Immune-regulatory properties carried by human amnion epithelial cells: Focus on the role of HLA-G and adenosinergic ectoenzymes.

Human amnion epithelial cells (hAEC) can be efficiently isolated from full-term amnion membrane and have been gaining recognition as advanced medical products. Such cells originate directly from the embryo during the early phase of development and exert a crucial function in the establishment of a tolerogenic environment, to avoid maternal immune rejection. Amnion cell immuno-modulation may be exploited, but additional efforts are required to establish the mechanisms underlying such capacity. The way to fully clarify such an issue is so far long. Here we overview current knowledge on the effects on innate or adaptive immune cells offered by intact hAEC or secreted mediators, pinpointing the mechanisms to date elucidated by our group and others. We move from the description of hAEC general features to molecular intermediaries generating effects directly or indirectly on immune cells. We focus on the role of non-canonical HLA class I molecules, with emphasis on HLA-G, but expand such analysis on adenosinergic mediators, cytokines, and hAEC-derived microvesicles. Finally, we report the ongoing clinical trials exploiting hAEC multipotency and immune modulation.

Different Polyphenolic Parenchyma Cell and Phloem Axial Resin Duct-Like Structure Formation Rates in Cupressus sempervirens Clones Infected with Seiridium cardinale.

The aim of this study was the characterization of constitutive and induced defense mechanisms in the bark tissues of Cupressus sempervirens before and after infection with the bark fungus Seiridium cardinale, which is responsible for cypress canker disease. The time-course development of polyphenolic parenchyma (PP) cells and phloem axial resin duct (PARD)-like structures in the phloem was investigated in two C. sempervirens clones, one resistant and one susceptible to the disease, through anatomical and histological observations carried out by light microscope during a 19-day trial. PP cells were constitutively more abundant in the canker-resistant clone (R clone) compared with the susceptible clone (S clone), whereas PARD-like structures were not present in the bark of untreated plants of both clones. PP cells increased in both clones as a response to infection, but in the R clone, they were more abundant 5 and 12 days after inoculation. After inoculation, PARD-like structures appeared in the phloem after 5 days in the R clone and only after 12 days in the S clone. Even the number of cells surrounding the PARD-like structures was higher in the R clone 5 and 12 days after inoculation compared with the S clone. These observations demonstrate a faster phloem response of the R clone in the early phase of the infection. This may slow down initial growth of the fungus, contributing to the resistance mechanism.

Canonical and non-canonical adenosinergic pathways.

Adenosine (ADO) is an immunosuppressive molecule with multiple functions in different human organs. ADO is released through the concerted action of surface molecules endowed with enzymatic functions, that belong to two different adenosinergic pathways. The canonical pathway is started by CD39, that converts ATP to AMP. On the other hand, the non-canonical pathway metabolizes NAD+ to ADPR, through the action of CD38. The latter byproduct is then converted to AMP by CD203a/PC-1. Both pathways converge to CD73, that fully degrades AMP to the final product ADO. In this Review we take into account the most relevant finding regarding the expression of ectoenzymes belonging to both adenosinergic pathways in different cell types, including regulatory cell subsets and neoplastic cells. Moreover, we summarize the role of these molecules in different physiological and pathological settings. Finally, we discuss potential therapeutic application of specific inhibitors of ectoenzymes and/or ADO receptors.

Functional insights into nucleotide-metabolizing ectoenzymes expressed by bone marrow-resident cells in patients with multiple myeloma.

