Chromosome 10 and RET gene copy number alterations in hereditary and sporadic Medullary Thyroid Carcinoma

About 30% of hereditary Medullary Thyroid Carcinoma (MTC) have been demonstrated to harbour imbalance between mutant and wild-type RET alleles.We studied the RET copy number alterations (RET CNA) in 65 MTC and their correlation with RET mutation and patients’ outcome.Fluorescence in situ Hybridization and Real-time PCR revealed RET CNA in 27.7% MTC but only in a variable percentage of cells. In sporadic MTC, RET CNA were represented by chromosome 10 aneuploidy while in hereditary MTC by RET amplification. A significant higher prevalence of RET CNA was observed in RET mutated MTC (P = 0.003). RET CNA was also associated to a poorer outcome (P = 0.005). However, the multivariate analysis revealed that only RET mutation and advanced clinical stage correlated with the worst outcome.In conclusion, 30% MTC harbour RET CNA in variable percentage of cells suggesting cell heterogeneity. RET CNA can be considered a poor prognostic factor potentiating the poor prognostic role of RET mutation.     

Multiple endocrine neoplasia type 2

Multiple endocrine neoplasia type 2 (MEN2) is an hereditary disease with a prevalence of 1/5000. Three phenotypic variants have been identified: MEN2A associates medullary thyroid carcinoma (MTC) to pheochromocytoma in about 20-50% of cases and to primary hyperparathyroidism in 5-20% of cases; MEN2B associates MTC to pheochromocytoma in 50% of cases, to marphanoid habitus and to mucosal and digestive ganglioneuromatosis whereas in familial isolated medullary thyroid carcinoma (FMTC), the other components of the disease are absent. In MEN2, natural history of the disease and a common embryologic origin (neural crest) may explain the phenotypes observed in the organ involved, beginning from the stage of hyperplasia to adenoma and cancer. MEN2 is an inherited autosomal dominant disease with a complete penetrance, related to germline mutation in the proto-oncogene RET. MTC represent the most frequent circumstance of diagnosis. Pheochromocytoma and HPT may reveal the disease unfrequently and are systematically associated to undiagnosed MTC which is present yet. Analysis of the RET gene allows to confirm the diagnosis of MEN2 by identifying the causal germline mutation. Management of MEN2 patients include thyroidectomy associated to cervical central and bilateral lymph nodes dissection for MTC, unilateral adrenalectomy for unilateral pheochromocytoma or bilateral adrenalectomy when both glands are involved, and selective resection of pathologic parathyroid glands for HPT. Familial genetic screening detects at risk subjects who will develop the disease and allows to manage them at the earliest stage of the disease by perform early or prophylactic thyroidectomy such giving them the best chance of cure. Prognosis of MEN2 is mainly related to the stage-dependant prognosis of MTC, thus pointing the necessity of a complete thyroid surgery for index cases with MTC and the earliest thyroidectomy for screened at risk subjects.     

Medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) is developed from thyroid C cells that secrete calcitonin (CT). MTC represents 5-10% of thyroid cancers with a 1-2% incidence in nodular thyroid diseases. Diagnosis is usually made by a solitary nodule often associated to nodal metastasis and confirmed by a high basal CT level which represents its biological marker. MTC may present as a sporadic form and in about 30% of case as a familial form as a part of multiple endocrine neoplasia syndrome, an hereditary dominant inherited disease related to germline mutation of the proto-oncogene RET. Both biological (CT) and genetic (RET) markers allows the optimal diagnosis and treatment of MTC; the former allows screening and early diagnosis of MTC by routinely CT measurements in nodular thyroid diseases that make the adequate and complete surgery required to be performed. The former leads to diagnose familial MTC and to identify at risk subjects in whom early or prophylactic surgery may be performed. Treatment of MTC is based on the complete surgical resection: total thyroidectomy associated to central and laterocervical nodal dissection. For locally advanced or metastatic MTC, complete cervical surgery is required and needs to be associated to other systemic treatments: as chemotherapy is not very efficient, radioimmunotherapy and RET target gene therapy (mainly tyrosine kinase inhibitors) appears as possible valuable therapeutic options for the future. Prognosis of MTC is mainly related to both the stage of the disease and the extend of the initial surgery. Ten-year survival is about 80% when the patients are not surgically cured and reaches 95% when the biological marker CT is normalized after surgery.     

