Combinatorial immune checkpoint blockade increases myocardial expression of NLRP-3 and secretion of H-FABP, NT-Pro-BNP, interleukin-1ß and interleukin-6: biochemical implications in cardio-immuno-oncology.

Background: Immune checkpoint blockade in monotherapy or combinatorial regimens with chemotherapy or radiotherapy have become an integral part of oncology in recent years. Monoclonal antibodies against CTLA-4 or PD-1 or PDL-1 are the most studied ICIs in randomized clinical trials, however, more recently, an anti-LAG3 (Lymphocyte activation gene-3) antibody, Relatlimab, has been approved by FDA in combination with Nivolumab for metastatic melanoma therapy. Moreover, Atezolizumab is actually under study in association with Ipilimumab for therapy of metastatic lung cancer. Myocarditis, vasculitis and endothelitis are rarely observed in these patients on monotherapy, however new combination therapies could expose patients to more adverse cardiovascular events. Methods: Human cardiomyocytes co-cultured with human peripheral blood lymphocytes (hPBMCs) were exposed to monotherapy and combinatorial ICIs (PD-L1 and CTLA-4 or PD-1 and LAG-3 blocking agents, at 100 nM) for 48 h. After treatments, cardiac cell lysis and secretion of biomarkers of cardiotoxicity (H-FABP, troponin-T, BNP, NT-Pro-BNP), NLRP3-inflammasome and Interleukin 1 and 6 were determined through colorimetric and enzymatic assays. Mitochondrial functions were studied in cardiomyocyte cell lysates through quantification of intracellular Ca++, ATP content and NADH:ubiquinone oxidoreductase core subunit S1 (Ndufs1) levels. Histone deacetylases type 4 (HDAC-4) protein levels were also determined in cardiomyocyte cell lysates to study potential epigenetic changes induced by immunotherapy regimens. Results: Both combinations of immune checkpoint inhibitors exert more potent cardiotoxic side effects compared to monotherapies against human cardiac cells co-cultured with human lymphocytes. LDH release from cardiac cells was 43% higher in PD-L1/CTLA-4 blocking agents, and 35.7% higher in PD-1/LAG-3 blocking agents compared to monotherapies. HDAC4 and intracellular Ca++ levels were increased, instead ATP content and Ndufs1 were reduced in myocardial cell lysates (p < 0.001 vs. untreated cells). Troponin-T, BNP, NT-Pro-BNP and H-FABP, were also strongly increased in combination therapy compared to monotherapy regimen. NLRP3 expression, IL-6 and IL-1ß levels were also increased by PDL-1/CTLA-4 and PD-1/LAG-3 combined blocking agents compared to untreated cells and monotherapies. Conclusions: Data of the present study, although in vitro, indicate that combinatorial immune checkpoint blockade, induce a pro- inflammatory phenotype, thus indicating that these therapies should be closely monitored by the multidisciplinary team consisting of oncologists, cardiologists and immunologists.

Atypical presentations of ectopic pregnancies in a rural tertiary care hospital-A case series.

In spite of advances in medical sciences, diagnosis of ectopic pregnancy eludes the clinician. In this case series, we describe three variants of life-threatening pregnancies. The successful management of these cases hinged on a strong index of suspicion and early detection. Heterotopic pregnancy is the occurrence of two pregnancies in different implantation sites at the same time, which is uncommon. Heterotopic pregnancy is uncommon in natural conception, despite its prevalence in assisted reproductive technologies. A strong index of suspicion and timely laparotomy was life-saving for the woman. Caesarean scar pregnancy is a form of ectopic pregnancy in which an aberrant pregnancy is implanted at the hysterotomy site of a prior caesarean procedure. Unexplained excessive bleeding at the time of medical termination of pregnancy prompted us to explore and make a timely diagnosis of caesarean scar pregnancy. Finally, tubal choriocarcinoma is a very aggressive, extremely rare trophoblastic tumour that can be either gestational or non-gestational. High serum beta HCG raised the suspicion and timely laparotomy confirmed the diagnosis. An alert clinician who takes timely decisions can avert life-threatening complications in these pregnancies.

