Use of transcranial low-intensity focused ultrasound for targeted delivery of stem cell-derived exosomes to the brain.

The blood-brain barrier (BBB) presents a significant challenge for targeted drug delivery. A proposed method to improve drug delivery across the BBB is focused ultrasound (fUS), which delivers ultrasound waves to a targeted location in the brain and is hypothesized to open the BBB. Furthermore, stem cell-derived exosomes have been suggested as a possible anti-inflammatory molecule that may have neural benefits, if able to pass the BBB. In the present study, transcranial low-intensity focused ultrasound (LIFU), without the use of intravenous microbubbles, was assessed for both (1) its ability to influence the BBB, as well as (2) its ability to increase the localization of intravenously administered small molecules to a specific region in the brain. In vivo rat studies were conducted with a rodent-customized 2 MHz LIFU probe (peak pressure = 1.5 MPa), and injection of labeled stem cell-derived exosomes. The results suggested that LIFU (without microbubbles) did not appear to open the BBB after exposure times of 20, 40, or 60 min; instead, there appeared to be an increase in transcytosis of the dextran tracer. Furthermore, the imaging results of the exosome study showed an increase in exosome localization in the right hippocampus following 60 min of targeted LIFU.

Pulmonary rehabilitation in lung cancer.

Non-small-cell lung cancer (NSCLC) represents a very severe disease, being its incidence increasingly reported and, nowadays, successfully treatable only when surgery is deemed to be feasible. Furthermore, the disease and the clinical effects related to the complementary therapies (radio and/or chemotherapy) may strongly affect, frequently with dramatic clinical side effects, the patient's ability to endure physical exercise. In such context, the PR(PR), which has already been proved to be useful and effective in other diseases such as COPD, could play a pivotal role. The aim of this review article is, therefore, to analyze the pertinent data recently reported in English literature in order to highlight the role of rehabilitation as complementary therapy in the management of patients with NSCLC. The evidence currently available suggests that, when surgery is indicated, PR is a safe and feasible option, both during pre-operative and post-operative timing.The safety and feasibility of rehabilitation are proven even in inoperable patients, although to date, little evidence has been reported on its role in the overall management of such complex diseases.

RHAMM mRNA expression in proliferating and migrating cells of the developing central nervous system.

Extracellular matrix components can influence cell behaviour by modulating a wide variety of events. In particular, the glycosaminoglycan hyaluronan is involved in many processes of the normal and pathological adult cells and it is essential for embryonic development. Two main HA receptors have been characterized in vertebrate developing embryos: CD44 and RHAMM. These receptors display completely different characteristics apart from their ability to bind hyaluronan. RHAMM is still the most mysterious hyaluronan receptor as it can act as cell surface receptor but it can also be localized in the cytoplasm or in the cell nucleus, displaying both hyaluronan dependent and independent functions. In particular, the role of RHAMM during embryogenesis is still largely unclear. We reported a detailed gene expression analysis of RHAMM during Xenopus laevis development comparing its mRNA distribution with that of the hyaluronan synthases and CD44 genes, in order to provide a first insight into the possible role of RHAMM during vertebrate embryogenesis. Our findings point out that RHAMM mRNA displays a specific distribution in proliferating regions of the developing neural tube and retina where synthesis of hyaluronan is not detected. On the contrary, RHAMM expression correlates with the expression of hyaluronan synthase-1 and hyaluronan-receptor CD44 gene expression in migrating cranial neural crest. These results suggest that during the central nervous system development RHAMM could be involved in cell proliferation and migration processes both in a hyaluronan independent and dependent manner.

Infections after simultaneous pancreas and kidney transplantation: a single-center experience.