Human myeloma cells grow in a hypoxic acidic niche in the bone marrow. Cross talk among cellular components of this closed niche generates extracellular adenosine, which promotes tumor cell survival. This is achieved through the binding of adenosine to purinergic receptors into complexes that function as an autocrine/paracrine signal factor with immune regulatory activities that i) down-regulate the functions of most immune effector cells and ii) enhance the activity of cells that suppress anti-tumor immune responses, thus facilitating the escape of malignant myeloma cells from immune surveillance. Here we review recent findings confirming that the dominant phenotype for survival of tumor cells is that where the malignant cells have been metabolically reprogrammed for the generation of lactic acidosis in the bone marrow niche. Adenosine triphosphate and nicotinamide-adenine dinucleotide extruded from tumor cells, along with cyclic adenosine monophosphate, are the main intracellular energetic/messenger molecules that serve as leading substrates in the extracellular space for membrane-bound ectonucleotidases metabolizing purine nucleotides to signaling adenosine. Within this mechanistic framework, the adenosinergic substrate conversion can vary significantly according to the metabolic environment. Indeed, the neoplastic expansion of plasma cells exploits both enzymatic networks and hypoxic acidic conditions for migrating and homing to a protected niche and for evading the immune response. The expression of multiple specific adenosine receptors in the niche completes the profile of a complex regulatory framework whose signals modify multiple myeloma and host immune responses.

Microvesicles released from multiple myeloma cells are equipped with ectoenzymes belonging to canonical and non-canonical adenosinergic pathways and produce adenosine from ATP and NAD.

Multiple myeloma (MM) derives from malignant transformation of plasma cells (PC), which accumulate in the bone marrow (BM), where microenvironment supports tumor growth and inhibits anti-tumor immune responses. Adenosine (ADO), an immunosuppressive molecule, is produced within MM patients' BM by adenosinergic ectoenzymes, starting from ATP (CD39/CD73) or NAD+ [CD38/CD203a(PC-1)/CD73]. These ectoenzymes form a discontinuous network expressed by different BM cells. We investigated the expression and function of ectoenzymes on microvesicles (MVs) isolated from BM plasma samples of patients with MM, using asymptomatic forms of monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM) as controls. The percentage of MVs expressing ectoenzymes at high levels was higher when derived from MM patients than controls. BM CD138+ PC from MM patients expressed high levels of all ectoenzymes. Paired MVs samples confirmed a higher percentage of MVs with high ectoenzymes expression in MM patients than controls. Pooled MVs from MM patients or controls were tested for ADO production. The catabolism of ATP, NAD+, ADPR and AMP to ADO was higher in MVs from MM patients than in those from controls. In conclusion, our results confirmed the hypothesis that MVs in MM niche are main contributor of ADO production. The ability of MVs to reach biological fluids strongly support the view that MVs may assume diagnostic and pathogenetic roles.

The Role of Extracellular Adenosine Generation in the Development of Autoimmune Diseases.

Adenosine (ADO) is an immunosuppressive molecule, which suppresses the immune responses by interacting with specific receptors expressed by immune effector cells. ADO is produced from ATP through the enzymatic activities of CD39 and CD73. Alternatively, ADO can be generated starting from NAD+, which is metabolized by the concerted action of CD38, CD203a/PC-1, and CD73. The role of ADO in immunity has been characterized in the last years in physiology and in pathological settings. This review examines a panel of reports focused on the functions of ADO in the context of human autoimmune/inflammatory diseases and the selected animal models. The final aim is to consider the role of adenosinergic ectoenzymes and ADO receptors as novel therapeutic targets for selected diseases.

Extended low-dose dexamethasone suppression test for diagnosis of atypical Cushing's syndrome in dogs.

The purpose of this study was to evaluate extension of the low-dose dexamethasone suppression (LDDS) test from 8 h to 12 h to detect possible hypercortisolemia associated with atypical hyperadrenocorticism (AHAC). Twelve client-owned dogs were enrolled in the study: 6 healthy dogs (group 1) and 6 dogs with suspected AHAC (group 2). Baseline EDTA plasma samples were collected for endogenous ACTH determination using an immunoradiometric assay. Serum samples were collected before and at 4, 8, 10, and 12 h post-administration of 0.01 mg/kg dexamethasone IV for cortisol concentration determination via chemiluminescent assay. Mean endogenous ACTH concentration did not differ between groups (group 1: 22.4 pg/mL, group 2: 20.0 pg/mL; P > 0.2). Mean baseline cortisol concentration also did not differ significantly between groups (group 1: 3.03 µg/dL, group 2: 4.95 µg/dL; P > 0.2) nor was there any difference in mean cortisol concentration between the groups at any other time point (P > 0.2). The cortisol concentration from 1 dog in group 2 suppressed to 0.7 µg/dL at 8 h but increased to 1.5 µg/dL at 10 h and 3.7 µg/dL at 12 h post-dexamethasone. Based on results of this study, use of an extended LDDS test could not differentiate between healthy dogs and dogs with AHAC. Diagnosis of AHAC should continue to be based on prior established criteria until new testing has been identified.

