ER) is clinically relevant in designing therapeutic strategies. The relative importance of the two types of estrogen receptors (ER-alpha and ER-beta) in human breast cancers in pre- and post-menopausal women has not been properly defined. To determine the possible association between the expression of estrogen receptor and serum estradiol levels in pre- and post-menopausal women with breast cancer. 44 patients with invasive ductal carcinoma of the breast were studied and a breast tissue biopsy was taken. ER-alpha and ER-beta were detected by immunocytochemistry. Serum levels of estradiol and estrone were measured by radioimmunoassay and FSH was measured using IRMA. We studied 21 pre- and 23 post-menopausal women with breast carcinoma. Examining the number of cases with tumors positive for ER, we found no differences in the frequency of ER-alpha between pre- and post-menopausal women, but ER-beta decreased marginally after menopause (p < 0.051). In cases with tumors positive for ER, the proportion of cells positive for ER-alpha was similar post-menopausally (53.95%) and pre-menopausally (57.21%), but for ER-beta the number of positive cells decreased significantly after menopause (p < 0.051). In pre-menopausal women there was a correlation between serum estradiol levels and ER-beta; in post-menopausal women there was a correlation between serum FSH levels and ER-alpha. These results indicate that estradiol levels in women with mammary carcinoma are related to ER-beta expression in the breast tumor tissue.

Telomerase activity, estrogen receptors (alpha, beta), Bcl-2 expression in human breast cancer and treatment response.

BACKGROUND: The mechanism for maintaining telomere integrity is controlled by telomerase, a ribonucleoprotein enzyme that specifically restores telomere sequences, lost during replication by means of an intrinsic RNA component as a template for polymerization. Among the telomerase subunits, hTERT (human telomerase reverse transcriptase) is expressed concomitantly with the activation of telomerase. The role of estrogens and their receptors in the transcriptional regulation of hTERT has been demonstrated. The current study determines the possible association between telomerase activity, the expression of both molecular forms of estrogen receptor (ERalpha and ERbeta) and the protein bcl-2, and their relative associations with clinical parameters. METHODS: Tissue samples from 44 patients with breast cancer were used to assess telomerase activity using the TRAP method and the expression of ERalpha, ERbeta and bcl-2 by means of immunocytochemical techniques. RESULTS: Telomerase activity was detected in 59% of the 44 breast tumors examined. Telomerase activity ranged from 0 to 49.93 units of total product generated (TPG). A correlation was found between telomerase activity and differentiation grade (p = 0.03). The only significant independent marker of response to treatment was clinical stage. We found differences between the frequency of expression of ERalpha (88%) and ERbeta (36%) (p = 0.007); bcl-2 was expressed in 79.5% of invasive breast carcinomas. We also found a significant correlation between low levels of telomerase activity and a lack of ERbeta expression (p = 0.03). CONCLUSION: Lower telomerase activity was found among tumors that did not express estrogen receptor beta. This is the first published study demonstrating that the absence of expression of ERbeta is associated with low levels of telomerase activity.

Stimulation of luteinizing hormone-releasing hormone release from perifused hypothalamic fragments by phospholipase A2.

LHRH release is dependent on the availability of calcium, and prostaglandin E2 is a potent releaser of LHRH. Therefore, we investigated the role of phospholipase A2 (PLA2) on the release of LHRH from the hypothalamus. Four rat hypothalami were perifused with Krebs-Ringer buffer, and after a 60-min preincubation period, PLA2 was applied during 10 min. The LHRH response was determined by RIA of 10-min fractions collected for the next 60 min. PLA2 induced LHRH release in a dose-related manner at amounts of 2, 10, and 50 U. Omission of Ca++ from the medium using EGTA eliminated the PLA2 effect. Indomethacin treatment increased rather than diminished the PLA2 stimulation. Perifusion with melittin, an activator of PLA2, also increased LHRH release. These results are interpreted as a demonstration that PLA2 has a role in the release of LHRH and that a different route of the cyclooxygenase may be involved besides the well known mediation of prostaglandin E2.

Participation of the Na+/H+ exchanger in the phospholipase-A2 activation of luteinizing hormone-releasing hormone release in rat hypothalamic fragments.

The role of the Na+/H+ exchanger in the phospholipase-A2 (PLA2) stimulation of LHRH release was investigated using in vitro incubations of rat hypothalamic fragments. It was found that monensin, the Na+/H+ ionophore, increased LHRH release in a dose-related manner. That effect diminished in the absence of calcium as well as after the addition of 2,4'-dibromoacetophenone, a blocker of PLA2 action. Amiloride, a blocker of the Na+/H+ exchanger, did not alter the effect of monensin. However, amiloride significantly diminished the effect of melittin, an activator of PLA2 action. LHRH release under PLA2 did not change when amiloride was added to the incubation medium. Lysophosphatidylcholine also increased LHRH release. These results were interpreted as evidence of the participation of Na+/H+ exchange in PLA2 activation in the release of LHRH in rat hypothalamic fragments. A role of lysophospholipids in this process is also suggested.

