Integrin function and regulation in development

Integrins are a large family of membrane receptors, consisting of alpha and beta subunits, that play a pivotal role in the interaction of cells with the extracellular matrix. Such interaction regulates the organization of cells in organs and tissues during development as well as cell differentiation and proliferation. We have shown that unfertilized oocytes express integrins that might be important during fertilization. We also analyzed nervous system and muscle tissue development showing that integrin expression is precisely regulated during organization of these tissues. The results indicate that two distinct integrin alpha subunits mediate the outgrowth of processes in nerve and glial cells. Alpha1 integrin, a laminin receptor, is up-regulated by nerve growth factor and other differentiation stimuli and is involved in neurite extension by nerve cells. In contrast, process extension by glial cells is likely to involve the alphaV integrin. Moreover, the latter integrin subunit is also transiently expressed in muscle of the embryo body where it localizes predominantly at developing myotendinous junctions. After birth this integrin disappears and is substituted by the alpha7 subunit. At the same time, important changes also occur in the expression of the associated beta subunit. In fact, the beta1A isoform which is expressed in fetal muscles, is substituted by beta1D. These isoforms are generated by alternative splicing and differ in only a few amino acid residues at the COOH terminus of the protein. This region of the molecule is exposed at the cytoplasmic face of the plasma membrane and is connected to the actin filaments. Our results show that beta1D, which is expressed only in striated muscle tissues, binds to both cytoskeletal and extracellular matrix proteins with an affinity higher than beta1A. Thus, beta1D provides a stronger link between the cytoskeleton and extracellular matrix necessary to support mechanical tension during muscle contraction. These results indicate that cells can regulate their interactions with the extracellular matrix by changing their expression of alpha integrin subunits and thus ligand specificity, or by more subtle changes involving alternative usage of different cytoplasmic domains. The important role of both alpha and beta integrin subunit cytoplasmic domains during development is further illustrated by the analysis of targeted mutations which we have generated by homologous recombination in mice.     

Detection of Brucella canis in a dog in Italy

Brucella spp. is a worldwide zoonotic pathogen. Infection by Brucella canis in dogs is endemic in the Southern USA and in Central and South America, but it appears sporadically in other parts of the world, including Europe. Tissue samples from a dog with chronic prostatitis, discospondylitis and locomotor problems were subjected to clinical and laboratory examinations. B. canis was detected by PCR in biological fluids and tissues of the animal, while antibodies to B. canis were found in the serum, providing additional strong evidence for the circulation of B. canis in Italy.     

Therapeutic Efficacy and Immunological Response of CCL5 Antagonists in Models of Contact Skin Reaction

Skin-infiltrating T-cells play a predominant role in allergic and inflammatory skin diseases such as atopic dermatitis, psoriasis and allergic contact dermatitis. These T-cells are attracted by several chemotactic factors including the chemokine CCL5/RANTES, a CC chemokine inducing both the migration and activation of specific leukocyte subsets. CCL5 has been found to be associated with various cell-mediated hypersensitive disorders such as psoriasis, atopic dermatitis and irritant contact dermatitis. We have used two antagonists, the first, Met-CCL5, a dual CCR1/CCR5 antagonist and the second, a variant in which GAG binding is abrogated, ⁴⁴AANA⁴⁷-CCL5, which acts as a dominant negative inhibitor of CCL5. The antagonists were tested in two models of contact skin reaction. The first, irritant contact dermatitis (ICD) is a pathological non-specific inflammatory skin condition arising from the release of pro-inflammatory cytokines by keratinocytes in response to haptens, usually chemicals. The second, contact hypersensitivity (CHS) is a T-cell dependent model, mimicking in part the T-cell-mediated skin diseases such as psoriasis. In both models, the CCL5 antagonists showed therapeutic efficacy by reducing swelling by 50% as well as the reduction of soluble mediators in homogenates derived from challenged ears. These results demonstrate that blocking the receptor or the ligand are both effective strategies to inhibit skin inflammation.     

Environmental implications of skeletal micro-density and porosity variation in two scleractinian corals

The correlations between skeletal parameters (bulk density, micro-density and porosity), coral age and sea surface temperature were assessed along a latitudinal gradient in the zooxanthellate coral Balanophyllia europaea and in the azooxanthellate coral Leptopsammia pruvoti. In both coral species, the variation of bulk density was more influenced by the variation of porosity than of micro-density. With increasing polyp age, B. europaea formed denser and less porous skeletons while L. pruvoti showed the opposite trend, becoming less dense and more porous. B. europaea skeletons were generally less porous (more dense) than those of L. pruvoti, probably as a consequence of the different habitats colonized by the two species. Increasing temperature had a negative impact on the zooxanthellate species, leading to an increase of porosity. In contrast, micro-density increased with temperature in the azooxanthellate species. It is hypothesized that the increase in porosity with increasing temperatures observed in B. europaea could depend on an attenuation of calcification due to an inhibition of the photosynthetic process at elevated temperatures, while the azooxanthellate species appears more resistant to variations of temperature, highlighting possible differences in the sensitivity/tolerance of these two coral species to temperature changes in face of global climate change.     