Presence of Hepatitis E Virus in a RED Deer (Cervus elaphus) Population in Central Italy.

Hepatitis E is an acute human disease caused by the hepatitis E virus (HEV). In addition to humans, HEV has been detected in several animal species and is recognized as a zoonotic pathogen. Pigs, wild boar and deer can be reservoir. In this study, we evaluated HEV prevalence in a free-living red deer (Cervus elaphus) population in central Italy by detecting virus-specific antibodies and RNA in sera. A total of 35 of 251 red deer sera were positive for anti-HEV IgG. HEV RNA was detected in 10 of 91 sera examined. Two genomic fragments targeted by diagnostic PCRs in the capsid region were sequenced, both matching with genotype 3 HEV. Overall results confirmed the occurrence of HEV infection in deer also in Italy.

Repetitive use of levosimendan for treatment of chronic advanced heart failure: clinical evidence, practical considerations, and perspectives: an expert panel consensus.

BACKGROUND: The intravenous inodilator levosimendan was developed for the treatment of patients with acutely decompensated heart failure. In the last decade scientific and clinical interest has arisen for its repetitive or intermittent use in patients with advanced chronic, but not necessarily acutely decompensated, heart failure. Recent studies have suggested long-lasting favourable effects of levosimendan when administered repetitively, in terms of haemodynamic parameters, neurohormonal and inflammatory markers, and clinical outcomes. The existing data, however, requires further exploration to allow for definitive conclusions on the safety and clinical efficacy of repetitive use of levosimendan. METHODS AND RESULTS: A panel of 30 experts from 15 countries convened to review and discuss the existing data, and agreed on the patient groups that can be considered to potentially benefit from intermittent treatment with levosimendan. The panel gave recommendations regarding patient dosing and monitoring, derived from the available evidence and from clinical experience. CONCLUSIONS: The current data suggest that in selected patients and support out-of-hospital care, intermittent/repetitive levosimendan can be used in advanced heart failure to maintain patient stability. Further studies are needed to focus on morbidity and mortality outcomes, dosing intervals, and patient monitoring. Recommendations for the design of further clinical studies are made.

A new device for the care of Congenital Central Hypoventilation Syndrome patients during sleep.

Congenital Central Hypoventilation Syndrome (CCHS) is a genetic disease that causes an autonomous nervous system dysregulation. Patients are unable to have a correct ventilation, especially during sleep, facing risk of death. Therefore, most of them are mechanically ventilated during night and their blood oxygenation is monitored, while a supervisor keeps watch over them. If low oxygen levels are detected by the pulse-oximeter, an alarm fires; the supervisor deals with the situation and, if there is neither a technical problem nor a false alarm, wakes the subject, as CCHS patients usually recover from hypoxia when roused from sleep. During a single night multiple alarms may occur, causing fractioned sleep for the subject and a lasting state of anxiety for supervisors.

Complementary IL-23 and IL-27 anti-tumor activities cause strong inhibition of human follicular and diffuse large B-cell lymphoma growth in vivo.

