Patrizio Sale

Early Detection of Fabry Cardiomyopathy by Tissue Doppler Imaging

Background—Fabry cardiomyopathy is diagnosed by detection of left ventricular hypertrophy (LVH) in patients with &agr;-Galactosidase A deficiency. Conventional noninvasive tools are unable to provide a preclinical diagnosis allowing prompt institution of enzymatic therapy. Methods and Results—We studied three groups of patients: 10 patients with causal mutations for Fabry disease and LVH, 10 mutation-positive patients without LV, and 10 healthy relatives without causal mutations and no LVH. All patients with LVH and 6 patients with Fabry disease without LVH with complex repetitive ventricular arrhythmias underwent biventricular endomyocardial biopsy to assess cardiac involvement. In all patients 2-dimensional echocardiography with tissue Doppler analysis in the pulsed Doppler mode was performed: systolic (Sa), early diastolic (Ea), and late diastolic (Aa) velocities were measured, and the Ea/Aa ratio and the dimensionless parameter E/Ea were computed at both corners of the mitral annulus. Histology and electron microscopy studies showed glycosphingolipid deposits in all cases. All mutation-positive patients had significant reduction of Sa, Ea, and Aa velocities at both corners of the mitral annulus compared with normal control subjects. Ea/Aa ratio was significantly lower and E/Ea ratio significantly higher in mutation-positive patients than in control subjects. Patients with LVH showed significantly lower contraction and relaxation tissue Doppler velocities, lower Ea/Aa ratio, and higher E/Ea ratio in comparison with mutation-positive patients with no LVH. Conclusions—Fabry cardiomyopathy is characterized by reduced myocardial contraction and relaxation tissue Doppler velocities, detectable even before development of LVH. Tissue Doppler imaging can provide a preclinical diagnosis of Fabry cardiomyopathy, allowing early institution of enzyme replacement therapy.

Tumor-Associated Tn-MUC1 Glycoform Is Internalized through the Macrophage Galactose-Type C-Type Lectin and Delivered to the HLA Class I and II Compartments in Dendritic Cells

The type of interaction between tumor-associated antigens and specialized antigen-presenting cells such as dendritic cells (DCs) is critical for the type of immunity that will be generated. MUC1, a highly O-glycosylated mucin, is overexpressed and aberrantly glycosylated in several tumor histotypes. This results in the expression of tumor-associated glycoforms and in MUC1 carrying the tumor-specific glycan Tn (GalNAcalpha1-O-Ser/Thr). Glycopeptides corresponding to three tandem repeats of MUC1, enzymatically glycosylated with 9 or 15 mol of GalNAc, were shown to specifically bind and to be internalized by immature monocyte-derived DCs (iDCs). Binding required calcium and the GalNAc residue and was competed out by GalNAc polymer and Tn-MUC1 or Tn-MUC2 glycopeptides. The macrophage galactose-type C-type lectin (MGL) receptor expressed on iDCs was shown to be responsible for the binding. Confocal analysis and ELISA done on subcellular fractions of iDCs showed that the Tn-MUC1 glycopeptides colocalized with HLA class I and II compartments after internalization. Importantly, although Tn-MUC1 recombinant protein was bound and internalized by MGL, the glycoprotein entered the HLA class II compartment, but not the HLA class I pathway. These data indicate that MGL expressed on iDCs is an optimal receptor for the internalization of short GalNAcs carrying immunogens to be delivered into HLA class I and II compartments. Such glycopeptides therefore represent a new way of targeting the HLA class I and II pathways of DCs. These results have possible implications in designing cancer vaccines.

