Increased susceptibility to Yersinia enterocolitica Infection of Tff2 deficient mice.

TFF2 is one of the members of the trefoil factor family, known for its role in protection of gastrointestinal epithelia upon injury; however, recent studies suggest that TFF2 could also play an important role in the immune system. In the present study Tff2 deficient and wild type mice were infected by Y. enterocolitica which resulted in a lethal outcome in all Tff2 deficient mice, but not in WT animals. Yersinia invaded Peyer's patches more efficiently as shown by high bacterial titers in the KO mice while wild type mice displayed lower titers and a visible bacterial accumulation in the intestine. Bacterial accumulation in Peyer's patches of Tff2 deficient mice was accompanied by increased recruitment of macrophages. While an increased level of MAC-1 positive cells was observed in the spleens of both Tff2 deficient and WT mice at third day post infection, bacterial dissemination to liver, lung and kidneys was observed only in Tff2 knock-out mice. Analysis of the cellular composition of spleen did not reveal any substantial alteration to WT animals, suggesting possible disregulation of hemopoietic cells involved in immune response to Y. enterocolitica. These new data indicate that Tff2 plays an important role in immune response by protecting the organism from consequences of infection and that Tff2 knock-out mice react adversely to bacterial infections, in this case specifically to Y. enterocolitica.

Plasmid vector-linked maturation of natural killer (NK) cells is coupled to antigen-dependent NK cell activation during DNA-based immunization in mice.

Plasmid DNA vaccines serve in a wide array of applications ranging from prophylactic vaccines to potential therapeutic tools against infectious diseases and cancer. In this study, we analyzed the mechanisms underlying the activation of natural killer (NK) cells and their potential role in adaptive immunity during DNA-based immunization against hepatitis B virus surface antigen in mice. We observed that the mature Mac-1(+) CD27(-) NK cell subset increased in the liver of mice early after DNA injection, whereas the number of the less mature Mac-1(+) CD27(+) NK cells in the liver and spleen was significantly reduced. This effect was attributed to bacterial sequences present in the plasmid backbone rather than to the encoded antigen and was not observed in immunized MyD88-deficient mice. The activation of NK cells by plasmid-DNA injection was associated with an increase in their effector functions that depended on the expressed antigen. Maturation of NK cells was abrogated in the absence of T cells, suggesting that cross talk exists between NK cells and antigen-specific T cells. Taken together, our data unravel the mechanics of plasmid vector-induced maturation of NK cells and plasmid-encoded antigen-dependent activation of NK cells required for a crucial role of NK cells in DNA vaccine-induced immunogenicity.

Accumulation and activation of natural killer cells in local intraperitoneal HIV-1/MuLV infection results in early control of virus infected cells.

Natural killer (NK) cells are important effectors in resistance to viral infections. The role of NK cells in the acute response to human immunodeficiency virus 1 (HIV-1) infected cells was investigated in a mouse model based on a HIV-1/murine leukemia virus (MuLV) pseudovirus. Splenocytes infected with HIV-1/MuLV were injected intraperitoneally and local immunologic responses and persistence of infected cells were investigated. In vivo depletion with an anti-NK1.1 antibody showed that NK cells are important in resistance to virus infected cells. Moreover, NK cell frequency in the peritoneal cavity increased in response to infected cells and these NK cells had a more mature phenotype, as determined by CD27 and Mac-1 expression. Interestingly, after injection of HIV-1/MuLV infected cells, but not MuLV infected cells, peritoneal NK cells had an increased cytotoxic activity. In conclusion, NK cells play a role in the early control of HIV-1/MuLV infected cells in vivo.

PU.1 and C/EBPalpha/beta convert fibroblasts into macrophage-like cells.