Interleukin (IL)-23 and IL-27 are pro-inflammatory cytokines that share functional and structural similarities and may exert anti-tumor activities against solid and hematological malignancies. Here, we asked whether IL-23 and IL-27, alone or in combination, may act directly against human follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) cells. In this study, we demonstrated for the first time that human primary FL and DLBCL cells expressed complete and functional IL-23 and IL-27 receptors (R) and that IL-23 and IL-27 exerted anti-tumor activities in vitro and in vivo through different and complementary mechanisms. In vivo studies using severe combined immunodeficiency /non-obese diabetic mice-injected subcutaneously with human SU-DHL-4 cell line revealed that IL-23 inhibited directly tumor-cell proliferation, whereas IL-27 impaired the angiogenic program of lymphoma cells resulting in strong reduction of cell growth. In addition, combined treatment of IL-23 and IL-27 amplified the anti-tumor effects in vivo as compared with administration of each cytokine alone. These anti-tumor mechanisms were confirmed by in vitro experiments performed with primary lymphoma cells and cell lines. Our results strongly encourage the development of future clinical trials to evaluate the toxicity and efficacy of the IL-23 and IL-27 in lymphoma patients.

Interferon-gamma and IL-10 may protect from allergic polysensitization in children: preliminary evidence.

BACKGROUND: A functional defect of T regulatory cells (Treg) has been proposed as pathogenic mechanism of allergic reaction. Polysensitization is a common feature of allergic patients. AIM OF THE STUDY: It was to investigate the possible role of Treg-Th1 cytokines, in the development of new sensitizations in childhood. METHODS: Forty monosensitized (MS) children with allergic rhinitis were evaluated and followed-up for 2 years. New sensitizations were investigated. IL-10 and IFN-gamma were evaluated in in vitro experiments. RESULTS: Children remaining MS showed significant higher production of both IL-10 and IFN-gamma. CONCLUSION: This preliminary study provided evidence that IL-10 and IFN-gamma production could be defective in allergic children prone to develop polysensitization.

Immunohistochemical detection of aetiological agents of proliferative and necrotizing pneumonia in italian pigs.

Proliferative and necrotizing pneumonia (PNP) is a form of interstitial pneumonia that occurs in weaning and post-weaning pigs. PNP is characterized by hypertrophy and hyperplasia of type II pneumocytes and coagulative necrosis and granular debris within alveolar spaces. Canadian and European studies suggest that the porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are the main causes of the disease, but Aujezsky's disease virus (ADV) and swine influenza virus (SIV) have also been considered as potential aetiological agents. An immunohistochemical study was carried out on the lungs of 28 Italian pigs with PNP in order to evaluate the role of PRRSV, PCV2 and ADV in PNP lesions. PRRSV infection was identified in the lungs of 11 pigs, PCV2 in the lungs of four pigs and coinfection with both viruses in the lungs of eight pigs. Neither virus was detected in the lungs of the remaining five pigs. ADV antigen was not detected in any sample. The principle aetiological agent of PNP in Italy therefore appears to be PRRSV. Coinfection with PRRSV and PCV2 is characterized by more severe microscopical changes in affected lungs.

Application of a protocol for the diagnosis of postweaning multisystemic wasting syndrome in Italy.

Samples of superficial inguinal and bronchial lymph nodes, ileum, tonsil and lung were taken from three to five pigs on each of 61 farms with a clinical history of postweaning multisystemic wasting syndrome (PMWS). The samples were examined histologically and by immunohistochemistry for porcine circovirus type 2 (PCV-2). PMWS was diagnosed in two stages: first, an evaluation of the haematoxylin and eosin-stained sections that identified the cases in which the characteristic PCV-2 cytoplasmic inclusion bodies were apparent, and secondly, a conclusive step in which immunohistochemistry was applied to confirm PMWS in the cases in which there were positive immunohistochemical results that coincided with lesions indicative of PMWS in at least one of the lymphoid and/or lung tissues. The location of PCV-2 in specific lesions (cell depletion in lymphoid organs and interstitial pneumonia) confirmed PMWS in 45 of the 61 farms, 31 of which were also infected with porcine reproductive and respiratory syndrome virus. The lymphoid tissues were more reliable than the lungs for the diagnosis of PMWS, both in individual pigs and in groups of pigs, and farm diagnoses based on a group of pigs were more reliable than diagnoses based on single pigs.

Primary pulmonary spindle cell tumour (haemangiopericytoma) in a dog.

