Immortalization and characterization of a new canine mammary tumour cell line FR37-CMT.

Here we describe the establishment of a new canine mammary tumour (CMT) cell line, FR37-CMT that does not show dependence on female hormonal signaling to induce tumour xenografts in NOD-SCID mice. FR37-CMT cell line has a stellate or fusiform shape, displays the ability to reorganize the collagen matrix, expresses vimentin, CD44 and shows the loss of E-cadherin which is considered a fundamental event in epithelial to mesenchymal transition (EMT). The up-regulation of ZEB1, the detection of phosphorylated ERK1/2 and the downregulation of DICER1 and miR-200c are also in accordance with the mesenchymal characteristics of FR37-CMT cell line. FR37-CMT shows a higher resistance to cisplatin (IC50 >50 µM) and to doxorubicin (IC50 >5.3 µM) compared with other CMT cell lines. These results support the use of FR37-CMT as a new CMT model that may assist the understanding of the molecular mechanisms underlying EMT, CMT drug resistance, fostering the development of novel therapies targeting CMT.

Liposomes versus lipid nanoparticles: comparative study of lipid-based systems as oryzalin carriers for the treatment of leishmaniasis.

Main-stay in treatment of leishmaniasis relies on chemotherapy but none of the current drugs combines high activity and low toxicity at affordable costs. Dinitroanilines are a new class of drugs with proved in vitro antileishmanial activity. However the development of their pharmaceutical formulations has been compromised by low water solubility and low accumulation in diseased organs. These limitations can be overcome by incorporation in lipid-based nanoformulations such as liposomes and solid lipid nanoparticles. In previous work this strategy was already followed with the incorporation of a dinitroaniline, oryzalin, resulting in the improvement of the biodistribution profile. The present work aims at demonstrating the in vitro and in vivo therapeutic activity of these oryzalin nanoformulations, and establishing a systematic comparison of both systems. After oryzalin incorporation suitable physicochemical properties for parenteral administration were obtained. Nanoformulations revealed reduced cytotoxicity and haemolytic activity when compared with free-oryzalin, while retaining the in vitro intracellular activity. Therapeutic activity, assessed in a murine model of visceral leishmaniasis, was evaluated in terms of number of administrations, dose-response and influence of the lipid excipient. Results demonstrate the superiority of both oryzalin nanoformulations on the reduction of parasitic burden in liver and spleen as compared to the control group (84 to 91%) and similar to Glucantime. A strong reduction in ED50 values (3 to 65 fold) as compared to free-oryzalin was also obtained, depending on the organ and nanoformulation used. Both oryzalin nanoformulations are potential candidates as therapeutic agents against visceral leishmaniasis.

The CTLA4 +49 A/G polymorphism is not associated with susceptibility to type 1 diabetes mellitus in the Portuguese population.

CTLA4 genetic polymorphisms have been associated with type 1 diabetes. We genotyped 207 patients and 249 controls for the most frequently investigated polymorphism of the CTLA4 gene (+49A/G (rs231775)). No significant differences were observed, suggesting that this polymorphism is not strongly associated with type 1 diabetes in the Portuguese population.

Sources of hepatic glucose production by 2H2O ingestion and Bayesian analysis of 2H glucuronide enrichment.

The contribution of gluconeogenesis to hepatic glucose production (GP) was quantified after (2)H(2)O ingestion by Bayesian analysis of the position 2 and 5 (2)H-NMR signals (H2 and H5) of monoacetone glucose (MAG) derived from urinary acetaminophen glucuronide. Six controls and 10 kidney transplant (KTx) patients with cyclosporine A (CsA) immunosuppressant therapy were studied. Seven KTx patients were lean and euglycemic (BMI = 24.3 +/- 1.0 kg/m(2); fasting glucose = 4.7 +/- 0.1 mM) while three were obese and hyperglycemic (BMI = 30.5 +/- 0.7 kg/m(2); fasting glucose = 7.1 +/- 0.5 mM). For the 16 spectra analyzed, the mean coefficient of variation for the gluconeogenesis contribution was 10% +/- 5%. This uncertainty was associated with a mean signal-to-noise ratio (SNR) of 79:1 and 45:1 for the MAG H2 and H5 signals, respectively. For control subjects, gluconeogenesis contributed 54% +/- 7% of GP as determined by the mean and standard deviation (SD) of individual Bayesian analyses. For the lean/normoglycemic KTx subjects, the gluconeogenic contribution to GP was 62% +/- 7% (P = 0.06 vs. controls), while hyperglycemic/obese KTx patients had a gluconeogenic contribution of 68% +/- 3% (P < 0.005 vs. controls). These data suggest that in KTx patients, an increased gluconeogenic contribution to GP is strongly associated with obesity and hyperglycemia.