Simultaneous pancreas-kidney transplantation (SPKT) is now an accepted therapy for patients with insulin-dependent diabetes mellitus. However, SPKT has an high rate of morbidity and mortality, mainly for infection. From October 1986 to June 2008, in our center 54 patients (18 female; 36 male) affected by diabetes and end-stage renal disease underwent SPKT. The mean duration of diabetes mellitus was 25 +/- 4 years. Only 4 patients had not been treated by dialysis before SPKT. Three operative techniques were used: duct injection (n = 5), bladder diversion (n = 14), and enteric diversion (n = 39). The kidneys were always placed into the left retroperitoneal space. The pancreas was placed extraperitoneally in 5 patients. Thirty-four recipients are alive, including 30 with function of both grafts. Six patients died during the first year after transplantation. Infectious complications were the main cause of death in 3 subjects whereas 98 infections were diagnosed in 51 patients. All patients were treated with immunosuppressive agents: steroids associated with calcineurin inhibitors and mycophenolic acid, or azathioprine. Antibody induction was used in 41 patients with anti-interleukin-2 monoclonal antibody or antithymocyte globulin. We detected 41 episodes of cytomegalovirus infection: systemic (n = 38), bladder (n = 2), and duodenal (n = 1). The 51 bacterial infections were systemic: (n = 10); urinary tract: (n = 22); pulmonary (n = 11); wound (n = 5); intestinal (n = 3). The 5 fungal infections were gastrointestinal tract (n = 3); and arteritis (n = 2). Some patients experienced more than 1 type of infection. The predominant etiology of the systemic infections was bacterial. In conclusion, infectious complications were the main causes of morbidity after SPKT. An early diagnosis of infection, particularly fungal complications, is essential. We recommend administration of broad-spectrum prophylactic antibiotics, antifungals, and antiviral agents.

Giant mid-esophageal diverticulum. Conservative treatment of postoperative leakage.

Mid-esophageal diverticula are rare entities. Only symptomatic patients usually receive surgical treatment. Esophageal leakage is one of the most common complications after these procedures. Though in literature, operative management is the preferred treatment for esophageal fistula, conservative approach is described in case of small leaks. We report a case of an operated giant mid-esophageal diverticulum complicated with an esophageal fistula. The patient underwent a surgical treatment and recovered completely.

Hypertension and diabetes mellitus are associated with cardiovascular events in the elderly without cardiovascular disease. Results of a 15-year follow-up in a Mediterranean population.

BACKGROUND AND AIMS: Epidemiological prospective data on cardiovascular (CV) events in elderly subjects from Mediterranean populations are lacking. We aimed to investigate 15-year incidence of CV events and to evaluate the association with CV risk factors in an elderly Mediterranean population. METHODS AND RESULTS: The population of a small Sicilian village were enrolled, visited and a blood sample was drawn at baseline. CV events were recorded in the 15 years of follow-up. From 1351 subjects (75% of the resident population); 315 were in the age range 65-85 years; 266 subjects free from CV disease were analysed. Seventy-seven CV events were recorded in 73 out of 266 subjects, with a 19.7% rate (in 10 years). Hypertension (HTN) (hazards ratio=2.1) and diabetes mellitus (DM) (hazards ratio=1.8) were independently associated with CV events. Subjects with both DM and HTN showed a lower survival free of CV events compared to those with DM or HTN. CONCLUSIONS: In a 15-year follow-up of an elderly Mediterranean population free from CV disease, diabetes mellitus and hypertension were related to CV events. The control of risk factors in the elderly needs to be reinforced to achieve better results in terms of CV prevention.

Preliminary results of liver transplantation for hepatocellular carcinoma among allocation organ policy strategies, neoadjuvant treatments, and intention-to-treat analysis.

We retrospectively evaluated the impact of our strategy for patients with hepatocellular carcinoma (HCC) according to an intention-to-treat analysis and drop-out probability. We evaluated only patients within the Milan criteria. We analyzed the outcomes of neoadjuvant strategies for HCC, organ allocation policy, and systematic application of strategies to increase the deceased donor pool as the current tendency to expand transplantability criteria for those patients. Kaplan-Meier survival probability rates at 1, 3, and 5 years according to an intention-to-treat analysis were 87.02%, 74.53%, and 65.93% for transplanted patients (n=108), and 50%, 14.29%, and 14.29% for the excluded or waiting list group (n=13), respectively (P< .0001). Drop-out risk at 3, 6, and 12 months was 2.40%, 8.59%, and 16.54%, respectively. During the same period, the mortality probability rates at 3, 6, and 12 months among patients without HCC awaiting orthotopic liver transplantation (OLT) were 3.60%, 9.50%, and 18.34%, respectively. Drop-out rate was lower among patients treated before OLT (P< .0001). On the basis of the neoadjuvant treatment results to reduce drop-out risk, we suggest avoiding the high priority for the HCC cohort, particularly within the first 6 months from entrance on the waiting list, because this approach can reduce the chances of patients with end-stage liver disease (ESLD) alone.

Renal transplant compartment syndrome: a case report.

