Wireless Middleware Solutions for Smart Water Metering.

While smart metering applications have initially focused on energy and gas utility markets, water consumption has recently become the subject of increasing attention. Unfortunately, despite the large number of solutions available on the market, the lack of an open and widely accepted communication standard means that vendors typically propose proprietary data collection solutions whose adoption causes non-trivial problems to water utility companies in term of costs, vendor lock-in, and lack of control on the data collection infrastructure. There is the need for open and interoperable smart water metering solutions, capable of collecting data from the wide range of water meters on the market. This paper reports our experience in the development and field testing of a highly interoperable smart water metering solution, which we designed in collaboration with several water utility companies and which we deployed in Gorino Ferrarese, Italy, in collaboration with CADF (Consorzio Acque Delta Ferrarese), the water utility serving the city. At the core of our solution is SWaMM (Smart Water Metering Middleware), an interoperable wireless IoT middleware based on the Edge computing paradigm, which proved extremely effective in interfacing with several types of smart water meters operating with different protocols.

The CD33xCD123xCD70 Multispecific CD3-Engaging DARPin MP0533 Induces Selective T Cell-Mediated Killing of AML Leukemic Stem Cells.

The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC) transplantation. The disease is driven by leukemic stem cells (LSCs), which are characterized by clonal heterogeneity and resistance to conventional therapy. These cells are therefore believed to be a major cause of progression and relapse. We designed MP0533, a multispecific CD3-engaging designed ankyrin repeat protein (DARPin) that can simultaneously bind to three antigens on AML cells (CD33, CD123, and CD70), aiming to enable avidity-driven T cell-mediated killing of AML cells coexpressing at least two of the antigens. In vitro, MP0533 induced selective T cell-mediated killing of AML cell lines, as well as patient-derived AML blasts and LSCs, expressing two or more target antigens, while sparing healthy HSCs, blood, and endothelial cells. The higher selectivity also resulted in markedly lower levels of cytokine release in normal human blood compared to single antigen-targeting T-cell engagers. In xenograft AML mice models, MP0533 induced tumor-localized T-cell activation and cytokine release, leading to complete eradication of the tumors while having no systemic adverse effects. These studies show that the multispecific-targeting strategy used with MP0533 holds promise for improved selectivity toward LSCs and efficacy against clonal heterogeneity, potentially bringing a new therapeutic option to this group of patients with a high unmet need. MP0533 is currently being evaluated in a dose-escalation phase 1 study in patients with relapsed or refractory AML (NCT05673057).

Investigating the characteristics of residential end uses of water: A worldwide review.

A detailed characterization of residential water consumption is essential for ensuring urban water systems' capability to cope with changing water resources availability and water demands induced by growing population, urbanization, and climate change. Several studies have been conducted in the last decades to investigate the characteristics of residential water consumption with data at a sufficiently fine temporal resolution for grasping individual end uses of water. In this paper, we systematically review 114 studies to provide a comprehensive overview of the state-of-the-art research about water consumption at the end-use level. Specifically, we contribute with: (1) an in-depth discussion of the most relevant findings of each study, highlighting which water end-use characteristics were so far prioritized for investigation in different case studies and water demand modelling and management studies from around the world; and (2) a multi-level analysis to qualitatively and quantitatively compare the most common results available in the literature, i.e. daily per capita end-use water consumption, end-use parameter average values and statistical distributions, end-use daily profiles, end-use determinants, and considerations about efficiency and diffusion of water-saving end uses. Our findings can support water utilities, consumers, and researchers (1) in understanding which key aspects of water end uses were primarily investigated in the last decades; and (2) in exploring their main features considering different geographical, cultural, and socio-economic regions of the world.

Protection from reinfection in "Candidatus Mycoplasma turicensis"-infected cats and characterization of the immune response.

