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The majority of malignant cells in chronic lymphocytic leukaemia (CLL) circulate in the peripheral blood whereas small lymphocytic lymphoma (SLL) cells reside in tissues. The aim of this study was to detect differences in chemokine receptor expression, DNA single nucleotide polymorphism (SNP) microarray analysis and proteomic profiling to help elucidate why the cells remain in their respective environments. We identified by flow cytometric studies of chemokine receptors and DNA SNP microarray analysis significant differences between cells from CLL and SLL patients. Proteomic analysis revealed two potential markers (m/z 3091 and 8707) to distinguish the two disorders. There was a significantly greater expression of leucocyte trafficking receptor CXCR3 (CD183) and migration and homing receptor CXCR4 (CD184), and significantly lower expression of cell adhesion molecule integrin α4 chain (CD49d), on CLL cells, compared with SLL cells. Conversely, SNP microarrays revealed greater numbers of copy-neutral loss of heterozygosity chromosomal aberrations, as well as gross chromosomal aberrations, in the SLL group, compared with the CLL group. These findings revealed that there was a significantly greater expression of trafficking, migration and homing receptors and significantly lower expression of adhesion molecules on CLL cells than on SLL cells, and that SLL may be a more progressive disease than CLL, with a more complex genotype.

This study set out to check the quantitative and qualitative properties of peripheral CD4+CD25+CD49d- T regulatory (CD49d- Treg) cells in type 2 diabetes mellitus (T2DM) patients. This work comprised 35 newly diagnosed patients and 35 healthy controls (HCs). The frequency of FoxP3 expressing CD49d- Treg cells was determined by flow cytometry. The gene expression of FoxP3 and CD49d was assessed by real-time PCR. Suppression assays with purified CD49d- Treg cells and CD4+CD25- T conventional (Tconv) cells were done by flow cytometry. The supernatants of Tconv/CD49d- Treg co-cultures were tested for IFN-γ, IL-4, IL-17, and IL-10 using ELISA. The frequency of CD49d- Treg cells (by both CD4+CD25+CD49d- and CD4+CD25++CD49d- phenotypes) observed to be reduced in patients versus HCs. In the patients, decreased protein and gene expression of FoxP3 was seen in CD49d- Treg cells. Suppressive potency of CD49d- Treg cells to inhibit Tconv cells proliferation was diminished, and inversely related to fasting plasma glucose and hemoglobin A1c in the patients. Tconv cells from T2DM patients released higher amount of IL-17 and lower concentration of IL-10 versus HCs. In Tconv/CD49d- Tregs co-cultures, decreased IL-17 and increased IL-10 levels were seen in HCs, but not T2DM patients. CD49d- Treg cells from the patients have a fundamental defect and Treg cells fail to inhibit the aggressive inflammatory responses.

SummaryExtensive transcriptional and ontogenetic diversity exists among normal tissue-resident macrophages, with unique transcriptional profiles endowing the cells with tissue-specific functions. However, it is unknown whether the origins of different macrophage populations affect their roles in malignancy. Given potential artifacts associated with irradiation-based lineage tracing, it remains unclear if bone-marrow-derived macrophages (BMDMs) are present in tumors of the brain, a tissue with no homeostatic involvement of BMDMs. Here, we employed multiple models of murine brain malignancy and genetic lineage tracing to demonstrate that BMDMs are abundant in primary and metastatic brain tumors. Our data indicate that distinct transcriptional networks in brain-resident microglia and recruited BMDMs are associated with tumor-mediated education yet are also influenced by chromatin landscapes established before tumor initiation. Furthermore, we demonstrate that microglia specifically repress Itga4 (CD49D), enabling its utility as a discriminatory marker between microglia and BMDMs in primary and metastatic disease in mouse and human.