Hypocorticism and interrenal hyperplasia are not directly related to masculinization in XX mas(-1)/mas(-1) carp, Cyprinus carpio

This study reports on a homozygous XX male strain of common carp (E5), which fail to mount a normal cortisol stress response. Earlier classical genetic analysis had indicated that masculinization of E5 fish was caused by a putative recessive mutation (mas(-1)/mas(-1)). Hypocorticism in E5 fish was studied to investigate if it was related to masculinization. Head-kidney tissues isolated from E5 fish showed a low cortisol-producing capacity in vitro, and also demonstrated a reduced sensitivity to stimulation with ACTH, when compared with an isogenic XY male carp strain (STD). There was no strain difference in androgen production by head-kidney tissues in vitro. E5 fish exhibited significant hyperplasia of the interrenal tissue (adrenal homologue of teleost fish) located in the head-kidney. Conversion of pregnenolone was significantly lower in E5 head-kidney homogenates, compared to STD homogenates, however, no strain difference was found in the conversion of 17alpha-hydroxyprogesterone into cortisol. Gonad homogenates incubated with pregnenolone showed no strain difference in conversion to corticosteroids and androgens. Results indicate that the interrenal hyperplasia and hypocorticism in this strain of carp may be due to a dysfunction of the 17alpha-hydroxylase activity of the enzyme P450c17 in the interrenal, but that this defect may not be the primary factor resulting in masculinization of these XX genotypes.     

Recognizing and treating myocarditis in recent-onset systemic sclerosis heart disease: Potential utility of immunosuppressive therapy in cardiac damage progression

Objectives

Scleroderma heart disease is a major risk of death in systemic sclerosis (SSc). Mechanisms underlying myocardial damage are still unclear. We performed an extensive study of SSc patients with recent-onset symptoms for heart disease and examined the efficacy of immunosuppressive therapy.

Methods

A cohort of 181 SSc patients was enrolled. Of these, 7 patients newly developed clinical symptoms of heart disease (heart failure, chest pain, and palpitation); all of them showed mild but persistent increase in cardiac enzymes. These patients underwent Holter ECG, 2D-echocardiography, perfusional scintigraphy, delayed-enhancement-cardiac magnetic resonance (DE-CMR), coronary angiography, and endomyocardial biopsy. Patients were treated for at least 12 months and followed-up for 5 years.

Results

Ventricular ectopic beats (VEBs) were found in 4 patients, wall motion abnormalities in 3, pericardial effusion in 6, and DE in CMR in 6 with T2-hyperintensity in 2. In all patients, histology showed upregulation of endothelium adhesion molecules and infiltration of activated T lymphocytes, with (acute/active myocarditis in 6) or without (chronic/borderline myocarditis in 1) myocyte necrosis. Parvovirus B19 genome was detected in 3. None showed occlusion of coronary arteries or microvessels. Compared with SSc controls, these patients more often had early disease, skeletal myositis, c-ANCA/anti-PR3 positivity, VEBs, pericardial effusion, and systolic and/or diastolic dysfunction. Immunosuppressive therapy improved symptoms and led to cardiac enzyme negativization; however, 2 patients died of sudden death during follow-up.

Conclusions

Myocarditis is a common finding in SSc patients with recent-onset cardiac involvement. Its early detection allowed to timely start an immunosuppressive treatment, preventing cardiac damage progression in most cases.     

New insights about cross-reactive epitopes of six trypanosomatid genera revealed that Crithidia and Leptomonas have antigenic similarity to L. (L.) chagasi

We investigated whether ELISA using crude antigens from insect and plant trypanosomatids, which are non-pathogenic and easily cultivated in large scale, has the same positivity data as Leishmania (Leishmania) chagasi, the etiological agent of human visceral leishmaniasis (VL) or canine leishmaniasis (CanL), or as Trypanosoma cruzi, the etiological agent of Chagas disease (CD). The antigens from Crithidia fasciculata, Crithidia luciliae, and Leptomonas seymouri showed 100% cross-reactivity with VL and CanL samples, with no statistically titers differences from L. (L.) chagasi, however, 34% (17/50) of VL samples revealed higher titers using the insect trypanosomatids than the homologous antigen. On the other hand, antigens from Strigomonas culicis, Angomonas deanei, and Phytomonas serpens showed low cross-reactivity with VL and CanL samples. The sera from patients with American tegumentary leishmaniasis showed low levels of cross-reactivity with all trypanosomatids investigated, even with L. (L) chagasi, without titers dissimilarity among them. These parasites were also worthless as antigen source for detection of CD cases, which required homologous antigens to reach 100% positivity. This study showed, by ELISA, that crude extract of Crithidia and Leptomonas have epitopes similar to L. (L.) chagasi, which supports the idea of using them as antigens source for the serodiagnosis of visceral leishmaniasis.     