"Candidatus Mycoplasma turicensis" (CMt) is a hemoplasma species of felids. Recent evidence has shown that cats that overcome bacteremia may be protected from reinfection. The purposes of this study were to (1) re-inoculate ostensibly recovered cats, (2) evaluate the immune response and (3) assess CMt tissue loads. Fifteen specified pathogen-free cats were subcutaneously inoculated with CMt: 10 cats (group A) had previously undergone bacteremia and recovered, and 5 naïve cats (group B) served as controls. CMt infections were monitored by real-time PCR using blood and tissue, and the humoral immune response was assessed using DnaK ELISA. Cytokine mRNA expression levels were measured by real-time PCR, and lymphocyte subsets were detected by flow cytometry. The cats in group A were protected from reinfection (no detectable bacteremia) and showed a transient decrease in antibodies. Eosinophilia was noted in cats from group A. The cats from group B became PCR-positive and seroconverted. All of the tissues analyzed from the cats in group B but none of the tissues analyzed from the cats in group A were CMt PCR-positive. Significant changes were observed in the expression of tumor necrosis factor-α, interferon-γ, interleukin-4 and the Th2/Th1 ratio in both groups. The cats from group A occasionally showed higher numbers of CD4+, CD8+, CD4+CD25+ and CD5+MHCII+ T lymphocytes than the control cats. In conclusion, this study describes, for the first time, the occurrence of immunological protection within the same hemoplasma species. Furthermore, the immune response during CMt infections appeared to be skewed toward the Th2 type.

Stimulation with a class A CpG oligonucleotide enhances resistance to infection with feline viruses from five different families.

Domestic cats are commonly affected by viral pathogens that induce lengthy infections with fatal outcomes. Prevention of viral propagation is of primordial importance in shelters and catteries, where cats from different backgrounds have narrow contacts. Oligonucleotides (ODN) containing cytosine-phosphate-guanosine motifs of class A (CpG-A) are highly potent synthetic inducers of innate antiviral mechanisms. The aim of this study was to test their ability to modulate innate immune responses and prevent viral replication as stand-alone agents in the domestic cat. CpG-A stimulation of feline peripheral blood mononuclear cells (PBMCs) enhanced their proliferation, increased the presence of co-stimulatory molecules on their surface and influenced their gene expression profiles in an antiviral orientation. Incubation of the supernatants of CpG-A stimulated PBMCs with feline cell lines of epithelial and fibroblastic origin induced expression of the antiviral myxovirus resistance (Mx) gene in these target cells, which also showed enhanced resistance to feline viruses from five distinct families, namely Coronaviridae, Herpesviridae, Caliciviridae, Parvoviridae, and Retroviridae. Most importantly, subcutaneous administration of CpG-A in domestic cats systemically increased the expression of Mx, reaching maximal levels within 24 h. Plasma from treated cats could furthermore inhibit viral replication in vitro. Altogether, our data highlight the promising potential of CpG-A to induce a preventive antiviral state in the cat and to protect feline populations against a broad range of virus infections.

Glycoprotein D of bovine herpesvirus 5 (BoHV-5) confers an extended host range to BoHV-1 but does not contribute to invasion of the brain.

Bovine herpesvirus 1 (BoHV-1) and BoHV-5 are closely related pathogens of cattle, but only BoHV-5 is considered a neuropathogen. We engineered intertypic gD exchange mutants with BoHV-1 and BoHV-5 backbones in order to address their in vitro and in vivo host ranges, with particular interest in invasion of the brain. The new viruses replicated in cell culture with similar dynamics and to titers comparable to those of their wild-type parents. However, gD of BoHV-5 (gD5) was able to interact with a surprisingly broad range of nectins. In vivo, gD5 provided a virulent phenotype to BoHV-1 in AR129 mice, featuring a high incidence of neurological symptoms and early onset of disease. However, only virus with the BoHV-5 backbone, independent of the gD type, was detected in the brain by immunohistology. Thus, gD of BoHV-5 confers an extended cellular host range to BoHV-1 and may be considered a virulence factor but does not contribute to the invasion of the brain.

Inhibition of herpes simplex virus type 1 replication by adeno-associated virus rep proteins depends on their combined DNA-binding and ATPase/helicase activities.