Cell-adhesion molecules (CAMs) dynamics in Multiple Sclerosis (MS) patients have been widely studied after Natalizumab (NTZ) introduction. However, their temporal dynamics after NTZ withdrawal (NTZ-W) has not been described. We prospectively evaluate changes in the expression levels of CAMs (CD49d, CD29, L-Selectin and CD11a) involved in T cell migration of 22 MS patients after NTZ-W. CD49d, CD29 and CD11a expression experienced a continuous increase expression two months after NTZ-W and Cd49d expression at month six after NTZ-W correlated to NTZ treatment duration, both in CD45+CD4+ and CD45+CD8+. CD49d expression up to month three after NTZ-W was related to MS activity in CD45+CD8+ at the end of the study. Results from this study suggest that patients with a longer NTZ treatment are more susceptible to present a "molecular rebound" after NTZ-W. CD49d determination may be a useful tool to closely monitor MS activity in patients who interrupt NTZ.

Release of immature neutrophils from bone marrow into circulation accompanies acute inflammatory states. Previous studies have identified a number of antigens that are differentially expressed on mature neutrophils and immature forms. We analyzed expression of these antigens on circulating neutrophils of influenza virus infection patients. We identified a minor neutrophil subpopulation with decreased granularity and the antigen surface expression pattern (CD35(-)/CD49d(+)) which defines metamyelocytes. CD35(-)/CD49d(+) neutrophils were detected in 35% of the patients but not in healthy individuals and correlated positively with blood metamyelocyte count. The sorted CD35(-)/CD49d(+) neutrophils displayed morphological features of metamyelocytes. Compared with a major (CD35(+)/CD49d(-)) neutrophil subpopulation, CD35(-)/CD49d(+) neutrophils demonstrated significantly diminished functional capacities (both phagocytosis and respiratory burst). Thus, CD35(-)/CD49d(+) neutrophils represent a phenotypically and functionally distinct subpopulation of the cells and can potentially be used to quantify immature neutrophil release in inflammation.

The cryopreservation of peripheral blood mononuclear cells (PBMCs) is a routine research laboratory process, enabling long-term storage of primary patient blood samples. Retrospective analysis of these samples has the potential to identify markers that may be associated with prognosis and response to treatment. To draw valid biological conclusions from this type of analysis, it is essential to ensure that any observed changes are directly related to the pathology of the disease rather than the preservation process itself. Therefore, we have investigated 15 cell surface markers that are relevant to chronic lymphocytic leukemia (CLL) on matched fresh and thawed samples to determine the effect of cryopreservation on their detection. We found that the number of CLL cells positive for the markers CD22, CD40, CD49d, CD54, CD69, and CXCR3 was decreased significantly after cryopreservation. In addition, the mean fluorescence intensity of 10 of the 15 markers changed significantly after cryopreservation. These findings demonstrate that care must be taken when interpreting this type of analysis on thawed samples.

Xenotransplantation of rat bone marrow cells (BMC) into immunodeficient (SCID) mice generates chimeric mice susceptible to paralytic autoimmune CNS inflammation. Herein, we identified a disease relevant subset of transplantable BMC lacking expression of CD11b/c and CD49d. Moreover, disease susceptibility was enhanced in the presence of non-myelin specific T-cells. Only the CD11b/c negative population of BM retained the capability to populate the blood, spleen and spinal cord of recipients and matured after transplant to express CD11b/c. These results indicate non-myelin T cells in combination with integrin negative BM represent pre-pathogenic determinants of an enhanced disease susceptibility to myelin reactive T cells.

