Antiparasitic Effects of Asteraceae Species Extracts on Echinococcus granulosus s.s.

Cystic echinococcosis is a zoonotic disease caused by the parasite Echinococcus granulosus sensu lato (s.l.), which is worldwide distributed and causes long-lasting infections in animals and humans. The existing treatment is limited to the use of benzimidazoles, mainly albendazole (ABZ). However, it has unwanted side effects and its efficacy is about 50%. The Asteraceae family includes plants that have therapeutic applications (medicinal species) and has an important role in new drug development. The species belonging to a different genus of this family show a wide range of anti-inflammatory, antimicrobial, antioxidant, hepatoprotective, and antiparasitic activities, among others. The aim of the present study was to evaluate the in vitro efficacy of extracts of four Asteraceae species against protoscoleces of E. granulosus sensu stricto (s.s.). On the other hand, the Stevia aristata extract was assessed on the murine cyst of E. granulosus (s.s.) and the efficacy of S. aristata extract was investigated in a murine model of CE. Stevia satureiifolia, S. aristata, Grindelia pulchella, and G. chiloensis extracts at 100 µg/mL caused a decrease in protoscoleces viability; however, S. aristata extract produced the greatest in vitro protoscolicidal effect. After 20 days of treatment with the highest concentration (100 µg/mL) of S. aristata extract, protoscoleces viability decreased to 0%. The tegumental changes observed by scanning electron microscopy were consistent with the reduction in vitality. The collapse of the germinal layer was registered in 60 ± 5.8% and 83.3 ± 12.0% of cysts treated during 4 days with 50 and 100 µg/ml, respectively. The half maximal effective concentration (EC50) value of the S. aristata extract against E. granulosus (s.s.) cysts was 47.86 µg/mL (96 h). The dosage of infected animals with the 50 mg kg-1 dose of S. aristata extract resulted in a significant reduction in cyst weight in comparison with the control group. In conclusion, S. aristata extract was demonstrated to exert a marked effect, both in vitro and in the murine model.

Anthelmintic activity of Stevia multiaristata extract against Echinococcus granulosus sensu stricto.

Cystic echinococcosis is a zoonotic disease caused by the larval stage of the parasite Echinococcus granulosus sensu lato. The available anti-parasitic treatment is mostly limited to a continuous administration of albendazole. However, due to its numerous side-effects and efficacy of around 50%, there is a need to find new drugs to improve the treatment for this disease. In the current study, the in vitro and in vivo efficacy of a Stevia multiaristata extract against E. granulosus sensu stricto (s.s.) was demonstrated. Stevia multiaristata extract (100 and 50 µg mL−1) caused a quick viability decrease on protoscoleces which was consistent with the observed tegumental alterations. Loss of turgidity was detected in 95 ± 3.4% of cysts incubated with S. multiaristata extract during 2 days (100 µg mL−1) and the collapse of the germinal layer was observed in 60 ± 9.3% of cysts treated with 100 µg mL−1 of the S. multiaristata extract during 4 days. The half maximal effective concentration value was 69.6 µg mL−1 and the selectivity index for E. granulosus s.s. cysts was 1.9. In this clinical efficacy study, the treatment of infected mice with the S. multiaristata extract (50 mg kg−1) caused a significant decrease in the weight of the cysts compared with the control group. These results coincided with the tissue damage observed in the cysts at the ultrastructural level. In conclusion, we observed high protoscolicidal and cysticidal effects, and significant reduction in the weight of the cysts in experimentally infected mice following treatment with the S. multiaristata extract.

Levothyroxine and insulin requirement in autoimmune polyglandular type 3 syndrome: a real-life study.

