An integrated approach to control Cystic Echinococcosis in southern Italy.

Cystic echinococcosis (CE) is a severe zoonosis, caused by the larval stage of the tapeworm Echinococcus granulosus. This helminth infection is of increasing public health and socio-economic concern due to the considerable morbidity rates that cause economic losses both in the public health sector and in the livestock industry. Control programmes against E. granulosus are considered long-term actions which require an integrated approach and high expenditure of time and financial resources. Since 2010, an integrated approach to control CE has been implemented in a highly endemic area of continental southern Italy (Campania region). Innovative procedures and tools have been developed and exploited during the control programme based on the following strategies: i) active and passive surveillance in livestock (using geospatial tools for georeferencing), ii) diagnosis in dogs (using the FLOTAC techniques and molecular analysis), iii) targeted treatment of farm dogs (using purpose-built confinement cages), iv) early diagnosis in livestock (by ultrasonography), v) surveillance in humans (through hospital discharge records analysis), vi) monitoring the food chain (analysing raw vegetables), vii) outreach activities to the general public (through dissemination material, e.g. brochures, gadgets, videos, virtual reality). Over eight years, the integrated approach and the new strategies developed have resulted in a noteworthy reduction of the parasite infection rates in livestock (e.g. up to 30 % in sheep). The results obtained so far highlight that using a one health multidisciplinary and multi-institution effort is of pivotal importance in preparing CE control programmes at regional level and could be extended to other endemic Mediterranean areas.

The role of 3D and speckle tracking echocardiography in cardiac amyloidosis: a case report.

Cardiac amyloidosis (CA) is a disorder characterized by amyloid fibrils deposition in cardiac interstitium; it results in a restrictive cardiomyopathy with heart failure (HF) and conduction abnormalities. The "gold standard" for diagnosis of CA is myocardial biopsy but possible sampling errors and procedural risks, limit it's use. Magnetic resonance (RMN) offers more information than traditional echocardiography and allows diagnosis of CA but often it's impossible to perform. We report the case of a man with HF and symptomatic bradyarrhythmia that required an urgent pacemaker implant. Echocardiography was strongly suggestive of CA but wasn't impossible to perform an RMN to confirm this hypothesis because the patient was implanted with a definitive pacemaker. So was performed a Speckle Tracking Echocardiography (STE) and a 3D echocardiography: STE allows to differentiate CA from others hypertrophic cardiomyopathy by longitudinal strain value < 12% and 3D echocardiography shows regional left ventricular dyssynchrony with a characteristic temporal pattern of dispersion of regional volume systolic change. On the basis of these results, finally was performed an endomyocardial biopsy that confirmed the diagnosis of CA. This case underlines the importance of news, noninvasive techniques such as eco 3D and STE for early diagnosis of CA, especially when RMN cannot be performed.

CD8beta endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes.

The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8beta chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8alphabeta, but not CD8alphaalpha or soluble CD8alphabeta, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8beta endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8beta constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2K(d), and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8beta, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56(lck). In addition, the cytoplasmic portion of CD8beta mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8alphabeta partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56(lck) in rafts, which in turn phosphorylates CD3 and initiates T cell activation.

Determination of p185 and adenylosuccinate lyase (ASL) activity in preneoplastic colon lesions and intestinal mucosa of human subjects.

OBJECTIVES: The HER2 gene has been found amplified in a number of human adenocarcinoma leading to elevated levels of expression of its encoded product, p185 protein. Because little information is available on the tissue and tumor specificity of this gene product, we studied the expression of p185 protein in preneoplastic colon lesions. Adenylosuccinate lyase (ASL, EC 4.3.2.2) is known to increase in malignancies such as colorectal, breast, and prostate cancer. In order to evaluate the potential of ASL as a tumor marker, its activity was determined and compared with the expression of p185. DESIGN AND METHODS: p185 was determined by an immunohistochemical procedure in patients with the preneoplastic lesions. ASL activity was evaluated in intestinal mucosa adjacent to colorectal cancers (patient group A) and in preneoplastic colorectal lesions (group B). The enzyme activity was evaluated in dialyzed supernatants, following the disappearance of substrate (adenylosuccinate AMP-S) and the formation of product (adenosine 5'-monophosphate-AMP), separated by high performance liquid chromatography. RESULTS AND CONCLUSIONS: Increased expression of p185 and elevated ASL activity were observed in tubular and tubulo-villous adenoma and may, therefore, be associated with the early stages of colorectal cancer.

