Continuously fluctuating selection reveals extreme granularity and parallelism of adaptive tracking.

Temporally fluctuating environmental conditions are a ubiquitous feature of natural habitats. Yet, how finely natural populations adaptively track fluctuating selection pressures via shifts in standing genetic variation is unknown. We generated high-frequency, genome-wide allele frequency data from a genetically diverse population of Drosophila melanogaster in extensively replicated field mesocosms from late June to mid-December, a period of ∼12 generations. Adaptation throughout the fundamental ecological phases of population expansion, peak density, and collapse was underpinned by extremely rapid, parallel changes in genomic variation across replicates. Yet, the dominant direction of selection fluctuated repeatedly, even within each of these ecological phases. Comparing patterns of allele frequency change to an independent dataset procured from the same experimental system demonstrated that the targets of selection are predictable across years. In concert, our results reveal fitness-relevance of standing variation that is likely to be masked by inference approaches based on static population sampling, or insufficiently resolved time-series data. We propose such fine-scaled temporally fluctuating selection may be an important force maintaining functional genetic variation in natural populations and an important stochastic force affecting levels of standing genetic variation genome-wide.

Osteoblast role in the pathogenesis of rheumatoid arthritis.

In the pathogenesis of several rheumatic diseases, such as rheumatoid arthritis, spondyloarthritis, osteoarthritis, osteoporosis, alterations in osteoblast growth, differentiation and activity play a role. In particular, in rheumatoid arthritis bone homeostasis is perturbed: in addition to stimulating the pathologic bone resorption process performed by osteoclasts in course of rheumatoid arthritis, proinflammatory cytokines (such as Tumor Necrosis factor-α, Interleukin-1) can also inhibit osteoblast differentiation and function, resulting in net bone loss. Mouse models of rheumatoid arthritis showed that complete resolution of inflammation (with maximal reduction in the expression of pro-inflammatory factors) is crucial for bone healing, performed by osteoblasts activity. In fact, abnormal activity of factors and systems involved in osteoblast function in these patients has been described. A better understanding of the pathogenic mechanisms involved in osteoblast dysregulation could contribute to explain the generalized and focal articular bone loss found in rheumatoid arthritis. Nevertheless, these aspects have not been frequently and directly evaluated in studies. This review article is focused on analysis of the current knowledge about the role of osteoblast dysregulation occurring in rheumatoid arthritis: a better knowledge of these mechanisms could contribute to the realization of new therapeutic strategies.

Effects of a probiotic (SLAB51™) on clinical and histologic variables and microbiota of cats with chronic constipation/megacolon: a pilot study.

Chronic constipation (CC) and idiopathic megacolon (IMC) occur frequently in cats. The aim of the study was to investigate the effects of a multi-strain probiotic (SLAB51™) in constipated cats (n=7) and in patients with megacolon and constipation (n=3). Ten pet cats with a diagnosis of chronic constipation, non-responsive to medical management received orally 2×1011 bacteria daily for 90 days. For microbiota analysis, selected bacterial groups were analysed by qPCR. Histological samples in megacolons were evaluated for interstitial cells of Cajal (ICC), enteric neurons, and neuronal apoptosis. Biopsies were compared at baseline (T0) and after the end of treatment (T1), and with those obtained from healthy control tissues (archived material from five healthy cats). Constipated cats displayed significantly lower ICC, and cats with idiopathic megacolon had significantly more apoptotic enteric neurons than controls. After treatment with SLAB51™, significant decreases were observed for feline chronic enteropathy activity index (FCEAI) (P=0.006), faecal consistency score, and mucosal histology scores (P<0.001). In contrast, a significant increase of ICC was observed after probiotic therapy. Lactobacillus spp. and Bacteroidetes were increased significantly after treatment (comparing constipated cats before and after treatment, and control healthy cats to constipated cats after treatment), but no other differences in microbiota were found between healthy controls and constipated cats. Treatment with SLAB51™ in cats with chronic constipation and idiopathic megacolon showed significant clinical improvement after treatment, and histological parameters suggest a potential anti-inflammatory effect of SLAB51™, associated with a reduction of mucosal infiltration, and restoration of the number of interstitial cells of Cajal.

