Analisi di espressione di microRNA in Tumore a Cellule Giganti: identificazione dei target con ruolo di potenziali biomarcatori

Il Tumore a Cellule Giganti dell’osso (TCG) è una rara neoplasia che rappresenta il 5% dei tumori di natura ossea; sebbene venga considerato un tumore a decorso benigno può manifestare caratteri di aggressività locale dando origine a recidive locali nel 10-25% dei casi, e nel 2-4% dei casi metastatizza a livello polmonare. In questo studio è stata valutata l’espressione dei miRNA mediante miRNA microarray in 10 pazienti affetti da TCG, 5 con metastasi e 5 liberi da malattia; sono stati riscontrati miRNA differenzialmente espressi tra i 2 gruppi di pazienti e la successiva validazione mediante Real Time PCR ha confermato una differenza significativa per il miR-136 (p=0.04). Mediante analisi bioinformatica con il software TargetScan abbiamo identificato RANK e NF1B come target del miR-136 e ne abbiamo studiato l’espressione mediante Real Time PCR su una più ampia casistica di pazienti affetti da TCG, metastatico e non, evidenziando una maggior espressione di NF1B nel gruppo di pazienti metastatici, mentre RANK non ha dimostrato una differenza significativa. L’analisi di Western Blot ha rilevato una maggiore espressione di entrambe le proteine nei pazienti metastatici rispetto ai non metastatici. Successivamente è stato condotto uno studio di immunoistochimica su TMA di 163 campioni di pazienti affetti da TCG a diverso decorso clinico che ha dimostrato una maggiore e significativa espressione di entrambe i target nei pazienti con metastasi rispetto ai non metastatici; le analisi di popolazione mediante Kaplan-Meier hanno confermato la correlazione tra over-espressione di RANK, NF1B e ricaduta con metastasi (p=0.001 e p<0.0005 rispettivamente). Lo studio di immunoistochimica è stato ampliato alle proteine maggiormente coinvolte nell’osteolisi che risultano avere un significato prognostico; tuttavia mediante analisi di ROC, la co-over-espressione di RANK, RANKL e NF1B rappresenta il migliore modello per predire la comparsa di metastasi (AUC=0.782, p<0.0005).

Evolutionary genetics of lactase persistence in Eurasian human populations

Although ability to digest lactose generally declines after weaning in all mammals, in some human populations it persists also in adult individuals, a condition named lactase persistence (LP). Studies on the prevalence of the LP phenotype in worldwide human populations have shown that the frequency of this trait is highly variable in different ethnic groups, appearing to be positively correlated with the importance of milk in the diet. In particular, several single-nucleotide polymorphisms (SNPs) in the proximity of the LCT gene have been proved to be associated with LP. Nevertheless, few studies have till now analyzed genetic variation underlying LP in a wide set of Eurasian populations and, especially, in the Italian one. In the present study, we thus typed 40 SNPs surrounding the LCT gene in more than 1,000 samples from Italian and Arabic peninsulas to investigate patterns of LP-related genetic diversity in two regions which have played a pivotal role in the recent human evolutionary history according to their geographical position and historical/archaeological records. Our results underline a high and complex variability of the explored genomic region in both studied populations. In particular, a clear diversification of Northern Italian groups from the rest of the peninsula, was observed, with the formers being genetically more similar to Northern European populations than to Southern Italians. These observation are consistent with known decreasing pattern of LP from Northern to Southern Italy and suggest the possibility of an independent evolution of LP-associated genotypes in Northern Italy. A similar scenario was observed in the Arabian peninsula, with Dhofari Arabs from Southern Oman and Yemeni clustering together with respect to Arabs from Northern Oman and the subgroup of Omanis of Asian origin which appeared instead to be genetically closer to Europeans than to the rest of Arabic groups.

High numbers of DC-SIGN+ dendritic cells in lesional skin of cutaneous T-cell lymphoma

The role of dendritic cells (DCs) in disease progression of primary cutaneous T-cell lymphoma (CTCL) is not well understood. With their unique ability to induce primary immune responses as well as immunotolerance, DCs play a critical role in mediation of anti-tumor immune responses. Tumor-infiltrating DCs have been determined to represent important prognostic factors in a variety of human tumors.