Impact of Phyllantus niruri and Lactobacillus amylovorus SGL 14 in a mouse model of dietary hyperoxaluria.

Hyperoxaluria is a pathological condition which affects long-term health of kidneys. The present study evaluates the impact of the combination of Lactobacillus amylovorus SGL 14 and the plant extract Phyllantus niruri (namely Phyllantin 14™) on dietary hyperoxaluria. Safety and efficacy of Phyllantin 14 have been evaluated in vivo. Mice C57BL6 fed a high-oxalate diet were compared to mice fed the same diet administered with Phyllantin 14 by gavage for 6 weeks. Control mice were fed a standard diet without oxalate. No adverse effects were associated to Phyllantin 14 supplementation, supporting its safety. Mice fed a high-oxalate diet developed significant hyperoxaluria and those administered with Phyllantin 14 showed a reduced level of urinary oxalate and a lower oxalate-to-creatinine ratio. Soluble and insoluble caecal oxalate were significantly lower in treated group, a finding in agreement with the colonisation study, i.e. mice were colonised with SGL 14 after 3 weeks. Microbiota analysis demonstrated that both oxalate diet and Phyllantin 14 can differently modulate the microbiota. In conclusion, our findings suggest that Phyllantin 14 supplementation represents a potential supportive approach for reducing urinary oxalate and/or for enhancing the efficacy of existing treatments.

Enzymatic quantification of total serum bile acids as a monitoring strategy for women with intrahepatic cholestasis of pregnancy receiving ursodeoxycholic acid treatment: a cohort study.

OBJECTIVE: To evaluate enzymatic total serum bile acid quantification as a monitoring strategy for women with intrahepatic cholestasis of pregnancy (ICP) treated with ursodeoxycholic acid (UDCA). DESIGN: Cohort. SETTING: One UK university hospital. POPULATION: 29 ICP cases treated with UDCA. METHODS: Serial samples were collected prospectively throughout gestation. Total serum bile acids were measured enzymatically and individual bile acids by high-performance liquid chromatography-tandem mass spectrometry. Data were log-transformed and analysed with random effects generalised least square regression. MAIN OUTCOME MEASURES: The relationship between enzymatic total bile acid measurements and individual bile acid concentrations after UDCA treatment. RESULTS: In untreated women, cholic acid was the principal bile acid (51%) and UDCA concentrations were <0.5%, whereas UDCA constituted 60% (IQR 43-69) of serum bile acids following treatment and cholic acid fell to <20%. Changes in the total bile acid measurement reflected similar alterations in the concentrations of the pathologically elevated bile acids, e.g. a two-fold increase in enzymatic total bile acids is accompanied by approximately a two-fold increase in cholic acid and chenodeoxycholic acid at most UDCA doses (P < 0.001). Most of the effects of UDCA on cholic acid occur in the first week of treatment (60% relative reduction, P = 0.025, 95% CI 0.2-0.9, from 10 micromol/l (4.7-17.6) to 3.5 micromol/l (1.4-7.5). CONCLUSION: Ursodeoxycholic acid becomes the main component of the bile acid measurement after treatment. Enzymatic total bile acid assays are good predictors of both cholic acid and chenodeoxycholic acid, the primary bile acids that are raised prior to treatment. TWEETABLE ABSTRACT: Ursodeoxycholic acid constitutes approximately 60% of the bile acid measurement and reduces pathological cholic acid in treated women.

Longevity and population age structure of the arroyo southwestern toad (Anaxyrus californicus) with drought implications.