FTY720, a sphingosine-1-phosphate receptor agonist, is the archeotype of a new class of immune modulators, which redirects lymphocytes from the peripheral blood into secondary lymphatic tissue. Previously, it was shown that FTY720 differentially decreases peripheral T-cells, expressing specific chemokine and adhesion receptors. Here, we investigated the effect of single doses FTY720 on peripheral B-cells expressing CD62L, CD11a, CD49d and CXCR4 in stable human renal allograft recipients. Peripheral blood lymphocytes were isolated by Ficoll density centrifugation and stained with monoclonal antibodies against CD3 or CD19 and CD62L, CD11a, CD49d, CXCR4 to determine the percentage of these T- and B-cell subpopulations. Total lymphocyte counts were measured by routine laboratory diagnostics to calculate absolute lymphocyte subset counts. In FTY720 treated patients, total lymphocyte counts decreased by 31.8% (0.25–2 mg) and 60.4% (3.5 mg), and total T-cell counts by 38.8% (0.25–2 mg) and 70.9% (3.5 mg). In comparison, total B-cell counts decreased by 32.2% (0.25–2 mg) and 61.1% (3.5 mg). The reduction of CD62L+ B-cells was less pronounced as compared to CD62L+ T-cells (0.25–2 mg: 15.7% vs. 57.3%; 3.5 mg: 57.2% vs. 86.9%). CD11a+ B-cells decreased by 15.4% (0.25–2 mg) and 57.1% (3.5 mg), and CD49d+ B-cells by 15.0% (0.25–2 mg) and 56.7% (3.5 mg). CXCR4+ B-cells decreased by 19.9% (0.25–2 mg) and 57.2% (3.5 mg). In vitro experiments showed that FTY720 did not change the mean expression of CD62L, CD11a, CD49d and CXCR4 on CD19+ B-cells. In conclusion FTY720 treatment reduces B-cells expressing CD62L to a significant lesser degree than T-cells expressing CD62L.

The α4β1-integrin (very late antigen-4 (VLA-4), CD49d/CD29) is an adhesion receptor involved in the interaction of lymphocytes, dendritic cells, and stem cells with the extracellular matrix and endothelial cells. This and other integrins have the ability to regulate their affinity for ligands through a process termed “inside-out” signaling that affects cell adhesion avidity. Several mechanisms are known to regulate integrin affinity and conformation: conformational changes induced by separation of the C-terminal tails, divalent ions, and reducing agents. Recently, we described a fluorescent LDV-containing small molecule that was used to monitor VLA-4 affinity changes in live cells (Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S., and Sklar, L. A. (2001) J. Biol. Chem. 276, 48670–48678). Using the same molecule, we also developed a fluorescence resonance energy transfer-based assay to probe the “switchblade-like” opening of VLA-4 upon activation. Here, we investigated the effect of reducing agents on the affinity and conformational state of the VLA-4 integrin simultaneously with cell activation initiated by inside-out signaling through G protein-coupled receptors or Mn2+ in live cells in real time. We found that reducing agents (dithiothreitol and 2,3-dimercapto-1-propanesulfonic acid) induced multiple states of high affinity of VLA-4, where the affinity change was accompanied by an extension of the integrin molecule. Bacitracin, an inhibitor of the reductive function of the plasma membrane, diminished the effect of dithiothreitol, but had no effect on inside-out signaling. Based on this result and differences in the kinetics of integrin activation, we conclude that conformational activation of VLA-4 by inside-out signaling is independent of and additive to reduction-regulated integrin activation.

Objective Dendritic cells (DC) play a key role in initiation of immune responses. In vitro modified acute myeloid leukemia (AML) blasts acquire certain specific features of DC and are suggested as a potential source of anti-leukemia vaccines. AML-DC have been characterized in terms of costimulatory molecule expression and cytokine production. In contrast, migratory capacity of AML-DC, which is a major attribute of DC required for their in vivo function, remains unknown. Here we present data on adhesion properties and profile of integrin expression of AML-DC. Materials and methods Blasts from nine patients were used to generate AML-DC by calcium ionophore treatment. Adhesion of AML-DC to the major components of the extracellular matrix and the profile of integrin expression was studied using flow cytometry. Results Similar to their normal counterparts, calcium ionophore-induced AML-DC acquired the ability to bind to fibronectin and in 4 of 7 studied cases to bind to denatured collagen. Adhesion to native collagen remained unchanged during DC-type differentiation of AML blasts. AML-DC and DC obtained from monocytes of healthy donors expressed CD49d, CD49e, α v β 3 , and α v β 5 . However, AML-DC from 3 of 8 patients down-regulated CD49d, which plays an important role in cell-to-cell and cell-to-matrix interactions and normally is coexpressed with CD83. Conclusion The results provide further evidence that AML blasts can be induced to display functional properties characteristic for DC and may prove useful for in vivo delivery and presentation of tumor antigens to the immune system. Abnormal CD49d expression and variability in AML-DC adhesion to denatured collagen indicate that motility of AML-DC from individual patients may vary, and a customized approach is essential for evaluating leukemic cell feasibility for vaccine design.

