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PubMed1999

Vascular cell adhesion molecule-1-mediated matrix metalloproteinase-2 induction in peripheral blood T cells is up-regulated in patients with HTLV-I-associated myelopathy.

We investigated whether matrix metalloproteinase-2 (MMP-2) is induced in peripheral blood T cells after their contact with tumor necrosis factor-alpha (TNF-alpha)-stimulated glioblastoma cell line (T98G), expressing vascular cell adhesion molecule-1 (VCAM-1), in patients with HTLV-I-associated myelopathy (HAM) compared to control patients with other neurological disorders (OND). Gelatin zymography revealed that the incremental ratio of gelatinolytic activity of MMP-2 in culture supernatants derived from T cells cocultured with TNF-alpha-stimulated T98G to that of supernatants derived from cultures of T cells alone was significantly higher in HAM patients than in control patients with OND. Immunoblot analysis of immunoprecipitates of culture supernatant showed that increased gelatinolytic activity of MMP-2 was due to increased production of MMP-2 protein in T cells. Increased gelatinolytic activity of MMP-2 in T cells of HAM patients was blocked by pretreatment of TNF-alpha-stimulated T98G with anti-VCAM-1 antibody before coculture with T cells, indicating that MMP-2 induction was VCAM-1-mediated. Although no significant differences were noted in the percentage of VLA-4-positive cells in cultured T cells between HAM patients and control patients with OND, our results indicate that VCAM-1-mediated MMP-2 induction is up-regulated in T cells of HAM patients.

Adult↗
PubMed2000

Interaction of alpha4-integrin with VCAM-1 is involved in adhesion of encephalitogenic T cell blasts to brain endothelium but not in their transendothelial migration in vitro.

Based on the observation that antibodies directed against alpha4-integrin and its counterreceptor VCAM-1 inhibit inflammatory cell recruitment into the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE), it has been concluded that alpha4-integrin/VCAM-1 interaction plays a critical role in T cell interaction with the blood-brain barrier (BBB) endothelium. In order to define the exact role of alpha4-integrin and VCAM-1 in T cell recruitment across the BBB endothelium we set up in vitro studies, where we investigated the interaction of freshly activated autoaggressive T cell blasts with the brain endothelial cell line bEnd5. A large panel of blocking antibodies directed against the alpha4-, beta1- and beta7-integrin subunits or against the alpha4beta7-heterodimer and against endothelial VCAM-1 significantly reduced adhesion of encephalitogenic T cell blasts to brain endothelium. However, the very same antibodies did not influence transendothelial migration of autoaggressive T cell blasts across a bEnd5 monolayer. Our in vitro observations, therefore, suggest that in vivo alpha4/VCAM-1 interactions are not involved in transendothelial migration of encephalitogenic T cells across the BBB but rather mediate earlier steps of T cell/BBB-interaction such as firm adhesion.

Animals↗
PubMed2000

Suppression of hepatic lesions in a murine graft-versus-host reaction by antibodies against adhesion molecules.

BACKGROUND/AIMS: The injection of parental CD4+ T cells into major histocompatibility complex (MHC) class II disparate F1 hybrid mice induced an autoimmune graft-versus-host reaction (GVHR) which is analogous to autoimmune liver diseases. The interaction of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and very late antigen-4 (VLA-4) has been known to be profoundly involved in the trafficking of lymphocytes into the inflammatory tissues. The aim of this study was to clarify the role of VLA4 or VCAM-1 in the development of GVHR-induced hepatic lesions in our model. METHODS: B6 T spleen cells were injected into (B6.C-H-2bm12xB6) F1 mice intravenously. Anti-VLA-4 mAbs and/or anti-VCAM-1 mAbs were injected intraperitoneally at a dose of 2.5 mg/kg of each mAbs per body weight of mouse. We examined the changes in GVHR-induced hepatic lesions, serum levels of antimitochondrial antibodies (AMA) and cytokine mRNA expressions of liver-infiltrating lymphocytes using H.E. and immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR), respectively. RESULTS: Hepatic lesions of anti-VLA-4 mAbs-treated mice were inhibited compared with those of GVHR mice. However, the administration of mAbs did not interfere with the induction of splenomegaly, the invasion of CD4+, CD8+, B220+, or Mac-1+ cells around bile ducts, nor the production of AMA. Liver-infiltrating CD4+ T cells obtained from these treated mice did not alter the expression of T helper (Th)1 and Th2 cytokine mRNA. CONCLUSION: The results suggest that treatment with antibodies against these adhesion molecules could inhibit the infiltration of lymphocytes without affecting the Th1/Th2 balance. The blockade of VLA-4-mediated cell infiltration into the liver in this model may have a possible novel therapeutic role of VLA-4 mAbs.