Threshold-dependent effects on peripheral nerve in vivo excitability properties in the rat

Various factors, including maturity, have been shown to influence peripheral nerve excitability measures, but little is known about differences in these properties between axons with different stimulation thresholds. Multiple nerve excitability tests were performed on the caudal motor axons of immature and mature female rats, recording from tail muscles at three target compound muscle action potential (CMAP) levels: 10%, 40% ("standard" level), and 60% of the maximum CMAP amplitude. Compared to lower target levels, axons at high target levels have the following characteristics: lower strength-duration time constant, less threshold reduction during depolarizing currents and greater threshold increase to hyperpolarizing currents, most notably to long hyperpolarizing currents in mature rats. Threshold-dependent effects on peripheral nerve excitability properties depend on the maturation stage, especially inward rectification (Ih), which becomes inversely related to threshold level. Performing nerve excitability tests at different target levels is useful in understanding the variation in membrane properties between different axons within a nerve. Because of the threshold effects on nerve excitability and the possibility of increased variability between axons and altered electric recruitment order in disease conditions, excitability parameters measured only at the "standard" target level should be interpreted with caution, especially the responses to hyperpolarizing currents.

Demonstration of epitope-spreading phenomena in bullous pemphigoid: results of a prospective multicenter study

Bullous pemphigoid (BP), the most common autoimmune subepidermal bullous disease, is associated with an autoantibody response to BP180 and BP230, two components of junctional adhesion complexes in human skin promoting dermo-epidermal cohesion. Retrospective analyses demonstrated that these autoantigens harbor several epitopes targeted by autoaggressive B and T cells. The aim of this prospective multicenter study was to assess the evolution of IgG autoantibodies in 35 BP patients over a 12-month observation period. Epitope-spreading (ES) events were detected in 17 of 35 BP patients (49%). They preferentially occurred in an early stage of the disease and were significantly related to disease severity at diagnosis. Moreover, in three patients, spreading of IgG reactivity to intracellular epitopes of BP180 and BP230 was preceded by recognition of the BP180 ectodomain. Finally, IgG reactivity with extracellular epitopes of BP180 and intracellular epitopes of BP230 correlated with the severity of BP in disease course. These findings support the idea that IgG recognition of the BP180 ectodomain is an early and crucial event in BP disease, followed by variable intra- and intermolecular ES events, which likely shape the individual course of BP.

Interaction of pregnancy and autoimmune rheumatic disease

During pregnancy, the fetus represents a natural allograft that is not normally rejected. While the maternal immune system retains the ability to respond to foreign antigens, tolerance mechanisms are up-regulated to protect the fetus from immunologic attacks by the mother. The profound immunologic adaptations during and after pregnancy do influence maternal autoimmune rheumatic diseases in several ways. One is triggering the onset of a rheumatic disease in the post partum period, the other influencing disease activity of established rheumatic disease. The review will discuss the mechanisms of increased susceptibility of rheumatoid arthritis (RA) in the first year post partum with a specific emphasis on the role of fetal cells or antigens persisting in the maternal circulation (so called microchimerism). Furthermore, the different influences of pregnancy on established rheumatic diseases will be highlighted. A marked beneficial effect of pregnancy is observed on RA whereas several other rheumatic diseases as ankylosing spondylitis (AS) and systemic lupus erythematosus (SLE) show either no particular effect or an aggravation of symptoms during pregnancy. Differences emerging in regard to modulation of disease symptoms during pregnancy seem related to response to hormones, the type of cytokine profile and immune response prevailing as well as further downstream interactions of molecular pathways that are important in disease pathogenesis.

Effects of dietary supplementation with synthetic vitamin D-3 and 25-hydroxycholecalciferol on blood calcium and phosphate levels and performance in laying hens

We investigated the effects of different dietary vitamin D regimen on selected blood parameters in laying hens. Supplementation with vitamin D-3 only was compared with a combination of vitamin D-3 and its metabolite 25-hydroxy-cholecalciferol (25(OH)D-3). Blood concentrations of total calcium, phosphate and 25 (OH)D-3 were determined. Four thousand one-day-old LSL chicks were split in two treatment groups and distributed to eight pens. The control group was given a commercial animal diet containing 2800 IU synthetic vitamin D-3 in the starter feed and 2000 IU synthetic vitamin D-3 in the pullet feed. The experimental group was fed the same commercial diet in which half the synthetic vitamin D-3 content had been substituted with 25(OH)D-3 (Hy center dot D (R)). At 18 weeks of age, pullets were transferred to the layer house. At the ages of 11, 18 and 34 weeks, between 120 and 160 blood samples were collected from both the control and the experimental groups, respectively. The experimental group had higher levels of 25 (OH)D-3 than the control group at all three ages. Serum calcium levels did not differ between the treatment groups at any age. With the onset of laying, calcium levels rose significantly. Whereas blood serum concentration at 18 weeks was 3 mmol/L in both treatment groups, it increased to 8.32 mmol/L in the control group and to 8.66 mmol/L in the experimental group at week 34. At weeks 11 and 34, phosphate was significantly lower in the experimental group. In conclusion, HyD (R) significantly affected serum phosphate and 25(OH)D-3 levels. No effects of (25(OH)D-3 supplementation on performance, shell quality and fractures of keelbones were found.