The arroyo southwestern toad is a specialized and federally endangered amphibian endemic to the coastal plains and mountains of central and southern California and northwestern Baja California. It is largely unknown how long these toads live in natural systems, how their population demographics vary across occupied drainages, and how hydrology affects age structure. We used skeletochronology to estimate the ages of adult arroyo toads in seven occupied drainages with varying surface water hydrology in southern California. We processed 179 adult toads with age estimates between 1 and 6 years. Comparisons between skeletochronological ages and known ages of PIT tagged toads showed that skeletochronology likely underestimated toad age by up to 2 years, indicating they may live to 7 or 8 years, but nonetheless major patterns were evident. Arroyo toads showed sexual size dimorphism with adult females reaching a maximum size of 12 mm greater than males. Population age structure varied among the sites. Age structure at sites with seasonally predictable surface water was biased toward younger individuals, which indicated stable recruitment for these populations. Age structures at the ephemeral sites were biased toward older individuals with cohorts roughly corresponding to higher rainfall years. These populations are driven by surface water availability, a stochastic process, and thus more unstable. Based on our estimates of toad ages, climate predictions of extreme and prolonged drought events could mean that the number of consecutive dry years could surpass the maximum life span of toads making them vulnerable to extirpation, especially in ephemeral freshwater systems. Understanding the relationship between population demographics and hydrology is essential for predicting species resilience to projected changes in weather and rainfall patterns. The arroyo toad serves as a model for understanding potential responses to climatic and hydrologic changes in Mediterranean stream systems. We recommend development of adaptive management strategies to address these threats.

Purification and characterization of ribosomal proteins L27 and L30 having antimicrobial activity produced by the Lactobacillus salivarius SGL 03.

AIMS: The aim of this study was to investigate the antimicrobial potential of proteins secreted by a new strain of Lactobacillus salivarius. METHODS AND RESULTS: The secretome of L. salivarius SGL 03 strain was analysed by gel-assisted fractionation and MS/MS to identify low-molecular-mass proteins. This strategy allowed us to identify 10 secreted proteins. Then, a combination of heterologous expression and agar well diffusion was used to characterize them as to their antimicrobial activity, mechanisms of action and stability. Our findings indicate that L27 and L30 proteins of the 50S ribosomal subunit have antimicrobial activity against Streptococcus pyogenes, Streptococcus uberis and Enterococcus faecium. In addition, both proteins are bactericidal against S. pyogenes and maintain their antimicrobial activity after different protease treatments, at acidic pH, after heat treatment, and if stored in a refrigerated ambient at least at 4°C. CONCLUSIONS: The overall results demonstrated that the L27 and L30 ribosomal proteins are of interest as new antimicrobial molecules to prevent the growth of S. pyogenes, S. uberis and E. faecium. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results provide the first insight into the extra-ribosomal activity of L27 and L30 secreted proteins of L. salivarius. This study demonstrated the capacity of L. salivarius SGL 03 to produce antimicrobial molecules and suggested this strain as a promising probiotic candidate.

Gold-iron oxide dimers for magnetic hyperthermia: the key role of chloride ions in the synthesis to boost the heating efficiency.

With the aim of producing Au-Fe x O y dimers with outstanding heating performances under magnetic hyperthermia conditions applicable to human patients, here we report two synthesis routes, a two-pot and a one-pot method. The addition of chloride ions and the absence of 1,2-hexadecanediol (HDDOL), a commonly used chemical in this synthesis, are the key factors that enable us to produce dimers at low temperature with crystalline iron oxide domains in the size range between 18-39 nm that is ideal for magnetic hyperthermia. In the case of two-pot synthesis, in which no chloride ions are initially present in the reaction pot, dimers are obtained only at 300 °C. In order to lower the reaction temperature to 200 °C and to tune the size of the iron oxide domain, the addition of chloride ions becomes the crucial parameter. In the one-pot method, the presence of chloride ions from the start of the synthesis (as counter ions of the gold salt precursor) enables a prompt formation of dimers directly at 200 °C. In this case, the reaction time is the main parameter used to tune the iron oxide size. A record value of specific absorption rates (SARs) up to 1300 W gFe-1 at 330 kHz and 24 kA m-1 was measured for dimers with an iron oxide domain of 24 nm in size.

Eco-morphological attributes and feeding habits in coexisting characins.

The head morphology and feeding habits of pairs of characin species (family Characidae) that coexist in four different coastal rainforest streams were analysed. Coexisting species differed in size, but were very similar in eco-morphological attributes. Gut analyses revealed differences in feeding preferences for each coexisting species, indicating resource partitioning. A pattern of organization in species pairs that was repeated in the four studied streams was noticed. The pattern consisted of one slightly larger species with a feeding preference for items of allochthonous origin and another smaller species with a preference for autochthonous items. The hypothesis that small morphological differences enable the current coexistence of those species pairs was proposed. Furthermore, the results show ecological equivalence among different species in the studied streams.