Animals↗
PubMedDate not supplied

Binding of human leukocytes to fibronectin is augmented by an anti-CD44 mAb (TL-1) and blocked by another anti-CD44 mAb (Hermes-3) but not by anti-VLA-4/VLA-5 mAbs.

Fibronectin (FN) forms meshworks in extracellular spaces, and it plays an important role in cellular trafficking. Lymphoid cells are activated by binding to FN of the VLA-4 and VLA-5 receptors. CD44 also acts as a receptor of FN, but the mechanism and physiologic regulation of their binding are poorly understood. We have developed an anti-CD44 monoclonal antibody (mAb) (TL-1) in which lymphoid cells are activated and form homotypic cell aggregation. In this study, we found that the adhesion of CEM, HSB2, and LAD lymphoid cells to FN was augmented by TL-1 treatment and was apparently blocked by another anti-CD44 mAb (Hermes-3), but TL-1 Fab' fragments treatment did not induce FN-binding. A similar phenomenon is reported in the binding of the CD44 molecule to HA. This augmentation was not inhibited by the CS1 and RGD peptides of FN or by anti-VLA-4 and -VLA-5 mAbs; it was energy-dependent and associated with cytoplasmic actin filaments. Tl-1 treatment did not alter the cell surface expression of CD44 molecules. These findings above suggested that activated and/or altered cell surface distribution of CD44 molecules via a conformational change augmented the avidity of its binding to FN, which may be similar to lymphocyte-hyaluronate and lymphocyte-endothelial cell binding. As the Hermes-3 binding site is also involved in the interaction between lymphocytes and endothelial cells, activation of lymphocytes via CD44 molecules may facilitate the binding of lymphocytes to endothelial cells, extravasation, and migration to inflammatory sites rich in FN.

Adjuvants, Immunologic↗
PubMed1996

Expression of the adhesion molecules ICAM-1, VCAM-1, and E-selectin and their ligands VLA-4 and LFA-1 in chronic venous leg ulcers.

BACKGROUND: Leukocyte binding to endothelial cells (ECs) is thought to contribute to the pathogenesis of leg ulcers caused by chronic venous insufficiency. In other systems, such binding is mediated by the interaction of adhesion molecules such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule- (VCAM-1) and E-selectin (on ECs), and leukocyte function-associated antigen-1(LFA-1) and very late activated antigen-4 (VLA-4) (on Leukocytes). OBJECTIVE: Our purpose was to determine whether an increased expression of these adhesion molecules contributes to the pathogenesis of chronic venous insufficiency. METHODS: Twenty-seven biopsy specimens of inflamed dermatoliposclerotic skin adjacent to venous leg ulcers were stained immunohistochemically with monoclonal antibodies against ICAM-1, VCAM-1, LFA-1, VLA-4, and E-selectin. Staining intensity was compared with that of normal skin. RESULTS: Specimens of leg ulcers caused by chronic venous insufficiency showed increased expression of ICAM-1 and VCAM-1 but not of E-selectin on The expression of LFA-1 and VLA-4 on perivascular leukocytes was increased dramatically in comparison to healthy skin. CONCLUSION: Upregulation of ICAM-1 and VCAM-1 on ECs may contribute to the increased adherence and extravasation of LFA-1 and VLA-4-positive leukocytes in chronic venous insufficiency.

Aged↗
PubMed1997

Relation between expression of adhesion molecules and clinical behavior in cutaneous follicle center cell lymphomas.