PURPOSE: To evaluate factors influencing the insulin and levothyroxine requirement in patients with autoimmune polyglandular syndrome type 3 (APS-3) vs. patients with type 1 diabetes mellitus (T1DM) and autoimmune hypothyroidism (AH) alone, respectively. METHODS: Fifty patients with APS-3, 60 patients with T1DM and 40 patients with AH were included. Anthropometric, clinical and biochemical parameters were evaluated in all patients. Insulin requirement was calculated in patients with APS-3 and T1DM, while levothyroxine requirement was calculated in APS-3 and AH. RESULTS: Patients with APS-3 showed higher age (p = 0.001), age of onset of diabetes (p = 0.006) and TSH (p = 0.004) and lower total insulin as U/day (p < 0.001) and U/Kg (p = 0.001), long-acting insulin as U/day (p = 0.030) and U/kg (p = 0.038) and irisin (p = 0.002) compared to T1DM. Patients with APS-3 had higher waist circumference (p = 0.008), duration of thyroid disease (p = 0.020), levothyroxine total daily dose (p = 0.025) and mcg/kg (p = 0.006), triglycerides (p = 0.007) and VAI (p = 0.010) and lower age of onset of thyroid disease (p = 0.007) than AH. At multivariate analysis, levothyroxine treatment and VAI were associated with insulin and levothyroxine requirement in APS-3, respectively. VAI was independently associated with insulin requirement in T1DM. Circulating irisin levels were independently associated with levothyroxine requirement in AH. CONCLUSION: Patients with APS-3 show lower insulin requirement and higher levothyroxine requirement than T1DM and AH alone, respectively. Levothyroxine treatment and VAI affect insulin and levothyroxine requirement, respectively, in APS-3. In T1DM, adipose tissue dysfunction, indirectly expressed by high VAI, is associated with an increased insulin requirement, while circulating irisin levels influence the levothyroxine requirement in AH.

Germline investigation in male breast cancer of DNA repair genes by next-generation sequencing.

PURPOSE: In order to better define the breast cancer (BC) genetic risk factors in men, a germline investigation was carried out on 81 Male BC cases by screening the 24 genes involved in BC predisposition, genome stability maintenance and DNA repair mechanisms by next-generation sequencing. METHODS: Germline DNAs were tested in a custom multi-gene panel focused on all coding exons and exon-intron boundaries of 24 selected genes using two amplicon-based assays on PGM-Ion Torrent (ThermoFisher Scientific) and MiSeq (Illumina) platforms. All variants were recorded and classified by using a custom pipeline. RESULTS: Clinical pathological data and the family history of 81 Male BC cases were gathered and analysed, revealing the average age of onset to be 61.3 years old and that in 35 cases there was a family history of BC. Our genetic screening allowed us to identify a germline mutation in 22 patients (23%) in 4 genes: BRCA2, BRIP1, MUTYH and PMS2. Moreover, 12 variants of unknown clinical significance (VUS) in 9 genes (BARD1, BRCA1, BRIP1, CHEK2, ERCC1, NBN, PALB2, PMS1, RAD50) were predicted as potentially pathogenic by in silico analysis bringing the mutation detection rate up to 40%. CONCLUSION: As expected, a positive family history is a strong predictor of germline BRCA2 mutations in male BC. Understanding the potential pathogenicity of VUS represents an extremely urgent need for the management of BC risk in Male BC cases and their own families.

Visceral adiposity index and DHEAS are useful markers of diabetes risk in women with polycystic ovary syndrome.

OBJECTIVE: On the basis of the known diabetes risk in polycystic ovary syndrome (PCOS), recent guidelines of the Endocrine Society recommend the use of an oral glucose tolerance test (OGTT) to screen for impaired glucose tolerance (IGT) and type 2 diabetes (T2DM) in all women with PCOS. However, given the high prevalence of PCOS, OGTT would have a high cost-benefit ratio. In this study, we identified, through a receiver operating characteristic analysis, simple predictive markers of the composite endpoint (impaired fasting glucose (IFG) or IGT or IFG+IGT or T2DM) in women with PCOS according to the Rotterdam criteria. DESIGN: We conducted a cross-sectional study of 241 women with PCOS in a university hospital setting. METHODS: Clinical, anthropometric, and metabolic (including OGTT) parameters were evaluated. The homeostasis model assessment of insulin resistance (HOMA2-IR), the Matsuda index of insulin sensitivity, and the oral dispositional index and visceral adiposity index (VAI) were determined. RESULTS: Out of 241 women included in this study, 28 (11.6%) had an IFG, 13 (5.4%) had IGT, four (1.7%) had IFG+IGT, and four (1.7%) had T2DM. Among the anthropometric variables examined, the VAI had a significantly higher C-statistic compared with BMI (0.760 (95% CI: 0.70-0.81) vs 0.613 (95% CI: 0.54-0.67); P=0.014) and waist circumference (0.760 (95% CI: 0.70-0.81) vs 0.619 (95% CI: 0.55-0.68); P=0.028). Among all the hormonal and metabolic serum variables examined, DHEAS showed the highest C-statistic (0.720 (95% CI: 0.65-0.77); P<0.001). CONCLUSIONS: In addition to fasting glucose, the VAI and DHEAS may be considered useful tools for prescreening in all women with PCOS without the classical risk factors for diabetes.

[Our trend in conservative surgery in differentiated carcinoma of the thyroid]. / Nostro orientamento sugli interventi conservativi nel carcinoma differenziato della tiroide.