An unusual case of early double kidney transplant dysfunction due to abdominal compartment syndrome is herein reported. A 62-year-old woman on peritoneal dialysis underwent dual kidney transplantation. The grafts were positioned extraperitoneally in both iliac possae using standard techniques. Surgical procedures and immediate postoperative period were uneventful. The urine output was immediate and the creatinine decreased, but in a few days she developed severe ascites with reduced urine output, increased creatinine, and progressive changes on Doppler ultrasound. The patient underwent paracentesis: the kidney function recovered as well as the Doppler ultrasound. Kidney biopsy was negative for rejection or renal pathology. Graft dysfunction was related to the presence of ascites. A catheter inserted in the abdomen measured intra-abdominal pressure (IAP) of 14 mm Hg. IAP correlated with renal function showing that IAP probably explained renal flow modifications.

Differential tissue expression of a calpastatin isoform in Xenopus embryos.

This study is aimed at demonstrating the role played by a calpastatin isoform (Xcalp3) in Xenopus embryos. A specific monoclonal antibody (mAb) was raised against a glutathione S-transferase (GST)-Xcalp3 fusion protein and characterized by immunoblotting and confocal fluorescence microscopy on stage 20-36 embryos. Under these conditions, calpastatin reactivity is associated with a major 110kDa protein fraction and preferentially expressed by notochord and somitic cells. In notochord cells, anti-calpastatin reactive sites were initially restricted to the luminal space of the vacuoles and later became diffused throughout the cytoplasm. In contrast, anti-calpastatin reactive sites in somitic cells were initially diffused throughout the cytoplasm and became restricted to a few intracellular granules in the later developmental stages. At the ultrastructural level, notochord cells appeared as flattened discs containing several vacuoles and numerous electron-dense granules. During transition from stages 26 to 32, electron-dense granules were gradually reduced in number as vacuoles enlarged in size and losed their calpastatin reactivity. Electron-dense granules were also present in myoblast cells and their number gradually reduced during development. To determine whether these observations bear any causal relationship to the calpain/calpastatin system, a number of Xenopus embryos were examined both ultrastructurally and histochemically following exposure to a specific calpain inhibitor (CI3). Under these conditions, Xenopus embryos exhibited an altered right-left symmetry and an abnormal axial shortening. In CI3-treated stage 32 embryos, notochord cells had a reduced vacuolar extension and exhibited at the same time an increase in granular content. The overall morphology of the somites was also distorted and myoblasts were altered both in shape and granular content. Based on these findings, it is concluded that the calpain/calpastatin may play an important role in the control of notochord elongation and somite differentiation during Xenopus embryogenesis.

[Cystic lymphangioma of the adult: our experience and review of literature]. / Linfangioma cistico dell'adulto: nostra esperienza e revisione della letteratura.

Cystic lymphangioma is an uncommon benign pathology, usually reported in children, rarely in adult. Its embryopathogenesis is still controversial: it seems to arise from the lymphatic vessels, mainly in the cervico-cranial district. It is macroscopically characterised by multiple cystic non-communicating concamerations. Definitive diagnosis used to be intraoperative and was usually an unexpected finding. Nowadays, with modern imaging technologies, CT and MRI, diagnosis can be assumed before intervention even though certain diagnosis can still be reached only with histological examination. Imaging techniques can help for a precise mapping of the lesion and definition of its limits with the other structures, improving therapeutic success. Various therapeutical options are reported in literature, but complete surgical excision is still considered the best approach and the most successful. The Authors report their experience and review the literature on cystic lymphangioma in adult.

Anaerobic degradation of BTEX in a packed-bed reactor.

A bench-scale horizontal-flow anaerobic immobilized biomass (HAIB) reactor was assayed aiming to verify its performance in degrading benzene, toluene, ethylbenzene and xylene (BTEX). A 138 ml HAIB reactor filled with polyurethane foam matrices containing immobilized anaerobic biomass was initially fed with synthetic substrate containing protein, carbohydrates and lipids. Thereafter, BTEX degradation was evaluated in the presence of the co-solvents ethanol and lineal alkylbenzene sulphonate (LAS), in two sequential experiments. The inlet BTEX concentration ranged from 1.3 to 27.0 mg/L of each compound and outlet concentrations were lower than 0.1 mg BTEX/L for both the experiments with ethanol and LAS. An active enriched microbial consortium was observed in the reactor, containing BTEX-degraders, and also acetogenic, acetotrophic and hydrogenotrophic microorganisms. The results from the most probable number (MPN) tests indicated a decrease in the number of methanogenic archae, while the number of anaerobic microorganisms in the biofilm was maintained during the experimental period. Methanogenic archae were found to represent less than 0.5 % of the total anaerobic organisms in the biomass inside the reactor.