Adeno-associated virus (AAV) has previously been shown to inhibit the replication of its helper virus herpes simplex virus type 1 (HSV-1), and the inhibitory activity has been attributed to the expression of the AAV Rep proteins. In the present study, we assessed the Rep activities required for inhibition of HSV-1 replication using a panel of wild-type and mutant Rep proteins lacking defined domains and activities. We found that the inhibition of HSV-1 replication required Rep DNA-binding and ATPase/helicase activities but not endonuclease activity. The Rep activities required for inhibition of HSV-1 replication precisely coincided with the activities that were responsible for induction of cellular DNA damage and apoptosis, suggesting that these three processes are closely linked. Notably, the presence of Rep induced the hyperphosphorylation of a DNA damage marker, replication protein A (RPA), which has been reported not to be normally hyperphosphorylated during HSV-1 infection and to be sequestered away from HSV-1 replication compartments during infection. Finally, we demonstrate that the execution of apoptosis is not required for inhibition of HSV-1 replication and that the hyperphosphorylation of RPA per se is not inhibitory for HSV-1 replication, suggesting that these two processes are not directly responsible for the inhibition of HSV-1 replication by Rep.

Flt3 ligand-treated neonatal mice have increased innate immunity against intracellular pathogens and efficiently control virus infections.

Flt-3 ligand (FL), a hematopoetic growth factor, increases the number of dendritic cells (DCs), B cells, and natural killer cells in adult mice but the effect in neonates was unknown. We show that FL treatment of newborn mice induced a >100-fold increase in the innate resistance against infection with herpes simplex virus type 1 and Listeria monocytogenes. This resistance required interferon (IFN)-alpha/beta for viral and interleukin (IL)-12 for bacterial infections. Long-term survival after viral but not bacterial infection was increased approximately 100-fold by FL treatment. After treatment, CD11c(+)/major histocompatibility complex type II(+) and CD11c(+)/B220(+) DC lineage cells were the only cell populations increased in the spleen, liver, peritoneum, and skin. DC induction was independent of IFNs, IL-2, -4, -7, -9, -15, and mature T and B cells. The data suggest that FL increases the number of DCs in neonates and possibly in other immune-compromised individuals, which in turn improves IFN-alpha/beta- and IL-12-associated immune responses.

Partial sequencing and expression of genes involved in glucose metabolism in adipose tissues and skeletal muscle of healthy cats.

Impaired insulin sensitivity is increasingly recognised in cats, but sequences of genes involved in insulin-signalling are largely undetermined in this species. In this study, extended feline mRNA sequences were determined for the adiponectin, glucose transporter-1 (GLUT1), GLUT4, peroxisome proliferative activated receptor-gamma1 (PPARgamma1), PPARgamma2, plasminogen activator inhibitor-1 (PAI-1), monocyte chemoattractant protein-1 (MCP-1) and insulin receptor genes. Conserved dog-specific primers identified from human-dog mRNA alignments were used to amplify feline cDNA in the polymerase chain reaction (PCR). The feline sequences determined by this method were used to design feline-specific primers suitable for real-time PCR for quantification of gene expression in insulin sensitive tissues of healthy cats. Partial sequences of feline mRNAs had 86-95% identity with dog and human genes. Expression of adiponectin, GLUT1, GLUT4, PPARgamma1, PPARgamma2, PAI-1 and insulin receptor mRNA was detected and quantified in subcutaneous and visceral fat and skeletal muscle, whereas MCP-1 mRNA was detected in adipose tissue but not in skeletal muscle. Further characterisation of genes related to glucose metabolism in cats will provide additional insights into insulin-signalling mechanisms in this species.

Stable transduction of bovine TLR4 and bovine MD-2 into LPS-nonresponsive cells and soluble CD14 promote the ability to respond to LPS.