Introduction and Establishment of Pissodes castaneus (Coleoptera: Curculionidae) in the Andean Patagonia of Argentina.

The pine weevils that occur in plantations of Pinus spp. in Andean Patagonia of Argentina belong to the species Pissodes castaneus (De Geer), a Eurasian endemic species, according to the identification based on molecular and morphological characters. Sequences of the mitochondrial Cytochrome oxidase subunit I and nuclear genes (28 S rDNA and ITS2) were obtained for individuals of 13 afforestations, covering the entire distribution area of the established populations in the Andean Patagonia of Argentina. Sequence comparison with representative species of the genus (European, North American, and Chinese species) shows that Patagonian specimens are conspecific to those of P. castaneus sequenced from Europe. Phylogenetic analyses indicate that all terminals from Patagonia form a monophyletic unit without evident subclades, eliminating the possibility of existence of more than one species of Pissodes Germar in this area, including cryptic ones. Moreover, the very low genetic divergence between the Patagonian populations suggests that it is plausible that P. castaneus was introduced into Patagonia from just one location. Mitochondrial DNA analysis shows that Patagonian terminals group together with a French haplotype and are clearly separated from other P. castaneus individuals represented in our sample, and reveal that established populations in Andean Patagonia originated via a limited introduction.

Necessity of therapy for post-thyroidectomy hypocalcaemia: a multi-centre experience.

PURPOSE: Hypoparathyroidism is one of the most common and most feared complications of total thyroidectomy (TT). The aim of this study is to detect possible markers that may facilitate early tracing of hypocalcaemia-prone patients in order to reduce clinical cost by optimizing patient discharge and to avoid unnecessary treatment. METHODS: Over an 18-month period, 995 patients, 23 % male and 77 % female, aged 52.9 ± 13.4 years, underwent TT in ten Lombardy hospitals. The following parameters were analyzed: calcaemia before and 12-24 and 48 h after surgery, pre- and post-operative parathyroid hormone (PTH) at 24 h and pre-operative 25OH vitamin D. RESULTS: Mortality was nil and morbidity was 22.4 %. Mean 24-h calcaemia and PTH were 2.17 ± 0.15 mmol/l and 31.81 ± 20.35 pg/ml, respectively; mean 24-h PTH decay was 36.7 ± 34.12 %. Four hundred seventy-three (47.5 %) patients were hypocalcaemic at discharge; 142 of whom had transient hypoparathyroidism that became permanent in 27. Patients developing hypocalcaemia had significantly higher values of PTH and calcium decay. At multiple logistic regression, only 24-h calcium decay, PTH drop and the presence of symptoms and parathyroid auto-grafting were significantly related to hypoparathyroidism. The association of these factors had a 99.2 % negative predictive value (NPV) for the development of hypoparathyroidism. A 70 % PTH drop had a 93.75 NPV for transient hypoparathyroidism. A 12 % calcaemia decay had a 95.7 NPV for hypoparathyroidism. CONCLUSIONS: Hypocalcaemic asymptomatic patients with less than 70 % PTH and 12 % calcaemia decay may be safely discharged without treatment. Symptomatic patients and those with parathyroid grafting should receive calcium and vitamin D.

Three-dimensional coherent diffractive imaging on non-periodic specimens at the ESRF beamline ID10.

The progress of tomographic coherent diffractive imaging with hard X-rays at the ID10 beamline of the European Synchrotron Radiation Facility is presented. The performance of the instrument is demonstrated by imaging a cluster of Fe2P magnetic nanorods at 59 nm 3D resolution by phasing a diffraction volume measured at 8 keV photon energy. The result obtained shows progress in three-dimensional imaging of non-crystalline samples in air with hard X-rays.

Assembly-mediated interplay of dipolar interactions and surface spin disorder in colloidal maghemite nanoclusters.