AIM OF THE STUDY: Report as contribution to the controversy between supporters of total thyroidectomy versus "less than total" thyroidectomy. MATERIALS AND METHODS: 42 patient operated on over six years; 35 treated with total thyroidectomy, 7 with lobohystmectomy. RESULTS: In the patients who underwent total thyroidectomy we observed recurrent nerve lesions in 5.7%, hypoparathyroidism in 14.3% and 1 lymph nodal relapse (it was a cancer stay III); in patients who underwent lobohystmectomy, we observed 1 temporary recurrent nerve palsy (14.2%) and 1 lymph nodal relapse (14.2%). DISCUSSION: The choice between total thyroidectomy and lobohystermectomy depends upon different goals: reduction in risk of relapse in total thyroidectomy, to minimize complications in lobohystmectomy. In our series the risk of lymph nodal relapse seems to depend more on biological characters of the tumour than surgical tech of lymphadenectomy; however, this occurrence does not change prognosis. CONCLUSIONS: In our experience, potential multifocality of the disease, low risk of hyatrogenic lesions and easy postoperatory management make total thyroidectomy the our preferred technique. Informed consensus is mandatory in order to involve the patients to the best choice.

The acute phase response in Sicilian patients with boutonneuse fever admitted to hospitals in Palermo, 1992-1997.

OBJECTIVES: To study the modifications of some components of the acute phase response (APR) in Sicilian patients with boutonneuse fever (BF) caused by Rickettsia conorii. METHODS: Sera from 500 Sicilian patients with confirmed BF were studied at the time of diagnosis and every week after treatment, and after recovery for the presence of various inflammatory mediators. Tumour necrosis factor alpha (TNFalpha), interleukin(IL)-6, IL-1alpha, IL-8, soluble TNF receptors (sTNF-R) and sIL-6R were assayed by commercially ELISA kits. C3, C4, factor B, C-reactive protein (CRP), fibrinogen, ceruloplasmin (Cp) and alpha(1)-antitrypsin (AAT) were assayed by a rate nephelometry. RESULTS: Interferon gamma (IFNgamma), IL-6, TNFalpha, and IL-10 cytokines were significantly modified, whereas IL-1 and IL-8 were not detectable in the blood in any phase of infection. sTNF-RI, sTNF-RII and sIL-6 were significantly increased in the first 2 weeks of infection, but sTNF-R levels were not related to the plasma levels of TNFalpha, whereas sIL-6 was directly related to serum IL-6 concentrations. C3, C4, factor B and CRP were significantly increased in the first 2 weeks of infection, but afterwards returned to the normal range, even though CRP was still high in the third week and C3 persisted high after the fourth week. Fibrinogen was high only in the first week in relation to the injury to the endothelial cells (ECs). The anti-inflammatory proteins, Cp and AAT, were extremely high in the first 2 weeks of infection acting as a buffer of APR activation. CONCLUSIONS: These results suggest that R. conorii is able to elicit, after invasion and proliferation in the ECs, the activation of APR. Further work is required to establish if active inhibitory mechanisms are operating during APR, or if there is a spontaneous decay in the initiation events.

Proliferating cell nuclear antigen labelling index in localised pigmented villo-nodular synovitis and its relationship to the size of nodules.

Proliferating cell nuclear antigen (PCNA) is one of the cell cycle-related proteins directly involved in DNA synthesis. It is a marker of cellular proliferation and has been shown to correlate with ploidy and proliferative activity of cells. Its expression has been used to estimate the growth fraction of human cancer and its prognostic value. Pigmented villo-nodular synovitis (PVNS) is characterised by a nodular lesion in the paratendinous synovial tissue or, less frequently, in a joint. Whether PVNS is a neoplastic or inflammatory lesion remains controversial. We have studied immunohistochemical PCNA expression with pc10 monoclonal antibody in 16 paraffin sections, in 16 cases of localised PVNS, or giant cell tumour of tendon sheath. We have found significant correlation between the size of the lesions and PCNA-LI (labelling index).

Bioactivity and glycosylation of circulating prolactin in various physiological and pathological conditions.