Genome-wide prediction of childhood asthma and related phenotypes in a longitudinal birth cohort

Childhood wheezing and asthma vary greatly in clinical presentation and time course. The extent to which phenotypic variation reflects heterogeneity in disease pathways is unclear.

Inflammasome-dependent IFN-γ drives pathogenesis in Streptococcus pneumoniae meningitis

The pathology associated with Streptococcus pneumoniae meningitis results largely from activation of immune-associated pathways. We systematically investigated the production of IFN subtypes, as well as their influence on pathology, in a mouse model of S. pneumoniae meningitis. Despite the occurrence of a mixed IFN type I/II gene signature, no evidence for production or involvement of type I IFNs in disease progression was found. In contrast, type II IFN (IFN-γ) was strongly induced, and IFN-γ(-/-) mice were significantly protected from severe disease. Using intracellular cytokine staining and targeted cell-depletion approaches, NK cells were found to be the dominant source of IFN-γ. Furthermore, production of IFN-γ was found to be dependent upon ASC and IL-18, indicating that an ASC-dependent inflammasome pathway was responsible for mediating IFN-γ induction. The influence of IFN-γ gene deletion on a range of processes known to be involved in bacterial meningitis pathogenesis was examined. Although neutrophil numbers in the brain were similar in infected wild-type and IFN-γ(-/-) mice, both monocyte recruitment and CCL2 production were less in infected IFN-γ(-/-) mice compared with infected wild-type controls. Additionally, gene expression of NO synthase was strongly diminished in infected IFN-γ(-/-) mice compared with infected controls. Finally, bacterial clearance was enhanced in IFN-γ(-/-) mice, although the underlying mechanism remains unclear. Together, these data suggest that inflammasome-dependent IFN-γ contributes via multiple pathways to pathology during S. pneumoniae meningitis.

Hibernacula Microclimate and White-nose Syndrome Susceptibility in the Little Brown Myotis (Myotis lucifugus)

The objective of this project was to determine the relationship between hibernacula microclimate and White-nose Syndrome (WNS), an emerging infectious disease in bats. Microclimate was examined on a species scale and at the level of the individual bat to determine if there was a difference in microclimate preference between healthy and WNS-affected little brown myotis (Myotis lucifugus) and to determine the role of microclimate in disease progression. There is anecdotal evidence that colder, drier hibernacula are less affected by WNS. This was tested by placing rugged temperature and humidity dataloggers in field sites throughout the eastern USA, experimentally determining the response to microclimate differences in captive bats, and testing microclimate roosting preference. This study found that microclimate significantly differed from the entrance of a hibernaculum versus where bats traditionally roost. It also found hibernaculum temperature and sex had significant impacts on survival in WNS-affected bats. Male bats with WNS had increased survivability over WNS-affected female bats and WNS bats housed below the ideal growth range of the fungus that causes WNS, Geomyces destructans, had increased survival over those housed at warmer temperatures. The results from this study are immediately applicable to (1) predict which hibernacula are more likely to be infected next winter, (2) further our understanding of WNS, and (3) determine if direct mitigation strategies, such as altering the microclimate of mines, will be effective ways to combat the spread of the fungus.

Canine microglial cells: stereotypy in immunophenotype and specificity in function?

Microglial cells represent the endogenous immune system of the central nervous system (CNS). Upon pathological insults they reveal their immunological potential aimed at regaining homeostasis. These reactions have long been believed to follow a uniform and unspecific pattern which is irrespective to the underlying disease entity. Evidence is growing that this view seriously underrates microglial competence as the defenders of the CNS. In the present study, microglial cells of 47 dogs were examined ex vivo by means of flow cytometry. Ex vivo examination included immunophenotypic characterization using eight different surface markers and functional studies such as phagocytosis assay and the reactive oxygen species (ROS) generation test. The dogs were classified according to their histopathological diagnoses in disease categories (controls, canine distemper virus (CDV) induced demyelination, other diseases of the CNS) and results of microglial reaction profiles were compared. Immunophenotypic characterization generally revealed relative high conformity in the microglial disease response among the different groups, however the functional response was shown to be more specific. Dogs with intracranial inflammation and dogs with demyelination showed an enhanced phagocytosis, whereas a significant up-regulation of ROS generation was found in dogs with demyelination due to CDV infection. This strongly suggests a specific response of microglia to infection with CDV in the settings of our study and underlines the pivotal role of microglial ROS generation in the pathogenesis of demyelinating diseases, such as canine distemper.