The serotonin 5-HT2B receptor from the puffer fish Tetraodon fluviatilis: cDNA cloning, genomic organization and alternatively spliced variants.

We cloned the 5-HT2B serotonin receptor from the puffer fish Tetraodon fluviatilis. Two cDNAs differing in length because of the use of alternative polyadenylation sites were isolated. We partly characterized the genomic organization of the 5-HT2B gene and we found two introns conserved in position between the puffer fish and mammals. In addition, four splice variants which would generate truncated forms of the receptor were detected.

mRNA expression of serotonin receptors of type 2C and 5A in human resting lymphocytes.

We investigated the presence of mRNA for serotonin receptors of type 2C (5-HT(2C)) in resting lymphocytes by means of RT-PCR and Southern blotting analyses, given their possible role in the pathophysiology of anxiety and eating disorders. At the same time, we explored also the presence of the specific mRNA for 5-HT(5A) receptors, a novel subtype for which still no functional data exist. Healthy subjects and patients with obsessive-compulsive or bipolar disorders were included in the study. The results showed the presence of the specific mRNAs for both 5-HT(2C) and 5-HT(5A) receptors in resting lymphocytes of the three groups of subjects. An additional band was also observed after the amplification of the 5-HT(5A) cDNA in each sample. These findings, while revealing the presence of 5-HT(2C) and 5-HT(5A) receptor mRNAs in an easily available tissue, can be considered preliminary for future quantitative analyses in patients with different psychiatric conditions.

Ectopic Hoxa2 induction after neural crest migration results in homeosis of jaw elements in Xenopus.

Hox genes are required to pattern neural crest (NC) derived craniofacial and visceral skeletal structures. However, the temporal requirement of Hox patterning activity is not known. Here, we use an inducible system to establish Hoxa2 activity at distinct NC migratory stages in Xenopus embryos. We uncover stage-specific effects of Hoxa2 gain-of-function suggesting a multistep patterning process for hindbrain NC. Most interestingly, we show that Hoxa2 induction at postmigratory stages results in mirror image homeotic transformation of a subset of jaw elements, normally devoid of Hox expression, towards hyoid morphology. This is the reverse phenotype to that observed in the Hoxa2 knockout. These data demonstrate that the skeletal pattern of rhombomeric mandibular crest is not committed before migration and further implicate Hoxa2 as a true selector of hyoid fate. Moreover, the demonstration that the expression of Hoxa2 alone is sufficient to transform the upper jaw and its joint selectively may have implications for the evolution of jaws.

Identification of different forms of calpastatin mRNA co-expressed in the notochord of Xenopus laevis embryos.

We isolated three Xenopus cDNA clones, Xcalp1, Xcalp2 and Xcalp3, which encode different forms of calpastatin mRNA. Compared to the canonical form of mammalian calpastatin, the predicted Xcalp3 protein contained a very long N-terminal domain L and an additional inhibitory domain. The other two deduced calpastatin proteins were truncated forms, both lacking domain L and containing four (Xcalp2) and two (Xcalp1) inhibitory domains, respectively. The presence of Xcalp1, Xcalp2 and Xcalp3 transcripts was detected by in situ hybridization in the notochord from the embryonic stage 20 to stage 36, afterwards the expression was only present in the growing tailbud. As shown by RT-PCR, the three calpastatin mRNAs were also expressed in the adult brain.

Distribution and cellular localization of the serotonin type 2C receptor messenger RNA in human brain.

The regional and cellular distribution of serotonin type 2C receptor messenger RNA was investigated in autopsy samples of human brain by in situ hybridization histochemistry. The main sites of serotonin receptor type 2C messenger RNA expression were the choroid plexus, cerebral cortex, hippocampus, amygdala, some components of the basal ganglia, the substantia nigra, the substantia innominata and the ventromedial hypothalamus, suggesting that this receptor might be involved in the regulation of different brain functions. Interestingly, in all regions examined, the serotonin type 2C receptor messenger RNA was always restricted to subpopulations of cells, suggesting a specific role, perhaps determined by regionality. A comparison of the in situ hybridization results with those previously obtained by means of radioligand binding experiments suggested that in most of the areas analysed the serotonin type 2C receptors were located at axon terminals.