Contribution of HLA class II genes to end stage renal disease in mexican patients with type 2 diabetes mellitus.

To analyze the contribution of MHC class II genes in type 2 diabetes mellitus (DM) with end stage renal disease (ESRD), we examined the distribution of HLA-DRB1, DQA1, DQB1 loci in Mexican Mestizos of Central Mexico, using PCR-SSOP and PCR-SSP. Three groups were included: 47 type 2 diabetic ESRD patients; 42 patients with ESRD and 50 type 2 DM patients with no kidney complication. The results were compared with those of 101 controls of the same area. The median since DM was first diagnosed, was 18 years prior to the onset of ESRD. The frequencies of DRB1*1502 and DQB1*0501 were increased in DM patients with ESRD (p = 0.004; RR = 7.4, CI = 1.5-37; EF = 0. 13; p = 0.007; RR = 2.9, CI = 2.3-3.5, EF = 0.21, respectively). In contrast, DRB1*0407 was decreased in the same group (p = 0.0008, RR = 0.2; CI = 0.035-0.70, PF = 0.19). Diabetic patients with DRB1*1502 are 8.8 times more likely to develop ESRD, independently of the duration time of DM. DRB1*1502 contributes to the susceptibility to ESRD while DRB1*0407 is involved in protection. The residue at DRB1-74 differs in these alleles: DRB1*0407 has glutamic acid and DRB1*1502 has an alanine, suggesting that this substitution may be important for both, peptide anchoring and for presentation to the T cells.

Symptoms at perimenopausal period: its association with attitudes toward sexuality, life-style, family function, and FSH levels.

We studied the association of symptoms with attitudes toward sexuality, life-style, family functioning, and follicle-stimulating hormone (FSH) levels in perimenopausal women. We collected data on depression, anxiety, nonspecific index of depression, and the empty nest syndrome in 222 women with a mean age of 47.7 (47.2-48.2, 95% CI) years (102 of them were at menopause). Family functioning was evaluated with the McMaster model, and attitudes toward sexuality with questions about women's opinions on sexuality. Scores of depression were higher in women with more than 1 year of menopause. Attitudes to sexuality was significantly associated with all four symptoms. For family function, affective involvement, control of behavior, the roles of the members in the family, and communication were associated with some symptoms. Serum FSH was associated with body mass index, abdomen/hip ratio, and depression. We concluded that: (1) Attitudes toward sexuality was the main regressor for symptoms; (2) the function of the family is also associated with some symptoms; and (3) FSH levels may be lower in obese menopausal women and higher in women with depression or anxiety.

Symptoms at the menopausal and premenopausal years: their relationship with insulin, glucose, cortisol, FSH, prolactin, obesity and attitudes towards sexuality.

The symptoms of 81 premenopausal and 70 menopausal women were studied to determine the association with obesity, attitudes towards sexuality (ATS), and diverse hormone values: fasting and postprandial glucose (FG, PG) and insulin (FI, PI), cortisol, prolactin, follicle-stimulating hormone (FSH). The mean age of the women studied was 49.1 years. The frequency of symptoms was 35.4% for depression, 34.3% for nonspecific symptoms of depression (NSSD), 38.6% for empty nest syndrome (ENS), and 42.3% for anxiety. NSSD, ENS, FSH and cortisol levels all possessed higher values at late-menopausal stage. A multiple regression analysis revealed the following results; NSSD was associated to ATS (negative); sleep alterations were correlated to prolactin, FSH, PI/PG, FI/FG and waist/hip ratio; FSH was associated with both a decreased sexual interest and depression. In the study of hormone levels it was found that cortisol, insulin and FI/FG were associated with ATS; PI, cortisol, FI/FG and PI/PG were associated with body mass index (BMI) and FSH; prolactin and FI/FG were associated with age. We concluded that: (1) data indicative of insulin resistance correlated to both depression and sleep alterations; (2) overweight is related to NSSD, sleep alterations, and hormonal changes.

Age at menopause in women with type 2 diabetes mellitus.