Quinoline resistance associated polymorphisms in the pfcrt, pfmdr1 and pfmrp genes of Plasmodium falciparum in Iran

Plasmodium falciparum resistance to chloroquine (CQ) has been documented in Iran since the early 1980s and has since gradually increased. Iran is therefore reviewing its national drug policy for malaria control. We describe the prevalence of single nucleotide polymorphisms (SNP) associated with quinoline drug resistance in south eastern Iran. Pre-treatment blood from patients with uncomplicated but symptomatic P. falciparum infection was analysed. Polymorphisms at codons 76, 152, 163 and 220 of the pfcrt gene (chloroquine resistance transporter) and at codons 86, 184, 1034, 1042 and 1246 of the pfmdr1 gene (multidrug resistance) were determined by PCR-RFLP and sequencing. In addition, SNPs on a recently described multidrug resistance protein (pfmrp) and a microsatellite (MS-4760) in the pfnhe-1 (sodium hydrogen exchanger) gene associated with quinoline and quinine resistance, respectively, were investigated for the first time in field samples not from Thailand. pfcrt 76T was found in 99% and pfmdr1 86Y in 72% of the samples. pfmrp 191H and 437S associated with decreased quinoline response were found to be absolutely linked at a frequency of 13.6%. The pfnhe-1 MS-4760 one repeat allele associated to quinine response in vitro was also detected. Sequencing of the pfcrt 72-76 haplotype revealed that SVMNT was the most common allele as previously observed in India. This suggests that pfcrt found in the Iranian P. falciparum population may have the same origin as in the P. falciparum populations in India but different from that normally found in south east Asia. In conclusion, the frequencies of quinoline resistance associated gene polymorphisms in this region suggest a population that has been significantly selected for by the long use of CQ.     

Molecular evolution of adiponectin in Carnivora and its mRNA expression in relation to hepatic lipidosis

Adiponectin is a novel adipocyte-derived hormone with low circulating concentrations and/or mRNA expression in obesity and non-alcoholic fatty liver disease (NAFLD). The adiponectin mRNA of several Carnivora species was sequenced to enable further gene expression studies in this clade with potential experimental species to examine the connections of hypoadiponectinemia to hepatic lipidosis. In addition, adiponectin mRNA expression was studied in the retroperitoneal fat of the American mink (Neovison vison), as hepatic lipidosis with close similarities to NAFLD can be rapidly induced to the species by fasting. The mRNA expression was determined after overnight-7 d of food deprivation and 28 d of re-feeding and correlated to the liver fat %. The homologies between the determined carnivoran mRNA sequences and that of the domestic dog were 92.2-99.1%. As the mRNA expression was not affected by short-term fasting and did not correlate with the liver fat %, there seems to be no clear connection between adiponectin and the development of lipidosis in the American mink. In the future, the obtained sequences can be utilized in further studies of adiponectin expression in comparative endocrinology.     

Prevalence of antibiotic resistance among Acinetobacter baumannii isolates from Aleppo, Syria

This study describes and analyzes Acinetobacter baumannii antibiotic susceptibly profile in Aleppo, Syria, thus providing vital information for guiding treatment of A baumannii infections. Two hundred sixty nonrepetitive A baumannii isolates were studied over 3.5 years. Resistance rates are at the higher end of globally reported levels. Newer cephalosporins and β-lactamase-resistant agents are becoming practically ineffective. Better activity is limited to carbapenems and colistin, which elicited the highest susceptibility levels.     

Changed gene expression for candidate ageing genes in long-lived Bicyclus anynana butterflies