BACKGROUND: Primary cutaneous follicle center cell lymphomas (PCFCCLs) of the head or trunk have a much better prognosis than morphologically similar large-cell lymphomas on the legs or follicle center cell lymphomas involving the skin secondarily (SCFCCLs). Recent studies suggest a relation between the expression of adhesion molecules and clinical behavior of malignant B-cell lymphomas. OBJECTIVE: Our purpose was to investigate a potential relation between the expression of adhesion molecules and clinical behavior of these three prognostically different groups of cutaneous B-cell lymphomas. METHODS: Immunohistochemical studies with a selected panel of monoclonal antibodies against adhesion molecules were performed on 10 PCFCCLs on the head or trunk, five PCFCCLs of the legs, and seven SCFCCLs. Expression of adhesion molecules was correlated with clinical and follow-up data. RESULTS: PCFCCLs of the head and trunk expressed ICAM-1 (80%) and LFA-1 (50%) much more frequently than PCFCCLs of the legs (40% and 20%, respectively) and SCFCCLs (14% and 14%, respectively). VLA-4 was expressed in 60% of PCFCCLs of the legs, but not by the PCFCCLs of the head or trunk. Absence of both ICAM-1 and LFA-1 on the neoplastic B cells correlated with a poor prognosis (seven of nine patients died of systemic lymphoma). In contrast, none of the patients with expression of LFA-1 or ICAM-1 have died of lymphoma thus far. CONCLUSION: Our results suggest a relation between the expression of adhesion molecules and the differences in clinical behavior between different groups of primary and secondary cutaneous follicle center cell lymphomas.

Adult↗
PubMed1998

Additional recognition sites in the C-terminal heparin-binding domain of fibronectin promote adhesion of PMA-treated U937 cells.

Recently we have shown an evidence that a peptide, corresponding to residues Gln1892 to Gly1910, from the C-terminal heparin-binding domain of fibronectin promotes adhesion of phorbol-12-myristate 13-acetate (PMA)-treated U937 cells and binds to both integrin alpha 4 beta 1 and glycosaminoglycans on U937 cells surface. We present additional adhesion-promoting sites to PMA-treated U937 cells present in the C-terminal heparin-binding domain of fibronectin. Three synthetic peptides (residues Ala1819 to Lys1830, designated E5; Thr1828 to Gly1940, E4; and Lys1946 to Leu1963, D1) were active to inhibit adhesion of PMA-treated U937 cells to the 29-kDa fragment comprising the C-terminal heparin-binding domain of fibronectin. Scrambled versions of these peptides had no inhibitory activity on this adhesion. The IgG-conjugated peptides (IgG-E5, IgG-E4, and IgG-D1) were also active and supported adhesion to an extent comparable to that of the 29-kDa fragment. The adhesion of PMA-treated U937 cells on these three IgG-conjugated peptides was only inhibited by glycosaminoglycans and not by integrin alpha 4 beta 1. These results indicate that additional adhesion-promoting sequences are present in the C-terminal heparin-binding domain of fibronectin and that the activity of these peptides depends on peptide sequence, mainly the result of net charges or net hydropathy indices.

Amino Acid Sequence↗
PubMedDate not supplied

The effect of dithiothreitol on VLA-4 detection in peripheral blood and induced sputum cells.

BACKGROUND: Induced sputum has been shown to be a reliable technique for investigating airway inflammation non-invasively. Flow cytometry could provide useful information in this area. However, the viscosity of the sample entails the use of a mucolytic agent. Dithiothreitol (DDT) is the most frequently used agent although it could affect detection of different inflammatory markers. METHODS: To measure the effect of DDT on the detection of certain adhesion molecules in eosinophils and lymphocytes, sputum was induced from seven non-smoking asthmatic and non-asthmatic subjects treated with 0.1 M DDT. The samples were analyzed by flow cytometry. Whole blood samples from the same subjects were also processed with DTT and analyzed by flow cytometry. RESULTS: Very late activated antigen-4 (VLA-4) levels on eosinophils in intracellular and surface staining were much lower than expected. VLA-4 on lymphocytes was also altered but less so than on eosinophils. VLA-4 levels were also decreased on blood cells after DTT treatment. No abnormalities were found in the detection of CD29 on eosinophils and the beta7-chain in lymphocytes. CONCLUSIONS: Flow cytometry could be used as a complementary method to induced sputum in the investigation of airway inflammation. However, DTT could interfere with the detection of some inflammatory markers, as is the case with VLA-4.

Adult↗
PubMed2000

Regulation of megakaryocytopoiesis in an in vitro stroma model: preferential adhesion of megakaryocytic progenitors and subsequent inhibition of maturation.