The signal response of IkappaB alpha is regulated by transferable N- and C-terminal domains.

IkappaB alpha retains the transcription factor NF-kappaB in the cytoplasm, thus inhibiting its function. Various stimuli inactivate IkappaB alpha by triggering phosphorylation of the N-terminal residues Ser32 and Ser36. Phosphorylation of both serines is demonstrated directly by phosphopeptide mapping utilizing calpain protease, which cuts approximately 60 residues from the N terminus, and by analysis of mutants lacking one or both serine residues. Phosphorylation is followed by rapid proteolysis, and the liberated NF-kappaB translocates to the nucleus, where it activates transcription of its target genes. Transfer of the N-terminal domain of IkappaB alpha to the ankyrin domain of the related oncoprotein Bcl-3 or to the unrelated protein glutathione S-transferase confers signal-induced phosphorylation on the resulting chimeric proteins. If the C-terminal domain of IkappaB alpha is transferred as well, the resulting chimeras exhibit both signal-induced phosphorylation and rapid proteolysis. Thus, the signal response of IkappaB alpha is controlled by transferable N-terminal and C-terminal domains.

Critical role for lysines 21 and 22 in signal-induced, ubiquitin-mediated proteolysis of I kappa B-alpha.

The NF-kappa B transcription factor induces rapid transcription of many genes in response to a variety of extracellular signals. NF-kappa B is readily activated from normally inhibited cytoplasmic stores by induced proteolytic degradation of I kappa B-alpha, a principal inhibitor of this transcription factor. Following the inhibitor's degradation, NF-kappa B is free to translocate to the nucleus and induce gene transcription. The I kappa B-alpha inhibitor is targeted for degradation by signal-induced phosphorylation of two closely spaced serines in its NH2 terminus (Ser32 and Ser36). Proteolytic degradation appears to be carried out by proteasomes which can recognize ubiquitinated intermediates of the I kappa B-alpha inhibitor. We provide evidence which supports a ubiquitin-mediated mechanism. Amino acid substitutions of two adjacent potential ubiquitination sites in the NH2 terminus of I kappa B-alpha (Lys21 and Lys22) almost completely block the rapid, signal-induced degradation of the mutant protein, while they do not interfere with induced phosphorylation. The mutant I kappa B-alpha also does not permit signal-induced activation of NF-kappa B bound to it. The data suggest that ubiquitination at either of the two adjacent lysines (21 and 22) is required for degradation following induced phosphorylation at nearby serines 32 and 36. Such dependence on ubiquitination of specific sites for protein degradation is unusual. This mechanism of degradation may also apply to I kappa B-beta, an inhibitor related to and functionally overlapping with I kappa B-alpha, as well as to cactus, an I kappa B homolog of Drosophila.

Production of inflammatory cytokines by Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines spontaneously originated from the peripheral blood of patients with human immunodeficiency virus (HIV) infection.

In this study we have raised spontaneous Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines (LCL) from the peripheral blood of human immunodeficiency virus (HIV)-infected individuals and of control patients with primary EBV infections. These LCLs were also raised in the presence of the viral inhibitor phosphonoformate (PFA); under these conditions, the in vitro infection of bystander B lymphocytes with EBV released in culture by in vivo infected B cells is inhibited. Thus, the latter LCLs are likely to represent the progeny of B cells latently infected by EBV in vivo. The LCLs raised in the presence or absence of PFA had the same phenotypic features, type of EBV latency, and growth pattern irrespective of whether they had been raised from HIV-seropositive individuals or patients with primary EBV infections or had been generated by infecting normal B cells in vitro. Studies on the production of inflammatory cytokines were conducted by Northern blotting or by determining the cytokine concentrations in the cell supernatants by immunoassays or bioassays. Three of eight LCLs from HIV-seropositive patients released TNF alpha and 5/5 released TNF beta, IL6 was present in the supernatants of 1/8 LCLs, and IL1 alpha and IL1 beta were not detected in any culture supernatant. No differences were noticed in the patterns of cytokine secretion among the LCLs from HIV-seropositive patients and in those raised from patients with primary EBV infections or obtained by infecting normal B cells in vitro with EBV. It is tempting to speculate that abnormally expanded EBV-harboring B cells in HIV-seropositive patients may participate in the pathogenesis of certain clinical manifestations by releasing inflammatory cytokines; some of these cytokines might also contribute to the in vivo spreading of HIV infection. However, the spontaneous LCLs from HIV-seropositive individuals do not display abnormal features compared to latently EBV-infected LCLs from other sources despite the high frequency of EBV-driven lymphoproliferative disorders observed in AIDS patients.