Complement activation is critical in the development of local mucosal damage and inflammation as well as of remote organ injury after mesenteric ischemia/reperfusion. To further define the role of C5 activation in local and remote tissue injury, C5 deficient (C5 −/− ) and wild-type control (C5 +/+ ) mice treated with an anti-C5 mAb were subjected to sham or ischemia followed by up to 4 h of reperfusion. The development of local (intestinal) and remote (lung) injury was associated with the expression of CD49d on the surface of circulating blood neutrophils and with VCAM on the endothelial cells of intestinal and lung vessels. Because CD49d heterodimerizes with integrin β1 on the surface of neutrophils and can bind VCAM on the endothelium, we propose that complement activation causes organ damage by upregulating molecules that lead to inappropriate homing of neutrophils.

1. Purpose/Background Up to 76% of pancreatic resections show histologically positive resection margins leaving patients with residual tumor load after surgery. Cold Physical Plasma (CPP) has shown antitumor effects inducing apoptosis. The application of CPP after resection of pancreatic cancer could significantly decrease the local microscopic tumor load. Yet, it is unknown whether plasmas may support tumor metastasis. Hence, the aim of this study was to characterize the influence of CPP on possible tumor cell spread. The cell adhesion profile of four different human pancreatic cancer cell lines was analyzed. In addition, the effects of CPP on Epithelial-to-Mesenchymal-Transition (EMT) of pancreatic cancer cells were investigated, especially the relation between ZEB-1 and E-Cadherin. To complete the in-vitro studies, the chicken chorioallantoic membrane model was used as an in-vivo model. In relation to preliminary experiments, the cell adhesion molecules as well as the tumor cell spread were characterized for each cell line. 2. Methods Using flow cytometry, the pancreatic cancer cell lines PA-TU-8988-T, PA-TU-8988-S, MIA-PaCa-2, PANC-1 were analyzed for CD324 (E-Cadherin), CD326 (EpCam), CD49b (Integrin α-2) and CD49d (Integrin α-4) as well as ZEB-1 after 60sec of CPP treatment. Furthermore, metabolic activity was investigated. All experiments were carried out in a 96-well plate in which adherent cells and supernatants (possibly containing floating cells) were observed. CPP treatment was performed employing the atmospheric pressure argon plasma jet kINPen 11. Examining the mechanic influence of the gas flow was done employing argon gas treatment without plasma. 3. Results/Conclusion CPP reduced viability and metastasis potential of aggressive pancreatic carcinoma cell lines, whereas less aggressive cell lines were less affected. PATU-T showed significantly decreased viability after 60s of CPP treatment compared to controls (p= 0.0304), whereas PATU-S was less affected (p=0.3372). CPP-treatment increased the expression of E-Cadherin (p=0.0001) in PATU-T and MIA-PaCa-2 (p=0.0001) compared to untreated controls. This might affect potential metastasis in these cell lines whereas PATU-S and PANC-1 were not significantly affected. Analyzing the number of viable cells after 4 days (as offspring of detached but viable floating tumor after plasma treatment) in all four cell lines, no changes between treated and non-treated cells were observed (p=0.8478). These results confirm the antitumor effect of Cold Physical Plasma in pancreatic cancer. Furthermore, the results are promising, that CPP is a safe treatment option which may reduce local reoccurrence and moreover has no promoting effects on tumor cell spread.