Long-term outcomes of direct acting antivirals in post-transplant advanced hepatitis C virus recurrence and fibrosing cholestatic hepatitis.

Long-term functional outcomes of sofosbuvir-based antiviral treatment were evaluated in a cohort study involving 16 Italian centres within the international compassionate use programme for post-transplant hepatitis C virus (HCV) recurrence. Seventy-three patients with cirrhosis (n=52) or fibrosing cholestatic hepatitis (FCH, n=21) received 24-week sofosbuvir with ribavirin±pegylated interferon or interferon-free sofosbuvir-based regimen with daclatasvir/simeprevir+ribavirin. The patients were observed for a median time of 103 (82-112) weeks. Twelve of 73 (16.4%) died (10 non-FCH, 2 FCH) and two underwent re-LT. Sustained virological response was achieved in 46 of 66 (69.7%): 31 of 47 (66%) non-FCH and 15 of 19 (79%) FCH patients. All relapsers were successfully retreated. Comparing the data of baseline with last follow-up, MELD and Child-Turcotte-Pugh scores improved both in non-FCH (15.3±6.5 vs 10.5±3.8, P<.0001 and 8.4±2.1 vs 5.7±1.3, P<.0001, respectively) and FCH (17.3±5.9 vs 10.1±2.8, P=.001 and 8.2±1.6 vs 5.5±1, P=.001, respectively). Short-treatment mortality was higher in patients with baseline MELD≥25 than in those with MELD<25 (42.9% vs 4.8%, P=.011). Long-term mortality was 53.3% among patients with baseline MELD≥20 and 7.5% among those with MELD<20 (P<.0001). Among deceased patients 75% were Child-Turcotte-Pugh class C at baseline, while among survivors 83.9% were class A or B (P<.0001). Direct acting antivirals-based treatments for severe post-transplant hepatitis C recurrence, comprising fibrosing cholestatic hepatitis, significantly improve liver function, even without viral clearance and permit an excellent long-term survival. The setting of severe HCV recurrence may require the identification of "too-sick-to-treat patients" to avoid futile treatments.

Phase I trial of the oral smoothened inhibitor sonidegib in combination with paclitaxel in patients with advanced solid tumors.

Purpose To establish a recommended phase II dose (RP2D) for the oral smoothened inhibitor sonidegib in combination with paclitaxel; secondary objectives include evaluation of safety, tolerability, markers of Hedgehog (Hh) signaling and preliminary antitumor activity. Methods Patients with advanced solid tumors were enrolled in cohorts of escalating sonidegib dose levels (400mg, 600mg and 800mg orally, once daily on days 1-28) in combination with paclitaxel 80 mg/m2 on days 1, 8 and 15 in 4-weekly cycles. Dose-limiting toxicities (DLTs) were assessed using CTCAE v4. Once the RP2D was defined, patients with advanced ovarian carcinoma were treated at this dose level in an expansion phase. Biomarkers of Hh signaling were assessed by immunohistochemistry in archival tissue and antitumor activity evaluated using RECIST 1.1. Results 18 patients were treated: 3 at 400 mg, 3 at 600 mg and 12 at 800 mg sonidegib. Only one patient treated at 800 mg presented a DLT (prolonged neutropenia resulting in failure to receive 75% of the planned sonidegib dose). However, 4 of 12 patients treated at 800 mg had their sonidegib dose reduced for toxicity after cycle 1. Hh biomarker (SHH, Patched, SMO and GLI1) staining did not correlate with clinical activity. Best response was partial response in 3 patients (2 ovarian, 1 breast cancer) and stable disease >4 cycles in 3 patients (2 ovarian, 1 anal cancer). Conclusions The combination of sonidegib and paclitaxel is tolerable and evidence of antitumor activity was identified. The RP2D of sonidegib was 800 mg in combination with paclitaxel 80mg/m2.