OBJECTIVE: Considering that chronic diseases such as diabetes mellitus (DM) may determine premature ovarian failure by various mechanisms, we studied the age at menopause in women without diabetes and in women with type 2 DM. DESIGN: We studied 409 women without diabetes and 404 patients with type 2 DM, selected from 45 to 55 years of age, for analysis with the status quo method. The age at menopause was calculated with a logistic regression on the proportions of menopausal women for each age group. RESULTS: In the groups, 172 women without diabetes and 207 women with diabetes had menopause. The regression procedure gave a median age of 49.7 +/- SD 1.7 years for the whole group, 49.6 +/- 1.6 years for the nondiabetic group, and 49.8 +/- 1.7 years for women with diabetes. Women without diabetes were 1.4 years younger, but this factor did not have an influence on the results. Smoking habits, vegetarianism, and somatometric variables were similar in both groups, except for waist/hip and abdomen/hip ratios, larger in the group of women with diabetes. The mean for years since diagnosis in patients < 45 years of age was 4.9 years. For older patients, the figure increased to 8.9 years. CONCLUSIONS: No difference for age at menopause was found between women without diabetes and women with type 2 diabetes who were 5 to 8 years since the diagnosis was made.

Gonadotropins and prolactin serum levels during the perimenopausal period: correlation with diverse factors.

We studied 490 women aged 35 to 55 years randomly selected from the urban population of León, México. The mean age for the onset of menopause, calculated with a probit regression was 48.5 years. The median of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) increased with the rate of menopause women, but the 10th percentile for both hormones did not increase above 15 IU/L in older groups. Meanwhile, prolactin did not change. In menopause women FSH correlated positively with cigarette smoking, and negatively with body mass index. Luteinizing hormone had negative correlation with the number of pregnancies. Most symptoms studied were not associated with menopause or gonadotropins levels, except hot flushes, and the empty nest syndrome. It was concluded that during menopause, FSH increases with the smoking habit, and decreases with overweight.

The effect of hormone replacement therapy on blood pressure and cardiovascular risk factors in menopausal women with moderate hypertension.

OBJECTIVE: To assess the effect of injectable hormone replacement therapy (HRT) vs a placebo in hypertensive menopausal women. DESIGN AND METHODS: Prospective randomised double-blind study over 90 days. Fifty-five menopausal women with mild to moderate hypertension, stopped their antihypertensive medication and were studied for 2 weeks. Diastolic BP increased to over 105 mm Hg in five patients who were not included in the study. The remaining patients were randomly allocated to three groups: placebo (PL), estradiol valeranate 10 mg (E), and estradiol valeranate 4 mg plus prasterone enantate 200 mg (E+P). A further five patients were excluded from the study for different reasons. RESULTS: Standing and recumbent BP decreased in the PL group but did not change in the E and E+P patients. No change was observed in the serum levels of total cholesterol or low density lipoprotein (LDL)-cholesterol at the end of the trial. Plasma renin activity (PRA), aldosterone and insulin levels decreased during the study; PRA fell more significantly in the E+P group, the aldosterone reduction was highly significant in both hormone treated groups, specially the E+P group. Plasma insulin decreased in all groups and FSH levels were lower in the two treatment groups. CONCLUSIONS: In a randomised controlled trial no rise in BP was found after 90 days in hypertensive women with two forms of HRT. There was an unexpected fall in BP in those women allocated to placebo injections.

Gonadotropins at menopause: the influence of obesity, insulin resistance, and estrogens.

UNLABELLED: Obese, postmenopausal women have lower FSH levels. To determine whether this is due to higher estrogen exposure, we compared feedback gonadotropin sensitivity and its relation to insulin resistance in four groups of obese and lean, postmenopausal women. Group one was treated with 400 mg troglitazone (TG) daily for two weeks; 150 clomiphene citrate (CC) was added daily for the second week. Group two received 150 mg CC daily for a week. Group three received 1000 mg metformin (MET) daily for two weeks, with 120 mg raloxifene (RAL) added during the second week. Group four received 120 mg RAL for a week. Before and after each period, a serum pool was obtained from samples taken every minute during a 10 ml interval. The women recruited for this study were categorized as obese or lean based on BMI >/= 29 or BMI < 29, respectively. Obese, menopausal women had lower FSH (45.5 IU/l) and LH (16.2 IU/l) values than those of lean (64.1 IU/l and 23.0 IU/l), but the obese menopausal women had higher leptin, DHEAS, glucose, insulin, and HOMA-IR levels. Log [FSH] was associated with BMI (r = -0.53, P < 0.000001) and number of pregnancies (r = -0.37, P = 0.0009). TG treatment did not change HOMA-IR or gonadotropin levels, but DHEAS and androstenedione levels decreased significantly. CC alone or together with TG, diminished FSH (-7.9 and -9.2) and LH (-2.5 and -3.6) concentrations, with a greater reduction in lean women. MET reduced glucose and the HOMA-IR index without affecting gonadotropin or steroid levels. CONCLUSIONS: obese, menopausal women have lower FSH levels due to greater estrogen exposure, by mechanisms unrelated to insulin resistance.