Neutrophil surface molecules function in part as biological sensors. Surface antigens undergo several changes during neutrophilic maturation to accommodate the cell's function. Surface antigens may appear with neutrophilic maturation, such as CD16b, CD35, and CD10; disappear with maturation, such as CD49d and CD64; be maintained during maturation, such as CD32, CD59, and CD82; or disappear with maturation but reappear after neutrophilic extravasation, such as CD49b. This article reviews the alterations in surface antigen expression during normal neutrophilic granulopoiesis.

Mycosis fungoides and Sézary syndrome represent the most frequent forms of cutaneous T cell lymphoma. Both are characterized by skin infiltrating and/or circulating malignant cells displaying a CD4+CD7– phenotype in the majority of cases. Because an expansion of CD4+CD7– cells may also be found in inflammatory dermatoses or in the aging process, we evaluated, by flow cytometry, the relationship between CD7 expression and the distribution of differentiation/activation or homing antigens on peripheral blood lymphocytes from 36 cutaneous T cell lymphoma patients and from healthy donors. CD4+CD7– cells were increased in all patients with cutaneous T cell lymphoma. As a consequence, the CD7+/– ratio was reduced in stage I–II mycosis fungoides (3.96 vs 6.55 in healthy donors), and inverted in stage III–IV MF and Sézary syndrome (0.28 and 0.12 respectively). In the late stage of disease, the CD7+/– inverted ratio was strictly related to the expression of CD15s, CD60, and CD45R0, and the lack of expression of CD26 and CD49d. Interestingly, in leukemic patients, this phenotype was also associated with peculiar morphologic (large size) or phenotypical (CD3dim expression) characteristics. Furthermore, a progressive reduction of circulating CD8+ cells was also seen throughout all stages of disease. The presence of these populations in cutaneous T cell lymphoma at late phases of disease and Sézary syndrome suggests that all of these molecules may play an important part in the activation pathway and skin homing of circulating T cells in lymphoproliferative disorders. Therefore, this may constitute a distinctive feature in cutaneous T cell lymphoma patients with more aggressive characteristics.

Experimental allergic encephalomyelitis (EAE) is facilitated in resistant BALB/c mice by intraperitoneal infection with an avirulent Semliki Forest virus (SFV-A7). Viral infection increases the incidence of EAE from 15-30% to 60-90% and speeds up appearance of paralysis from 24 to 14 days. In this paper, we describe treatment of virus-facilitated EAE with monoclonal antibodies (mAbs) against leukocyte and/or endothelial cell adhesion molecules. Therapy with mAb against ICAM-1 (intercellular adhesion molecule-1) had a modest effect, but caused hemorrhagic brain and spinal cord lesions. Therapy with mAb against Mac-1 (alpha M beta 2-integrin) was well tolerated but had no effect. Therapy with mAb against VLA-4 (alpha 4 beta 1-integrin) was safe, diminished both clinical and histopathological signs of EAE, decreased induction of VCAM-1 (vascular cell adhesion molecule-1) on brain vessels and diminished infiltration of VLA-4+ cells into the brain. The amount of viral antigen in the brain was not altered. We conclude that facilitation of leukocyte entry into the brain is a major mechanism for viral facilitation of EAE in the BALB/c mouse, and that facilitation can be inhibited by anti-adhesion therapy. This may have implications for treatment of relapses triggered by viral infections in multiple sclerosis.