The interaction of bovine cells with lipopolysaccharide (LPS) was explored using human embryo kidney (HEK) 293 cell line stably transduced with bovine toll-like receptor-4 (TLR4) alone or in combination with bovine MD-2. These lines and mock-transduced HEK293 cells were tested by flow cytometry for LPS-fluorescein isothiocyanate (LPS-FITC) binding, nuclear factor kappa B (NFkappaB) activation, interleukin-8 (IL-8) production and interferon-beta mRNA expression/interferon (IFN) type I production. Whereas bovine TLR4 was sufficient to promote binding of high concentrations of LPS-FITC, both bovine TLR4 and MD-2 were required for activation by LPS, as assessed by NFkappaB activation and IL-8 production. Induction of IFN bioactivity was not observed in doubly transduced HEK293 cells, and no evidence for IFN-beta mRNA induction in response to LPS was obtained, although cells responded by IFN-beta mRNA expression to stimulation by Sendai virus and poly-inosinic acid-poly-cytidylic acid (poly(I:C)). Cells stably transduced with both bovine TLR4 and bovine MD-2 responded to LPS by IL-8 production, in decreasing order, in the presence of fetal bovine serum (FCS), of human serum, and of human serum albumin (HSA). The reduced activity in the presence of HSA could be restored by the addition of soluble CD14 (sCD14) but not of LPS binding protein (LBP). This is in contrast to macrophages which show a superior response to LPS in the presence of HSA when compared with macrophages stimulated by LPS in the presence of FCS. This suggests that macrophages but not HEK293 cells express factors rendering LPS stimulation serum-independent. Stably double-transduced cells reacted, in decreasing order, to LPS from Rhodobacter sphaeroides, to LPS from Escherichia coli, to synthetic lipd-IVa (compound 406), to diphosphoryl-lipid-A (S. minnesota) and to monophosphoryl-lipid-A (S. minnesota). They failed to react to the murine MD-2/TLR4 ligand taxol. This resembles the reactivity of bovine macrophages with regard to sensitivity (ED(50)) and order of potency but is distinct from the reactivity pattern of other species. This formally establishes that in order to react to LPS, cattle cells require serum factors (e.g. sCD14) and cell-expressed factors such as MD-2 and TLR4. The cell lines described are the first of a series expressing defined pattern recognition receptors (PRR) of bovine origin. They will be useful in the study of the interaction of the bovine TLR4-MD-2 complex and Gram-negative bovine pathogens, e.g. the agents causing Gram-negative bovine mastitis.

Evidence for dissociation of TLR mRNA expression and TLR agonist-mediated functions in bovine macrophages.

Toll-like receptors are of key importance in the recognition of and response to infectious agents by cells of the innate immune system. TLR mRNA expression and TLR-mediated functions were determined in bovine macrophages (MPhi) infected with bovine viral diarrhea virus (BVDV) or stimulated with interferon-gamma (IFN-gamma) in order to see whether they are correlated under these conditions. As parameters quantitative real time RT-PCR (QRT-PCR) for TLR2, TLR3 and TLR4, NO and TNF production were measured. Triggering of bovine MPhi with bona fide TLR2 and TLR4 agonists (lipopolysaccharide, lipoteichoic acid, peptidoglycan, lipopetide) led to NO and TNF production but neither TLR3 nor TLR9 agonists (double-stranded RNA, CpG DNA) showed this effect. The mRNA expression of TLR2, TLR3 and TLR4 was neither influenced by MPhi costimulation with IFN-gamma nor by MPhi preinfection with BVDV nor by the ligands themselves. However, NO production induced by TLR2 or TLR4 agonists was strongly modulated either by IFN-gamma costimulation or BVDV preinfection. Thus costimulation of MPhi with IFN-gamma resulted in an increase of both NO synthesis and TNF expression by cells stimulated simultaneously by TLR2 or TLR4 agonists. Preinfection of bovine MPhi by BVDV resulted in upregulation of TLR2- and TLR4-mediated NO synthesis. Collectively, these data show that TLR-mediated functions may be modulated by viral infection or activation via IFN-gamma of MPhi whereas the mRNA concentrations of relevant TLR members were not significantly influenced. Thus, the amount of TLR2, TLR3 and TLR4 mRNA transcripts is stable at least under the conditions tested. More importantly, modulation of TLR-mediated responses was dissociated from mRNA expression of TLR members.

Time-dependent alterations in gene expression of interleukin-8 in the bronchial epithelium of horses with recurrent airway obstruction.

OBJECTIVE: To evaluate time-dependent alterations in gene expression of chemokines in bronchial epithelium of recurrent airway obstruction (RAO)-affected horses and whether alterations resulted from increases in gene expression of interleukin (IL)-17 in cells isolated from bronchoalveolar lavage fluid (BALF). ANIMALS: 8 RAO-susceptible horses and 9 control horses. PROCEDURE: In 2 experiments, both groups of horses were evaluated after being maintained on pasture and after being stabled and fed dusty hay for 1, 14, 35, and 49 days (experiment 1) or 14 and 28 days (experiment 2). In experiment 1, gene expression of IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and Toll-like receptor 4 (TLR4) in epithelium and IL-8, IL-17, and TLR4 in BALF cells was measured. In experiment 2, bronchial biopsy specimens were evaluated for IL-8 immunoreactivity. RESULTS: In RAO-susceptible horses after 14 days of challenge exposure, there was a 3- and 10-fold increase in gene expression of IL-8 for epithelial and BALF cells and an increase in IL-8 immunoreactivity in epithelial cells. Challenge exposure failed to alter gene expression of CXCL1, GM-CSF, G-CSF, and TLR4 in epithelial cells of any horses at any time point. During challenge exposure, gene expression of BALF cell IL-17 was downregulated in control horses (day 1) and upregulated in RAO-affected horses (day 35). CONCLUSIONS AND CLINICAL RELEVANCE: Epithelial-derived IL-8 may promote airway neutrophilia, but the inciting stimulus is unlikely to be IL-17 because upregulation of this gene is subsequent to that of IL-8 in epithelial cells.