OBJECTIVE: Studies of megakaryocytic progenitor cell interactions have focused on single receptor-ligand interactions using isolated components of the extracellular matrix. To approach a physiologic condition, we studied megakaryocytic development of human progenitor cells cultured on two stromal cell lines and on human bone marrow stroma. MATERIALS AND METHODS: Human CD34(+) cells were cocultured with stromal layers in the presence of thrombopoietin. Megakaryocytes were quantified by monoclonal antibodies against glycoprotein (GP) IIb/IIIa (CD41) and GPIX (CD42a). Megakaryocytic clonogenic capacity (burst-forming unit-megakaryocyte and colony-forming unit-megakaryocyte) was determined using fibrin clot assays. RESULTS: After 6 days, a higher percentage of megakaryocytes and more megakaryocytic colonies were recovered from the adherent cell fraction compared to the nonadherent cell fraction. In contrast, significantly more granulocytic and erythroid colonies were recovered from the nonadherent cell fraction. Repeated replating of nonadherent cells onto fresh stroma showed a decline in megakaryocytic recovery of the remaining adherent cells, pointing toward selective adhesion of megakaryocytic progenitors. This was supported further by the finding that burst-forming unit and colony-forming unit megakaryocytes were preferentially recovered from the adherent cell fraction at 24 hours. No effect of blocking the beta(1) integrins VLA-4 and VLA-5 on human progenitor cells was observed. A higher expression of CD42a antigen and a higher percentage of morphologically recognizable polyploid megakaryocytes were found when cells were grown in noncontact cultures compared to when grown adhered to stroma. CONCLUSION: In contrast to granulocytic and erythroid progenitors, both very early and more mature megakaryocytic progenitors are preferentially located in the adherent fraction in an in vitro stromal model, leading to inhibition of maturation of megakaryocytes. This suggests that the presence of stroma components in ex vivo expansion cultures, aimed at preservation and expansion of megakaryocytic progenitors, might be a prerequisite.

Animals↗
PubMed2001

Chemokine stromal cell-derived factor-1alpha modulates VLA-4 integrin-dependent adhesion to fibronectin and VCAM-1 on bone marrow hematopoietic progenitor cells.

Stromal cell-derived factor-1alpha (SDF-1alpha) is a potent chemoattractant for hematopoietic progenitor cells (HPC), suggesting that it could play an important role during their migration within or to the bone marrow (BM). The integrin VLA-4 mediates HPC adhesion to BM stroma by interacting with CS-1/fibronectin and VCAM-1. It is required during hematopoiesis and homing of HPC to the BM. As HPC migration in response to SDF-1alpha might require dynamic regulation of integrin function, we investigated if SDF-1alpha could modulate VLA-4 function on BM CD34(hi) cells.CD34(hi) BM cells and hematopoietic cell lines were tested for the effect of SDF-1alpha on VLA-4-dependent adhesion to CS-1/fibronectin and VCAM-1, as well as to BM stroma. CD34(hi) BM cells that adhered to VLA-4 ligands after SDF-1alpha treatment were characterized in colony-forming and long-term culture-initiating cell (LTC-IC) assays.SDF-1alpha rapidly (1 minute) and transiently upregulated the adhesion of CD34(hi) BM cells and hematopoietic cell lines to both CS-1/fibronectin and VCAM-1, and to BM stromal cells. The upregulation of VLA-4-dependent cell adhesion by SDF-1alpha targeted primitive LTC-IC as well as committed CD34(hi) cells. SDF-1alpha-triggered enhancement in VLA-4 function was inhibited by pertussis toxin (PTx) and cytochalasin D, indicating the involvement of G(i) protein downstream signaling and an intact cytoskeleton. Instead, activation of p44/42 MAP kinases by SDF-1alpha did not functionally correlate with enhancement of VLA-4-dependent cell adhesion. Modulation of VLA-4-mediated CD34(hi) BM cell adhesion by SDF-1alpha could play a key role in their migration within and to the BM and therefore influence their proliferation and differentiation.

Animals↗
PubMed2001

Combining G-CSF with a blockade of adhesion strongly improves the reconstitutive capacity of mobilized hematopoietic progenitor cells.

