Michela Cassanello

Arginase 2 is expressed by human lung cancer, but it neither induces immune suppression, nor affects disease progression

In human prostate cancer, Arginase 2 (ARG2) and nitric oxide synthase (NOS) are concomitantly expressed by tumor cells, and induce tumor immune escape via peroxynitrite‐dependent Tyrosine nitrosylation. Since there were no data regarding this immune suppressive mechanism in other tumor types, and an evaluation of its clinical relevance in human tumors had still to be provided, we have investigated presence and clinical relevance of ARG2 and NOS expression in lung cancer. No evidence of NOS expression was found, no significant NOS enzymatic activity was detected. Instead, ARG2 protein was expressed by tumor cells. In a cohort of 120 patients, the amount of ARG2‐positive tumor cells was significantly higher in small cell lung cancers (SCLC) than in non‐small cell lung cancers (NSCLC). Large cell undifferentiated carcinomas had twice ARG2 than the other NSCLC subtypes. ARG2 expression was increased in Grade 3 tumors, as compared to Grades 1 and 2. However, no relationship was found with tumor size and stage, and with patient survival. Indeed, the enzyme was active, since the Arginine catabolite Ornithine was produced, but Arginine depletion was not attained. In addition, nitrotyrosine was not found in tumor tissue. Accordingly, when tumor cells isolated from lung cancer were incubated with activated autologous T cells, no inhibition of proliferation was detected. Our results indicate that ARG2 is expressed in lung cancer, but it does not induce tumor immune escape and does not affect disease progression, most probably due to the lack of concomitant NOS expression.

Albuminuria and glomerular damage in mice lacking the metabotropic glutamate receptor 1

The metabotropic glutamate (mGlu) receptor 1 (GRM1) has been shown to play an important role in neuronal cells by triggering, through calcium release from intracellular stores, various signaling pathways that finally modulate neuron excitability, synaptic plasticity, and mechanisms of feedback regulation of neurotransmitter release. Herein, we show that Grm1 is expressed in glomerular podocytes and that a glomerular phenotype is exhibited by Grm1(crv4) mice carrying a spontaneous recessive inactivating mutation of the gene. Homozygous Grm1(crv4/crv4) and, to a lesser extent, heterozygous mice show albuminuria, podocyte foot process effacement, and reduced levels of nephrin and other proteins known to contribute to the maintenance of podocyte cell structure. Overall, the present data extend the role of mGlu1 receptor to the glomerular filtration barrier. The regulatory action of mGlu1 receptor in dendritic spine morphology and in the control of glutamate release is well acknowledged in neuronal cells. Analogously, we speculate that mGlu1 receptor may regulate foot process morphology and intercellular signaling in the podocyte.

Why do premature newborn infants display elevated blood adenosine levels

Our preliminary data show high levels of adenosine in the blood of very low birth weight (VLBW) infants, positively correlating to their prematurity (i.e. body weight class). This prompted us to look for a mechanism promoting such impressive adenosine increase. We hypothesized a correlation with oxygen challenge. In fact, it is recognized that either oxygen lack or its excess contribute to the pathogenesis of the injuries of prematurity, such as retinopathy (ROP) and periventricular white matter lesions (PWMI). The optimal concentration of oxygen for resuscitation of VLBW infants is currently under revision. We propose that the elevated adenosine blood concentrations of VLBW infants recognizes two sources. The first could be its activity-dependent release from unmyelinated brain axons. Adenosine in this respect would be an end-product of the hypometabolic VLBW newborn unmyelinated axon intensely firing in response to the environmental stimuli consequent to premature birth. Adenosine would be eventually found in the blood due to blood-brain barrier immaturity. In fact, adenosine is the primary activity-dependent signal promoting differentiation of premyelinating oligodendrocyte progenitor cells (OPC) into myelinating cells in the Central Nervous System, while inhibiting their proliferation and inhibiting synaptic function. The second, would be the ecto-cellular ATP synthesized by the endothelial cell plasmalemma exposed to ambient oxygen concentrations due to premature breathing, especially in lung. ATP would be rapidly transformed into adenosine by the ectonucleotidase activities such as NTPDase I (CD39), and NT5E (CD73). An ectopic extra-mitochondrial aerobic ATP synthetic ability was reported in many cell plasma-membranes, among which endothelial cells. The potential implications of the cited hypotheses for the neonatology area would be great. The amount of oxygen administration for reviving of newborns would find a molecular basis for its assessment. VLBW infants may be regarded as those in which premature exposure to ambient oxygen concentrations and oxidative stress causes a premature functioning of the extra-mitochondrial oxidative phosphorylation primarily in axons and endothelium. Adenosine may become a biomarker of prematurity risk, whose implications further studies may assess.

Validity of β-D-glucosidase activity measured in dried blood samples for detection of potential Gaucher disease patients.