Cytokine induction in pulmonary airways of horses with heaves and effect of therapy with inhaled fluticasone propionate.

Work in humans and laboratory animals has identified a central role for cytokines and chemokines in development and persistence of lower airway inflammation. The objectives of this study were to determine interleukin (IL)-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha induction in bronchoalveolar lavage (BAL) of control horses and horses with heaves both during remission and exacerbation of the disease, and to determine the effect of therapy with inhaled fluticasone propionate on the cytokine profile of horses with heaves. IL-1 beta and TNF-alpha mRNA expression was significantly higher in horses with heaves after exposure to moldy hay compared to either values obtained during clinical remission or to healthy controls. IL-8 mRNA expression and protein concentrations were significantly higher in horses with heaves than in controls. Both IL-4 and IFN-gamma mRNA expression was increased at various times in heaves-susceptible horses compared to controls. IL-2, IL-5 and IL-10 mRNA expression was not detected in BAL cells of either group. Therapy with inhaled fluticasone propionate after induction of a severe heaves exacerbation resulted in complete resolution of clinical signs, normalization of pulmonary function tests, and significant decrease in BAL neutrophilia. This was associated with a significant decrease in IL-4 mRNA expression and increase in IFN-gamma/IL-4 ratio in horses with heaves. These results demonstrate the clinical efficacy of inhaled fluticasone propionate for the treatment of heaves and suggest a role for cytokines in the development of lower airway inflammation in heaves-susceptible horses.

Increased adenosine concentration in bronchoalveolar lavage fluid of horses with lower airway inflammation.

Several reports have suggested a role for adenosine in the pathogenesis of chronic airway conditions and this has led to new therapeutic strategies to limit airway inflammation. In this study, detectable levels of adenosine in bronchoalveolar lavage (BAL) samples from 11 horses with non-infectious lower-airway inflammation and 14 healthy controls are reported, with significantly higher values in horses with airway inflammation. Although these increased levels did not correlate with changes in neutrophil percentage in BAL, a positive association between adenosine levels and signs of lower airway inflammation (clinical score) was observed. These novel findings support the hypothesis that adenosine may contribute to bronchoconstriction and also act as a pro-inflammatory mediator in the bronchoalveolar milieu of horses with airway inflammation. Further investigation of this axis could lead to new approaches for the treatment of highly prevalent lower airway inflammatory conditions in the horse.

Feline pancreatic islet-like clusters and insulin producing cells express functional Toll-like receptors (TLRs).

Toll-like receptors (TLRs) are cellular receptors that recognize molecules derived from pathogens, endogenous molecules generated after cellular stress, and free fatty acids. TLR activation leads to a proinflammatory reaction that is fundamental in the initiation of an innate immune response and subsequent adaptive responses but also can damage tissues. TLRs are not only expressed within the immune system, but also in most other organ systems including the pancreas. TLR4 is expressed in pancreatic ß-cells of rodents and humans and its stimulation affects insulin secretion in response to glucose. A low-grade inflammation is often associated with disturbed performance of ß-cells and insulin resistance, the cardinal metabolic event of type-2 diabetes. Feline diabetes mellitus shares many similarities with type-2 diabetes in humans. Our objective was to elucidate the role of TLRs in feline pancreatic islets and islet-like clusters (ILC) that consist of islets with their adjacent tissue. We tested whether TLRs are triggered by their agonists and lead to the expression of inflammatory cytokines. We confirmed the expression of all known feline TLRs in pancreas and ILC. Furthermore, stimulation with TLR agonists increased IL-6 mRNA and protein content and the expression of other proinflammatory cytokines indicating a clear proinflammatory response. The reactivity to TLR ligands was stronger in ß-cell enriched populations obtained after sorting by FACS indicating that inflammatory stimuli can also be generated within ß-cells. We conclude that the microenvironment of feline ß-cells harbor the potential for inflammatory reactions, that can be initiated by molecules released from bacteria or viruses or other molecules recognized by TLRs. Therefore infections associated with bacteriemia and viremia can induce inflammation in islets and damage the endocrine pancreatic tissue.