Haemangiopericytoma is a soft tissue sarcoma believed to originate from pericytes. These tumours are commonly located on the skin and subcutaneous tissue of dogs and are most commonly found on the limbs. To the authors' knowledge, primary lung haemangiopericytomas have not been previously described in dogs. This case report describes the diagnostic evaluation and treatment of a primary haemangiopericytoma of the lung in a 10-year-old male, neutered, Siberian husky dog. Staging of the tumour was performed using a computed tomography scan of the thorax and a computed tomography-guided fine-needle aspiration biopsy of the lesion. Treatment was a right caudal lobectomy from a right lateral approach. No regional lymph node changes were noted on computed tomography or intraoperative assessments. Histopathology confirmed a spindle cell tumour that stained positive for vimentin and negative for desmin and S-100.

Catastrophic relapse of Evans syndrome five years after allogeneic BMT notwithstanding full donor chimerism. Terminal hemolytic-uremic syndrome.

A patient with severe Evans syndrome received an allo-BMT from his HLA-identical sister on November, 2000. Full marrow and blood donor chimerism were achieved only after 5 donor lymphocyte infusions (DLI), and coincided with complete clinical remission and disappearence of auto-antibodies. Five years later, hemolytic anemia recurred with rapid increase of serum bilirubin to over 50 mg%: he responded to combined therapy, but died on day +17 from admission of an acute hemolytic uremic syndrome (HUS). All circulating blood cells, including erythrocytes, were 100% donor. Ex vivo cultured and expanded T and B cells from the peripheral blood were also 100% donor. The supernatants from B cell cultures, containing either IgM or IgG, did not react with a panel of erythrocytes. Thus in this typical autoimmune disease with a predominant B cell pathogenesis the donor immune system resulted "innocent of autoimmunity". The persistence of long-lived recipient autoreactive plasma-cell lines in survival niches, still producing autoantibodies, may be hypothesized for this and similar cases. The postulated graft-versus-autoimmunity (GVA) effect was apparently not sufficient to eradicate autoimmunity in this patient.

Ten cases of feline mesothelioma: an immunohistochemical and ultrastructural study.

In the cat only 10 cases of mesothelioma, mainly of the peritoneum, have been previously reported. This paper describes a further 10 cases, eight pleural and two peritoneal, in males and females aged 1-17 years. Histologically, five tumours were epithelial, three fibrosarcomatous and two biphasic. Immunohistochemical markers used in human pathology for the identification of mesotheliomas include vimentin, cytokeratin (CK) AE1/AE3, HBME-1, CK 5/6, calretinin, thrombomodulin, carcinoembryonic antigen (CEA), CD15, E-cadherin and desmin. All 10 feline mesotheliomas were positive for vimentin and CK AE1/AE3, six were positive for HBME-1, two for CK5/6, three for CEA and four for E-cadherin. All were negative for desmin and calretinin. Antibodies to thrombomodulin and CD15 failed to cross-react with feline tissues. Electron microscopy, performed in four cases, revealed microvillar structures, desmosomes and intracytoplasmic lumina, confirming its value as a diagnostic tool. The study showed that mesothelial marker antibodies commonly used in human patients can be used for the diagnosis of feline mesothelioma, preferably as a panel of antibodies rather than only one.

Deciduoid peritoneal mesothelioma in a dog.

Deciduoid mesothelioma is a rare variant of epithelial mesothelioma, up to now only described in human pathology, which bears remarkable cytomorphologic resemblance to the endometrium of pregnancy, termed decidua. A case of peritoneal mesothelioma with deciduoid features in a 10-year-old, female dog is reported. Multiple whitish-gray nodules (1-5 mm in diameter) in parietal peritoneum and mesentery were histologically composed of large, proliferating, polygonal or ovoid cells with an abundant eosinophilic, glassy cytoplasm. Immunohistochemical evaluation indicated that the neoplastic cells coexpressed cytokeratin and vimentin with strong and diffuse cytoplasmic staining, and ultrastructural analysis showed long and slender mesothelial-type microvilli; these findings confirmed the mesothelial origin of the tumor.