Vedolizumab for the treatment of Crohn's disease.

INTRODUCTION: Crohn's disease (CD) is an immune-mediated condition characterized by inflammation of the gut tissue, associated with progressive damage of the affected intestinal tract and possible complications. A treat-to-target approach is strongly advocated, consisting of early and aggressive inflammatory control. However, a great proportion of affected subjects lack response or are intolerant to conventional therapy. Even though the first-line biologic therapy targeting tumor necrosis factor-alfa (TNF-α) is associated with improvement of inflammation in some patients, others do not respond at first or lose response over time. These findings brought about the possibility of different mechanisms being involved in perpetuating the chronic inflammatory state. Novel drugs targeting different inflammatory pathways have been studied in CD, specifically addressed to leucocyte trafficking. Areas covered: We aim to review the relevant data available in the literature and briefly summarize the efficacy and safety profile of vedolizumab in the treatment of CD. Expert commentary: Vedolizumab has shown, from pivotal and real-life data, significant clinical benefit among CD patients, in addition to a singular safety profile. Future studies will provide helpful data concerning its use in special situations.

Flow Cytometry-Defined CD49d Expression in Circulating T-Lymphocytes Is a Biomarker for Disease Progression in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) affects 1:3500-1:5000 male births, and is caused by X-linked mutations in the dystrophin gene, manifested by progressive muscle weakness and wasting due to the absence of dystrophin protein, leading to degeneration of skeletal muscle. DMD patients are clinically heterogeneous and the functional phenotype often cannot be correlated with the genotype. Therefore, defined reliable noninvasive biomarkers aiming at predicting if a given DMD child will progress more or less rapidly will be instrumental to better design inclusion of defined patients for future therapeutic assays. We recently showed that CD49d expression levels in blood-derived T-cell subsets can predict disease progression in DMD patients. Herein we describe in detail the methodology to be applied for defining, through four-color flow cytometry, the membrane expression levels of the CD49d (the α4 chain of the integrins α4ß1 and α4ß7) in circulating CD4+ and CD8+ T cell subsets. Since we have also shown that this molecule can also be placed as a potential target for therapeutics in DMD, we also describe the cell migration functional assay that can be applied to test potential CD49d inhibitors that can modulate their ability to cross endothelial or extracellular matrix (ECM) barriers.

Altered red cell and platelet adhesion in hemolytic diseases: Hereditary spherocytosis, paroxysmal nocturnal hemoglobinuria and sickle cell disease.

OBJECTIVES: Intravascular hemolysis may have important pathophysiological consequences, such as the induction of cellular adhesion and vasculopathy. We compared the adhesive properties of red cells (RBC) and platelets in hereditary spherocytosis (HS), paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease (SCD) patients. DESIGN AND METHODS: The adhesion of RBC and platelets, from patients and healthy subjects, was determined using static adhesion assays. RBC surface markers were characterized by flow cytometry and lactate dehydrogenase (LDH), plasma hemoglobin (pHb) and TNF-α were assayed in serum/plasma samples. RESULTS: pHb levels were elevated in all three hemolytic diseases, indicating the incidence of intravascular hemolysis. RBC adhesion and TNF-α were augmented in HS and SCD, but not in PNH. Reticulocyte counts were raised in the three diseases, but were higher in HS and SCD than in PNH; high expressions of CD71, CD36 and CD49d were observed on SCD RBC, while CD71 alone was increased on HS and PNH RBC. Splenectomy was associated with reversals of increased pHb, RBC adhesion, reticulocytes, RBC marker expression and inflammation in HS. In contrast, platelet adhesion was elevated in SCD and PNH, but not HS. Platelet adhesion correlated significantly with serum LDH, but not pHb, in the hemolytic disease cohort; interestingly, LDH did not correlate with reticulocytes or pHb levels. CONCLUSIONS: Results indicate that extravascular, rather than intravascular, hemolysis (and ensuing RBC production) may contribute to elevations in RBC adhesive properties in HS and SCD, while mechanisms peculiar to each disease may augment platelet adhesion in SCD and PNH.

Immunomodulatory drugs (natalizumab), worsening of multiple sclerosis, rebound effect and similitude.