Combined GSTM1 and GSTT1 null genotypes are associated with a lower risk of papillary thyroid cancer.

Individual susceptibility to cancer is influenced by polymorphisms of genes encoding drug-metabolizing enzymes such as the glutathione S-transferases (GST). The null polymorphisms of the GSTM1 and GSTT1 genes have been associated to a modified risk of several cancers but studies of thyroid cancer have produced conflicting results. The aim of this study was to investigate the relationship between these polymorphisms and the risk of papillary thyroid cancer (PTC). A total of 188 patients with PTC and 247 controls were genotyped using a PCR-based assay. Odds ratios (OR) and 95% confidence intervals (CI) for each homozygous null genotype were determined. The frequency of each of the GSTM1 and GSTT1 null genotypes did not differ significantly between patients and controls (OR=0.83, 95%CI: 0.56-1.21; p=0.328; and OR=0.66, 95%CI: 0.39-1.12; p=0.123, respectively), but the frequency of individuals that had the combined GSTM1 null/GSTT1 null genotypes was significantly lower in the patient group (OR=0.50, 95%CI: 0.26-0.97; p=0.040). The GSTM1 null genotype was associated with a lower risk of advanced cancer stages (III/IV) (OR=0.50, 95%CI: 0.26-0.96; p=0.036) and the GSTT1 null genotype was associated with a lower risk of the follicular variant of PTC (OR=0.31, 95%CI: 0.10-0.97; p=0.044). These results suggest that GSTM1 and GSTT1 null genotypes are weak, yet possible, modifiers of the risk of PTC. This protective effect may be due to a role of the GSTM1 and GSTT1 encoded enzymes in the metabolic activation of putative thyroid carcinogens or in other pathways involved in thyroid carcinogenesis.

Efficacy of the liposome trifluralin in the treatment of experimental canine leishmaniosis.

Liposomes are used as carriers to deliver drugs and to treat diseases where infection is localised in the mononuclear phagocyte system cells, as is the case of leishmaniosis. Trifluralin is a dinitroaniline with proved anti-Leishmania activity in vitro. The efficacy of liposomal trifluralin (LIP/TFL) was studied in the treatment of experimental canine leishmaniosis through quantification of parasite burden using the limiting dilution assay, follow-up of anti-Leishmania antibodies by indirect fluorescent immunoassay and cytokine expression by Reverse Transcriptase-PCR, in the bone marrow, lymph nodes, skin and peripheral blood mononuclear cells in 5 female beagle dogs. After treatment, dogs showed a general remission of clinical signs related to parasite burden reduction and Th1 cytokine mRNA expression, but there was no significant decrease in antibody levels. Alternative treatment schemes with LIP/TFL are necessary to achieve optimal results.

[Fetal growth and glycemic control in type 1 diabetes pregnancy]. / Crescimento fetal e controlo glicémico em grávidas diabéticas tipo 1.

INTRODUCTION: Conflicting results have been reported with respect to the relationship between direct or indirect measures of glycemic control in mothers with type 1 diabetes and macrosomia. OBJECTIVE: To evaluate the frequency of LGA babies in type 1 diabetic pregnancies and analyse the influence of some maternal characteristics and glucose control in oversized babies. MATERIAL AND METHODS: A retrospective study of 18 pregnant women with type 1 diabetes mellitus was performed. It was divided in two groups: group 1 (G1- n=9)--pregnant women with LGA babies and group 2 (G2- n=9)--pregnant women with AGA (Appropriate weight for gestational age) babies. We evaluate the follow parameters: HbA1c in the third trimester of pregnancy, fasting and 1 h postprandial capillary glucose levels, pregestational BMI, maternal age, duration of Diabetes mellitus, weight gain during pregnancy, microvascular diabetes complications (retinopathy and nefropathy), and type of delivery. We defined LGA birth weight over the 90 centile. RESULTS: LGA babies occurred in 50% of gestations. We did not find any statistical differences in maternal age, diabetes mellitus duration, pregestational BMI, weight gain during pregnancy, microvascular diabetes complications, HbA1c levels (medium value in the two groups 6,5%). The glucose fasting values were higher in G1: 95,7 +/- 31.7 mg/ dl, vs G2: 83.3 +/- 17.1 mg/dl without, however, reaching statistically significant differences. There was statically differences in postprandial glucose values G1: 160.3 +/- 60.2 mg/dl vs G2: 111.9 +/- 27.1 mg/dl -- p= 0.043. CONCLUSIONS: The frequency of LGA babies was elevated 50% in type 1 diabetic pregnancies, although normal HbA1c values. Thus we conclude that the 1 h postprandial glucose levels should be considered a strong predictor of fetal growth.