Candidate genes for the regulation of lifespan have emerged from studies that use mutants and genetically manipulated model organisms. However, it is rarely addressed whether these genes contribute to lifespan variation in populations of these species that capture natural standing genetic variation. Here, we explore expression variation in three candidate ageing genes, Indy, sod2, and catalase, in Bicyclus anynana, a butterfly with well understood ecology.We used lines established from natural populations and artificially selected for increased adult starvation resistance. They show a considerable increase in adult lifespan under both starvation and optimal food conditions. We measured adult butterflies of various ages, under a range of optimal and starvation diets, from two selected populations and one unselected control population.In all lines, Indy and catalase are up-regulated in response to starvation while this is not evident for sod2. Under starvation, Indy and catalase are up-regulated in, while this is not evident for sod2. Under optimal food conditions, Indy is down-regulated at a later age, with Indy expression showing relatively high inter-individual variation. We find differences between the selected lines and the unselected line. Under starvation conditions, expression is higher for catalase in one, and for sod2 in both selected lines. Importantly, sod2 expression is also higher in the selected populations under optimal food conditions.We conclude that sod2, but not Indy, is involved in the response to artificial selection for increased starvation resistance. The role of catalase is less clear because of the differences between the two selected lines. Moreover, sod2 appears to be a candidate gene that underpins the genetic correlation between starvation resistance and longevity. Our study indicates that some, but not all, genes identified through mutant screens in other organisms may underpin standing genetic variation for ageing-related traits in stocks of Bicyclus butterflies established from natural populations. Clearly, this needs to be investigated in other organisms as well, especially in the organisms to which mutants screens were applied. This information will narrow down the list of genes that underpin variation in lifespan and ageing in extant populations of organisms, and which may serve as candidate genes in humans.     

Stress-induced facilitation of the cortisol response in 17alpha-hydroxylase deficient XX mas-1/mas-1 carp (Cyprinus carpio)

Facilitation of the stress response results from a reduction of the inhibitory effects of circulating corticosteroids, allowing an animal to respond to a novel stressor. In this study, the existence of a facilitated cortisol stress response in normal (STD) and 17alpha-hydroxylase deficient XX mas-1/mas-1 (E5) carp was investigated. E5 carp had previously been characterized as having a low cortisol response to stress. Fish were subjected to either cortisol feeding or daily-acute stress, from 45 until 140 days post-hatch (dph) and were then subjected to a novel net-confinement stressor at 141 dph. Growth of E5 fish was reduced in both the daily-acute stress and cortisol-fed groups, but STD fish were only affected by daily-acute stress. Cortisol feeding had no effect on the stress response of STD fish but daily-acute stress significantly inhibited the response to a subsequent novel stressor. In contrast, daily-acute stress facilitated the cortisol stress response of E5 fish to a novel stressor, while cortisol feeding inhibited the cortisol response. Facilitation was accompanied by significant enlargement of the head-kidney tissue (which contains the steroidogenic interrenal tissue) in E5 fish. To our knowledge this is the first report of stress-induced facilitation in a lower vertebrate.     

Laboratory and field evaluation of Teknar HP-D, a biolarvicidal formulation of Bacillus thuringiensis ssp. israelensis, against mosquito vectors

Larvicidal efficacy of Teknar HP-D, an improved biolarvicidal formulation of Bacillus thuringiensis ssp. israelensis (Bti), against Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti was determined in the laboratory, and in field the efficacy of the formulation was tested against Cx. quinquefasciatus breeding in cesspits, unused wells and drains. The toxicity of the formulation to Gambusia affinis (larvivorous fish), Notonecta sp. and Diplonychus indicus (water bugs) was also evaluated in the laboratory. Teknar HP-D was field tested at three recommended dosages, 1, 1.5 and 2l/ha, selecting five habitats for each dosage. Another five habitats were kept untreated as controls. Ae. aegypti showed greatest susceptibility to the Bti toxin in the laboratory. In cesspits, all the three dosages caused >80% reduction of pupal recruitment up to day 6 post-treatment, indicating that a weekly application at the lowest would be necessary for sustained control. The residual activity of the formulation was longer in unused wells, causing >80% reduction of pupal recruitment for 17 days from the day of treatment. In controlling pupal recruitment the three dosages produced equal effect. Application of Teknar HP-D at 1 l/ha once in three weeks is therefore recommended to control Cx. quinquefasciatus in unused wells. However, in drains, >80% reduction of pupal recruitment was observed for only 3 days and hence, application of Teknar HP-D at 2 l/ha that caused significantly higher level of reduction twice in a week at 3-day interval is necessary. At dosages from 0.032 to 3.2 mg/l (ppm), Teknar HP-D was non-toxic to Gambusia fish. The two predatory water bugs, Notonecta sp. and Diplonychus indicus that fed on the surviving larvae of Cx. quinquefasciatus exposed to the sub-lethal doses (LC(50) and LC(80)) of Teknar HP-D were safe with out having any mortality.     