Association of sexual activity and bacteriuria in women with non-insulin-dependent diabetes mellitus.

A group of 215 patients with non-insulin-dependent diabetes mellitus (NIDDM) (147 women and 68 men) were selected by home interview, and accepted to undergo clinical examination including urine culture, in order to determine the frequency of bacteriuria and its possible associations with clinical and laboratory findings. Bacteriuria was found in 17.7% of women and 1.5% of men. Univariate and multivariate analysis performed for the group of women revealed sexual intercourse and pyuria as the only factors associated with the frequency of bacteriuria. It was concluded that asymptomatic bacteriuria may be associated with sexual activity in women with NIDDM.

Adherence to treatment and social support in patients with non-insulin dependent diabetes mellitus.

We carried out a cross-sectional study to investigate factors associated with adherence to diet and medication in non-insulin-dependent diabetes mellitus (NIDDM) patients. A total of 200 patients not seeking treatment from clubs for diabetics from two hospitals in León, Mexico, accepted inclusion. Patients interviewed had a mean age of 58.8 (53.3-56.4, 95% C.I.) years. We evaluated adherence to diet and medication, knowledge on diabetes, social support, family's structure and functioning (with a modified McMaster model), metabolic control, and complications. Stepwise multiple regression procedure showed that adherence to diet was associated with years since diagnosis (p = 0.003) and with social support (p = 0.007). Adherence to medication was associated with social support (p = 0.002), and the age of the spouse (p = 0.016). Adherence to medication was lower in patients from families with rigid control than in the group with Laissez-faire type of control (p = 0.010) or the group with flexible control (p = 0.002). Social support was lower in the group with chaotic control than that in the group with flexible control (p < 0.001). Compliance to diet was associated with peripheral neuropathy and plasma creatinine, and adherence to medication with plasma glucose and peripheral neuropathy. We concluded that (1) adherence to treatment in NIDDM patients is associated with social support; (2) some aspects related to the family, such as the age of the spouse and the control of behavior, were also associated with compliance to treatment; and (3) it is important for the practicing physicians, and for institutional programs, to consider factors associated with adherence to treatment.

Menopause in normal and uncomplicated NIDDM women: physical and emotional symptoms and hormone profile.

OBJECTIVE: To compare the physical characteristics, emotional symptoms and metabolic conditions of menopausal women with and without non insulin dependent diabetes mellitus (NIDDM). METHODS: We studied 100 menopausal women 45-72 years of age, 51 with and 49 without NIDDM, in a cross-sectional design. Biological characteristics were collected and emotional symptoms were assessed with a modified Hamilton and Bech-Rafaelsen scale, scoring depression, anxiety, non specific symptoms of depression (NSSD) and the empty nest syndrome (ENS). Weight, body mass index (BMI), waist/hip and abdomen/hip ratios and percent of body fat were registered. The sulfoconjugated form of the dehydroepiandrosterone (DHEAS), follicle stimulating hormone (FSH), cortisol and fasting, as well as postprandial insulin/glucose ratios, were measured in blood. RESULTS: Women with NIDDM had earlier mean age for menopause, more central obesity and less peripheral fat; they had also more prevalent emotional symptoms than non diabetic menopausal women. In women with NIDDM, symptoms were associated with years since diagnosis and with BMI. In non diabetic menopausal women schooling and attitudes to sexuality were associated with symptoms. FSH was inversely associated with BMI in both diabetic and non diabetic women; postprandial insulin/glucose ratio was correlated with central obesity in the group without NIDDM and cortisol with sitting systolic blood pressure (SBP) in the group with NIDDM. CONCLUSION: The diagnosis of NIDDM and its metabolic conditions were associated with an increased frequency of some symptoms in menopausal women.

Personality profiles and socioeconomic factors in contraceptive use.

The correlation of contraceptive use with socioeconomic factors and personality profiles was examined in a sample of 3060 women by a questionnaire based on the diagnostic criteria of the DMS III. Use of safer contraceptives correlated with several socioeconomic factors, among them, the schooling of the wife had higher significance and explained most of the variance contained in other factors. Women unprotected did not show a distinct personality profile. Indices of compulsion and phobias were significantly different between groups of contraceptive use. Users of injectables showed higher scores of compulsion, whereas IUD users had higher scores of phobias. These results were interpreted to mean that personality profiles partially explain the preference for certain methods.