Earlier work has shown that the transcription factor C/EBPalpha induced a transdifferentiation of committed lymphoid precursors into macrophages in a process requiring endogenous PU.1. Here we have examined the effects of PU.1 and C/EBPalpha on fibroblasts, a cell type distantly related to blood cells and akin to myoblasts, adipocytes, osteoblasts, and chondroblasts. The combination of the two factors, as well as PU.1 and C/EBPbeta, induced the up-regulation of macrophage/hematopoietic cell surface markers in a large proportion of NIH 3T3 cells. They also up-regulated these markers in mouse embryo- and adult skin-derived fibroblasts. Based on cell morphology, activation of macrophage-associated genes, and extinction of fibroblast-associated genes, cell lines containing an attenuated form of PU.1 and C/EBPalpha acquired a macrophage-like phenotype. The lines also display macrophage functions: They phagocytose small particles and bacteria, mount a partial inflammatory response, and exhibit strict CSF-1 dependence for growth. The myeloid conversion is primarily induced by PU.1, with C/EBPalpha acting as a modulator of macrophage-specific gene expression. Our data suggest that it might become possible to induce the transdifferentiation of skin-derived fibroblasts into cell types desirable for tissue regeneration.

Urokinase plasminogen activator receptor, beta 2-integrins, and Src-kinases within a single receptor complex of human monocytes.

The glycosylphosphatidylinositol (GPI)-anchored membrane protein urokinase plasminogen activator-receptor (uPA-R; CD87) is one of the key molecules involved in migration of leukocytes and tumor cells. uPA bound to uPA-R provides the cell proteolytic potential used for degradation of extracellular matrix. uPA-R is also involved in induction of cell adhesion and chemotaxis. Here, we provide a molecular explanation for these uPA-R-related cellular events. By size fractionation of monocyte lysate and affinity isolation on its natural ligand uPA, we demonstrate uPA-R as a component of a receptor complex of relatively large size. Reprecipitation and immunoblotting techniques allowed us to detect the protein tyrosine kinases (PTKs) p60fyn, p53/56lyn, p58/64hck, and p59fgr as components of this "uPA-R complex". Activation of monocytes even with enzymatically inactivated uPA resulted in induction of tyrosine phosphorylation, suggesting modulation of uPA-R-associated PTKs upon ligand binding. In spite of their presence in large complexes, we did not find the GPI-linked proteins CD14, CD58, and CD59 in the uPA-R complex, which indicates the presence of different receptor domains containing GPI-linked proteins in monocytes. However, we identified the leukocyte integrins LFA-1 and CR3 as components of the uPA-R complex as indicated by coisolation of these molecules, as well as by cocapping and comodulation of uPA-R and leukocyte integrins on the monocyte surface. The assemblage of uPA-R, PTKs and membrane spanning beta 2-integrins in one receptor complex indicates functional cooperation. In regard to the involvement of these molecules in pericellular proteolysis, signal transduction, as well as adhesion and chemotactic movement, we suggest uPA-R complex as a potential cellular device for cell migration.

Aberrant high expression of B lymphocyte chemokine (BLC/CXCL13) by C11b+CD11c+ dendritic cells in murine lupus and preferential chemotaxis of B1 cells towards BLC.

We observed here that the expression of B lymphocyte chemokine (BLC/CXCL13) was markedly enhanced in the thymus and kidney in aged (NZB x NZW)F1 (BWF1) mice developing lupus nephritis, but not in similarly aged NZB and NZW mice. BLC-positive cells were present in the cellular infiltrates in the target organs with a reticular pattern of staining. CD11b+CD11c+ dendritic cells were increased in the thymus and spleen in aged BWF1 mice and identified as the major cell source for BLC. CD4+ T cells as well as B cells were dramatically increased in the thymus in aged BWF1 mice, whereas no increase was observed in aged NZB and NZW mice. B1/B2 ratio in the thymus was significantly higher than those in the spleen and peripheral blood in aged BWF1 mice. Interestingly, BLC showed preferential chemotactic activity for B1 cells derived from several mouse strains, including nonautoimmune mice. Cell surface CXCR5 expression on B1 cells was significantly higher than that on B2 cells. Thus, aberrant high expression of BLC by myeloid dendritic cells in the target organs in aged BWF1 mice may play a pivotal role in breaking immune tolerance in the thymus and in recruiting autoantibody-producing B cells in the development of murine lupus.

The development of murine plasmacytoid dendritic cell precursors is differentially regulated by FLT3-ligand and granulocyte/macrophage colony-stimulating factor.