Clinical relevance of action observation in upper-limb stroke rehabilitation: A possible role in recovery of functional dexterity. A randomized clinical trial

Objective. A randomized controlled observer-blind trial was designed to evaluate the effectiveness of action observation as an add-on treatment to the standard rehabilitation of upper-limb function, early after stroke. Methods. Stroke survivors (N = 102) were consecutively recruited from 13 centers 30 days (±7) after a first-ever stroke and randomly assigned to the experimental (EG) or control group (CG). EG participants watched video footage of daily routine tasks (actions) carried out with the upper limb in order to prepare to imitate the presented action. At the end of each sequence, a therapist prompted the patient to perform the same movement for 2 minutes, providing help when needed. Static images without animals or human beings were shown to the CG. At the end of each sequence, the CG executed movements that simulated the shoulder and elbow joint mobilization activities performed by the EG. Results for the Fugl-Meyer test, Frenchay Arm test, Box and Block test (BBT), Modified Ashworth Scale, and Functional Independence Measure Motor items were recorded before treatment (T0), after 4 weeks of treatment (T1), and at the follow-up visit 4 to 5 months after the conclusion of treatment (T2). Results. An improvement over time was appreciated on all measures of impairment and functional ability with both treatment programs. A Time × Treatment interaction emerged from the generalized estimating equations analysis of BBT, showing significant T0–T1 and T0–T2 differences in favor of EG. Conclusion. This multicenter trial endorses the use of action observation in upper-extremity rehabilitation, along with a role for the mirror neuron system in poststroke recovery.

Nitric oxide synthases in normal and benign hyperplastic human prostate: Immunohistochemistry and molecular biology

The expression of nitric oxide synthase (NOS) isoforms has been investigated in normal (three subjects) and benign hyperplastic prostate (ten patients) by immunohistochemistry and reverse transcriptase‐polymerase chain reaction (RT‐PCR). The inducible NOS (iNOS or NOS‐2) is not detected in normal prostate, while it is expressed in the prostate of all benign prostatic hyperplasia (BPH) patients, even in the absence of prostatitis or systemic signs of an inflammatory condition. This suggests that sex hormones may be involved in iNOS induction and that there may be a role for NO in the pathogenesis of BPH. Constitutive NOSs (nNOS and eNOS) are expressed in both normal and hyperplastic prostate and are co‐expressed in epithelial cells. eNOS, however, is present mainly in the basal layer cells; nNOS seems abundantly expressed in the more superficial cells of the affected prostate. This indicates that the switching between the two constitutive isoforms may be part of the usual process of cell differentiation from the basal to the secretory layer of the epithelium. Copyright

Pro-inflammatory gene expression in solid glioblastoma microenvironment and in hypoxic stem cells from human glioblastoma

BackgroundAdaptation to hypoxia and consequent pro-inflammatory gene expression of prostate and breast carcinomas have been implicated in the progression toward cancer malignant phenotype. Only partial data are available for the human tumor glioblastoma multiforme (GBM). The aim of our study was to analyze the hypoxic and pro-inflammatory microenvironment in GBMs and to demonstrate that in a stem/progenitor cell line derived from human glioblastoma (GBM-SCs), hypoxia activates a coordinated inflammatory response, evidencing an invasive and migratory phenotype.MethodsFrom each of 10 human solid glioblastomas, clinically and histopathologically characterized, we obtained three surgical samples taken from the center and the periphery of the tumor, and from adjacent host normal tissue. Molecular and morphological analyses were carried out using quantitative real-time PCR and western blot (WB). GBM stem and differentiated cells were incubated under hypoxic conditions and analyzed for pro-inflammatory gene expression and for invasive/migratory behavior.ResultsA panel of selected representative pro-inflammatory genes (RAGE and P2X7R, COX2, NOS2 and, PTX3) were analyzed, comparing tumor, peritumor and host normal tissues. Tumors containing leukocyte infiltrates (as assessed using CD45 immunohistochemistry) were excluded. Selected genes were overexpressed in the central regions of the tumors (i.e. in the more hypoxic areas), less expressed in peripheral regions, and poorly expressed or absent in adjacent normal host tissues. Western blot analysis confirmed that the corresponding pro-inflammatory proteins were also differently expressed. Hypoxic stem cell lines showed a clear time-dependent activation of the entire panel of pro-inflammatory genes as compared to differentiated tumor cells. Biological assays showed that invasive and migratory behavior was strengthened by hypoxia only in GBM stem cells.ConclusionsIn human solid glioblastoma we have observed a coordinated overexpression of a panel of pro-inflammatory genes as compared to host normal tissue. We have also evidenced a similar pattern of overexpressed genes in GBM-SCs after hypoxic treatment, showing also a gain of invasive and migratory function that was lost when these stem cells differentiated. We suggest that, as has been previously described for prostatic and mammary carcinoma, in human glioblastoma acquisition of a proinflammatory phenotype may be relevant for malignant progression.