Exercise intensity and pacing strategy of a 5-km indoor race walk during a World Record attempt: a case study

The aim of this case study was to describe the physiological and regulatory processes, by means of heart rate (HR) monitoring and pacing strategy, in a top-level race walker (age: 32 years; height: 1.76 m; body mass: 62 kg; training volume: 130-150 km·wk) who was focused on the attainment of the 5-km indoor race walk (RW) World Record. The HRmean was 185 ± 14.9 b·min, with an HRmean/HRmax ratio of 0.96. Almost the whole race (91.8%) was performed to an intensity ≥90% of the HRmax; lower intensity work was negligible (8.1%). The race profile was a reverse J-shaped pacing curve; in fact, the athlete completed the first 1,000 m in the fastest time, slowing during the middle 3,000 m, and increasing the speed during the final 1,000 m of the race. Despite the attempt failed (the athlete performed only the 2009 World leading performance, 18 minutes 23 seconds 47 tenths), these data suggest that a more linear strain distribution for the entire performance would be optimal instead of a fast-start strategy, which leads to a drastic decrement of the walking velocity. Moreover, this study supports the use of HR monitoring combined with the regulation of the effort to understand the physiological and regulatory processes during an indoor RW event.     

Endothelin-1 response to mental stress in early ischemic lesions of the extremities due to systemic sclerosis

We studied circulating levels of endothelin-1, catecholamines and nitric oxide after a mental arithmetic test in 14 patients with early ischemic lesions of the extremities due to systemic sclerosis and slightly impaired peripheral vascular flow. The test induced an increase (P < 0.01) in blood pressure, heart rate, endothelin-1 and catecholamine levels, whereas it did not change the low basal levels of nitric oxide. In healthy subjects (n = 20) the test significantly (P < 0.01) decreased endothelin-1 without affecting nitric oxide. The low basal levels of nitric oxide and the high plasma concentration of endothelin-1 after psychological stress cannot be explained by an impaired release from the limited ischemic lesions alone. This suggests a diffuse microvascular derangement that aggravates the course of peripheral microvascular ischemic lesions.     

Genetic polymorphisms of Fas (CD95) and Fas ligand (CD178) influence the rise in CD4+ T cell count after antiretroviral therapy in drug-naïve HIV-positive patients

Fas and Fas ligand (FasL) are the main genes that control cell death in the immune system. Indeed, they are crucial for the regulation of T lymphocyte homeostasis because they can influence cell proliferation. A strong debate exists on the importance of Fas/FasL system during HIV infection, which is characterized by the loss of CD4+ T cells directly, or indirectly, caused by the virus. To investigate whether the genetic background of the host plays a role in the immunoreconstitution, we studied the influence of different Fas and FasL polymorphisms on CD4+ T lymphocyte count and plasma viral load following initiation of highly active antiretroviral therapy (HAART) in drug-naïve HIV+ patients. We studied 131 individuals, who were compared to 136 healthy donors. Statistical analysis was performed by using X ² test, Fischer's Exact Test, and analysis for repeated measurements. The group of HIV+ patients had an unexpected lower frequency of FasLnt169 polymorphism (delT allele) than healthy controls (p=0.039). We then observed no significant differences in the immune reconstitution, in terms of CD4+ T cell increase, when the influence of single alleles of the gene Fas or FasL was considered. However, the combination of some polymorphisms of Fas or FasL significantly influenced CD4+ T cell production and viral load decrease, showing that these genes can play a role in the immunoreconstitution triggered by antiretroviral therapy.     

4-hydroxynonenal and TGF-beta1 concur in inducing antiproliferative effects on the CaCo-2 human colon adenocarcinoma cell line

4-Hydroxynonenal (HNE) has been demonstrated to exert its antiproliferative effect by up-regulating the c-Jun-N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family (MAPKs). Transforming growth factor-beta1 (TGF-beta1) is the major negative regulatory factor in controlling cell proliferation, and Smads are its intracellular transducers. Recent data on human colon adenocarcinoma has shown a low HNE content paralleled by a marked alteration of TGF-beta1 levels within the tumor mass. The two events appear related because of the demonstrated marked ability of HNE to up-regulate expression and synthesis of TGF-beta1; the combined decreases of HNE and TGF-beta1 found in cancer cells provide a favorable condition for neoplastic progression. Furthermore, HNE is likely able to interact with the cytokine to enhance apoptosis and increase intracellular reactive oxygen species (ROS) formation in the CaCo-2 colon carcinoma cell line. The probable mechanism whereby HNE and TGF-beta1 interact to induce apoptosis is through cross-talk between the main signaling pathways of the two molecules (JNK and Smads), and the observed ROS production might only contribute to amplifying the apoptotic pathways. The network between the two signaling pathways here involved is now under investigation.     

Oxysterol-induced up-regulation of MCP-1 expression and synthesis in macrophage cells

To investigate the proinflammatory potential of cholesterol and cholesterol oxidation products (oxysterols), which are present in oxidized low-density lipoproteins, foam cells, and fibrotic plaque, we used an in vitro model mimicking the challenge of macrophage cells by the cholesterol accumulating within the central core of atheroma. A biologically representative oxysterol mixture was shown to be potentially able to sustain a chronic inflammatory process within the vascular wall by up-regulating the expression of defined proinflammatory genes. In particular, expression and synthesis of the major chemokine for monocytes/macrophages, namely monocyte chemotactic protein-1 (MCP-1), were consistently increased when cells of the macrophage lineage (U937 cell line) were incubated with this mixture. On the contrary, an identical concentration of unoxidized cholesterol in no case modified expression or synthesis of the chemokine. Up-regulated expression and synthesis of MCP-1 by the oxysterol mixture was clearly dependent on a net increment of phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor kappaB (NF-kappaB) nuclear binding. The results indicate that cholesterol may contribute to the progression of atherosclerotic lesions by strongly up-regulating crucial proinflammatory factors like MCP-1, but only after having been oxidized to oxysterols.