Effect of hyperlipidemia on 11ß-hydroxysteroid-dehydrogenase, glucocorticoid receptor, and leptin expression in insulin-sensitive tissues of cats.

Glucocorticoid (GC) action depends on GC plasma concentration, cellular GC receptor expression, and the pre-receptor hormone metabolism catalyzed by 11ß-hydroxysteroid dehydrogenase (11ß-HSD). 11ß-Hydroxysteroid dehydrogenase exists in 2 isoforms; 11ß-HSD1 converts inactive cortisone to cortisol, and 11ß-HSD2 converts cortisol to cortisone. Increasing evidence in humans and experimental animals suggests that altered tissue cortisol metabolism may predispose to diabetes mellitus (DM). Once DM is established, hyperglycemia and hyperlipidemia may further maintain the abnormal metabolism of cortisol. To gain further insight in this regard, healthy cats were infused for 10 d with lipids (n = 6) or saline (n = 5). At the end of the infusion period, tissue samples from adipose tissue (visceral, subcutaneous), liver, and muscle were collected to determine mRNA expression of 11ß-HSD1, 11ß-HSD2, and GC receptor by real-time reverse-transcriptase polymerase chain reaction; blood samples were collected to determine plasma cortisol and leptin concentrations. Lipid infusion resulted in greater 11ß-HSD1 expression and lower GC receptor expression in visceral and subcutaneous adipose tissue, and lower 11ß-HSD2 expression in visceral adipose tissue and liver. Plasma cortisol did not differ. Leptin and body weight increased in lipid-infused cats. In spite of comparable circulating cortisol levels, up-regulation of 11ß-HSD1 and down-regulation of 11ß-HSD2 expression may result in increased tissue cortisol concentrations in fat depots of hyperlipidemic cats. Down-regulation of GC receptor may represent a self-protective mechanism against increased tissue cortisol levels. In conclusion, hyperlipidemia has a profound effect on 11ß-HSD expression and supports the connection between high lipid concentrations and tissue cortisol metabolism.

Quantitative real-time PCR detection of insulin signalling-related genes in pancreatic islets isolated from healthy cats.

The cat has recently been proposed as a valuable model for type 2 diabetes mellitus (T2DM), because feline diabetes shares several similarities with the disease in humans. Impaired beta-cell function, decreased beta-cell mass, insulin resistance that is often related to obesity, and pancreatic amyloid deposition, are among these common features. In this study, and to further develop the cat as a model of T2DM, feline pancreatic islets were isolated and real-time PCR quantification of mRNA transcripts of genes central to beta-cell function and survival established. In particular, mRNA quantification systems were determined for insulin, the insulin enhancer pancreatic duodenal homeobox-1 (PDX-1), the insulin suppressor CCAAT/enhancer binding protein-beta (C/EBPbeta), glucose transporter isoform 2 (GLUT2), Fas receptor, the caspase-8 inhibitor FLIP (FLICE [caspase-8]-inhibitory protein) and two chemokines, interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1). Pancreatic islets were isolated by collagenase digestion from healthy cat donors. Partial feline mRNA sequences were determined for PDX-1, C/EBPbeta, GLUT2 and FLIP using primers identified from conserved regions of human, dog and rat mRNA. These novel and the previously available sequences (insulin, Fas receptor, IL-8 and MCP-1) were used to design feline-specific primers suitable for real-time PCR in isolated pancreatic islets. The adopted protocol of collagenase digestion yielded pancreatic islets that were frequently surrounded by acinar cells. Quantification of mRNA transcripts was simple and reproducible in healthy cats. Characterisation of genes related to insulin signalling in cats will prove useful to better understand the pathogenesis of feline diabetes and possibly of human T2DM.