Metabotropic glutamate receptors in stem/progenitor cells

Functional mGlu receptor subtypes are found in stem/progenitor cells, and regulate proliferation, differentiation, and survival of these cells. Activation of mGlu5 receptors supports self-renewal of embryonic stem cells, which are pluripotent cells isolated from the blastocyst capable of generating all the bodys cell lineages, including germ cells. Differentiation of embryonic stem cells into embryoid bodies is associated with the induction of mGlu4 receptors, the activation of which drives cell differentiation towards the mesoderm and endoderm lineages. Different mGlu receptor subtypes, mGlu3 and mGlu5 receptors in particular, are found in neural stem cells (stem cells resident in the CNS that give rise to neurons, astrocytes or oligodendrocytes) isolated from the developing brain or from regions of persistent neurogenesis of the adult brain (e.g. the subventricular zone lining the wall of the lateral ventricles). The evidence that activation of mGlu3 and mGlu5 receptors stimulates proliferation of these cells is particularly interesting because of the similarities between neural stem cells and putative cancer stem cells that support the growth of malignant gliomas. A link among mGlu receptors, stem cells and cancer is supported by the finding that mGlu4 receptors are expressed by cerebellar granule cell neuroprogenitors, which are the putative cells of origin of medulloblastomas. The study of mGlu receptors in stem/progenitor cells has potential applications in the optimisation of protocols of cell expansion and differentiation aimed at cell replacement strategies, and may gain new insights into the pathophysiology of neurodevelopmental disorders and brain tumours.

Hand Robotics Rehabilitation: Feasibility and Preliminary Results of a Robotic Treatment in Patients with Hemiparesis

Background. No strongly clinical evidence about the use of hand robot-assisted therapy in stroke patients was demonstrated. This preliminary observer study was aimed at evaluating the efficacy of intensive robot-assisted therapy in hand function recovery, in the early phase after a stroke onset. Methods. Seven acute ischemic stroke patients at their first-ever stroke were enrolled. Treatment was performed using Amadeo robotic system (Tyromotion GmbH Graz, Austria). Each participant received, in addition to inpatients standard rehabilitative treatment, 20 sessions of robotic treatment for 4 consecutive weeks (5 days/week). Each session lasted for 40 minutes. The exercises were carried out as follows: passive modality (5 minutes), passive/plus modality (5 minutes), assisted therapy (10 minutes), and balloon (10 minutes). The following impairment and functional evaluations, Fugl-Meyer Scale (FM), Medical Research Council Scale for Muscle Strength (hand flexor and extensor muscles) (MRC), Motricity Index (MI), and modified Ashworth Scale for wrist and hand muscles (AS), were performed at the beginning (T0), after 10 sessions (T1), and at the end of the treatment (T2). The strength hand flexion and extension performed by Robot were assessed at T0 and T2. The Barthel Index and COMP (performance and satisfaction subscale) were assessed at T0 and T2. Results. Clinical improvements were found in all patients. No dropouts were recorded during the treatment and all subjects fulfilled the protocol. Evidence of a significant improvement was demonstrated by the Friedman test for the MRC (P < 0.0123). Evidence of an improvement was demonstrated for AS, FM, and MI. Conclusions. This original rehabilitation treatment could contribute to increase the hand motor recovery in acute stroke patients. The simplicity of the treatment, the lack of side effects, and the first positive results in acute stroke patients support the recommendations to extend the clinical trial of this treatment, in association with physiotherapy and/or occupational therapy.