BACKGROUND: Homeopathic treatment is based on the principle of similitude ('like cures like') administering to sick individuals substances that cause similar symptoms in healthy individuals, employing the paradoxical or biphasic action of the organism as therapeutic response. This homeostatic, vital or secondary action of the organism is scientifically explained by the rebound effect of drugs, resulting in worsening of symptoms after enantiopathic treatment withdrawal. Natalizumab reduces relapses in patients with active multiple sclerosis (MS), but recent studies report severe worsening of MS after suspension of treatment, as a consequence of the rebound effect. METHOD: Extending this source of evidence, this work reviews research that demonstrates secondary worsening of MS after discontinuation of natalizumab, a human monoclonal antibody that suppresses the disease inflammatory activity as primary action. RESULTS: Several studies refer to the immune reconstitution inflammatory syndrome (IRIS) as a plausible explanation of reactivation of MS after withdrawal of natalizumab: a rebound effect or secondary action of the organism in response to the primary immunosuppression caused by the drug. CONCLUSION: Relapses of MS after discontinuation of natalizumab treatment indicate rebound of disease activity, supporting the homeopathic principle and warning healthcare professionals about this serious iatrogenic event.

Molecular evolution of α4 integrin binding site to lentiviral envelope proteins in new world primates.

Integrin epitopes encoded by ITGA4 exons 5 and 6 encompass the α4ß7 binding site to natural ligands and HIV-1 gp120. Functional assays of α4 variants of new world primates (NWP) showed reduced binding of several ligands, including the HIV-1 envelope, probably accounting for restriction phenotypes conferring resistance to lentiviral infection (Darc et al., 2011). In this paper, we have analyzed, by cloning and sequencing, the α4 domain polymorphisms present in 10 NWP species and four old world primates (including human). Analyses of differential selection at codon sites and along evolutionary lineages were carried out. We identified codons under positive selection, including polymorphic variations at codon 201, presumably convergent during NWP radiation and significant positive selection leading to a single allele (SagVar2).

Expression analysis of wound healing genes in human periapical granulomas of progressive and stable nature.

INTRODUCTION: Wound healing process involves the activation of extracellular matrix components, remodeling enzymes, cellular adhesion molecules, growth factors, cytokines and chemokines genes. However, the molecular patterns underlying the healing process at the periapical environment remain unclear. Here we hypothesized that endodontic infection might result in an imbalance in the expression of wound healing genes involved in the pathogenesis of periapical lesions. Furthermore, we suggest that differential expression of wound healing markers in active and latent granulomas could account for different clinical outcomes for such lesions. METHODS: Study samples consisted of 93 periapical granulomas collected after endodontic surgeries and 24 healthy periodontal ligament tissues collected from premolars extracted for orthodontic purposes as control samples. Of these, 10 periapical granulomas and 5 healthy periapical tissues were used for expression analysis of 84 wound healing genes by using a pathway-specific real-time polymerase chain reaction array. The remaining 83 granulomas and all 24 control specimens were used to validate the obtained array data by real-time polymerase chain reaction. Observed variations in expression of wound healing genes were analyzed according to the classification of periapical granulomas as active/progressive versus inactive/stable (as determined by receptor activator for nuclear factor kappa B ligand/osteoprotegerin expression ratio). RESULTS: We observed a marked increase of 5-fold or greater in SERPINE1, TIMP1, COL1A1, COL5A1, VTN, CTGF, FGF7, TGFB1, TNF, CXCL11, ITGA4, and ITGA5 genes in the periapical granulomas when compared with control samples. SERPINE1, TIMP1, COL1A1, TGFB1, and ITGA4 mRNA expression was significantly higher in inactive compared with active periapical granulomas (P < .001), whereas TNF and CXCL11 mRNA expression was higher in active lesions (P < .001). CONCLUSIONS: The identification of novel gene targets that curb the progression status of periapical lesions might contribute to a more accurate diagnosis and lead to treatment modalities more conducive to endodontic success.

Evidence for a regulatory role of alpha 4-integrins in the maturation of eosinophils generated from the bone marrow in the presence of dexamethasone.