Plasmacytoid predendritic cells or type 1 interferon (IFN)-producing cells (IPCs) have recently been identified in mice. Although culture systems giving rise to different murine dendritic cell subsets have been established, the developmental regulation of murine plasmacytoid IPCs and the culture conditions leading to their generation remain unknown. Here we show that large numbers of over 40% pure CD11c(+)CD11b(-)B220(+)Gr-1(+) IPCs can be generated from mouse bone marrow cultures with FLT3-ligand. By contrast GM-CSF or TNF-alpha, which promote the generation of CD11c(+)CD11b(+)B220(-) myeloid DCs, block completely the development of IPCs. IPCs generated display similar features to human IPCs, such as the plasmacytoid morphology, the ability to produce large amounts of IFN-alpha in responses to herpes simplex virus, and the capacity to respond to ligands for Toll-like receptor 9 (TLR-9; CpG ODN 1668), but not to ligands for TLR-4 (lipopolysaccharide [LPS]). Unlike human IPCs which produce little IL-12p70, mouse IPCs produce IL-12p70 in response to CpG ODN 1668 and herpes simplex virus. This study demonstrates that the development of murine CD11c(+)CD11b(-)B220(+)Gr-1(+) IPCs and CD11c(+)CD11b(+)B220(-) myeloid DCs is differentially regulated by FLT3-ligand and granulocyte/macrophage colony-stimulating factor. Human IPCs and mouse IPCs display different ability to produce IL-12p70. Large numbers of mouse IPCs can now be obtained from total bone marrow culture.

CD8alpha- CD11b+ dendritic cells present exogenous virus-like particles to CD8+ T cells and subsequently express CD8alpha and CD205 molecules.

Recombinant porcine parvovirus virus-like particles (PPV-VLPs) are particulate exogenous antigens that induce a strong, specific cytotoxic T lymphocyte (CTL) response in the absence of adjuvant. In the present report, we demonstrate in vivo that dendritic cells (DCs) present PPV-VLPs to CD8+ T cells after intracellular processing. PPV-VLPs are captured by DCs with a high efficacy, which results in the delivery of these exogenous antigens to 50% of the whole spleen DC population. In vivo, a few hours after injection, PPV-VLPs are presented exclusively to CD8+ T cells by CD8alpha- DCs, whereas 15 hours later they are presented mainly by CD8alpha+ DCs. After PPV-VLPs processing, a fraction of CD11b+ DCs undergo phenotypic changes, i.e., the up-regulation of CD8alpha and CD205 and the loss of CD4 molecules on their surface. The failure to detect mRNA coding for CD8alpha in CD11b+ DCs suggests that CD8alpha expression by these cells is not due to de novo synthesis. In recombination-activating gene knockout mice (Rag-/-), CD11b+ DCs did not express CD8alpha and PPV-VLPs presentation by CD8alpha+ DCs was severely diminished. These results indicate that both CD8alpha- and CD8alpha+ DCs play an important role in the induction of CTL responses by exogenous antigens, such as VLP.

A subpopulation of Mac-1 (CD11b/CD18) molecules mediates neutrophil adhesion to ICAM-1 and fibrinogen.

We report that a subpopulation (10%) of the Mac-1 (CD1 1b/CD18) molecules on activated neutrophils mediates adhesion to ICAM-1 and fibrinogen. We describe a novel mAb (CBRM1/5) that binds to an activation-specific neoepitope on a subset of Mac-1 molecules on neutrophils and monocytes after stimulation with chemoattractants or phorobol esters but does not recognize Mac-1 on resting myeloid cells. CBRM1/5 immunoprecipitates a subpopulation of Mac-1 molecules from detergent lysates of neutrophils, binds to immunoaffinity-purified Mac-1, and localizes to the I domain on the alpha chain of Mac-1. Because CBRM1/5 recognizes a fraction of Mac-1 on activated neutrophils, but still blocks Mac-1-dependent adhesion to fibrinogen and ICAM-1, we suggest that only a small subset of Mac-1 molecules is competent to mediate adhesion.

Diaryl-dithiolanes and -isothiazoles: COX-1/COX-2 and 5-LOX-inhibitory, *OH scavenging and anti-adhesive activities.