Up-regulation of pro-inflammatory genes as adaptation to hypoxia in MCF-7 cells and in human mammary invasive carcinoma microenvironment

The role of tumor cells in synthesizing pro‐inflammatory molecules is still controversial. Here we report that hypoxic treatment of the MCF‐7 human mammary adenocarcinoma cell line induced activation of hypoxia‐inducible factor 1α (HIF‐1α) and nuclear factor‐kappa B (NF‐κB). Importantly, hypoxia regulated expression of alarmin receptors such as the receptor for advanced glycation end products (RAGE) and the purinoreceptor (P2X7R), and up‐regulated inflammatory response (IR) genes such as the inducible enzymes nitric oxide synthase (NOS2), cycloxygenase (COX2), and the acute‐phase protein pentraxin‐3 (PTX3). Hypoxia also stimulated chemokine (C‐X‐C motif) receptor 4 (CXCR4) mRNA synthesis. In fact, the CXCR4 ligand stromal‐derived factor‐1α (SDF‐1α) increased invasion and migration of hypoxic MCF‐7 cells. Inhibition of HIF‐1α by chetomin and NF‐κB by parthenolide reduced mRNA and protein expression of the studied molecules and prevented invasion of hypoxic MCF‐7 cells. Moreover, solid invasive mammary tumor microenvironment was analyzed after laser‐capture microdissection (LCMD) comparing tumor versus host normal tissue. Nuclear translocation of HIF‐1α and NF‐κB and up‐regulation of IR, CXCR4, estrogen receptor α (ERα), and epithelial growth factor receptor (EGFR) was observed in tumor but not in host normal tissue in the absence of a local inflammatory leukocyte infiltrate. We conclude that under hypoxic conditions MCF‐7 cells acquire a pro‐inflammatory phenotype, and that solid human mammary carcinoma evidenced a similar activation of HIF‐1α, NF‐κB, and IR genes in malignant tumor cells as compared to the normal host tissues. We suggest a role for IR activation in the malignant progression of transformed cells.

Ovarian cancer cytoreduction induces changes in T cell population subsets reducing immunosuppression

Surgery is the primary therapeutic strategy for most solid tumours; however, modern oncology has established that neoplasms are frequently systemic diseases. Being however a local treatment, the mechanisms through which surgery plays its systemic role remain unknown. We have investigated the influence of cytoreduction on the immune system of primary and recurrent ovarian cancer. All ovarian cancer patients show an increase in CD4+CD25+FOXP3+ circulating cells (CD4 Treg). CD4/CD8 ratio is increased in primary tumours, but not in recurrent neoplasms. Primary cytoreduction is able to increase circulating CD4 and CD8 effector cells and decrease CD4 naïve T cells. CD4+ Treg cells rapidly decreased after primary tumour debulking, while CD8+CD25+FOXP3+ (CD8 Treg) cells are not detectable in peripheral blood. Similar results on CD4 Treg were observed with chemical debulking in women subjected to neoadjuvant chemotherapy. CD4 and CD8 Treg cells are both present in neoplastic tissue. Interleukin (IL)‐10 serum levels decrease after surgery, while no changes are observed in transforming growth factor‐β1 and IL‐6 levels. Surgically induced reduction of the immunosuppressive environment results in an increased capacity of CD8+ T cells to respond to the recall antigens. None of these changes was observed in patients previously subjected to chemotherapy or affected by recurrent disease. In conclusion, we demonstrate in ovarian cancer that primary debulking is associated with a reduction of circulating Treg and an increase in CD8 T‐cell function. Debulking plays a beneficial systemic effect by reverting immunosuppression and restoring immunological fitness.

Evidence for cell surface association between CXCR4 and ganglioside GM3 after gp120 binding in SupT1 lymphoblastoid cells

CXCR4 (fusin) is a chemokine receptor which is involved as a coreceptor in gp120 binding to the cell surface. In this study we provide evidence that binding of gp120 triggers CXCR4 recruitment to glycosphingolipid‐enriched microdomains. Scanning confocal microscopy showed a nearly complete localization of CXCR4 within GM3‐enriched plasma membrane domains of SupT1 cells and coimmunoprecipitation experiments revealed that CXCR4 was immunoprecipitated by IgG anti‐GM3 after gp120 pretreatment. These findings reveal that gp120 binding induces a strict association between CXCR4 and ganglioside GM3, supporting the view that GM3 and CXCR4 are components of a functional multimolecular complex critical for HIV‐1 entry.