[Acute thyroiditis]. / Tiroidites agudas.

We review the pathophysiology, clinical features and therapy of acute thyroiditis. Four cases are reported stressing the role of fine needle aspiration for the diagnosis of this clinical entity.

The hyperparathyroidism-jaw tumour syndrome in a Portuguese kindred.

The hyperparathyroidism-jaw tumour (HPT-JT) syndrome is an autosomal dominant disease characterized by the occurrence of parathyroid tumours and fibro-osseous tumours of the jaw bones. Some HPT-JT patients may also develop renal abnormalities, which include Wilms' tumours, hamartomas and polycystic disease. The HPT-JT gene has been mapped to chromosome 1q25-q31, and we report the clinical and genetic findings in a kindred from central Portugal. HPT-JT was observed in six members from three generations; all had primary hyperparathyroidism (five had parathyroid adenomas, one a parathyroid carcinoma). Ossifying jaw fibromas affecting the maxilla and/or mandible were observed in 5/6. Renal cysts (<2.5 cm) were observed in four. Genetic studies using 18 polymorphic loci from chromosome 1q25-q31, together with leukocyte DNA from 11 family members and tumour DNA from three parathyroids (two adenomas and one carcinoma), revealed loss of tumour heterozygosity in the parathyroid carcinoma only, and the retained haplotype was found to cosegregate with the disease in the six affected members. A new Portuguese kindred with the HPT-JT syndrome that maps to chromosome 1q25-q31 has been identified, and these findings will help in the further characterization of this inherited disorder.

[deficiency of growth hormone in children. Re-evaluation after therapeutic completion]. / Deficiência de GH nas crianças. Reavaliação após conclusão terapêutica.

OBJECTIVE: To assess GH secretion in young adults treated with GH replacement therapy in childhood. PATIENTS AND METHODS: From the 38 patients who concluded treatment with GH, we studied 20 (52.6%), 9 girls and 11 boys. Thirteen had Growth Hormone Deficiency (GHD)-65%, while 7 had Multiple Pituitary Hormone Deficiency (MPHD)-35%. The patients were retested within 6 months to 6 years after completing GH therapy. The mean age (+/- SD) at retesting was 18.1 +/- 2.6 years for those with GHD and 20.8 +/- 2.8 for those with MPHD. At reassessment we performed two provocative tests: insulin tolerance test (ITT) and clonidine test. RESULTS: Seven of the 20 patients retested, retained GH deficiency. Of the 13 patients with GHD, only one maintained the deficiency, while of the 7 patients with MPHD, 6 maintained the deficiency. CONCLUSION: Young adults with GH deficiency treated with this hormone should be retested in order to identify those who are truly GH insufficient adults and may benefit from replacement therapy.

[Kidney transplantation in patients with type I and type II diabetes mellitus]. / Transplantação renal em doentes com diabetes mellitus tipo I e tipo II.

A total of 618 patients with end-stage renal disease received kidney transplants between 1980 and September 1996. Twenty eight of them were diabetics. Better results were achieved for type 1 diabetic patients than for type 2 (mortality: 5.9% vs 27.3%; functioning graft: 88.2% vs 72.7%). The morbility was also higher in those patients (infections: 81.8% vs 29.4%; vascular complications: 45.5% vs 17.6%). Actuarial patient and graft survival were lower for type 2 than for non diabetic patients. For type 1 diabetics the results are similar to those for non diabetics. Better results can probably be achieved by restricting the selection criteria. The decision to transplant or maintain on dialysis should be made on a case by case basis.

[Craniocerebral imaging in children with short stature]. / Imagiologia crânio-encefálica nas crianças com baixa estatura.