BACKGROUND: Although eosinophils co-express multiple integrin receptors, the contributions of integrins to eosinophil development have not been explored. We previously described extensive aggregation and cytological immaturity in eosinophils developing in bone-marrow (BM) cultures exposed to dexamethasone. Here we examined the relationship of alpha 4 integrins with these effects of dexamethasone. OBJECTIVES: We evaluated: (a) the effects of exposure to dexamethasone in BM culture on eosinophil expression of alpha 4 integrin receptors and ligands; (b) the contribution of alpha 4 integrins to eosinophil aggregation and maturation. METHODS: Cultures were established with IL-5 (alone or with dexamethasone) for up to 7 days, and eosinophil production, alpha 4 integrin receptor/ligand expression, aggregation and morphology were evaluated before and after targeting alpha 4 integrin-dependent adhesions. Because prostaglandin E2 (PGE2) modifies the effects of dexamethasone on eosinophilopoiesis, PGE2 effects on alpha 4 integrin expression and function were also evaluated. RESULTS: Dexamethasone increased the yield of eosinophils up to day 7. The frequency of eosinophils expressing alpha 4, beta1 and beta 7 integrin receptors at day 7 was also increased by dexamethasone. Eosinophils also expressed the alpha 4 beta 1 ligand, VCAM-1. Dexamethasone increased the expression of alpha 4 integrin and VCAM-1 in aggregates containing eosinophils as early as day 3. PGE2, added up to day 3, modified the effects of dexamethasone to suppress the expression of alpha 4 integrin, decrease aggregation and promote cytological maturation of eosinophils recovered at day 7. Dissociation of immature eosinophils from clusters present at day 3 by reagents targeting alpha 4 or beta1 integrins or VCAM-1 also induced cytological maturation. The concordant effects of targeting alpha 4 integrins with drugs and antibodies support a relationship between alpha 4-mediated aggregation and maturational arrest. CONCLUSIONS: These observations support a novel role for alpha 4 integrin receptors and ligands in eosinophilopoiesis. In addition, increased alpha 4 expression following glucocorticoid exposure may contribute to the retention and accumulation of eosinophils in haemopoietic tissue.

Alterations in biomarkers of cardiovascular disease (CVD) in active acromegaly.

OBJECTIVES: In acromegalic patients, cardiovascular and metabolic comorbidities contribute to enhance mortality. Available data on the lipoprotein profile of these patients are controversial. Our aim was to characterize the lipoprotein profile and emergent biomarkers of cardiovascular disease in active acromegalic patients in comparison with sex- and age-matched healthy controls. PATIENTS: Eighteen patients with active acromegaly and 18 controls were studied. MEASUREMENTS: Glucose levels, hormonal status, lipoprotein profile and C reactive protein (CRP) were evaluated by standardized methods. Cholesteryl ester transfer protein (CETP) and lipoprotein-associated phospholipase A(2 )(Lp-PLA(2)) were measured by radiometric techniques, endothelin-1 and vascular cell adhesion molecule (VCAM)-1 by enzyme-linked immunosorbent assay, and leucocytes CD18, CD49d and CD54 by flow cytometry. RESULTS: After adjusting for body mass index (BMI), acromegalic patients presented a more atherogenic lipoprotein profile, consisting of higher levels of triglycerides and apolipoprotein B and alterations in the ratios which estimate insulin resistance and atherogenic risk. CETP activity was significantly increased in acromegalic patients as compared to controls (168 +/- 17 vs. 141 +/- 30% per ml h, respectively; P < 0.05). Endothelin-1 levels evidenced an increase in the patients' group (0.9 +/- 0.2 vs. 0.7 +/- 0.2 ng/l, respectively; P < 0.01) and showed positive and significant correlations with GH, IGF-1 and IGFBP-3 (r = 0.45, 0.42 and 0.44, respectively; P < 0.01 for all of them; with BMI as a fixed variable). Lymphocytes from acromegalic patients showed increased CD49d content (282 +/- 59 vs. 246 +/- 48 arbitrary units, respectively; P < 0.05). CONCLUSIONS: Taken together, the alterations described seem to contribute to constituting a state of higher propensity for the development of atherosclerotic cardiovascular disease, which adds to the presence of specific cardiomyopathy.

Human eosinophil adhesion and degranulation stimulated with eotaxin and RANTES in vitro: lack of interaction with nitric oxide.