Hyperglycaemia but not hyperlipidaemia decreases serum amylase and increases neutrophils in the exocrine pancreas of cats.

The goal of the study was to determine whether hyperglycaemia or hyperlipidaemia causes pancreatitis in cats and to assess the effect of excess serum glucose and lipids on amylase and lipase activity. Ten-day hyperglycaemic and hyperlipidaemic clamps were carried out in five and six healthy cats, respectively. Ten healthy cats received saline and served as controls. The activity of amylase was below the normal range in 4 of 5 hyperglycaemic cats by day 10. The activity of lipase did not vary in any of the cats. Samples of exocrine pancreas were normal on histological examination, but the number of tissue neutrophils was increased in hyperglycaemic cats (P<0.05). In a retrospective study 14 of 40 (35%) cats with naturally occurring diabetes mellitus had amylase activities below the reference range at the time of admission. Amylase activities normalised within 1 week of insulin therapy and subsequent glycaemic control. Lipase activity was increased in 26 of 40 (65%) diabetic cats and remained elevated despite glycaemic control. In conclusion, hyperglycaemia, but not hyperlipidaemia, increases pancreatic neutrophils in cats. However, because the histological morphology of the exocrine pancreas was normal, hyperglycaemia may play only a minor role in the pathogenesis of pancreatitis. Low amylase activities in diabetic cats may reflect an imbalance in glucose metabolism rather than pancreatitis.

Assessment of six different collagenase-based methods to isolate feline pancreatic islets.

Isolation of pancreatic islets is necessary to study the molecular mechanisms underlying beta-cell demise in diabetic cats. Six collagenase-based methods of isolation were compared in 10 cat pancreata, including single and double course of collagenase, followed or not by Ficoll centrifugation or accutase, and collagenase plus accutase. Morphometric analysis was performed to measure the relative area of islet and exocrine tissue. Islet specific mRNA transcripts were quantified in isolates by real-time PCR. The single and double course of collagenase digestion was successful in each cat and provided similar islet-to-exocrine tissue ratio. Quantities of insulin mRNA did not differ between the two methods. However, on histological examination either method yielded only approximately 2% of pure islets. The other methods provided disrupted islets or insufficient samples in 1-7 cats. Although pancreas digestion with single and double course of collagenase was superior, further studies are needed to improve islet isolation in cats.

Malignant catarrhal fever of cattle is associated with low abundance of IL-2 transcript and a predominantly latent profile of ovine herpesvirus 2 gene expression.

BACKGROUND: Malignant catarrhal fever (MCF) is a lethal disease of cattle, characterized by vasculitis, necrosis, and accumulation of activated, dysregulated cytotoxic lymphocytes in various tissues. Ovine gamma herpesvirus 2 (OvHV-2) is a causative agent of MCF, which may trigger the disease through immunopathogenic pathways. Lymphocytes are the main target of the virus. However, the pathogenic basis of the disease is still mysterious. METHODS/FINDINGS: We hypothesized that the gene expression patterns of OvHV-2 and the relative abundances of host cell transcripts in lymphnodes may be used to identify pathways that help to explain the pathogenesis of MCF. Therefore, viral and host cell gene expression patterns in lymph nodes of animals with MCF and healthy controls were analyzed by microarray. Two regions on the viral genome were transcriptionally active, one encoding an orthologue to the latency-associated nuclear antigen (ORF73) of other gamma herpesviruses, the other with no predicted open reading frame. A vast number of transcripts related to inflammatory processes, lymphocyte activation, cell proliferation and apoptosis were detected at different abundances. However, the IL-2 transcript was eminent among the transcripts, which were, compared to healthy controls, less abundant in animals with MCF. The ratio between CD4- and CD8-positive T-lymphocytes was decreased in the lymphnodes of animals with MCF compared to healthy controls. In contrast, the same ratio was stable, when peripheral blood lymphocytes were analyzed. CONCLUSIONS/SIGNIFICANCE: The phenotype of mice with a deficient IL-2-system almost perfectly matches the clinical signs observed in cattle with MCF, which feature a significantly decreased IL-2 transcript abundance, compared to healthy cattle. This supports the hypothesis that immunopathogenic events are linked to the pathogenesis of MCF. IL-2-deficiency may play an important role in the process. Therefore, this work opens new avenues for research on MCF.