Multiple posttranslational processes modify native PRL and result in the secretion of several PRL isoforms with different bioactivity. Since we observed that serum samples contain non-lactogenic substances able to interfere in Nb2 cell bioassay, in this study we extracted PRL molecules from sera of pregnant and non-pregnant normal adults, fetuses and patients with prolactinoma and evaluated the ability of partially purified PRL to stimulate Nb2 cell proliferation. The preliminary immunopurification of PRL samples, conferred good sensitivity and specificity to PRL biological assay. Whenever possible, bioactivity values were correlated with glycosylated-PRL levels (G-PRL), the major posttranslational modification known to reduce PRL bioactivity. The ratio of bioactive (B-) vs immunoreactive PRL (I-PRL) (B/I) in normal subjects was 0.9 +/- 0.1 (mean +/- SD), and not affected by TRH and sulpiride administration. PRL B/I ratio did not change during pregnancy, both in maternal (0.8 +/- 0.1) and fetal circulation (1.0 +/- 0.01). In patients with prolactinoma PRL B/I ratios (0.8 +/- 0.18) were in the normal range. However, in 2 women with microprolactinoma, with a clear discrepancy between high I-PRL levels and mild clinical features, a significantly reduced PRL B/I ratio was observed (0.51 +/- 0.08 and 0.52 +/- 0.1 respectively). Conversely, a woman with clear clinical features of hyperprolactinemia, but border-line elevated I-PRL levels had a PRL B/I ratio in the upper limit of normal range. No variation in G-PRL vs NG-PRL percentages was observed in all the cases studied. In conclusion, our data show that physiological and pathological conditions of hyperprolactinemia, including fetal life, are associated in the majority of cases, with the secretion of PRL molecules with unchanged mitogenic activity on Nb2 cells. Nb2 PRL bioassay may be an useful tool to explain the discrepancies between clinical features and immunoreactive PRL levels in some particular cases.

Use of cabergoline in the long-term treatment of hyperprolactinemic and acromegalic patients.

Cabergoline (Cab), a very potent and long-lasting dopaminergic compound, was administered to 26 women with pituitary microprolactinoma [mean serum PRL levels: 124.8 +/- 11.3 micrograms/l (+/- SE), range 62-300 micrograms/l] and 3 patients with GH-secreting pituitary adenoma (2 with associated PRL hypersecretion) for 12 and 24 months, respectively. In microprolactinomas, a stable normoprolactinemia was achieved in 96.1% of cases: in 13 women (50%) with the lowest dose of the drug (0.5 mg/week), and in other 12 patients (46.1%) with increasing doses up to 3 mg/week. All the oligomenorrheic/amenorrheic women, except one, restored regular and ovulatory menses. Two patients became pregnant. Pituitary abnormalities at high resolution-CT (HR-CT) scan disappeared in 13 of 19 patients (68.4%) after 12 months of therapy and this feature persisted in 8/13 cases (61.5%) 12 months after drug withdrawal. During Cab discontinuation (range: 3-60 months), mean serum PRL levels remained significantly lower than the basal ones. Six of 25 women are still without therapy. In 2 patients, normoprolactinemia persisted up to 38 and 60 months, respectively. Cab treatment was re-instituted in 13 patients because of the recurrence of hyperprolactinemia. Five patients were lost at follow up. In all the acromegalic patients, Cab (1-3 mg/week) normalized serum GH, IGF-I and PRL levels. A clear improvement in clinical symptoms was observed in all patients, but neuroradiological improvement in only one. Cab therapy was very well tolerated, as only seven patients complained of mild and transient side-effects and none had to stop treatment. In conclusion, Cab is an effective, safe, and well tolerated dopaminergic compound for the treatment of hyperprolactinemic disorders and the control of the clinical and hormonal features of dopamine-sensitive acromegalic patients.

CBP/p300 as a co-factor for the Microphthalmia transcription factor.

The Microphthalmia basic-Helix-Loop-Helix-Leucine Zipper (bHLH-LZ) transcription factor (Mi) plays a crucial role in the genesis of melanocytes; mice deficient for a functional (Microphthalmia) gene product lack all pigment cells. We show here that the Mi activation domain resides N-terminal to the DNA-binding domain and that as little as 18 amino acids are sufficient to mediate transcription activation. The minimal activation region of Mi is highly conserved in the related transcription factor TFE3 and is predicted to adopt an amphipathic alpha-helical conformation. This region of Mi is also highly conserved with a region of E1A known to be essential for binding the CBP/p300 transcription cofactor. Consistent with these observations, the Mi activation domain can interact in vitro with CBP specifically through a region of CBP required for complex formation with E1A, P/CAF and c-Fos, and anti p300 antibodies can co-immunoprecipitate Mi from both melanocyte and melanoma cell lines. In addition, co-transfection of a vector expressing CBP2 (aas 1621-1891) fused to the VP16 activation domain potentiated the ability of Mi to activate transcription, confirming the significance of the CBP-Mi interaction observed in vitro. These data suggest that transcription activation by Mi is achieved at least in part by recruitment of CBP. The parallels between transcription regulation by Microphthalmia in melanocytes and MyoD in muscle cells are discussed.