Plasma epinephrine and norepinephrine response to stimuli in autonomic neuropathy of type 2 diabetes mellitus.

The objective of this study was to examine epinephrine and norepinephrine plasma levels in patients with clinical type 2 diabetes mellitus, at different stages of autonomic neuropathy. Eighteen patients were classified in groups without (n = 6) and with early (n = 6), definite (n = 3) and severe (n = 3) neuropathy. Blood catecholamine levels were measured after the Valsalva maneuver, cold exposure and orthostatic tests. The norepinephrine basal levels were lower in patients with severe neuropathy (0.4 +/- 0.2 nmol/l), compared with the group with no neuropathy (1.3 +/- 0.5 nm/l, p = 0.034), or with early neuropathy (1.3 +/- 0.7 nm/l, p = 0.035). After the Valsalva maneuver, no increase was found in the group with severe alteration. In patients without neuropathy, cold exposure induced a peak of norepinephrine at 5 min (delta = 1.9 +/- 1.6 nmol/l). The increase was lower in groups with definite and severe damage. In patients with definite or moderate neuropathy, the orthostatic test induced minimal or no response. The epinephrine response to the maneuvers was not significant, and no differences were found among the groups. Norepinephrine basal levels and cold responses are diminished in patients with definite and severe autonomic neuropathy. This provides further evidence on their impaired response to stress. The comparable epinephrine levels in patients with or without autonomic neuropathy indicates that adrenal medullar function is not significantly altered.

Periodontal disease in non-insulin dependent diabetes mellitus (NIDDM). The effect of age and time since diagnosis.

The association of periodontal disease with diabetes was studied in non-insulin dependent diabetes mellitus (NIDDM) patients. In a cross-sectional design, 100 patients (46 males and 54 females) were selected in 4 groups according to age and years since diagnosis of diabetes. The groups were: group 1, > 55 years old, diabetes diagnosed > or = 5 years; group 2, < or = 55 years old, diabetes diagnosed > or = 5 years; group 3, > 55 years, diabetes diagnosed < 5 years; and group 4, < or = 55 years, diabetes diagnosed < 5 years. Buccal and lingual pockets were deeper and lingual and buccal recessions greater for groups diagnosed 5 or more years ago, (P < 0.0001). In groups diagnosed less than 5 years, higher recession indices were found for patients older than 55. The loss of buccal insertion was also greater for groups diagnosed 5 or more years ago (P < 0.0001). For groups diagnosed less than 5 years ago, the loss was greater in the group older than 55 (P = 0.01). There was a marginal difference in gingival bleeding among the 4 groups (P = 0.047). Post-hoc analysis showed differences between the 2 groups who had been diagnosed less than 5 years, with lower indices for patients < or = 55. All groups were statistically different regarding bone loss (P < 0.0001), with higher indices for the groups with older age, and for groups with 5 or more years since diagnosis. There was no difference in dental mobility among the 4 groups (P = 0.0981). It was concluded that years since diagnosis of diabetes is more significant than age for severity of periodontal disease in NIDDM.

Clinical and radiological improvement of periodontal disease in patients with type 2 diabetes mellitus treated with alendronate: a randomized, placebo-controlled trial.

BACKGROUND: Alendronate (ALN) is an aminobisphosphonate commonly used for osteoporosis in postmenopausal women. We studied the effect of ALN on bone loss prevention in type 2 diabetes mellitus patients with periodontal disease. METHODS: In a controlled double-blind, randomized study we evaluated prospectively diabetic patients paired by gender and years since diagnosis for 6 months. The study included 40 patients (20 men and 20 women), 50 to 60 years old, with more than 5 years since diagnosis of diabetes and established periodontitis. They were randomly allocated to alendronate (10 mg/daily) or placebo treatment for 6 months. The endpoints of treatment were: the distance between the alveolar bone border and the cemento-enamel-junction (CEJ) evaluated by means of digital radiographic imaging, a biochemical marker of bone resorption (urine N-telopeptide) (Ntx), and periodontal parameters. Metabolic control was assessed at baseline and after 6 months. RESULTS: Baseline and 6-month glycated hemoglobin levels were similar in both groups. Alendronate induced a significant decrease in NTx at 6 months (P = 0.006). Periodontal parameters improved in both groups. However, they were significantly better for the ALN treated group. Alveolar bone border-CEJ distance increased in the placebo, but decreased in the ALN group (P = 0.0003). CONCLUSIONS: In type-2 diabetic patients, alendronate induced more improvement in alveolar bone crest height than control therapy. No differences in urinary N-telopeptide or glycated hemoglobin were observed in this short-term randomized controlled pilot trial.