Of the several families of adhesion receptors involved in leukocyte-endothelial cell interactions, only the selectins have been shown to initiate leukocyte interaction under physiologic shear; indeed, beta 2 (CD18) intergrins responsible for neutrophil arrest are unable to engage without prior selectin-mediated rolling. In contrast, alpha 4 (CD49d) integrins are shown here to initiate lymphocyte contract ("tethering") in vitro under shear and in the absence of a selectin contribution. The alpha 4 integrin ligands MAdCAM-1 and VCAM-1 support loose reversible interactions including rolling, as well as rapid sticking and arrest that is favored following integrin activation. Moreover, alpha 4 beta 7 mediates L-selectin (CD62L)-independent attachment of blood-borne lymphocytes to lamina propria venules in situ. Scanning electron microscopy of alpha 4 beta 7hi lymphoid cells reveals that, like L-selectin, alpha 4 beta 7 is highly concentrated on microvillous sites of initial cellular contact, whereas the beta 2 integrin LFA-1 is excluded from villi. Thus, alpha 4 but not beta 2 integrins can initiate leukocyte adhesion under flow, a capacity that may be in part a function of topographic presentation on microvilli.

Time-dependent changes in the surface receptor expression of various maturational and integrin receptors on peripheral blood cells were studied in two healthy human volunteers following oral applications of thalidomide (Thd). In each measurement the receptor density was quantified by prior calibration of the flow cytometer with latex beads bearing a determined number of fluorescence molecules. The effects observed in the course of the Thd-treatment were practically identical or at least very similar in both the volunteers during four different trials, and were in accord with previous results obtained in large-scale studies (68 treated animals) with non-human primates. It should be stressed that no clear-cut changes were observed in the percentage or absolute numbers of primary lymphocyte subsets such as CD3, CD4 and CD20. After the first two doses of 7 mg Thd/kg body wt the CD18 (the common beta-chain of the beta 2-integrins) marker already decreased in surface density or was no longer detectable on granulocytes, monocytes and lymphocytes. This effect persisted throughout the treatment period and slowly subsided after discontinuation of treatment. With a few days lag phase, the surface density of CD54 (ICAM-1) on granulocytes increased and many cells previously not bearing this receptor newly acquired such surface markers. On monocytes however, the CD54 receptor was lost on many cells. Within the lymphocyte fraction a loss of the CD54 marker could be noted on CD4 cells but not on CD8 cells, where an increase of the receptor expression could be observed. Other markers, such as the alpha chains of the beta 1 integrins CD49b (VLA alpha 2) and CD49d (VLA alpha 4) showed contrasting reactions to the Thd-treatment. Whereas a pronounced loss of the receptor density of CD49d was observed and only few cells with high epitope density were left in the blood at the end of the complete dosing schedule, no such effect was observable on cells bearing the CD49b epitope. A distinct reduction of the number of receptors was also noticeable on L-selectin (Leu8) bearing cells. On CD4 positive lymphocytes, the majority of the described effects on the integrin and adhesion receptors was seen on cells bearing the CD45R0 maturational epitope. This functional receptor is strongly down-regulated and the pathway of CD45RA to CD45R0 maturation is apparently altered by Thd-treatment. These multiple changes we observed may explain the large variety of therapeutic effects experienced in the treatment with Thd.

The leukocyte integrin alpha 4 beta 1 (VLA-4, CD49d/CD29) is a receptor for the extracellular matrix protein fibronectin and the endothelial adhesion protein VCAM-1. We have analyzed the biosynthesis and post-translational modifications of the two subunits of this receptor complex. The alpha 4 subunit was initially synthesized as a single-chain polypeptide that underwent the formation of complex endoglycosidase H-resistant oligosaccharide side chains and which could be proteolytically cleaved into two noncovalently associated fragments. The level and rate of alpha 4 subunit cleavage was dependent on the cell studied. The T cell tumor line HPB-ALL expressed both intact and fragmented alpha 4 on the cell surface. The interleukin-2-dependent natural killer line NK 3.3 and long term interleukin-2-dependent activated T lymphocytes cleaved the alpha 4 polypeptide earlier and more efficiently than did HPB-ALL cells and did not have detectable levels of intact alpha 4 on the cell surface. The proteolysis of alpha 4 was blocked by treating cells with either the lysosomotrophic amine NH4Cl or the carboxylic ionophore monensin. The presence of complex N-linked oligosaccharides did not seem to be necessary for alpha 4 cleavage or for binding of the alpha 4 beta 1 complex to a synthetic peptide corresponding to the binding site for this receptor on fibronectin.