L-histidine/medroxyprogesterone acetate interaction modulates human breast cancer cell growth and progestin receptor expression in vitro.

The effect of different L-histidine concentrations on human mammary tumour cell (CG5) proliferation was studied to test the hypothesis of a role of histidine in modulating sex steroid-regulated cell proliferation. Cell growth was only possible in the 10(-5) M and 10(-2) M range, while its inhibition by medroxyprogesterone acetate was confined to the 10(-4) M and 10(-3) M range. 10(-3) M L-histidine enhanced the effect of medroxyprogesterone acetate in reducing the number of cells in the S phase. The results show also that 10(-3) M L-histidine favours progestin diffusion into cells and increases progestin receptors density. The present data are in line with previous observations of the effect of histidine on the growth of experimental animal tumours, add evidence that histidine concentration influences the control of cell proliferation by sex steroids, and suggest a possible use of histidine in association with progestational drugs in the treatment of human neoplasia.

Depression of CD4 T cell subsets and alteration in cytokine profile in boutonneuse fever.

Interferon (IFN)-gamma, interleukin (IL)-10, IL-6, and tumor necrosis factor (TNF)-alpha were significantly increased in sera from Sicilian patients with acute boutonneuse fever (BF) compared with those of healthy controls. IFN-gamma levels dropped sharply within the second week after infection. IL-6, IL-10, and TNF-alpha levels gradually declined; in convalescent patients only were they in the normal range. In contrast, peripheral blood mononuclear cells (PBMC) stimulated in vitro with phytohemagglutinin (PHA) produced low levels of IL-10 and IFN-gamma in acute BF that were compatible with the reduction in the levels of CD4+, CD4+/CD45RO+, and CD4+/CD45RA+ cells. In vitro production of TNF-alpha and IL-6 from PBMC stimulated with PHA was not significantly modified during the various phases of the infection compared with control PBMC, which could be due to the persistence of high levels of CD14+ monocytes compensating for the decrease in CD20+ B cells.

Prolactin responsiveness to peptide histidine methionine-27 in normal subjects and hyperprolactinemic patients.

In order to verify whether synthetic peptide histidine methionine (PHM-27) is able to induce serum prolactin (PRL) rise in normal subjects and to investigate its effect on PRL secretion in hyperprolactinemic conditions, PHM-27 (100 micrograms i.v. over 60 min at a rate of 3.3 micrograms/min) was given to 6 normal subjects and 11 hyperprolactinemic women, and serum PRL levels were measured before and at intervals up to 120 min after beginning the infusion. On a separate occasion, a saline infusion was administered to all these subjects as a control test. In normal subjects, PHM-27 caused PRL to increase from 7.8 +/- 1.4 micrograms/l (mean +/- SE) to 13 +/- 2.1 micrograms/l (p < 0.05), the peak occurring at 30 min, whereas it did not significantly modify serum PRL levels in 11 patients with prolactin-secreting adenomas. We also observed a significant difference of serum PRL pattern during PHM infusion when compared to saline in normal subjects. In contrast, in hyperprolactinemic states, PRL curves were similar in both tests. Continuous infusion of PHM-27 was very well tolerated and caused no important adverse events. These data suggest that PHM, like vasoactive intestinal peptide, can participate in the regulation of PRL release under physiological conditions and that the unsignificant PRL increase in PRL-secreting tumors may reflect abnormalities of the PRL secretion mechanism in these pathological states.

Primary amenorrhoea and infertility due to a mutation in the beta-subunit of follicle-stimulating hormone.

We report a woman with primary amenorrhoea and infertility associated with an isolated deficiency of pituitary follicle-stimulating hormone (FSH), but normal luteinizing hormone (LH) secretion. Ovulation was induced by administration of exogenous FSH and resulted in a successful pregnancy. Sequence analysis of the FSH beta-subunit gene indicated that she is homozygous for a two nucleotide frameshift deletion in the coding sequence. Her mother and son are heterozygous for this mutation. This deletion results in an alteration of amino acid codons 61-86 followed by a premature termination codon. The predicted truncated beta-subunit peptide lacks regions which are important for association with the alpha subunit and for binding to and activation of the FSH receptor. Abnormalities of FSH structure or function might be an under recognised but treatable cause of infertility.