[Pregnancy in patients with rheumatoid arthritis and inflammatory spondylarthropathies]

The activity of a rheumatic disease can be influenced by pregnancy and puerperium. Prospective studies have shown an improvement in joint involvement in rheumatoid arthritis in two thirds to three quarters of pregnancies. After birth, an exacerbation is common. In spondylarthropathies there is no relevant change in disease activity. The fetal outcome is not impaired in patients with rheumatoid arthritis and inflammatory spondylarthropathies. Every pregnancy in women with a rheumatic disease should be considered as high-risk, and such pregnancies require close collaboration between rheumatologists and obstetricians.

Gauss-Seidel Estimation of Generalized Linear Mixed Models with Application to Poisson Modeling of Spatially Varying Disease Rates

Generalized linear mixed models (GLMMs) provide an elegant framework for the analysis of correlated data. Due to the non-closed form of the likelihood, GLMMs are often fit by computational procedures like penalized quasi-likelihood (PQL). Special cases of these models are generalized linear models (GLMs), which are often fit using algorithms like iterative weighted least squares (IWLS). High computational costs and memory space constraints often make it difficult to apply these iterative procedures to data sets with very large number of cases. This paper proposes a computationally efficient strategy based on the Gauss-Seidel algorithm that iteratively fits sub-models of the GLMM to subsetted versions of the data. Additional gains in efficiency are achieved for Poisson models, commonly used in disease mapping problems, because of their special collapsibility property which allows data reduction through summaries. Convergence of the proposed iterative procedure is guaranteed for canonical link functions. The strategy is applied to investigate the relationship between ischemic heart disease, socioeconomic status and age/gender category in New South Wales, Australia, based on outcome data consisting of approximately 33 million records. A simulation study demonstrates the algorithm's reliability in analyzing a data set with 12 million records for a (non-collapsible) logistic regression model.

Marked disturbance of calcium homeostasis in mice with targeted disruption of the Trpv6 calcium channel gene

We report the phenotype of mice with targeted disruption of the Trpv6 (Trpv6 KO) epithelial calcium channel. The mice exhibit disordered Ca(2+) homeostasis, including defective intestinal Ca(2+) absorption, increased urinary Ca(2+) excretion, decreased BMD, deficient weight gain, and reduced fertility. Although our Trpv6 KO affects the closely adjacent EphB6 gene, the phenotype reported here is not related to EphB6 dysfunction. INTRODUCTIOn: The mechanisms underlying intestinal Ca(2+) absorption are crucial for overall Ca(2+) homeostasis, because diet is the only source of all new Ca(2+) in the body. Trpv6 encodes a Ca(2+)-permeable cation channel responsible for vitamin D-dependent intestinal Ca(2+) absorption. Trpv6 is expressed in the intestine and also in the skin, placenta, kidney, and exocrine organs. MATERIALS AND METHODS: To determine the in vivo function of TRPV6, we generated mice with targeted disruption of the Trpv6 (Trpv6 KO) gene. RESULTS: Trpv6 KO mice are viable but exhibit disordered Ca(2+) homeostasis, including a 60% decrease in intestinal Ca(2+) absorption, deficient weight gain, decreased BMD, and reduced fertility. When kept on a regular (1% Ca(2+)) diet, Trpv6 KO mice have deficient intestinal Ca(2+) absorption, despite elevated levels of serum PTH (3.8-fold) and 1,25-dihydroxyvitamin D (2.4-fold). They also have decreased urinary osmolality and increased Ca(2+) excretion. Their serum Ca(2+) is normal, but when challenged with a low (0.25%) Ca(2+) diet, Trpv6 KO mice fail to further increase serum PTH and vitamin D, ultimately developing hypocalcemia. Trpv6 KO mice have normal urinary deoxypyridinoline excretion, although exhibiting a 9.3% reduction in femoral mineral density at 2 months of age, which is not restored by treatment for 1 month with a high (2%) Ca(2+) "rescue" diet. In addition to their deranged Ca(2+) homeostasis, the skin of Trpv6 KO mice has fewer and thinner layers of stratum corneum, decreased total Ca(2+) content, and loss of the normal Ca(2+) gradient. Twenty percent of all Trpv6 KO animals develop alopecia and dermatitis. CONCLUSIONS: Trpv6 KO mice exhibit an array of abnormalities in multiple tissues/organs. At least some of these are caused by tissue-specific mechanisms. In addition, the kidneys and bones of Trpv6 KO mice do not respond to their elevated levels of PTH and 1,25-dihydroxyvitamin D. These data indicate that the TRPV6 channel plays an important role in Ca(2+) homeostasis and in other tissues not directly involved in this process.