Lactate released by inflammatory bone marrow neutrophils induces their mobilization via endothelial GPR81 signaling.

Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81-/- mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection.

Role of fructosamine-3-kinase in protecting against the onset of microvascular and macrovascular complications in patients with T2DM.

INTRODUCTION: Microangiopathic and macroangiopathic complications are the main cause of morbidity and mortality in the diabetic population. Numerous publications have highlighted the role of glycation in the onset of complications of diabetes. In this context, the detection of fructosamine-3-kinase (FN3K)-an enzyme capable of counteracting the effect of hyperglycemia by intervening in protein glycation-has attracted great interest. Several studies have linked FN3K genetic variability to its enzymatic activity and glycated hemoglobin (HbA1c) levels. Here, we investigated the role of FN3K polymorphisms in the development of microvascular and macrovascular complications of diabetes. RESEARCH DESIGN AND METHODS: The anthropometric and biochemical parameters, and any medical history of microangiopathic and macroangiopathic complications, were documented in a sample of 80 subjects with type 2 diabetes. All subjects were screened for FN3K gene and analyzed for the combination of three polymorphisms known to be associated with its enzymatic activity (rs3859206 and rs2256339 in the promoter region and rs1056534 in exon 6). RESULTS: The combination of allelic variants of FN3K polymorphisms resulted in 13 distinct genotypic variants within the cohort. Comparison between genotypes showed no significant differences in terms of demographic, anthropometric and biochemical parameters, risk markers and long-term complications, except for a higher age and vitamin E levels associated with the genotype presenting GG at position -385, TT at position -232, and CC at c.900 A. Evaluating the microangiopathic and macroangiopathic complications as a whole, we found that they appeared significantly less present in this genotype compared with all other genotypes (p=0.0306). CONCLUSIONS: The group of patients carrying the favorable allele for the three polymorphisms of the FN3K gene revealed less severe microangiopathy and macroangiopathy, suggesting a protective role of this genotype against the onset of the complications of diabetes.

Daily Onset of Light and Darkness Differentially Controls Hematopoietic Stem Cell Differentiation and Maintenance.

Hematopoietic stem and progenitor cells (HSPCs) tightly couple maintenance of the bone marrow (BM) reservoir, including undifferentiated long-term repopulating hematopoietic stem cells (LT-HSCs), with intensive daily production of mature leukocytes and blood replenishment. We found two daily peaks of BM HSPC activity that are initiated by onset of light and darkness providing this coupling. Both peaks follow transient elevation of BM norepinephrine and TNF secretion, which temporarily increase HSPC reactive oxygen species (ROS) levels. Light-induced norepinephrine and TNF secretion augments HSPC differentiation and increases vascular permeability to replenish the blood. In contrast, darkness-induced TNF increases melatonin secretion to drive renewal of HSPCs and LT-HSC potential through modulating surface CD150 and c-Kit expression, increasing COX-2/αSMA+ macrophages, diminishing vascular permeability, and reducing HSPC ROS levels. These findings reveal that light- and darkness-induced daily bursts of norepinephrine, TNF, and melatonin within the BM are essential for synchronized mature blood cell production and HSPC pool repopulation.

VLA-4 Affinity Assay for Murine Bone Marrow-derived Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) are defined by their functional ability to self-renew and to differentiate into all blood cell lineages. The majority of HSC reside in specific anatomical locations in the bone marrow (BM) microenvironment, in a quiescent non motile mode. Adhesion interactions between HSCs and their supporting BM microenvironment cells are critical for maintaining stem cell quiescence and protection from DNA damaging agents to prevent hematology failure and death. Multiple signaling proteins play a role in controlling retention and migration of bone marrow HSCs. Adhesion molecules are involved in both processes regulating hematopoiesis and stem- and progenitor-cell BM retention, migration and development. The mechanisms underlying the movement of stem cells from and to the marrow have not been completely elucidated and are still an object of intense study. One important aspect is the modification of expression and affinity of adhesion molecules by stem and progenitor cells which are required both for stem cell retention, migration and development. Adhesion is regulated by expression of the adhesion molecules, their affinity and avidity. Affinity regulation is related to the molecular binding recognition and bond strength. Here, we describe the in vitro FACS assay used in our research to explore the expression, affinity and function of the integrin α4ß1 (also termed VLA-4) for murine bone marrow retained EPCR+ long term repopulation HSC (LT-HSC) (Gur- Cohen et al., 2015 ).