OBJECTIVES Gaucher disease (GD) diagnosis relies on the demonstration of deficient β-D-glucosidase (GBA) activity in cellular homogenates. Diagnosis process, however, can be delayed as (i) some GD symptoms are non-specific; and (ii) diagnostic tests are performed in specialized laboratories. These difficulties negatively impact on timely access of patients to therapy. GBA assay in dried blood spots (DBS) represents a method facilitating early identification of patients who will be finally diagnosed with gold standard assay of nucleated cells. Aim of this study is to investigate the DBS analytical performance compared with gold standard method. DESIGN & METHODS A cross-sectional study started by comparing data of 50 DBS and 50 homogenate samples from the same subjects (25 known-GD and 25 controls). The subsequent phase examined 443 DBS samples. Along with these, 73 blood samples were sent for leukocyte separation and/or EBV-lymphoblast cell lines, and 1 skin biopsy for fibroblast cell lines. Overall the study included a total of 493 subjects. RESULTS While the results from this first validation group did not yield false positive/negative values, when the analysis was extended to 443 DBS, 14.4% (64 samples) of positive results was yielded. Among these, only 15 were confirmed as GD values with gold standard test. In addition, a thorough examination of some clinical data also revealed 2 false negative results which were confirmed by both enzymatic and molecular analyses. CONCLUSIONS DBS test could be useful as screening method although with cautions, whereas the standardized GBA assay should remain the gold standard for laboratory diagnosis of Gaucher disease.

Modulation of microglial activation by adenosine A2a receptor in animal models of perinatal brain injury

Neuroinflammation has a key role in the pathogenesis of perinatal brain injury. Caffeine, a nonspecific antagonist of adenosine receptors (ARs), is widely used to treat apnea of prematurity and has been linked to a decrease in the incidence of cerebral palsy in premature infants. The mechanisms explaining its neuroprotective effect have not yet been elucidated. The objective of this study was to characterize the expression of adenosine and ARs in two neonatal rat models of neuroinflammation and to determine the effect of A2aR blockade on microglial activation assessed through inflammatory cytokine gene expression. We have used two rat models of microglial activation: the gestational low protein diet (LPD) model, associated with chronic brain injury, and postnatal ibotenate intracerebral injections, responsible for acute excitotoxicity injury. Adenosine blood levels have been measured by Tandem Mass Spectrometry. The expression of ARs in vivo was assessed using qPCR and immunohistochemistry. In vivo models have been replicated in vitro on primary microglial cell cultures exposed to A2aR agonist CGS-21680 or antagonist SCH-58261. The effects of these treatments have been assessed on the M1/M2 cytokine expressions measured by RT-qPCR. LPD during pregnancy was associated with higher adenosine levels in pups at postnatal day 1 and 4. A2aR mRNA expression was significantly increased in both cortex and magnetically sorted microglial cells from LPD animals compared to controls. CD73 expression, responsible for extracellular production of brain adenosine, was significantly increased in LPD cortex and sorted microglia cells. Moreover, CD73 protein level was increased in ibotenate treated animals. In vitro experiments confirmed that LPD or control microglial cells exposed to ibotenate display an increased expression, at both protein and molecular levels, of A2aR and M1 markers (IL-1β, IL-6, iNOS, TNFα). This pro-inflammatory profile was significantly reduced by SCH-58261, which reduces M1 markers in both LPD and ibotenate-exposed cells, with no effect on control cells. In the same experimental conditions, a partial increased of M1 cytokines was observed in response to A2aR agonist CGS-21680. These results support the involvement of adenosine and particularly of its receptor A2aR in the regulation of microglia in two different animal models of neuroinflammation.

Transgenic mice overexpressing arginase 1 in monocytic cell lineage are affected by lympho-myeloproliferative disorders and disseminated intravascular coagulation.

Arginase (ARG) is a metabolic enzyme present in two isoforms that hydrolyze l-arginine to urea and ornithine. In humans, ARG isoform 1 is also expressed in cells of the myeloid lineage. ARG activity promotes tumour growth and inhibits T lymphocyte activation. However, the two ARG transgenic mouse lines produced so far failed to show such effects. We have generated, in two different genetic backgrounds, transgenic mice constitutively expressing ARG1 under the control of the CD68 promoter in macrophages and monocytes. Both heterozygous and homozygous transgenic mice showed a relevant increase in mortality at early age, compared with wild-type siblings (67/267 and 48/181 versus 8/149, respectively, both P < 0.005). This increase was due to high incidence of haematologic malignancies, in particular myeloid leukaemia, myeloid dysplasia, lymphomas and disseminated intravascular coagulation (DIC), diseases that were absent in wild-type mice. Atrophy of lymphoid organs due to reduction in T-cell compartment was also detected. Our results indicate that ARG activity may participate in the pathogenesis of lymphoproliferative and myeloproliferative disorders, suggest the involvement of alterations of L-arginine metabolism in the onset of DIC and confirm a role for the enzyme in regulating T-cell homeostasis.