Three series of non-steroidal anti-inflammatory drugs (NSAIDs) inhibiting the cyclooxygenase/5-lipoxygenase (COX/5-LOX) pathways as such as formation of hydroxyl radicals and adhesion were prepared: 4,5-diaryl isothiazoles, 4,5-diaryl 3H-1,2-dithiole-3-thiones and 4,5-diaryl 3H-1,2-dithiole-3-ones. The aim of the present study was to develop substances which can intervene into the inflammatory processes via different mechanisms of action as multiple target non-steroidal anti-inflammatory drugs (MTNSAIDs) with increased anti-inflammatory potential. The current lead 11a was evaluated in COX-1/2, 5-LOX and (*)OH scavenging in vitro assays and in a static adhesion assay where it proved to inhibit adhesion. Moreover, 11a treatment attenuated expression of macrophage adhesion molecule-1 (Mac-1) on extravasated polymorphonuclear leukocytes (PMNs) which indicates that the activation was reduced. The assays used are predictive for the in vivo efficacy of test compounds as shown for 11a in a peritonitis model of acute inflammation in mice. Thus, the novel 5-LOX/COX and (*)OH inhibitor 11a possesses anti-inflammatory activity that, in addition to COX/5-LOX inhibition, implicates effects on leukocyte-endothelial interactions.

IL-15 inhibits pre-B cell proliferation by selectively expanding Mac-1+B220+ NK cells.

Natural killer (NK) cells are the cells critical for inhibition of repopulation of allogenic bone marrow cells. However, it is not well known if NK cells affect autologous lymphopoiesis. Here, we observed that NK cells could inhibit pre-B cell proliferation in vitro driven by interleukin (IL)-7 in a manner dependent on IL-15. Interestingly, the great majority of expanding NK cells were Mac-1(+)B220(+), a recently identified potent interferon (IFN)-gamma producer. Indeed, IFN-gamma was produced in those cultures, and pre-B cells lacking IFN-gamma receptors, but not those lacking type I IFN receptors, were resistant to such an inhibition. Furthermore, even NK cells from mice lacking beta2-microglobulin, which were known to be functionally dampened, inhibited pre-B cell proliferation as well. Thus, activated NK cells, which were expanded selectively by IL-15, could potentially regulate B lymphopoiesis through IFN-gamma beyond the selection imposed upon self-recognition.

(2R,3R)-2-(3',4'-dihydroxybenzyl)-3-(3'',4''-dimethoxybenzyl)butyrolactone suppresses fMLP-induced superoxide production by inhibiting fMLP-receptor binding in human neutrophils.

This study investigated the mechanism underlying the inhibiting effect of (2R,3R)-2-(3',4'-dihydroxybenzyl)-3-(3'',4''-dimethoxybenzyl) butyrolactone (PP-6), a lignan from Piper philippinum, on superoxide anion production induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. Human neutrophils were stimulated with fMLP (1 microM), PMA (100 nM) or leukotriene B(4) (LTB(4); 1 microM) and induced superoxide anion release. PP-6 specifically inhibited fMLP-induced superoxide anion production in a concentration-dependent manner with an IC(50) value of 0.3+/-0.1 microM. Intracellular signaling caused by fMLP, PMA or LTB(4) were evaluated. PP-6 specifically inhibited fMLP-induced intracellular calcium mobilization and ERK (p42/p44), Akt and p38 phosphorylation. Moreover, PP-6 specifically inhibited fMLP-induced Mac-1 expression without affecting this caused by LTB(4) or PMA. PP-6 did not increase cAMP level in human neutrophils. PP-6 did not inhibit superoxide anion production by NaF (20 mM), a direct activator of G-protein, the target of the inhibitory action of PP-6 appears to be a component of the signal transduction pathway upstream of G-protein. PP-6 inhibited FITC-fMLP binding to neutrophils in a concentration-dependent manner with an IC(50) of 1.5+/-0.2 microM. PP-6 did not bring a parallel shift in the concentration response of fMLP-induced superoxide anion. Additionally, the inhibiting effect of PP-6 on fMLP-induced superoxide anion was reversed when PP-6 was washed out. These experimental results suggest that PP-6 exerts non-competitive and reversible antagonistic effect on fMLP receptor.