Bone marrow fibroblasts parallel multiple myeloma progression in patients and mice: in vitro and in vivo studies.

The role of cancer-associated fibroblasts (CAFs) has not been previously studied in multiple myeloma (MM). Here, cytofluorimetric analysis revealed higher proportions of bone marrow (BM) CAFs in patients with active MM (both at diagnosis and relapse) compared with patients in remission or those with monoclonal gammopathy of undetermined significance or deficiency anemia (controls). CAFs from MM patients produced increased levels of transforming growth factor-ß, interleukin-6, stromal cell-derived factor-1α, insulin-like growth factor-1, vascular endothelial growth factor and fibroblast growth factor-2 and displayed an activated and heterogeneous phenotype, which supported their origin from resident fibroblasts, endothelial cells and hematopoietic stem and progenitor cells via the endothelial-mesenchymal transition as well as mesenchymal stem cells via the mesenchymal transition, as both of these processes are induced by MM cells and CAFs. Active MM CAFs fostered chemotaxis, adhesion, proliferation and apoptosis resistance in MM cells through cytokine signals and cell-to-cell contact, which were inhibited by blocking CXCR4, several integrins and fibronectin. MM cells also induced the CAFs proliferation. In syngeneic 5T33MM and xenograft mouse models, MM cells induced the expansion of CAFs, which, in turn, promoted MM initiation and progression as well as angiogenesis. In BM biopsies from patients and mice, nests of CAFs were found in close contact with MM cells, suggesting a supportive niche. Therefore, the targeting of CAFs in MM patients may be envisaged as a novel therapeutic strategy.

Four proteins governing overangiogenic endothelial cell phenotype in patients with multiple myeloma are plausible therapeutic targets.

Bone marrow (BM) angiogenesis has an important role in the initiation and progression of multiple myeloma (MM). We looked at novel mechanisms of vessel formation in patients with MM through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with active MM (MMECs) and ECs of patients with monoclonal gammopathy of undetermined significance (MGECs) and of subjects with benign anemia (normal ECs). Four proteins were found overexpressed in MMECs: filamin A, vimentin, α-crystallin B and 14-3-3ζ/δ protein, not yet linked to overangiogenic phenotype. These proteins gave a typical distribution in the BM of MM patients and in MMECs versus MGECs, plausibly according to a different functional state. Their expression was enhanced by vascular endothelial growth factor, fibroblast growth factor 2, hepatocyte growth factor and MM plasma cell conditioned medium in step with enhancement of MMEC angiogenesis. Their silencing RNA knockdown affected critical MMEC angiogenesis-related functions, such as spreading, migration and tubular morphogenesis. A gradual stabilization of 14-3-3ζ/δ protein was observed, with transition from normal ECs to MGECs and MMECs that may be a critical step for the angiogenic switch in MMECs and maintenance of the cell overangiogenic phenotype. These proteins were substantially impacted by anti-MM drugs, such as bortezomib, lenalidomide and panobinostat. Results suggest that these four proteins could be new targets for the antiangiogenic management of MM patients.

Recurrence of pleomorphic adenoma of the palate after sixteen years: case report and an analysis of the literature.

Pleomorphic adenoma or mixed tumour (MT) is a benign neoplasia of slow growth and epithelial histogenesis. We report a particular case of recurring MT of the hard palate. A 39-year-old man came to us with a swelling of the hard half-palate. The patient, 19 years earlier, had had a small formation in the same place that, over a period of three years had slowly grown. Histology showed that it was an MT and it was promptly removed. Sixteen years after the operation, a small recurrence reappeared, reaching a diameter of 12 mm. The patient underwent a new excision. The case reported is of particular interest due to many aspects: the outbreak from the minor salivary glands; the male sex; the young age of the patient at the first sign of the tumour; the appearance of a recurrence after 16 years, not contemplated in literature; and finally, the rapid growth of the second appearance.