BACKGROUND: Airway eosinophilia is considered a central event in the pathogenesis of asthma. The toxic components of eosinophils are thought to be important in inducing bronchial mucosal injury and dysfunction. Previous studies have suggested an interaction between nitric oxide (NO) and chemokines in modulating eosinophil functions, but this is still conflicting. In the present study, we have carried out functional assays (adhesion and degranulation) and flow cytometry analysis of adhesion molecules (VLA-4 and Mac-1 expression) to evaluate the interactions between NO and CC-chemokines (eotaxin and RANTES) in human eosinophils. METHODS: Eosinophils were purified using a percoll gradient followed by immunomagnetic cell separator. Cell adhesion and degranulation were evaluated by measuring eosinophil peroxidase (EPO) activity, whereas expression of Mac-1 and VLA-4 was detected using flow cytometry. RESULTS: At 4 h incubation, both eotaxin (100 ng/ml) and RANTES (1000 ng/ml) increased by 133% and 131% eosinophil adhesion, respectively. L-NAME alone (but not D-NAME) also increased the eosinophil adhesion, but the co-incubation of L-NAME with eotaxin or RANTES did not further affect the increased adhesion seen with chemokines alone. In addition, L-NAME alone (but not D-NAME) caused a significant cell degranulation, but it did not affect the CC-chemokine-induced cell degranulation. Incubation of eosinophils with eotaxin or RANTES, in absence or presence of L-NAME, did not affect the expression of VLA-4 and Mac-1 on eosinophil surface. Eotaxin and RANTES (100 ng/ml each) also failed to elevate the cyclic GMP levels above baseline in human eosinophils. CONCLUSION: Eotaxin and RANTES increase the eosinophil adhesion to fibronectin-coated plates and promote cell degranulation by NO-independent mechanisms. The failure of CC-chemokines to affect VLA-4 and Mac-1 expression suggests that changes in integrin function (avidity or affinity) are rather involved in the enhanced adhesion.

Increased adhesive properties of neutrophils in sickle cell disease may be reversed by pharmacological nitric oxide donation.

Increased leukocyte adhesion to vascular endothelium contributes to vaso-occlusion in sickle cell disease. Since nitric oxide bioavailability is decreased in sickle cell disease and nitric oxide may inhibit leukocyte adhesion, we investigated whether stimulation of NO-signaling pathways can reduce the adhesive properties of neutrophils from sickle cell disease individuals (sickle cell diseaseneu). sickle cell diseaseneu presented greater adhesion in vitro to both fibronectin and ICAM-1 than control neutrophils. Co-incubation of sickle cell diseaseneu with the nitric oxide-donor agents, sodium nitroprusside and dietheylamine NONOate (DEANO), and the guanylate cyclase stimulator, BAY41-2272, all significantly reduced the increased adhesion to fibronectin/ICAM-1. Oxadiazolo[4,3-a]quinoxalin-1-one, a guanylate cyclase inhibitor, reversed sodium nitroprusside/DEANO-diminished adhesion to fibronectin, implicating cGMP-dependent signaling in this mechanism. Interestingly, intracellular cGMP was significantly higher in neutrophils from sickle cell disease individuals on hydroxyurea (sickle cell diseaseHUneu). Accordingly, sickle cell diseaseHUneu adhesion to fibronectin/ICAM-1 was significantly lower than that of sickle cell diseaseneu. Agents that stimulate the nitric oxide/cGMP-dependent pathway may have beneficial effects on leukocyte function if used in these subjects.

Cytokines produced by CD4+ T cells against a synthetic GTF-I(1301-1322) peptide of Streptococcus mutans in naturally sensitized humans.

Streptococcus mutans (S. mutans) is the main etiological agent in dental caries. Its virulence factors are the proteins PAc and glucosyltransferase (GTF), which are related to its physiopathogenia and have been used in research for a dental caries vaccine. It was reported that using experimental animal models, GTF-I(1301-1322) synthetic peptide from the GLU region of the GTFs has Tepitopes, induces production of serum antibodies in saliva and reduces the presence of caries, but little is known about the cellular response in naturally sensitized humans. The aim of this study was to observe whether GTF-I(1301-1322) peptide is capable of activating CD4+ T cells in PBMC from naturally sensitized humans, to classify the response and to establish the relationship with dental caries. The study was conducted on 30 individuals classified into the following 3 groups: active caries (AC), History of Caries (HC), and free of caries (H). A blood sample was drawn from each individual. Specific antigen stimulation and flow cytometry analyses were used to determine cells producing the cytokines IFN-gamma (type 1 cytokine) and IL-13 (type 2 cytokine). Cell memory response to GTF-I(1301-1322) peptide was found in naturally sensitized humans. Three different responses were detected: TH0, TH1, and NR. The percentage of CD4+ T cells producing the cytokines IFN-gamma (type 1 cytokine) was greater than the percentage producing IL-13 (p=0.006). No statistically significant differences were found among the three groups for the other variables studied (p < or = 0.05). In conclusion, specific cellular immune responses against the GTF-I(1301-1322) peptide of S. mutans does not differ between individuals who are naturally sensitized, caries- resistant or with caries.