Adhesion Assay for Murine Bone Marrow Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) are defined by their functional abilities to self-renew and to give rise to all mature blood and immune cell types throughout life. Most HSCs are retained in a non-motile quiescent state within a specialized protective microenvironment in the bone marrow (BM) termed the niche. HSCs are typically distinguished from other adult stem cells by their motility capacity. Movement of HSCs across the physical barrier of the marrow extracellular matrix and blood vessel endothelial cells is facilitated by suppression of adhesion interactions, which are essential to preserve the stem cells retained within their BM niches. Importantly, homing of HSCs to the BM following clinical transplantation is a crucial first step for the repopulation of ablated BM as in the case of curative treatment strategies for hematologic malignancies. The homing process ends with selective access and anchorage of HSCs to their specialized niches within the BM. Adhesion molecules are targets to either enhance homing in cases of stem cell transplantation or reduce BM retention to harvest mobilized HSCs from the blood of matched donors. A major adhesion protein which is functionally expressed on HSCs and is involved in their homing and retention is the integrin alpha4beta1 (Very late antigen-4; VLA4). In this protocol we introduce an adhesion assay optimized for VLA4 expressing murine bone marrow stem cells. This assay quantifies adherent HSCs by flow cytometry with HSC enriching cell surface markers subsequent to the isolation of VLA4 expressing adherent cells.

Possible role of fructosamine 3-kinase genotyping for the management of diabetic patients.

Diabetes mellitus is a global pandemic and continues to increase in numbers and significance. Several pathogenic processes are involved in the development of such disease and these mechanisms could be influenced by genetic, epigenetic and environmental factors. Non-enzymatic glycation reactions of proteins have been strongly related to pathogenesis of chronic diabetic complications. The identification of fructosamine 3-kinase (FN3K), an enzyme involved in protein deglycation, a new form of protein repair, is of great interest. FN3K phosphorylates fructosamines on the third carbon of their sugar moiety, making them unstable and causing them to detach from proteins, suggesting a protective role of this enzyme. Moreover, the variability in FN3K activity has been associated with some polymorphisms in the FN3K gene. Here we argue about genetic studies and evidence of FN3K involvement in diabetes, together with results of our analysis of the FN3K gene on a Caucasian cohort of diabetic patients. Present knowledge suggests that FN3K could act in concert with other molecular mechanisms and may impact on gene expression and activity of other enzymes involved in deglycation process.

Lack of relationship between the P413L chromogranin B variant and a SALS Italian cohort.

Chromogranins were reported to interact specifically with mutant forms of superoxide dismutase that are linked to amyotrophic lateral sclerosis (ALS). Particularly, a variation c.1238C>T (p.Pro413Leu) in the chromogranin B gene, CHGB, has been associated with an earlier age at onset in both familial and sporadic ALS in French/French-Canadian populations studied. The aim of our study was to evaluate the P413L chromogranin variation in Italian patients with sporadic ALS. The study included 366 Italian patients with sporadic ALS and 382 control subjects. Genotyping of the polymorphism P413L in the CHGB gene was performed and the clinical characteristics of patients were analyzed in relation to their genotype. Our study on a cohort of Italian patients with SALS and controls failed to confirm an increased frequency of the 413L variant in SALS patients. Furthermore, we did not confirm the previous observation of a difference of age at onset between T-allele carriers and non-carriers (median age of onset 58.5 vs. 60.2years of age, respectively). Our findings do not support the 413L variant as a risk factor for sporadic ALS in the Italian population.

PDCD10 gene mutations in multiple cerebral cavernous malformations.