OBJECTIVE: To analyse the type and frequency of cranial CT and NMR imaging anomalies in children of short stature. PATIENTS AND INTERVENTIONS: We studied 57 children of short stature with a mean age (+/-SD) of 10.1 +/- 3.8 years, 34 boys and 23 girls, all of them with auxometric criteria of GH deficiency. After studying the pituitary function and determination of karyotype in the girls, the children were classified in to five groups:-Isolated GHD (IGHD) (n = 32), multiple pituitary hormone deficiency (MPHD) (n = 6), neurosecretory dysfunction (NSD) (n = 8), Turner syndrome (n = 7) and idiopathic short stature (ISS) (n = 4). The imaging methods used were cranial CT or NMR. RESULTS: Of the 57 children studied the CT/NMR was abnormal in 37(64.9%) children. We found anomalies in 65.6% of IGHD patients, 62.5% of NSD patients, 100% of MPHD patients and 57.1% and 25% in the Turner s. patients and ISS patients respectively. The most frequent anomaly was hypoplastic pituitary found in 50% of IGHD patients, 37.5% of NSD patients and 33.3% of MPHD patients. None of the cases of Turner s. or ISS had hypoplastic pituitary. An empty sella was the second most frequent anomaly found in 7 patients (IGHD-3, MPHD-3, DNS-1). Of the 25 children in which NMR was performed, 8 had hypoplastic pituitary and stalk and 2 had interruption of the pituitary stalk and ectopic neurohypophysis. CONCLUSION: These results strengthen the necessity for CT/NMR imaging in children of short stature which, besides allowing identification of tumors, also permits the diagnosis of idiopathic GHD because of its frequent association with cranial imaging anomalies, mainly hypoplastic pituitary.

[The prevalence of hypertension in acromegaly]. / Prevalência da HTA na acromegália.

AIM: To estimate the prevalence of hypertension (HT) in a group of patients with acromegaly at the moment of diagnosis and after treatment. PATIENTS AND METHODS: Fifty-seven patients, 43 females and 14 males with a mean age of 45.19 +/- 11.9 years were studied retrospectively. In the last visit 9 patients (15.7%) were in remission and 47 (84.2%) had active acromegaly. We considered hypertensive the patients with systolic BP > or = 140 and/or diastolic BP > or = 90 mmHg. Hypertension was classified in four stages:- mild, moderate, severe and very severe. RESULTS: The prevalence of hypertension at the moment of diagnosis was 35%. The hypertensive patients had a mean age of 51.75 +/- 9.3 years and normotensive patients 41.65 +/- 11.6 years (p < 0.001). In females the prevalence of HT was 27.9% and in males it was 57% (p = NS). In hypertensive patients (n = 20), the mean BP was 159 +/- 15 (syst.)/97.2 +/- 9.8 (diast.), 16 patients (80%) had mild to moderate HT and the remainder had severe (n = 2) and very severe (n=1) HT. In the last visit, 22.2% of patients were cured and 46.8% of those with active acromegaly were hypertensive. None of the patients cured and initially normotensive developed HT; among those that were hypertensive (n = 3), 2 remained hypertensive and 1 became normotensive. Among patients with active acromegaly and initially normotensive, 7 developed HT 4.85 +/- 2.03 years later; of those hypertensive at diagnosis (n = 16), only one became normotensive. The last case was 27 years old. The patients that remained hypertensive had a mean age of 53.8 +/- 6.85 years (41-62 years). CONCLUSIONS: The prevalence of hypertension at the moment of diagnosis was 35%, similar to the majority of studies published and higher than the general population. The hypertensive patients were significantly older the normotensive patients and most of them had mild to moderate HT. We observed an increase in the prevalence of HT over the years in the cases that maintained active acromegaly. In our series only one of the three patients cured became normotensive, therefore, we concluded that HT in acromegaly is frequently irreversible. The chances of normalization seems higher in younger patients and probably with a shorter duration of acromegaly.

Insulin sensitivity and insulin secretion in lean and obese normal pregnant women.

An insulin-modified frequently sampled intravenous glucose tolerance test with minimal model analysis was performed in normal pregnant women between 28-32 weeks of gestation, to assess insulin sensitivity and insulin secretion. Insulin sensitivity in the pregnant group (no. 26) was reduced to approximately 50% that of nonpregnant group (no. 27) (p < 0.05). This increased insulin resistance was compensated by an enhancement of the first phase of insulin secretion, which was increased more than twofold in the pregnant women when compared with the nonpregnant women (p < 0.05). There was a trend toward greater insulin resistance and insulin secretion in the obese pregnant women (no. 7) as compared with the lean pregnant women (no. 19) although this difference was not statistically significant. Our findings confirm that late pregnancy is a state of physiologic insulin resistance compensated by an increase of insulin secretion.