Therapy with hydroxyurea is associated with reduced adhesion molecule gene and protein expression in sickle red cells with a concomitant reduction in adhesive properties.

Propagation of the vaso-occlusive process in sickle cell anaemia (SCA) is a complex process involving the adhesion of steady-state SCA patients red cells and reticulocytes to the vascular endothelium. The effect of hydroxyurea therapy (HUT) on the adhesive properties of sickle cells and the expression of adhesion molecule genes by erythroid cells of SCA individuals is not yet fully understood. The expressions of the CD36 gene and the VLA-4-integrin subunit genes, CD49d (alpha-subunit) and CD29 (beta-subunit), were compared in the reticulocytes of steady-state SCA patients and patients on HUT using real-time PCR. Basal adhesion of red cells from these subjects was also compared using static adhesion assays, as was surface protein expression, using flow cytometry. Basal sickle red cell adhesion to fibronectin was significantly greater than that of normal cells (P < 0.01); in contrast, HUT was associated with significantly lower levels (P < 0.01) of red cell adhesion that were similar to those of control cells; this decrease could not be justified solely by altered reticulocyte numbers in this population. Accordingly, flow cytometry demonstrated that reticulocytes from patients on HUT had significantly lower CD36 and CD49d surface expressions (P < 0.01) and, importantly, significantly lower expressions of the CD36, CD49d and CD29 genes (P < 0.05) than reticulocytes of SCA patients not on HUT. Taken together, data support the hypothesis that HUT reduces the adhesive properties of sickle cells and that this decrease appears to be mediated, at least in part, by a decrease in the gene and, consequently, surface protein expression of adhesion molecules such as VLA-4 and CD36.

Early inflammatory markers in elicitation of allergic contact dermatitis.

BACKGROUND: Allergic Contact Dermatitis (ACD) is regarded as a T-cell-mediated delayed-type hypersensitivity reaction. We studied the kinetics of the expression of CS-1 fibronectin, thymus and activation-regulated chemokine (CCL17/ TARC) and different chemokine receptors (CR) in skin biopsies from individuals suffering from back problems, with the antigen responsible of their contact dermatitis and an irrelevant antigen. METHODS: Samples were taken at 2, 10, and 48 hours for histological and immunohistochemical studies using monoclonal antibodies against human CS-1 fibronectin, CCL17, CD3, CD68, CD49d, CXCR3, CCR5, and CCR3. RESULTS: At positive antigen stimulated sites there was an early expression of CS-1 fibronectin (2 hours), followed by CCL17 and a later accumulation of alplha4/beta1+ (CD49d), CD3+, CD68+, CXCR3+ and CCR5+ mononuclear cells. At 48 hours, approximately 59 % of infiltrating cells were CXCR3+, 42% CCR5+, and only 14 % CCR3+. CONCLUSIONS: These results showed for the first time a very early expression of CS-1 fibronectin which preceded production of CCL17 in blood endothelial cells (BCEs) from patients' skin with ACD. The role of these molecules in recruitment of monocytes and effector T cells in ACD is discussed.

The "early' CD4+CD8+ stage of thymocte differentiation hallmarks the end of a strong positive correlation between extracellular matrix receptor expression and protein tyrosine phosphorylation.