Clinical trial: peg-interferon alfa-2b and ribavirin for the treatment of genotype-1 hepatitis C recurrence after liver transplantation.

BACKGROUND: Treatment of hepatitis C virus (HCV) recurrence after liver transplantation (LT) is difficult with low response rates. AIM: To assess the safety and efficacy of pegylated-interferon (PEG-IFN) alfa-2b + ribavirin (RBV) in patients with post-LT recurrent genotype-1 HCV and to establish stopping rules according to response. METHODS: Fifty-three patients with post-LT HCV recurrence were enrolled. Patients received PEG-IFN alfa-2b 1.0 micro/kg/week plus RBV 8-10 mg/kg/day for 24 weeks. Those with 'early virological response at week 24' (EVR24) continued treatment for 24 weeks (group A). Patients without EVR24 were randomized to continue (group B) or to discontinue (group C). RESULTS: Overall sustained virological response (SVR) was 26% (14/53). Alanine aminotransferase, rapid virological response, EVR12, EVR24, undetectable serum HCV-RNA at weeks 12 (cEVR12) and 24 (cEVR24) were related to SVR. cEVR12 and cEVR24 (OR: 14.7; 95% CI: 2.02-106.4) were independent predictors of SVR. All patients with SVR, had cEVR12. No patient in groups B and C achieved end-of-treatment response. One patient in group B had SVR. CONCLUSIONS: Pegylated-interferon alfa-2b was effective in one of four of patients with HCV genotype 1 after LT. Treatment should be discontinued in patients with no virological response at week 12. Further studies are needed to evaluate whether a longer treatment period may be beneficial in patients with > or =2 log10 drop in HCV-RNA at week 24.

High incidence of allograft dysfunction in liver transplanted patients treated with pegylated-interferon alpha-2b and ribavirin for hepatitis C recurrence: possible de novo autoimmune hepatitis?

BACKGROUND: Interferon may trigger autoimmune disorders, including autoimmune hepatitis, in immunocompetent patients. To date, no such disorders have been described in liver transplanted patients. METHODS: 9 of 44 liver transplanted patients who had been receiving pegylated-interferon alpha-2b and ribavirin for at least 6 months for hepatitis C virus (HCV) recurrence, developed graft dysfunction despite on-treatment HCV-RNA clearance in all but one case. Laboratory, microbiological, imaging and histological evaluations were performed to identify the origin of graft dysfunction. The International Autoimmune Hepatitis scoring system was also applied. RESULTS: In all cases infections, anastomoses complications and rejection were excluded, whereas the autoimmune hepatitis score suggested a "probable autoimmune hepatitis" (score from 10 to 14). Three patients developed other definite autoimmune disorders (overlap anti-mitochondrial antibodies (AMA)-positive cholangitis, autoimmune thyroiditis and systemic lupus erythematosus, respectively). In all cases, pre-existing autoimmune hepatitis was excluded. Anti-lymphocyte antibodies in immunosuppressive induction treatment correlated with the development of the disorder, whereas the use of granulocyte colony-stimulating factor to treat interferon-induced neutropenia showed a protective role. Withdrawal of antiviral treatment and treatment with prednisone resulted in different outcomes (five remissions and four graft failures with two deaths). CONCLUSIONS: De novo autoimmune hepatitis should be considered in differential diagnosis along with rejection in liver transplanted patients developing graft dysfunction while on treatment with interferon.

[Papillary carcinoma of the thyroid: our experience]. / Il carcinoma papillifero della tiroide: la nostra esperienza.