Spontaneous production of granulocyte colony-stimulating factor in vitro by human B-lineage lymphocytes is a distinctive marker of germinal center cells.

The ability of human B lymphocytes to produce granulocyte (G)-CSF in vitro was investigated. Highly purified tonsillar B cells were fractionated into large and small cells by a Percoll density gradient, cultured, and tested for G-CSF gene expression. Large B cells spontaneous produced G-CSF mRNA and protein, whereas small B cells did not, even after incubation with various stimuli. Immunophenotypic analyses showed that large B lymphocytes contained approximately 60 to 70% of cells with the characteristic surface markers of germinal center (GC) B cells (CD38+, CD10+, and surface IgG+). The remaining cells expressed CD39, CD23, and surface IgD and were presumably in vivo-activated follicular mantle zone B cells. Fractionation of the large B lymphocytes into CD39+, surface IgD+, and CD39-, surface IgD- cells showed that the latter, but not the former, cell type produced G-CSF spontaneously in culture. Stimulation of purified (CD39-, surface IgD-) GC B cells with a CD40 mAb alone or in combination with IL-4 increased G-CSF production. Because these stimuli rescued a large fraction of GC cells (up to 50%) from spontaneous apoptosis in vitro, the finding may suggest that prevention of apoptotic death resulted in an increased G-CSF production or that CD40 mab and/or IL-4 increased G-CSF gene expression in G-CSF-producing GC B cells. Malignant B cells purified from the invaded lymph nodes of three patients with follicular center cell lymphoma and three Burkitt lymphoma cell lines, which had an immunophenotype identical with that of normal GC B cells, spontaneously produced G-CSF in vitro, thus confirming the GC origin of the cytokine. Incubation of normal purified GC B cells with rG-CSF resulted in the rescue of GC B cells from apoptosis, suggesting that G-CSF may be used by GC B cells in an autocrine manner. This autocrine loop of production and response to G-CSF by GC B cells may be activated by stimuli such as those delivered via the surface CD40 molecule, that participate in the rescue of GC B cells from apoptosis.

Production of hematopoietic growth factors by human B lymphocytes: mechanisms and possible implications.

In this study we have investigated the ability of human B lymphocytes to produce granulocyte-macrophage colony stimulating factor (GM-CSF) and, in preliminary experiments, granulocyte CSF (G-CSF). The sources of human B cells were surgically removed tonsils from normal individuals and peripheral blood from patients with B cell chronic lymphocytic leukemia (B-CLL). Tonsil B lymphocytes were purified by E rosetting and complement-mediated cytotoxicity with selected monoclonal antibodies and subsequently fractionated by a Percoll density gradient into in vivo activated and resting cells. The latter cell fractions were subsequently cultured with or without stimuli. GM-CSF was detected by a bioassay, G-CSF by an enzyme-linked immunoassay. In vivo and in vitro activated B cells produced GM-CSF, whereas in vivo activated, but not in vitro activated, B lymphocytes produced G-CSF. These results were confirmed by Northern blot experiments with cDNA probes specific for GM-CSF and G-CSF genes. Many B cell suspensions from B-CLL patients produced GM-CSF or G-CSF only following Staphylococcus Aureus Cowan I (SAC) stimulation; in some cases, a spontaneous production or no production at all of the two cytokines was detected. The possible implications of these results for B cell physiology and for the pathogenesis of immunologically mediated diseases will be discussed.