Anthropophilic mosquitoes and malaria transmission in the eastern foothills of the central highlands of Madagascar

Malaria remains a major public health problem in Madagascar, as it is the first cause of morbidity in health care facilities. Its transmission remains poorly documented. An entomological study was carried out over 1 year (October 2003-September 2004) in Saharevo, a village located at an altitude of 900 m on the eastern edge of the Malagasy central highlands. Mosquitoes were sampled weekly upon landing on human volunteers and in various resting-places. Out of 5515 mosquitoes collected on humans, 3219 (58.4%) were anophelines. Eleven anopheline species were represented, among which Anopheles funestus, Anopheles gambiae, Anopheles arabiensis and Anopheles mascarensis. Out of 677 mosquitoes collected in bedrooms by pyrethrum spray catches and in Muirhead-Thomson pits, 656 (96.9%) were anopheline belonging to these four latter species. The proportion of mosquitoes that fed on human varied according to the resting-places and the mosquito species: 86% of An. funestus resting in bedrooms fed on humans, whereas only 16% of An. funestus and 0% of An. mascarensis resting in pits fed on humans. The proportion of anopheline mosquitoes infected with human Plasmodium was measured by circumsporozoite protein-ELISA: 10/633 An. funestus (1.58%), 1/211 An. gambiae s.l. (0.48%) and 2/268 An. mascarensis (0.75%). The annual entomological inoculation rate (number of bites of infected anophelines per adult) was estimated at 2.78. The transmission was mainly due to An. funestus and only observed in the second half of the rainy season, from February to May. These results are discussed in the context of the current malaria vector control policy in Madagascar.     

Phylogenetic relationships among species of Anopheles (Nyssorhynchus) (Diptera, Culicidae) based on nuclear and mitochondrial gene sequences

Phylogenetic relationships among 21 species of mosquitoes in subgenus Nyssorhynchus were inferred from the nuclear white and mitochondrial NADH dehydrogenase subunit 6 (ND6) genes. Bayesian phylogenetic methods found that none of the three Sections within Nyssorhynchus (Albimanus, Argyritarsis, Myzorhynchella) were supported in all analyses, although Myzorhynchella was found to be monophyletic at the combined genes. Within the Albimanus Section the monophyly of the Strodei Subgroup was strongly supported and within the Myzorhynchella Section Anopheles antunesi and An. lutzii formed a strongly supported monophyletic group. The epidemiologically significant Albitarsis Complex showed evidence of paraphyly (relative to An. lanei-Myzorhynchella) and discordance across gene trees, and the previously synonomized species of An. dunhami and An. goeldii were recovered as sister species. Finally, there was evidence of complexes in several species, including An. antunesi, An. deaneorum, and An. strodei.     

Identification of four members of the Anopheles funestus (Diptera: Culicidae) group and their role in Plasmodium falciparum transmission in Bagamoyo coastal Tanzania

The role of Anopheles funestus group in malaria transmission was investigated in Bagamoyo coastal Tanzania, in the process of characterizing the area as a malaria vaccine testing site. Mosquitoes were sampled inside houses and multiplex PCR was used to identify 649 specimens. The following species were found: A. funestus s.s. (84.3%), A. leesoni (13.6%), A. rivulorum (1.5%) and A. parensis (0.6%). Multiplex PCR of 147 blood-fed specimens showed that over half (57.1%) of the identifiable blood meals were taken from human hosts, and human blood index in A. funestus and A. leesoni was 55% and 82% respectively. Plasmodium falciparum infection rate determined by nested PCR was 11% in A. funestus s.s. Although the abundance was low, 26 specimens of A. leesoni, two of A. rivolurum and one of A. parensis were found positive for P. falciparum. The presence of four A. funestus species in Tanzania emphasizes the relevance to define precisely their spatial and temporal distribution, specific behaviour, ecology and exact role in malaria transmission.     

Identification of Tunisian Leishmania spp. by PCR amplification of cysteine proteinase B (cpb) genes and phylogenetic analysis

Discrimination of the Old World Leishmania parasites is important for diagnosis and epidemiological studies of leishmaniasis. We have developed PCR assays that allow the discrimination between Leishmania major, Leishmania tropica and Leishmania infantum Tunisian species. The identification was performed by a simple PCR targeting cysteine protease B (cpb) gene copies. These PCR can be a routine molecular biology tools for discrimination of Leishmania spp. from different geographical origins and different clinical forms. Our assays can be an informative source for cpb gene studying concerning drug, diagnostics and vaccine research.