Leptin receptor is elevated in carotid plaques from neurologically symptomatic patients and positively correlated with augmented macrophage density.

BACKGROUND: Carotid artery lesions from symptomatic patients are characterized by inflammation and neovascularization. The adipokine leptin promotes angiogenesis and activates inflammatory cells, and the leptin receptor (ob gene-encoded receptor), ObR, is expressed in advanced atherosclerotic lesions. The present study quantitatively analyzed ObR messenger RNA (mRNA) expression and immunoreactivity in carotid artery plaques from symptomatic and asymptomatic persons. Plaque angiogenesis, gene expression of vascular endothelial growth factor (VEGF), and macrophage density were also analyzed. METHODS: Carotid endarterectomy specimens were collected from 26 patients undergoing surgery for hemispheric cerebrovascular symptoms (n = 13) or progressive asymptomatic internal carotid stenosis (n = 13). A representative sample, including part of the most active site, was collected from each lesion and evaluated by real-time polymerase chain reaction analysis for ObR(long) and ObR(common) isoforms, VEGF(165), and macrophage adhesion molecule-1 (Mac-1) mRNA, and by immunohistochemistry for ObR, von Willebrand factor (vWF), and CD68 antigen expression. RESULTS: All plaques exhibited advanced atherosclerosis (American Heart Association class IV through VI). Transcript levels were preferentially elevated in symptomatic plaques for ObR(long) (P = .0006) and ObR(common) (P = .033), with a simultaneous upregulation of VEGF(165) (P = .001) and Mac-1 mRNA expression (P = .003). Immunohistochemical analysis confirmed a significant increase of ObR antigen levels (P = .011) and CD68-positive inflammatory cells (P = .049) in symptomatic plaques, whereas neovascularization, evident in all plaques, was similar in both groups (P = .7). CONCLUSION: The ObR(long) and ObR(common) genes are upregulated and their protein preferentially synthesized in clinically symptomatic carotid plaques. Moreover, ObR expression is positively correlated with augmentation of gene transcripts related to macrophage density and neovascularization. These data suggest that ObR(long) and ObR(common) may be linked with histologic features of carotid plaque instability, which are associated with cerebral ischemic symptoms.

Implication of adhesion molecules in inflammation of the common bile duct in patients with secondary cholangitis due to biliary obstruction.

BACKGROUND/AIMS: The aim of the present study was to investigate the participation of the adhesion molecules and their ligands in the inflammatory process in secondary cholangitis. METHODOLOGY: Biopsy specimens were collected from the common bile duct from 29 patients with extrahepatic bile obstruction. Immunohistochemistry was used to study the expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin, LFA-1, PECAM-1, Mac-1, and VLA-4). The patients were categorized into 2 groups - chronic exacerbated cholangitis and chronic sclerotic cholangitis. RESULTS: An increased ICAM-1 expression was demonstrated and also de novo VCAM-1 and E-selectin appearance on the endothelium of microvessels in chronic exacerbated cholangitis. The inflammatory cells were strongly LFA-1-, Mac-1- positive. In chronic sclerotic cholangitis the E-selectin expression persisted on vascular endothelium in the fibrous tissue and reduced inflammatory infiltrate showed LFA-1 and VLA-4 positivity. The newly formed vessels in the fibrous connective tissue were PECAM-1-positive. CONCLUSIONS: From these results, it could be concluded that in chronic exacerbated cholangitis with complete bile obstruction the firm adhesion is mediated by ICAM-1/LFA-1 and ICAM-1/Mac-1 and less by VCAM-1/VLA-4 pathways. In chronic sclerotic cholangitis, caused by incomplete obstruction, the firm adhesion was maintained by ICAM-1/LFA-1 and less by VCAM-1/VLA-4 pathways. It seems likely that PECAM-1 and VCAM-1/VLA-4 play additional roles in neutrophil recruitment.

Increased expression of the interleukin 1 receptor on blood neutrophils of humans with the sepsis syndrome.