Controlled assembly of single-crystal, colloidal maghemite nanoparticles is facilitated via a high-temperature polyol-based pathway. Structural characterization shows that size-tunable nanoclusters of 50 and 86 nm diameters (D), with high dispersibility in aqueous media, are composed of ∼13 nm (d) crystallographically oriented nanoparticles. The interaction effects are examined against the increasing volume fraction, φ, of the inorganic magnetic phase that goes from individual colloidal nanoparticles (φ = 0.47) to clusters (φ = 0.72). The frozen-liquid dispersions of the latter exhibit weak ferrimagnetic behaviour at 300 K. Comparative Mössbauer spectroscopic studies imply that intra-cluster interactions come into play. New insight emerges from the clusters' temperature-dependent ac susceptibility that displays two maxima in χ''(T), with strong frequency dispersion. Scaling-law analysis together with the observed memory effects suggests that a superspin-glass state settles-in at TB ∼ 160-200 K, while at lower-temperatures, surface spin-glass freezing is established at Tf ∼ 40-70 K. In such nanoparticle-assembled systems, with increased φ, Monte Carlo simulations corroborate the role of the inter-particle dipolar interactions and that of the constituent nanoparticles' surface spin disorder in the emerging spin-glass dynamics.

Plasticity in the diet of Astyanax taeniatus in a coastal stream from south-east Brazil.

In this work we aimed to characterise the diet of Astyanax taeniatus (Jenyns 1842) (Characiformes, Characidae) along the Mato Grosso stream and we hypothesised that the diet of this species would vary according to spatial, seasonal and ontogenetic differences. Specimens were collected every second month at three sites with different physical attributes. The feeding habits of 651 specimens were analysed using the Alimentary Index (IA(i)). Analysis of the diet showed an effect of spatial, temporal and ontogenetic factors. Vegetal allochthonous items were more important in the diet of Astyanax taeniatus in upstream sites while animal autochthonous items were more important downstream. Ontogenetic differences in the diet were significant only in upstream sites, where adults consumed a greater amount of vegetal matter than juveniles. These results reinforce the idea that Astyanax species are opportunistic and show trophic plasticity.

A novel hybrid organic/inorganic photonic crystal slab showing a resonance action at the band edge.

Here we propose and experimentally demonstrate a hybrid photonic crystal (PC) slab consisting of air rods in a nanocomposite prepared by incorporating CdSe/CdS core/shell NRs (NR) in a polymer. Since the styrene methyl acrylate based polymer (ZEP) is transparent in the visible spectral range and is an electron-sensitive material, it was chosen as the embedding matrix for the NRs. Scanning electron microscopy and luminance measurements were used to characterize the experimental structure. The vertical extraction of the light, by the coupling of the modes guided by the PC slab to the free radiation via Bragg scattering, consists of a narrow orange emission band at 592 nm with a full width at half-maximum (FWHM) of 17 nm. The original characteristics of hybrid materials based on polymers and colloidal NRs, able to combine the unique optical properties of the inorganic moiety with the processability of the host matrix, are extremely appealing in view of their technological impact on the development of new high performing optical devices such as organic light-emitting diodes, ultra-low threshold lasers and non-linear devices.

Feeding ecology of Hypostomus punctatus Valenciennes, 1840 (Osteichthyes, Loricariidae) in a costal stream from Southeast Brazil.

In the present study we aimed to compare the feeding ecology of Hypostomus punctatus from a coastal stream from Southeast Brazil with data previously published for the same study site before environmental changes. Feeding preferences were assessed through a sample of 138 specimens (67 from the dry and 71 from the rainy season) using the Index of Alimentary Importance (IAi). We registered five different food items (detritus, plant fragments, Diatoms, Chloroficeae and Cianobacteries) composing the species diet. Detritus was the most abundant one both during the rainy and dry seasons (IAirainny = 90.34 and IAidry = 96.30). No significant differences were registered for the volume of food items consumed during the rainy and dry seasons. The Frequency of Occurrence analysis showed that four (detritus, plant fragments, Diatoms and Chloroficeae) among the five all other consumed ones, were always frequent. Comparing our own results with those available for the study site, we suggest that the feeding habit of H. punctatus has changed according to environmental changes and that the species diet is strongly dependent upon environmental conditions.