Characterization of a cloned xenopus laevis serotonin 5-HT1A receptor expressed in the NIH-3T3 cell line.

In a previous work we isolated a Xenopus 5-HT1A receptor gene and now report the characterization of this receptor. The HindIII-XbaI fragment of this gene was cloned into the pcDNA I NEO vector and stably transfected into eukaryotic cells (NIH-3T3). To determine the specific 5-HT1A receptor binding, [3H]8-OH-DPAT was used as radioligand. The selective 5-HT1A receptor agonist bound only a single class of saturable high-affinity binding sites with pharmacological characteristics similar to those of the mammalian 5-HT1A receptor. The effects of X5-HT1A receptor activation on cell growth were also investigated in stably transfected NIH-3T3 cells. The 5-HT1A agonist 8-OH-DPAT was found to increase DNA synthesis and accelerated cell growth.

Distribution of the 5-HT5A serotonin receptor mRNA in the human brain.

The 5-HT5A receptor is a member of a new subfamily of serotonin [5-hydroxytryptamine (5-HT)] receptors recently cloned from the human and rodent brain. The role of this receptor in normal brain functions as well as its possible involvement in pathological states is still to be determined. We therefore studied the regional distribution and cellular localization of 5-HT5A receptor mRNA in human brain sections from autopsy samples by in situ hybridization histochemistry, in order to obtain anatomical information which might be useful in formulating hypotheses on possible functions subserved by this receptor in the central nervous system (CNS). Our results showed that the main sites of 5-HT5A mRNA expression were the cerebral cortex, hippocampus and cerebellum. In the neocortical regions, the 5-HT5A receptor mRNA was mainly distributed in the layers II-III and V-VI. In the hippocampus, the dentate gyrus and the pyramidal cell layer of the CA1 and CA3 fields expressed 5-HT5A mRNA at high levels. The broad distribution in the neocortex and hippocampus supports the view that the 5-HT5A receptor in these areas might be implicated in high cortical and limbic functions. The 5-HT5A mRNA was widely distributed in the cerebellum where it was highly expressed in the Purkinje cells, in the dentate nucleus and, at a lower level, in the granule cells. Since the cerebellum receives diffuse serotonergic afferents, this finding suggests that the 5-HT5A receptor may have an important role in mediating the effects of 5-HT on cerebellar functions.

Molecular structure of the rDNA intergenic spacer (IGS) in Triturus: implications for the hypervariability of rDNA loci.

Ribosomal DNA (rDNA) variation in the species Triturus vulgaris meridionalis (Amphibia, Urodela) is remarkable because of unusually high intraspecific variability in the number and distribution of ribosomal loci in the karyotype; in addition, portions of the intergenic spacer (IGS) are clustered at chromosomal loci where they are not associated with ribosomal 18S and 28S RNA genes. These clusters are referred to as extraribosomal, and they appear to consist mostly of repetitive BamHI elements. In this paper, we report the complete nucleotide sequence of an IGS of T. v. meridionalis; this structural analysis is aimed to get insight into the molecular mechanism(s) of spreading of the ribosomal cistrons as well as its possible functional significance. We found that the IGS of T. vulgaris has a modular structure: modular repetitive elements contain sequences possibly related to the regulation of transcription of the ribosomal units. In particular, both ribosomal and extraribosomal IGS elements contain presumptive enhancers. Interestingly, the enhancer-containing region is mostly conserved between ribosomal and extraribosomal elements, while mutations accumulate in a region characterized by repetitions of a simple sequence motif, that we consider as a possible recombination hotspot. Our data suggest that extraribosomal elements most probably originated from ribosomal enhancer-containing elements able to move independently from the ribosomal unit at novel chromosomal positions, perhaps with the aid of the simple repetitive motif. We argue that a similar mechanism may lead to the spreading of complete repetition units as well, giving rise to multiple, and variable, ribosomal sites. We propose that hypervariability in the number and distribution of the rDNA loci, as seen in T. vulgaris, is a further mechanism to ensure redundancy, which seems to be an intrinsic property of rDNA biology, the occurrence of IGS elements independently clustered at separate chromosomal loci being a by-product of this mechanism.