OBJECTIVE: Mobilization of hematopoietic progenitor cells is achieved mainly by application of growth factors and, more recently, by blockade of adhesion. In this report, we describe the advantages of a combined treatment with granulocyte colony-stimulating factor (G-CSF) and anti-VLA4 (CD49d)/anti-CD44 as compared to treatment with the individual components. MATERIALS AND METHODS: Mobilization by intravenous injection of anti-CD44, anti-VLA4, or G-CSF was controlled in spleen and bone marrow with regard to frequencies of multipotential colony-forming unit (C-CFU), marrow repopulating ability, long-term reconstitution, recovery of myelopoiesis, and regain of immunocompetence. RESULTS: Mobilization by anti-CD44 had a strong effect on expansion of early progenitor cells in the bone marrow, while the recovery in the spleen was poor. In anti-CD49d-mobilized noncommitted and committed progenitors, progenitor expansion was less pronounced, but settlement in the spleen was quite efficient. Thus, anti-CD44 and anti-CD49d differently influenced mobilization. Accordingly, mobilization and recovery after transfer were improved by combining anti-CD44 with anti-CD49d treatment. Mobilization by G-CSF was most efficient with respect to recovery of progenitor cells in the spleen. However, when transferring G-CSF-mobilized cells, regain of immunocompetence was strongly delayed. This disadvantage could be overridden when progenitor cells were mobilized via blockade of adhesion and when expansion of these mobilized progenitor cells was supported by low-dose G-CSF only during the last 24 hours before transfer. CONCLUSION: Mobilization of pluripotent progenitor cells via antibody blockade of CD44 or CD49d or via G-CSF relies on distinct mechanisms. Therefore, the reconstitutive capacity of a transplant can be significantly improved by mobilization regimens combining antibody with low-dose G-CSF treatment.

Animals↗
PubMed2001

Cell cycle activation of hematopoietic progenitor cells increases very late antigen-5-mediated adhesion to fibronectin.

Recent studies suggested that trafficking of hematopoietic progenitor cells is related to cell cycle status. We studied whether adhesion of progenitor cells to extracellular matrix proteins was modulated by cell cycle transit. Mobilized peripheral blood CD34+ cells were stimulated ex vivo for 48 hours with stem cell factor, flt-3 ligand, and thrombopoietin and fractionated by adhesion to fibronectin or vascular cell adhesion molecule-1 (VCAM-1). Adherent and nonadherent cells were assayed for cell cycle status, long-term culture-initiating cell frequency, and integrin function. Binding to fibronectin, but not to VCAM-1, displayed a cell cycle selectivity as the adherent fraction to fibronectin was enriched in cycling CD34+ cells and in cycling long-term culture-initiating cells compared to the nonadherent fraction. Combined cell cycle and phenotypic analysis showed that the expression of VLA-5 was upregulated during S/G2+M but that of VLA-4 remained constant. The selective binding of cycling CD34+ cells to fibronectin was reverted by anti-VLA-5 but not by anti-VLA-4 blocking antibodies. Also, cycling CD34+ cells preferentially adhered to the VLA-5 binding domain but not to the VLA-4 binding domain of fibronectin. Adhesion of cycling CD34+ cells to fibronectin was a reversible process modulated by cell cycle progression, because adherent cells could exit the cell cycle and return to a nonadhesive state within an additional 48-hour culture period. The results indicate that the enhanced binding capacity of cycling progenitor cells to fibronectin is mediated by VLA-5.

Adult↗
PubMed1999

Adhesion of hematopoietic progenitor cells to human bone marrow or umbilical vein derived endothelial cell lines: a comparison.