Diabetes in pregnancy: state of the art in the Mediterranean countries, Portugal.

In Portugal since 1980 the health care for diabetes in pregnancy has begun to be specialised and centralised, and the care is provided by multidisciplinary teams. At present there are eight diabetic teams in the main cities in the North, Centre and South of the country: three in Oporto, two in Coimbra, two in Lisbon and one in Almada. In our Center of the University Hospital, Coimbra, from 1980 to 1992 neonatal mortality dropped from 110/1000 to near 0/1000, macrosomia from 55% to 20%, neonatal hypoglycemia from 28% to 5.6%, RDS from 26% to 2.5%, congenital malformation from 9% to 2%. A "consensus on diabetes and pregnancy" to implement and give uniformity to health care was recently done (1996): a) criteria for pregnancy planning and management for pregestional diabetes (type 1 and 2) have been given; b) protocol of screening, diagnosis and management for gestational diabetes have been suggested; c) finally a recent (1995) epidemiologic report of a multicentric Portuguese survey has been reported.

Delivery in diabetic pregnancy.

Two hundred and twenty deliveries of diabetic pregnant women, occurring from 1990-1994 were studied: 186 gestational (GDM) and 34 pregestational diabetes (PGDM). Women who delivered during the year of 1994 were considered as control population (3615 births). Mode of delivery, planned delivery, delivery's gestational age, shoulder dystocia, macrosomia and large for gestational age (LGA) were investigated. Cesarean section and planned delivery were respectively 39% vs 20.5% and 51.6% vs 16% respectively in diabetic vs control women. Deliveries after 40 weeks were 29% in GDM, 3% in PGDM and 50% in control women. Macrosomia occurred in 17.7% of diabetic against the 8% of controls. Finally shoulder dystocia occurred in the 3.6% of diabetic women against the 0.3% of the control group. These data indicate that in our diabetic population there is a high rate of cesarean sections and planned deliveries, as well as macrosomia, LGA and shoulder dystocia. Obstetric decision to allow the delivery to term or near term was not enough to bring the rate of macrosomia and LGA close to the normal, which can be consequence of the diabetic control in pregnancy, in spite of intensive care intervention.

[Diabetes mellitus after renal transplant]. / Diabetes mellitus após transplante renal.

UNLABELLED: Posttransplant Diabetes Mellitus (PTDM) has traditionally been attributed to therapy with steroids, however with the introduction of Cyclosporine (cya) into clinical practice, an increase in the incidence of PTDM was noted. We studied the prevalence of PTDM in Renal transplant recipients and its clinical characteristics. MATERIALS AND METHODS: 355 patients were submitted to a renal transplant between 30/06/80 and 31/12/92. After stabilization of the doses of the immunosuppressive therapy we considered diabetic patients those with fasting glycemic values > or = 140 mg/dl and those with glycemic values between 100-140 mg/dl were given to an oral glucose tolerance test (WHO criteria 1985). RESULTS: Posttransplant diabetes mellitus occurred in 28 patients (7.9%), 11 females with a mean age of 53.36 +/- 12.75 years and 17 males with a mean age of 51.05 +/- 10.60 years. DISCUSSION: Due to the great prevalence of alterations in glucose tolerance and their adverse effects on the cardio-vascular system, we think that all renal transplant recipients should be screened.

[Pancreatic transplantation]. / Transplantação pancreática.

The AA. report on their very limited experience with whole pancreatic transplantation. For the moment this is the only experience in Portugal, the detailed report of the 3 cases may be of some help to other Portuguese groups interested in starting this therapeutical approach for insulin-dependent end-stage renal failure patients. The main comments focus on the third simultaneous pancreas-kidney transplantation, which failed for technical reasons, mainly related to less than good selection of both donor and recipient. In all the three cases the technique preferred was the duodenocystostomy. The 2 first cases are doing very well, free of insulin and with normal glucose metabolism, at 15 and 7 months after grafting. The AA. also make some considerations on the indications, complications and follow-up of patients with pancreas-kidney transplantation.