Feeding ecology of Hypostomus punctatus Valenciennes, 1840 (Osteichthyes, Loricariidae) in a costal stream from Southeast Brazil / Ecologia trófica de Hypostomus punctatus Valenciennes, 1840 (Osteichthyes, Loricariidae) de um riacho costeiro do sudeste do Brasil

In the present study we aimed to compare the feeding ecology of Hypostomus punctatus from a coastal stream from Southeast Brazil with data previously published for the same study site before environmental changes. Feeding preferences were assessed through a sample of 138 specimens (67 from the dry and 71 from the rainy season) using the Index of Alimentary Importance (IAi). We registered five different food items (detritus, plant fragments, Diatoms, Chloroficeae and Cianobacteries) composing the species diet. Detritus was the most abundant one both during the rainy and dry seasons (IAirainny = 90.34 and IAidry = 96.30). No significant differences were registered for the volume of food items consumed during the rainy and dry seasons. The Frequency of Occurrence analysis showed that four (detritus, plant fragments, Diatoms and Chloroficeae) among the five all other consumed ones, were always frequent. Comparing our own results with those available for the study site, we suggest that the feeding habit of H. punctatus has changed according to environmental changes and that the species diet is strongly dependent upon environmental conditions.

No presente estudo, tivemos como objetivo comparar a ecologia trófica de Hypostomus punctatus de um riacho costeiro do sudeste do Brasil com dados previamente publicados para a mesma localidade deste estudo, antes que tenha sofrido alterações ambientais. As preferências alimentares foram analisadas através da análise de 138 exemplares (67 da estação seca e 71 da estação chuvosa), utilizando-se o Índice de Importância Alimentar (IAi). Registramos cinco diferentes itens alimentares (detrito, fragmentos vegetais, Diatomáceas, Clorofíceas e Cianobactérias), compondo a dieta da espécie. Detrito foi o item mais abundante, tanto durante a estação chuvosa como durante a seca (IAichuva = 90,34 e IAiseca = 96,30). Não foram registradas diferenças significativas entre o volume consumido de cada um dos itens na estação seca e chuvosa. A análise da frequência de ocorrência mostrou que quatro (detrito, fragmento vegetal, Diatomáceas e Clorofíceas) entre os cinco itens consumidos foram sempre frequentes. Comparando nossos resultados com aqueles previamente publicados, sugerimos que o hábito alimentar de H. punctatus mudou de acordo com as alterações ambientais e que sua dieta é fortemente dependente das condições ambientais.

Multilocus microsatellite polymorphism analysis to characterize Leishmania infantum strains isolated in Sicily.

Different approaches are being developed to improve the differentiation of Leishmania genus using biochemical and molecular methods. In this study, 11 independent polymorphic microsatellites were used for the typing of strains of L. infantum isolated in Sicily. Polymerase chain reaction was employed to amplify the microsatellites contained in 12 DNA regions selected from among more investigated loci. A total of 51 isolates of L. infantum from dogs were tested by using the same locus panel. The products were successively analysed using an automatic sequence detector (ABI PRISM 3130 AB), to discover relevant microsatellite polymorphisms. It was possible to discriminate between MON-1 and non-MON-1 groups. Moreover, the method permitted to distinguish various genotypes of L. infantum isolates within each zymodema. Model- and distance-based analyses of the data set showed comparable results. The frequency of heterozygosity in the alleles analysed varied extremely between the different groups of isolates. As the method exhibits a high level of discrimination, it is suitable for characterization of closely related strains in epidemiological studies.

"For export only" medicines come back to Europe: a RP-LC method for the screening of six glucocorticoids in illegal and counterfeit anti-inflammatory and lightening creams.