Because of the potential importance of interleukin 1 (IL-1) in modulating inflammation and the observations that human blood neutrophils (PMN) express IL-1 receptors (IL-1R) and synthesize IL-1 alpha and IL-1 beta, we studied the IL-1R on blood PMN from a group of patients with the sepsis syndrome. We report a marked enhancement in the sites per cell of IL-1R expressed on sepsis-PMN of 25 consecutively studied patients compared to 20 controls (patient mean = 9,329 +/- 2,212 SE; control mean = 716 +/- 42 SE, respectively). There was no demonstrable difference in the Kd of IL-1R on sepsis-PMN (approximately 1 nM) as determined by saturation curves of 125I-IL-1 alpha binding and the IL-1R on sepsis-PMN had an apparent Mr approximately 68,000, a value like that of normal PMN. Cytofluorographic analysis indicated that the sepsis-PMN phenotype is a single homogeneous population with respect to IL-1R expression. In contrast, expression of the membrane complement receptor CR3 is not increased on sepsis-PMN. Similar increases in expression of IL-1R were not observed in various other inflammatory processes, including acute disseminated inflammation and organ failure not caused by infection, acute infection without organ failure, and immunopathologies such as active systemic lupus erythematosus and rheumatoid arthritis. Enhanced expression of IL-1R was not related simply to the state of myeloid stimulation. Increased expression of IL-1R on normal PMN was induced in vitro by incubating cells with recombinant human granulocyte-macrophage/colony-stimulating factor for 18 h and this response was inhibited by cycloheximide, suggesting the possibility that de novo synthesis of IL-1R might occur in PMN during the sepsis syndrome.

Interleukin-6- and leukemia inhibitory factor-induced terminal differentiation of myeloid leukemia cells is blocked at an intermediate stage by constitutive c-myc.

Interleukin-6 (IL-6) and leukemia inhibitory factor (LIF), two multifunctional cytokines, recently have been identified as physiological inducers of hematopoietic cell differentiation which also induce terminal differentiation and growth arrest of the myeloblastic leukemic M1 cell line. In this work, it is shown that c-myc exhibited a unique pattern of expression upon induction of M1 terminal differentiation by LIF or IL-6, with an early transient increase followed by a decrease to control levels by 12 h and no detectable c-myc mRNA by 1 day; in contrast, c-myb expression was rapidly suppressed, with no detectable c-myb mRNA by 12 h. Vectors containing the c-myc gene under control of the beta-actin gene promoter were transfected into M1 cells to obtain M1myc cell lines which constitutively synthesized c-myc. Deregulated and continued expression of c-myc blocked terminal differentiation induced by IL-6 or LIF at an intermediate stage in the progression from immature blasts to mature macrophages, precisely at the point in time when c-myc is normally suppressed, leading to intermediate-stage myeloid cells which continued to proliferate in the absence of c-myb expression.

Preadipocytes mediate lipopolysaccharide-induced inflammation and insulin resistance in primary cultures of newly differentiated human adipocytes.

Recent data suggest that proinflammatory cytokines secreted from adipose tissue contribute to the morbidity associated with obesity. However, characterization of the cell types involved in inflammation and how these cells promote insulin resistance in human adipocytes are unclear. We simulated acute inflammation using the endotoxin lipopolysaccharide (LPS) to define the roles of nonadipocytes in primary cultures of human adipocytes. LPS induction of the mRNA levels of proinflammatory cytokines (e.g. IL-6, TNF-alpha, and IL-1beta) and chemokines (e.g. IL-8, monocyte chemoattractant protein-1) occurred primarily in the nonadipocyte fraction of newly differentiated human adipocytes. Nonadipocytes were characterized as preadipocytes based on their abundant mRNA levels of preadipocyte markers preadipocyte factor-1 and adipocyte enhancer protein-1 and only trace levels of markers for macrophages and myocytes. The essential role of preadipocytes in inflammation was confirmed by modulating the degree of differentiation in the cultures from approximately 0-90%. LPS-induced proinflammatory cytokine/chemokine expression and nuclear factor-kappaB and MAPK signaling decreased as differentiation increased. LPS-induced cytokine/chemokine expression in preadipocytes was associated with: 1) decreased adipogenic gene expression, 2) decreased ligand-induced activation of a peroxisome proliferator activated receptor (PPAR)-gamma reporter construct and increased phosphorylation of PPARgamma, and 3) decreased insulin-stimulated glucose uptake. Collectively, these data demonstrate that LPS induces nuclear factor-kappaB- and MAPK-dependent proinflammatory cytokine/chemokine expression primarily in preadipocytes, which triggers the suppression of PPARgamma activity and insulin responsiveness in human adipocytes.