A substantial proportion of HIV + individuals suffer from HIV-associated neurocognitive impairment (NCI). Likely pathogenic mechanisms include neurotoxicity from persistent immune activation during suppressive antiretroviral therapy (ART). The investigation of activated, CNS-invading leukocytes in relation to HIV-associated NCI is in its infancy. We investigated lymphocyte migration into the CNS in 30 HIV + individuals with ( N = 14) and without NCI who participated in a CNS-penetrating antiretroviral therapy trial. Differentiation status of CD4 and CD8 memory T-cells (central memory, CM; effector memory, EM; CD45RA + EM, EMRA) and expression of chemokine receptor CXCR3 and integrin CD49d in peripheral blood and cerebrospinal fluid (CSF) were compared using flow cytometry. NCI was defined by a global deficit score (GDS) ⩾0.5 obtained from a comprehensive neuropsychological test battery. At baseline, greater proportions of highly differentiated and migration-marker-expressing memory T-cells were found in CSF compared to blood: higher %EM in CSF for CD4 and CD8 cells. Also, CSF-derived CD4 memory T-cells expressed greater levels of CXCR3 and CD49d ( p ’s p N = 16), %CD49d + CD4 EM changes in blood predicted the changes in GDS over time ( p

S A T U R D A Y 155 CD49d+ Neutrophils Connect the Viral and Hygiene Hypotheses Sheila S. Bhat, Desire Hunter, Erika Buell, Dorothy S. Cheung, MD, FAAAAI, Mitchell H. Grayson, MD, FAAAAI; Medical College of Wisconsin, Medical College of Wisconsin, Milwaukee, WI. RATIONALE: Two hypotheses have been proposed to explain the increase in atopic disease. The viral and hygiene hypotheses are well known; however, it is not known if they could both play a role in the same individual. We utilized a well-characterized mouse model where paramyxoviral infection with Sendai virus (SeV) leads to atopic disease. The development of this post-viral atopic disease depends upon accumulation of CD49d+ neutrophils in the airway. Respiratory exposure to lipopolysaccharide (LPS), however, elicits a strong recruitment of CD49d– neutrophils to the airway. We hypothesized that exposure to LPS in SeV infected mice would reduce the frequency of CD49d+ neutrophils, thus preventing development of atopic disease. METHODS: C57BL6 mice were inoculated intranasally with SeV and given LPS (1, 3, and 10mg) at 0, 24, 48, and 60 hours later. Three days post SeV inoculation bronchoalveolar lavage (BAL) was performed. Flow cytometry was performed to determine the frequency of CD49d+ and CD49dneutrophils in the BAL. RESULTS: Administering 10 mg LPS at 60 hours post SeV infection was most effective in reducing CD49d+ neutrophils (17.661.7% versus 2.460.2%, 10 mg LPS versus no LPS, N54-5 p

Thr183-Tyr185 (p 0.013), belonging to the MAP Kinase signaling pathway, and GSK-3alpha/beta Ser21-9 (p 0.0018) and DVL3 (p 0.04), both responsible for the maintenance of an active -catenin signalling. UM but not M CLL cells also displayed significant linkages between ERK1/2 Thr202-Tyr204, p90 RSK Ser380, and MEK1/2 Ser 217-221 (rho 0.5, p 0.002 in all comparisons), all proteins belonging to the MAP Kinase signaling pathway, thus reflecting an activated proliferation profile in this CLL subset. Conclusion: Overall, the results obtained suggest that specific signaling pathways are constitutively active in circulating CLL cells, particularly if they express CD49d, CD38 and an UM IGHV gene mutational status. This suggests a constitutive receptor engagement by CD49d/BCR-specific ligands allegedly present in blood/plasma. Experiments aimed at investigating the phosphoproteomic profiles of CLL cells whose surface receptors are engaged by specific ligands and/or autologous plasma components are currently ongoing.