Papillary carcinoma (PTC) is the most frequently occurring thyroid carcinoma. The aim of the present work is to perform a retrospective study to assess 172 patients with PTC who have been treated surgically, with radium-iodine (except microca) and LT4 from 1989 to 1999. The incidence of local, regional and systemic recurrence of the disease was verified together with the results at distance. Great importance on survival have preoperative cytological diagnosis, radical surgery and a modulated radio-iodine therapy. Age and tumor stage have been found as major prognostic factors, while no significant difference according to sex has been documented.

[Parotid tumors: a diagnostic and therapeutic protocol in 81 surgical cases]. / I tumori della parotide un protocollo diagnostico e terapeutico dopo 81 casi operati.

AIM: In the last five years we cured 81 patients divided in two groups: the first was managed inordinately, the second according to a diagnostic and therapeutic protocol. METHODS: The first 23 patients were differently evaluated, before surgery, by means of traditional sialography, US, CT, CT sialography, MR, larger bore needles and FNAB. Neoplasm enucleations, atipic resections, superficial and total conservative parotidectomies were performed. The following 58 patients were evaluated and cured according to a diagnostic and therapeutic protocol. RESULTS: Diagnostic and therapeutic time were shortened, patients endurance improved and cost lowered. DISCUSSION: We think that many diagnostic procedures are useless and delay operation that, if possible, must be performed according to principles of radical extirpation.

A static, closed and scaffold-free bioreactor system that permits chondrogenesis in vitro.

OBJECTIVE: To characterise in vitro engineered cartilaginous constructs made employing a novel static, scaffold-free and closed chamber system. DESIGN: Chondrocytes derived from slaughter age pigs (3-6 months) were seeded at high density (20 x 10(6)) into cylindrical chambers (1.0 x 0.5cm) and were maintained between an upper and a lower membrane (100 kDa) for 21 days and subsequently cultured in open culture for 7 additional days. RESULTS: Viable constructs produced were approximately 10 mmx2mm in size and were stable enough to be handled by surgical pincers. Histology and electron microscopy evaluations revealed a neo-cartilage structure of high cell density with a comprehensive extracellular matrix. Predominately collagen type II and negligible amounts of collagen types I and X were detected using RT-PCR and SDS-PAGE analyses. CONCLUSIONS: In this study, we provide evidence of a scaffold-free system that can produce immature hyaline-like cartilaginous constructs suitable for in vivo implantation, or that may be useful for in vitro studies of events related to the process of chondrogenesis.

Alternative messenger RNA splicing and enzyme forms of cathepsin B in human osteoarthritic cartilage and cultured chondrocytes.

OBJECTIVE: In previous studies, we suggested that cathepsin B, which is present at sites of cartilage remodeling in osteoarthritis (OA), may act as an antagonist of cartilage repair, an enhancer of the action of metalloproteinases, and a mediator of cartilage neovascularization and mineralization. Alternative splicing of cathepsin B pre-messenger RNA (pre-mRNA) and/or mRNA overexpression is a plausible regulatory mechanism. In the present study, we investigated the abundance of cathepsin B transcripts and the properties of cathepsin B protein in normal and OA cartilage, osteophytes, and cultured chondrocytes. METHODS: Cathepsin B mRNA splice variants containing the full-length transcript (CB) and the variants lacking either exon 2 (CB[-2]) or lacking exons 2 and 3 (CB[-2,3]) were measured by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot assays and were localized by in situ RT-PCR. Cathepsin B protein was analyzed by electrophoretic, Western blot, and chromatographic methods. RESULTS: The relative content of CB, CB(-2), and CB(-2,3) varied considerably in OA cartilage and osteophytes, with less variation in normal cartilage. The mean cathepsin B mRNA level was significantly higher in OA cartilage and osteophytes than in normal cartilage. Normal cultured chondrocytes attained cathepsin B mRNA levels similar to those in OA cartilage. Enzyme overexpression resulted in the secretion of procathepsin B, followed by activation to the proteolytically active form. CONCLUSION: The high levels of CB and CB(-2) are consistent with an overproduction of secreted procathepsin B in OA. Up-regulation of chondrocyte cathepsin B, which takes place at both the transcriptional and the translational level, suggests a leading role of the enzyme in the progression of OA.