Selective recruitment of src family kinase Hck by leukocyte integrin alphaMbeta2 but not alphaLbeta2 or alphaXbeta2.

Integrins are type I heterodimeric (alpha/beta) cell adhesion molecules. They trigger cell-signaling by recruiting cytosolic molecules to their cytoplasmic tails. Integrin alpha cytoplasmic tail contributes towards integrin function specificity, an important feature of integrins having different alpha subunits but sharing the same beta subunit. Herein, we show that the src family kinase Hck co-capped selectively with leukocyte integrin alpha(M)beta(2) but not alpha(L)beta(2) or alpha(X)beta(2). This was disrupted when the alpha(M) cytoplasmic tail was substituted with that of alpha(L) or alpha(X). Co-capping was recovered by alpha(L) or alpha(X) cytoplasmic tail truncation or forced separation of the alpha and beta cytoplasmic tails via salt-bridge disruption.

Quantitative analysis of complement receptors, CR1 (CD35) and CR3 (CD11b), on neutrophils improves distinction between bacterial and viral infections in febrile patients: comparison with standard clinical laboratory data.

There is an ongoing need for sensitive and specific markers of bacterial infection. In this prospective study, standard clinical laboratory data (neutrophil count, serum C reactive protein level, erythrocyte sedimentation rate) and quantitative flow cytometric analysis of neutrophil complement receptors, CR1 and CR3, were obtained from 289 hospitalized febrile patients. After microbiological confirmation or clinical diagnosis, 135 patients were found to have either bacterial (n = 89) or viral (n = 46) infection. The patient data was compared to 60 healthy controls. In bacterial infections, all measured variables were significantly increased, particularly the average amounts of CR1 and CR3 on neutrophils were over three-fold and two-fold higher, respectively, compared to viral infections and controls. We described a novel marker of local and systemic bacterial infections designated 'clinical infection score (CIS) point', which incorporates quantitative analysis of complement receptors on neutrophils and standard clinical laboratory data. CIS point varied between 0 and 8, and displayed 98% sensitivity and 97% specificity in distinguishing between bacterial and viral infections [average (S.D.); CIS points: 6.2 (1.7) vs. 0.6 (1.0); p < 0.001]. These findings suggest that the proposed CIS-based diagnostic test could potentially assist physicians in deciding whether antibiotic treatment is necessary.

Chemokine receptor CCR2 involvement in skeletal muscle regeneration.

Chemokines, signaling through the CCR2 receptor, are highly expressed in injured skeletal muscle. Their target specificity depends on the cellular expression of the specific receptors. Here we demonstrate that, in freeze-injured muscle, CCR2 co-localized with Mac-3, a marker of activated macrophages as well as with myogenin, a marker of activated muscle precursor cells. The degeneration/regeneration process in skeletal muscle of CCR2-/- and wild-type mice was not significantly different at day 3. However in contrast to the regenerated muscle of the wild-type mice, the muscle from CCR2-/- mice was characterized by impaired regeneration, inflammation, and fibrotic response at day 14, increased fat infiltration, fibrosis, and calcification at day 21, and impaired strength recovery until at least 28 days post-injury. Consistently, the increased expression of Mac-1 and TNF-alpha was prolonged in the injured muscle of CCR2-/- mice. The expression pattern of the myogenic factors MyoD and myogenin was similar for both types of mice, while NCAM, which is associated with the initiation of fusion of muscle precursor cells, was more increased in the injured muscle of CCR2-/- mice. In conclusion, the study delineates that signaling through CCR2 is involved in muscle precursor cell activities necessary for complete and rapid regeneration of injured skeletal muscle.