Production of granulocyte-macrophage colony-stimulating factor but not IL-3 by normal and neoplastic human B lymphocytes.

The ability of human B cells to produce granulocyte-macrophage (GM)-CSF and IL-3 was investigated. B cells, isolated from tonsils or from the peripheral blood of patients with B cell chronic lymphocytic leukemia using mAb and immune rosettes, were cultured with or without Staphylococcus aureus Cowan strain I. GM-CSF and IL-3 were measured in the culture supernatants using a bioassay based on the selective proliferative response of the MO7e megakaryoblastic cell line to IL-3 or GM-CSF. S. aureus Cowan I-stimulated normal B cells released measurable amounts of GM-CSF but not of IL-3 as determined in neutralization assays with specific mAb in the MO7e cell line test. Some of the unstimulated normal B suspensions also produced GM-CSF, albeit in lower quantities. When normal B cells were fractionated into small (resting) and large (activated) B cells by Percoll density gradients, spontaneous GM-CSF production was detected only in the large cell fractions, but small cells were induced to produce GM-CSF upon S. aureus Cowan I stimulation. On a per cell basis, tonsillar B cells were found capable of releasing more GM-CSF than activated peripheral blood monocytes. The amount of GM-CSF produced by B cells was always inferior to that released by stimulated peripheral blood T cells or NK cells. The purified B cell suspensions from all 14 B cell chronic lymphocytic leukemia patients studied released GM-CSF but not IL-3 in the culture supernatants, sometimes even in the absence of stimuli. Northern blot analysis on total or poly(A)+ RNA confirmed the presence of GM-CSF, but not of IL-3, mRNA transcripts in both normal and malignant B cells. The results of these studies support the notion that activated human B lymphocytes release sufficient GM-CSF to play a role in the control of both hematopoiesis and the inflammatory process.

A non-hormonal therapeutic alternative in cervico-vaginal dystrophies.

The pharmaco therapeutic effect of polydeoxyribonucleotide vaginal suppositories in two dosages (PDRN 1,65 mg and PDRN 5 mg)* on vaginal dystrophy was studied. 40 women, aged more than forty, in surgical or physiological climacteric with cervico-vaginal dystrophy were examined. The study was carried out according to the double-blind method, randomized, between groups. Both treatments produced important ameliorations of subjective symptomatology and objective signs. No significant difference emerged between the two preparations. PDRN was proved to be a valid alternative to local hormonal therapies.

Genotypic analysis of engraftment in thalassemia following bone marrow transplantation using synthetic oligonucleotides.

DNA hybridization with synthetic oligonucleotide probes was used to assess engraftment in 19 thalassemic patients who received bone marrow grafts from their respective healthy HLA-identical siblings. Three oligomers complementary to the tandem repetitive sequences of different hypervariable regions of human DNA were designed so as to produce simple RFLP (restriction fragment length polymorphism) patterns. Each probe hybridizes to one or two bands in HinfI-digested genomic DNA. The combined use of these three probes allowed a discrimination between all the HLA-identical siblings tested. Both donor-specific and recipient-specific DNA fragments existed in 18 out of the 19 sibling pairs studied. One pair possessed only a donor-specific fragment. DNA analysis at an early stage after the graft detected donor-specific fragments in 15 out of 19 patients, recipient-specific fragments in three patients and a mix of recipient and donor fragments in one patient. At a later stage this patient possessed donor-specific fragments only. Follow-up DNA analysis confirmed these findings. Thus 16 patients continued to display donor-specific fragments over 60 days post-transplant. These DNA data showed strong correlation with the clinical status of the patients as well as with other markers of engraftment including cytogenetics and hemoglobin synthesis. The patients who showed donor-specific fragments over 60 days have been free of thalassemic symptoms for over 300 days. Moreover, in 11 cases it was possible to predict the fate of the graft within 15 days after transplantation. In conclusion, the use of the three synthetic oligonucleotide probes provides a powerful tool in documenting engraftment in bone marrow transplantation.