"For export only" anti-inflammatory and lightening creams are medicinal products sold in African countries for their skin whitening action. In the last years, Rapid Alerts from European Medicinal Regulatory Agencies evidenced the presence of a large number of illegal and counterfeit anti-inflammatory products advertised for their whitening action on black skin in the European market. These drugs, containing glucocorticoids, are illegally sold in Europe in unauthorized ethno-cosmetics-shops and mainly bought by immigrants. This paper reports a new RP-LC method for the rapid simultaneous screening of six different active ingredients in anti-inflammatory and whitening products (creams, ointment and suspension): betamethasone dipropionate, dexamethasone, fluocinonide, fluocinolone acetonide, clobetasol propionate, methyl-prednisolone acetate. The method was developed and validated in view of its possible application in quality control laboratories, mainly those appointed to the control of illegal/counterfeit medicinal products. The associated measurement uncertainty was calculated from validation data. The method was then applied to the analysis of whitening products obtained from the Italian illegal market.

Occurrence of medullary thyroid carcinoma, bronchial carcinoid tumor, and papillary thyroid carcinoma in a family bearing the RET G691S polymorphism.

RET mutations play an important role in the development of human neuroendocrine tumors. The prevalence of the RET polymorphism G691S of exon 11 is higher in patients with medullary thyroid carcinoma (MTC) as compared to the general population. A weak association between RET polymorphisms and sporadic papillary thyroid carcinoma (PTC) has also been described. We hereby describe the association of MTC, bronchial carcinoid tumor, and PTC in a familial setting. A 75-yr-old woman developed MTC 7 yr after successful treatment of a bronchial carcinoid. Serum calcitonin was 12.9 pg/ml with a peak response to pentagastrin (151.0 pg/ml). The patient underwent total thyroidectomy and a genetic mutational analysis of the RET gene. Histological evaluation confirmed MTC with no evidence of lymph nodes involvement. After thyroidectomy serum calcitonin was <2.0 pg/ml. A germline missense mutation at codon 691 in exon 11 of the RET gene was found. The mutational analysis was extended to the patient's offspring, and her daughter was found to bear the G691S polymorphism of RET. Wild type RET gene was found in the son. The daughter, who showed a nodular goiter, autoimmune thyroiditis and normal serum calcitonin, also underwent thyroidectomy. Histologic examination of the thyroid revealed an incidental PTC. This is the first description of a bronchial carcinoid tumor occurring in association with MTC. The occurrence of apparently unrelated NET in the same subject, or within a family, should be regarded as a challenge for deeper investigations into the possible oncogenic role of this genetic alteration.

Nucleoside hydrolase DNA vaccine against canine visceral leishmaniasis.

The Nucleoside Hydrolase (NH36) is the main marker of the FML complex of Leishmania donovani, antigen of the licensed Leishmune® vaccine for prophylaxis of canine visceral leishmaniasis. As a DNA vaccine in mice, it induces a TH1 immune response. We vaccinated mongrel dogs with the VR1012NH36 vaccine for prophylaxis and immunotherapy against a high dose Leishmania chagasi infection (7 x 108 infective amastigotes). The untreated controls developed more symptoms, higher parasite/lymphocyte ratio, smaller DTH reactions, lower proportions of NH36-specific CD4+ cells and sustained NH36-specific CD8+ cell counts than dogs of the prophylaxis group. In the immunotherapy treated group, enlarged DTH reactions, enhanced CD4+ and sustained CD8+ lymphocyte proportions were also detected, however, without reduction of symptoms or parasite/lymphocyte ratio, indicating that the vaccine was sufficiently potent to prevent but not to control the disease. Both treatments determined higher survival rates. Anti-FML antibodies increased in vaccinated and control dogs while anti-NH36 antibodies were only increased in vaccinees (p= 0.000). The parasite load of an untreated survivor control dog (638.05 parasites) felt outside the IC95% of that of vaccinated dogs (32.02, IC95% 9.45-64.59) suggesting that both vaccination treatments succeeded in reducing the Leishmania infective burden. Accordingly, an untreated control dog showed lower levels of IFN γ-ß, IL-2, IL4 but not IL-10 ß actin-relative quantification. We conclude that the VR1012-NH36 vaccine induces strong prophylactic protection and a milder immunotherapeutic effect against a high dose canine experimental infection with Leishmania chagasi.