We used irradiation-induced thymic regression/reconstitution to study phosphotyrosine (PTyr) levels and expression of extracellular matrix receptors in thymocyte subsets by flow cytometry. High PTyr levels (PTyr(hi)) characterized cells from the CD4-CD8-(DN)CD25in/hi to the "early" CD4-CD8+(DP)CD25- stage. Correlation indexes (R) between the percentages of these PTyrhi cells and cells with up-regulated expression of alpha4 integrin (alpha4hi) were strongly positive (R= 0.91, P= 0.002, for DN; R= 0.98, P= 0.0001 for DP). At the "early" DP stage, R between PTyrhi cells and cells with up-regulated expression of alpha5 integrin and L-selectin (alpha5hi and L-sel(hi)) also rendered strongly positive (R>0.95, p<0.0003). "Late" expanding DP cells exhibited intermediate PTyr levels (PTyr(in)), associated with a down-regulation of the adhesion receptors assessed. Triple-labeling suggested that in most early CD3-/lo cells, alpha4hi and alpha5hi, but not L-sel(hi) expression preceded a PTyr(hi) content. CD3in/hi-enriched CD8+ cells were also PTyr(hi), but conversely to the immature ones exhibited a tendency for a negative R between PTyr(hi) and alpha4hi (R = -0.93, P = 0.067, n= 4) or alpha5hi cells (R = -0.77, P = 0.23, n = 4). CD4+ cells were either PTyr(hi) or PTyr(in), exhibiting a tendency for a positive R (R = 0.59, P = 0.124, n= 8) between PTyr(hi) and L-sel(hi) cells only. In conclusion, our results associate an up-regulation of alpha4 and alpha5 chains expression with PTyr(hi) levels and, as elsewhere published, with increased adhesion to fibronectin up to the "early" DP stage, but not afterwards.

alpha 4 Integrins and sialyl Lewis x modulation in chronic Chagas disease: further evidence of persistent immune activation.

We have previously shown that titers of soluble platelet selectin (s-P-selectin) and soluble vascular cell adhesion molecule-1 (s-VCAM-1) were increased in sera of patients with chronic Trypanosoma cruzi infection. In this study, we analyzed the expression of CD49d-integrins, that bind to VCAM-1, and sialyl Lewis x (SLe(x)), which binds selectins, in peripheral blood lymphocytes of 27 patients with Chagas' disease at different levels of disease severity. Patients with a mild form of Chagas' disease showed a lower number of CD49d(+) cells, in comparison with those with severe chronic cardiopathy. Decreased levels of CD49d(+) cells were detected in CD3(-) cell populations. Conversely, SLe(x) expression was found to be decreased in patients with severe Chagas' disease. Levels of soluble platelet endothelial cell adhesion molecule-1 (s-PECAM-1) were significantly increased in the plasma of patients with severe Chagas' disease while unaltered levels of MCP-1 were recorded. These data show that VCAM-1 and P-Selectin ligands are differentially expressed during the chronic phase of the Trypanosoma cruzi infection. These findings also reinforce a role of the P-selectin/SLe(x) adhesion pathway rather than very late antigen-4 (VLA-4)/VCAM-1, in the pathogenesis of Chagas' disease.

Blockade of either alpha-4 or beta-7 integrins selectively inhibits intestinal mast cell hyperplasia and worm expulsion in response to Nippostrongylus brasiliensis infection.

Mast cells are known to express high levels of alpha4 integrins including alpha4beta7 and are found in increased numbers in mucosal inflammation. Mast cell accumulation is particularly prominent in the intestine following nematode infection. The adhesion molecule requirements for this process have not yet been defined. The role of alpha4 and beta7 integrin chains in the intestinal mast cell hyperplasia following infection of rats with the nematode parasite Nippostrongylus brasiliensis was examined in this study. Rats were infected with N. brasiliensis larvae and treated with either anti-alpha4 (TA-2), anti-beta7 or isotype-matched control antibodies. The initial mast cell hyperplasia in response to N. brasiliensis infection was significantly inhibited by either anti-alpha4 or anti-beta7 treatment. In contrast, the intestinal eosinophil response to N. brasiliensis infection was not reduced at day 14 or day 16. Elevations in serum IgE levels due to N. brasiliensis infection were also not inhibited by anti-alpha4 or anti-beta7 antibody treatment. Anti-alpha4 antibody but not anti-beta7 antibody treatment also induced a small but significant decrease in the numbers of mast cells in tongue tissue. These data suggest a role for alpha4 integrins, in particular alpha4beta7, in the regulation of mast cell precursor migration to the intestine.