Cerebral cavernous malformations (CCMs) are vascular abnormalities that may cause seizures, intracerebral haemorrhages, and focal neurological deficits. Familial form shows an autosomal dominant pattern of inheritance with incomplete penetrance and variable clinical expression. Three genes have been identified causing familial CCM: KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3. Aim of this study is to report additional PDCD10/CCM3 families poorly described so far which account for 10-15% of hereditary cerebral cavernous malformations. Our group investigated 87 consecutive Italian affected individuals (i.e. positive Magnetic Resonance Imaging) with multiple/familial CCM through direct sequencing and Multiplex Ligation-Dependent Probe Amplification (MLPA) analysis. We identified mutations in over 97.7% of cases, and PDCD10/CCM3 accounts for 13.1%. PDCD10/CCM3 molecular screening revealed four already known mutations and four novel ones. The mutated patients show an earlier onset of clinical manifestations as compared to CCM1/CCM2 mutated patients. The study of further families carrying mutations in PDCD10/CCM3 may help define a possible correlation between genotype and phenotype; an accurate clinical follow up of the subjects would help define more precisely whether mutations in PDCD10/CCM3 lead to a characteristic phenotype.

De novo MGC4607 gene heterozygous missense variants in a child with multiple cerebral cavernous malformations.

Cavernous malformations are angiographically occult, low-pressure neurovascular lesions with distinct imaging and clinical characteristics; main clinical manifestations are seizure, focal neurological deficits and epileptic attacks. Here we describe the molecular characterization of an Italian child, a symptomatic patient, affected by multiple cerebral cavernous malformations, without a family history of the disease and harbouring a new MGC4607 gene mutation. We identified two de novo missense variants in exon 6 of the gene both present on the same allele (cis configuration). DNA analysis for KRIT1, and PDCD10 gene variation through direct sequencing and MLPA analysis excluded further mutations. STR multiplex assay, allele-specific analysis and DHPLC analysis were performed for a better genetic characterization. Our findings emphasize the importance of the genetic test in subjects presenting multiple cerebral cavernomas for an adequate counselling, as well as for disease management since early identification of genetic abnormalities enable patients to have their lesions removed before they haemorrhage and cause deficit and/or epilepsy.

Wide phenotypic spectrum of the TARDBP gene: homozygosity of A382T mutation in a patient presenting with amyotrophic lateral sclerosis, Parkinson's disease, and frontotemporal lobar degeneration, and in neurologically healthy subject.

Mutations in the TARDBP gene are described as a cause of autosomal dominant amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) with or without motor neuron involvement, and, recently, Parkinson's disease (PD). We hereby describe a family presenting the A382T mutation; two subjects were in the homozygous state, and two were in the heterozygous state. The index case, carrying the A382T mutation in the homozygous state, had an 8-year history of sporadic PD and 6 years later developed ALS and FTLD; his brother, carrying the same mutation in the homozygous state, and the other two family member carriers of the same mutation in the heterozygous state were without neurological signs and symptoms. This family confirms that mutation in transactive response (TAR)-DNA-binding protein 43 (TDP43), both the homozygous and the heterozygous state, may be found in subjects with different clinical conditions ranging from neurological disease to non-neurological disease. In addition, the aforementioned findings add to the debate for the ethical and psychological dilemmas about genetic counseling.

Mutation in the senataxin gene found in a patient affected by familial ALS with juvenile onset and slow progression.

We report an Italian male with juvenile onset familial disease characterized by progressive weakness and wasting of four limbs and prolonged survival. Diagnostic work-up revealed the diffuse involvement of central and peripheral motor neurons. Genetic analysis revealed a L389S mutation in the senataxin (SETX) gene.

Phenotypic heterogeneity in a SOD1 G93D Italian ALS family: an example of human model to study a complex disease.

We report different clinical expression in seven members of a large family with amyotrophic lateral sclerosis (ALS) and the G93D mutation in exon 4 of the Cu/Zn superoxide dismutase (SOD1) gene. The ALS clinical course in the proband showed an unusually fast progression of the disease compared to the paucisymptomatic presentation associated to this mutation in the two previously Italian families described. The remaining mutation carriers did not show the aggressive clinical course displayed by the proband. We selected few genes known to be ALS modifiers searching for genetic variants that could explain the wide phenotypic diversity within the family. Exclusion of causative genes such as TDP43, FUS, PGRN and VAPB was performed too. We believe that this kind of family with contrasting phenotypes of ALS may be considered an excellent human model to study the relationship between a wider genetic profile, including modifier genes, and the clinical expression of the disease. Therefore, the novelty of our approach is also represented by the study of a single family to reproduce a composite structure in which search for possible modifier genes/genetic variants linked to SOD1 mutated.