Homing of hematopoietic progenitor cells (HPC) to the bone marrow may be mediated by adhesion molecules specifically expressed on human bone marrow endothelial cells (HBMEC). This hypothesis suggests that HPC would preferentially bind to HBMEC compared to endothelial cells from other origins. In this study, HPC were allowed to adhere either to HBMEC cell lines or to human umbilical vein endothelial cells (HUVEC) in two different experimental set-ups. First, adherence was measured using a flow cytometric assay with three different colors identifying each cell population (HPC, HBMEC, HUVEC). HPC could adhere (in a competitive way) to the two endothelial cell lines under stirring conditions, which simulated adhesion under shear stress, as present in blood vessels. Because this assay requires relatively firm adhesion and the endothelial cells don't form a monolayer, we studied the same interactions under less stringent conditions. HPC were allowed to adhere to endothelial monolayers under gently rocking conditions. Differential adhesion of HPC to a set of endothelial cell lines did not correlate with the origin of the endothelial cells. Adhesion of HPC to both types of endothelial cells was inhibited in the presence of various combinations of monoclonal antibodies against the adhesion molecules VLA-4, CD18, and/or E-selectin. No indications were obtained for qualitative differences in the role of these molecules in adhesion of HPC to either HBMEC or HUVEC cell lines. In conclusion, no preferential adhesion of HPC to HBMEC compared to HUVEC cells was observed. This may be due to a lack of origin-specific differences between endothelial cells, implying that the specificity of homing is not regulated at the entrance of the bone marrow. Otherwise, the origin-specific differences between endothelial cells of different origins may be microenvironment-induced, rather then intrinsic, implying that care should be exercised with the use of endothelial cell lines in studies investigating the specificity of homing of HPC.

Antibodies, Monoclonal↗
PubMed2001

Astrocytic factors down-regulate the expression of major histocompatibility complex-class-II and intercellular adhesion molecule-1 on human monocytes.

Several factors contribute to the maintenance of central nervous system immune privilege and astrocytes have been identified as a major source of immunomodulatory cytokines. To investigate whether hematogenous monocytes are immunologically deactivated by astrocyte-derived factors human monocytes were stimulated with lipopolysaccharide or interferon (IFN)-gamma and treated with the supernatant from pure astrocyte cultures, interleukin (IL)-4, IL-10, or with IL-1-receptor antagonist (1L-1-RA). Flow cytometry demonstrated that the supernatant from astrocyte cultures was the most potent agent in reducing the levels of major histocompatibility complex (MHC)-class-II- as well as intercellular adhesion molecule-1-expression, whereas IL-4, IL-10, and IL-1-RA had only marginal effects. The expression of leukocyte function antigen-1 and very late antigen-4 was not modulated by either factor. In conclusion, astrocytes seem to provide soluble factors that have the capacity to deactivate hematogenous monocytes.

Astrocytes↗
PubMed2002

Long-term alterations of oral mucosa in radiotherapy patients.

PURPOSE: The aim of this investigation was to describe the alterations in oral mucosa after radiotherapy. METHODS AND MATERIALS: Biopsies were taken from patients before irradiation, at 60 Gy, and 6-12 months after radiotherapy. Histomorphological evaluation of the vessels was performed, and endothelial expression of ICAM-1, VCAM-1, and E-selectin was also evaluated, as well as distribution of LFA-1-, Mac-1-, VLA-4-, RM3/1-, 27E10-, and 25F9-bearing cells in the subepithelial tissue. RESULTS: The expression of ICAM-1 was downregulated after radiotherapy, whereas the percentage of LFA-1- and VLA-4-bearing cells increased. VCAM-1 remained at low levels. The subepithelial infiltration was still dominated by RM3/1-positive macrophages. The number of vessels decreased, while the lumina of the remaining vessels in the deeper connective layer increased. CONCLUSIONS: The late effects of radiotherapy are characterized by a decreased number of blood vessels and by significantly different expression patterns of the adhesion molecules studied, and of integrins and macrophage subpopulations compared to the conditions before irradiation and at 60 Gy.

Aged↗
PubMed1999

Irradiation induces increase of adhesion molecules and accumulation of beta2-integrin-expressing cells in humans.

PURPOSE: The purpose of our investigation was to describe the dose- and time-dependent histomorphologic alterations of the irradiated tissue, the composition of the infiltrate, and the expression patterns of various adhesion molecules. METHODS AND MATERIALS: We analyzed immunohistochemically alterations in oral mucosa in 13 head and neck cancer patients before radiotherapy and with 30 Gy and 60 Gy. All had oral mucosa irradiation, with a final dose of 60 Gy using conventional fractionation. Snap-frozen specimens were stained using the indirect immunperoxidase technique. Histomorphology was studied in paraffin-embedded sections. In addition, we determined the clinical degree of oral mucositis. RESULTS: Histomorphologic evaluation showed no vascular damage. Irradiation caused a steep increase of beta2-integrin-bearing cells (p < 0.01), whereas the percentage of beta1-integrin-positive cells remained at low levels. Additionally we found an increase in the expression of endothelial intercellular adhesion molecule-1 (ICAM-1) (p < 0.01) and E-selectin (p < 0.05), while endothelial vascular cell adhesion molecule-1 (VCAM-1) expression remained at very low levels. CONCLUSION: Our findings indicate that in radiation-induced oral mucositis there is no marked vascular damage until the end of radiotherapy. For recruitment of leukocytes, beta2 is more involved than beta1. Pharmaceuticals that block leukocyte adhesion to E-selectin or ICAM-1 may prevent radiation-mediated inflammation in oral mucosa.