Integrin alpha2beta1 mediates the cell attachment of the rotavirus neuraminidase-resistant variant nar3.

It was previously reported that integrins alpha2beta1, alpha4beta1, and alphaXbeta2 are involved in rotavirus cell infection. In this work we studied the role of integrin subunits alpha2, alpha4, and beta2 on the attachment of rotaviruses RRV and nar3 to MA104 cells. Integrin alpha2beta1 was found to serve as the binding receptor for the neuraminidase-resistant virus nar3, whereas the neuraminidase-sensitive strain RRV interacted with this integrin at a postattachment step. It was shown that nar3 binds alpha2beta1 through the DGE integrin-recognition motif located in the virus surface protein VP5. Integrin subunits alpha4 and beta2 do not seem to be involved in the initial cell binding of either virus.

Upregulated expression of fibronectin receptors underlines the adhesive capability of thymocytes to thymic epithelial cells during the early stages of differentiation: lessons from sublethally irradiated mice.

A 250-cGy whole-body gamma-radiation dose was used to induce thymus regression in mice, and to study the expression and function of extracellular matrix (ECM) receptors in distinct thymocyte subsets emerging during repopulation of the organ. The onset of regeneration was detected from day 2 to 3 postirradiation (P-Ir), when a remarkable increase in the absolute counts of CD3(-)CD25(hi)CD44(+) and CD3(-)CD25(in/hi)CD44(-) cells occurred. Enhanced expression of L-selectin, alpha4, and alpha5 integrin chains (L-selhi alpha4(hi) alpha5(hi)) was also exhibited by these cells. This pattern of expression was maintained until the CD4(+)CD8(+) (DP) young stage was achieved. Afterward, there was a general downregulation of these ECM receptors in DP as well as in CD4(+) or CD8(+) single positive (SP) thymocytes (L-selin alpha4(in) alpha5(in)). In some recently generated SP cells, alpha4 expression was downregulated before the alpha5 chain, and L-selectin was upregulated in half of more mature cells. The expression of the alpha6 integrin chain was downregulated only in maturing CD4(+) cells. Importantly, the increased expression of L-selectin and alpha4 and alpha5 chains in thymocytes was strongly correlated with their adhesiveness to thymic epithelial cells (TEC) in vitro. Blocking experiments with monoclonal antibody or peptides showed the following: (1) that the LDV rather than the REDV cell attachment motif in the IIIC segment of fibronectin is targeted by the alpha4 integrin during thymocyte/TEC adhesion; (2) that the RGD motif of the 120-kD fragment of fibronectin, a target for alpha5 integrin, has a secondary role in this adhesion; and (3) that the YIGSR cell attachment motif of the beta1 chain of laminin/merosin recognized by a nonintegrin receptor is not used for thymocyte adherence. In conclusion, our results show that an upregulated set of receptors endows CD25(+) precursors and cells up to the young DP stage with a high capability of interacting with thymic ECM components.

Role of eosinophilic airway inflammation in models of asthma.

Eosinophils play a central role in the establishment and outcome of bronchial inflammation in asthma. Animal models of allergy are useful to answer questions related to mechanisms of allergic inflammation. We have used models of sensitized and boosted guinea pigs to investigate the nature of bronchial inflammation in allergic conditions. These animals develop marked bronchial infiltration composed mainly of CD4+ T-lymphocytes and eosinophils. Further provocation with antigen leads to degranulation of eosinophils and ulceration of the bronchial mucosa. Eosinophils are the first cells to increase in numbers in the mucosa after antigen challenge and depend on the expression of alpha 4 integrin to adhere to the vascular endothelium and transmigrate to the mucosa. Blockage of alpha 4 integrin expression with specific antibody prevents not only the transmigration of eosinophils but also the development of bronchial hyperresponsiveness (BHR) to agonists in sensitized and challenged animals, clearly suggesting a role for this cell type in this altered functional state. Moreover, introduction of antibody against Major Basic Protein into the airways also prevents the development of BHR in similar model. BHR can also be suppressed by the use of FK506, an immunosuppressor that reduces in almost 100% the infiltration of eosinophils into the bronchi of allergic animals. These data support the concept that eosinophil is the most important pro-inflammatory factor in bronchial inflammation associated with allergy.