Environmental assessment of a site contaminated by organic compounds.

This paper presents a study on environmental assessment of an abandoned industrial area located in central Italy. Main production was refractory materials and compounds for treatment of industrial wastewater. The present work deals with a methodology for development of a sound sampling design, chemical characterization of soil samples, definition of the degree of site contamination according to law limits and evaluation of the fate and transport of contaminants by EPA simulation model (VLEACH 2.2a). Results indicate that toxic compounds (polycyclic aromatic hydrocarbons and plasticizers) are uniformly distributed in the contaminated site and only in one sampling point their concentrations exceed law limits. Modeling results confirm that contaminants migration to groundwater can be excluded, addressing for a site remediation limited to the surface layer.

Review article: the transmission of Helicobacter pylori from stomach to stomach.

The mode of transmission of Helicobacter pylori is largely unknown and is a matter of circumstantial evidence and speculation rather than fact. However, the principle evidence is of two sorts: the epidemiological data, providing evidence of possible risk factors associated with transmission, and the identification of potential sources from which H. pylori could be acquired. Evidence exists for several potential sources of infection and several possible modes of transmission, and it is feasible that the transmission of H. pylori varies according to the cultural and demographic circumstances. However, the most likely recognized source for H. pylori is the human stomach, although it is not known by what route the organism is transmitted to the stomach. Evidence suggests close personal contact is important and that acquisition occurs mainly in childhood. This article reviews the evidence for the source of infection and the route of transmission of H. pylori.

Characterization of L-carnitine transport into rat skeletal muscle plasma membrane vesicles.

Transport of L-carnitine into skeletal muscle was investigated using rat sarcolemmal membrane vesicles. In the presence of an inwardly directed sodium chloride gradient, L-carnitine transport showed a clear overshoot. The uptake of L-carnitine was increased, when vesicles were preloaded with potassium. When sodium was replaced by lithium or cesium, and chloride by nitrate or thiocyanate, transport activities were not different from in the presence of sodium chloride. However, L-carnitine transport was clearly lower in the presence of sulfate or gluconate, suggesting potential-dependent transport. An osmolarity plot revealed a positive slope and a significant intercept, indicating transport of L-carnitine into the vesicle lumen and binding to the vesicle membrane. Displacement experiments revealed that approximately 30% of the L-carnitine associated with the vesicles was bound to the outer and 30% to the inner surface of the vesicle membrane, whereas 40% was unbound inside the vesicle. Saturable transport could be described by Michaelis-Menten kinetics with an apparent Km of 13.1 microM and a Vmax of 2.1 pmol.(mg protein-1).s-1. L-Carnitine transport could be trans-stimulated by preloading the vesicles with L-carnitine but not with the carnitine precursor butyrobetaine, and was cis-inhibited by L-palmitoylcarnitine, L-isovalerylcarnitine, and glycinebetaine. On comparing carnitine transport into rat kidney brush-border membrane vesicles and OCTN2, a sodium-dependent high-affinity human carnitine transporter, cloned recently from human kidney also expressed in muscle, the Km values are similar but driving forces, pattern of inhibition and stereospecificity are different. This suggests the existence of more than one carnitine carrier in skeletal muscle.

Accurate diagnosis of Helicobacter pylori. Stool tests.

From the limited data available, it seems that the H. pylori stool assay represents a highly accurate diagnostic tool to detect H. pylori infection before and shortly after therapy. As a test that is noninvasive, accurate, simple, and cost-effective, the H. pylori stool assay has the potential to become the preferred diagnostic tool in many different clinical settings from epidemiologic studies to pediatric investigations, from pre-endoscopic screening policies to posttherapy monitoring.