Aged↗
PubMed2002

LC/MS/MS plasma assay for the peptidomimetic VLA4 antagonist I and its major active metabolite II: for treatment of asthma by inhalation.

In vitro and in animals, I is a potent and specific peptidomimetic for the potential treatment of airway inflammation in the pathogenesis of asthma. Preclinical studies indicated extensive conversion of I to an active metabolite II, and thus, a very sensitive assay for I and II was needed to support an inhalation ascending-dose study in man. The LC/MS/MS plasma/urine assay method (1.0 ml of sample) involves the following: liquid-liquid extraction of acidified plasma into pentane-ethyl acetate (90:10 v/v); evaporation of the organic extract, reconstitution into methanol; addition of water to the methanolic extract and freezing. After thawing, the extract is centrifuged and the clear supernatant injected for chromatography. Extract is chromatographed on a YMC ODS-AM column (50 x 2.0 mm). For detection, a Sciex 365 LC/MS/MS with an electrospray inlet and used in the positive ion, multiple reaction monitoring mode was used to monitor precursor-->fragment ions of m/z 709-->594 for I and m/z 513-->380 for II. The plasma assay was linear over the concentration range of 0.1-100 ng/ml in plasma for I and II. Accuracy and precision for I ranged from 97.9 to 102.1% of nominal with a 0.84-10.65% CV; similarly for II, 98.0-101.7% and 1.39-9.28% CV, respectively. Extraction recovery averaged 63.7% for I and 64.9% for II. This general assay methodology may be applied to assay small acidic peptides and peptidomimetics from biological fluids by LC/MS/MS.

Administration, Inhalation↗
PubMed1998

Increased expression of neutrophil and monocyte adhesion molecules LFA-1 and Mac-1 and their ligand ICAM-1 and VLA-4 throughout the acute phase of myocardial infarction: possible implications for leukocyte aggregation and microvascular plugging.

OBJECTIVES: This study sought to evaluate expression of adhesion molecules on neutrophils and monocytes throughout the acute phase of myocardial infarction. BACKGROUND: Neutrophil and monocyte counts increase within days from onset of acute myocardial infarction. Because leukocytes are recruited to the involved myocardial region, we postulated that these activated cells would display an increased expression of adhesion molecules necessary for effective endothelial transmigration. METHODS: We measured the expression of neutrophil and monocyte lymphocyte function associated antigen-1 (LFA-1), Mac-1, very late after activation antigen-4 (VLA-4) and intercellular adhesion molecule-1 (ICAM-1) by flow cytometry throughout the acute phase of acute myocardial infarction in 25 patients and 10 age-matched control subjects. RESULTS: Expression of Mac-1 on neutrophils increased significantly, whereas no expression of VLA-4 and ICAM-1 was detected. The expression of LFA-1, Mac-1, VLA-4 and ICAM-1 on the monocyte cell membrane in patients with an acute myocardial infarction was increased compared with that in control subjects by 22% (on day 7), 67%, 13% and 44% (all on day 4), respectively (all p < 0.001). Elevated density of monocyte-specific CD14 in the AMI versus the control group was also shown (30%, p < 0.001). CONCLUSIONS: Increased expression of neutrophil and monocyte adhesion molecules may contribute to their adhesion to endothelium in the ischemic territory. This adhesion could feasibly precipitate vasoconstriction or add a local thrombotic effect due to tissue factor expression secondary to Mac-1 engagement. In addition, the manifestation of increased density of LFA-1 and Mac-1 by activated leukocytes with monocytes also expressing ICAM-1 suggests that leukocytes may form microaggregates that could cause microvascular plugging. This mechanism may facilitate the occurrence of the "no-reflow" phenomenon or slow coronary filling after acute myocardial infarction.

Aged↗