The blood transcriptome prior to ovarian cancer diagnosis: A case-control study in the NOWAC postgenome cohort.

Epithelial ovarian cancer (EOC) has a 5-year relative survival of 50%, partly because markers of early-stage disease are not available in current clinical diagnostics. The aim of the present study was to investigate whether EOC is associated with transcriptional profiles in blood collected up to 7 years before diagnosis. For this, we used RNA-stabilized whole blood, which contains circulating immune cells, from a sample of EOC cases from the population-based Norwegian Women and Cancer (NOWAC) postgenome cohort. We explored case-control differences in gene expression in all EOC (66 case-control pairs), as well as associations between gene expression and metastatic EOC (56 pairs), serous EOC (45 pairs, 44 of which were metastatic), and interval from blood sample collection to diagnosis (≤3 or >3 years; 34 and 31 pairs, respectively). Lastly, we assessed differential expression of genes associated with EOC in published functional genomics studies that used blood samples collected from newly diagnosed women. After adjustment for multiple testing, this nested case-control study revealed no significant case-control differences in gene expression in all EOC (false discovery rate q>0.96). With the exception of a few probes, the log2 fold change values obtained in gene-wise linear models were below ±0.2. P-values were lowest in analyses of metastatic EOC (80% of which were serous EOC). No common transcriptional profile was indicated by interval to diagnosis; when comparing the 100 genes with the lowest p-values in gene-wise tests in samples collected ≤3 and >3 years before EOC diagnosis, no overlap in these genes was observed. Among 86 genes linked to ovarian cancer in previous publications, our data contained expression values for 42, and of these, tests of LIME1, GPR162, STAB1, and SKAP1, resulted in unadjusted p<0.05. Although limited by sample size, our findings indicated less variation in blood gene expression between women with similar tumor characteristics.

Exercise Increases Mucosal-associated Invariant T Cell Cytokine Expression but Not Activation or Homing Markers.

Mucosal-associated invariant T (MAIT) cells have properties of both the innate and adaptive immune systems but are an understudied population within exercise immunology. These lymphocytes aggregate at the mucous membranes, but it is unknown if submaximal exercise alters their circulating numbers or function. PURPOSE: To determine the MAIT cell response to submaximal exercise on activation and homing marker expression and stimulated cytokine production. METHODS: Twenty healthy, young, recreationally active males cycled for 40 min at 86% of VT after an overnight fast. Peripheral blood mononuclear cells were isolated and labeled to identify specific MAIT cell populations using flow cytometry. Cytokine production after stimulation was also determined. RESULTS: Mucosal-associated invariant T cells were 2.9% of T cells and increased to 3.9% after exercise and with recovery whereas cell numbers significantly increased by 91.5% after exercise before returning to resting levels. Chemokine and activation marker absolute cell number significantly increased while expression levels remained constant but the high levels of CCR5 may help direct MAIT cells to sites of inflammation. After stimulation, TNFα expression significantly increased after exercise before returning to baseline with a similar trend for IFNγ. CONCLUSIONS: The MAIT cell numbers undergo a partial biphasic response after submaximal exercise and appear to be preferentially mobilized within T cells; however, the magnitude of the submaximal response was attenuated relative to maximal exercise. Stimulated MAIT cells increase TNFα expression, indicating greater responsiveness to pathogens after acute exercise.

Antibodies to receptors are associated with biomarkers of inflammation and myocardial damage in heart failure.

INTRODUCTION: Naturally occurring antibodies are linked to inflammation, tissue injury and apoptosis, processes also linked to heart failure. Associations between antibodies, inflammation and myocardial damage, have not been elucidated in heart failure. OBJECTIVE: We investigated if 25 antibodies to receptors expressed in the cardiovascular system were associated with troponin-T, biomarkers of inflammation and clinical measures of disease severity, in patients with heart failure. METHODS: Antibodies in sera from patients (n=191) with ischemic (n=155) or non-ischemic (n=36) heart failure were measured with full-receptor sandwich enzyme-linked immunosorbent assays. All patients underwent coronary angiography with determination of left ventricular ejection fraction (LVEF) and left ventricular end-diastolic pressure (LVEDP). Measured biomarkers included troponin-T, C-reactive protein, erythrocyte sedimentation rate, fibrinogen and neopterin. RESULTS: Stabilin-1-antibodies correlated with troponin-T (ß 0.23 p=0.008), soluble endoglin-antibodies with erythrocyte sedimentation rate (ß 0.19, p=0.007) and fibrinogen (ß 0.28, p<0.001). Platelet-derived growth factor subunit ß-antibodies were associated with neopterin (ß 0.17, p=0.002). All antibodies were correlated (R 0.26 to 0.91) and formed 4 principal components (PCs). Patients with high CRP and high PC2 had higher NYHA class and patients with high troponin-T and high PC1 had lower LVEDP (interactions, all p<0.05). CONCLUSION: Antibodies to receptors are correlated and are associated with biomarkers of inflammation and myocardial damage, which further modifies their association with disease severity in heart failure. Their functional activity and immunological function, remain undecided.

Enhanced gut homing receptor expression of unswitched memory B cells in rheumatoid arthritis.

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Natalizumab and impedance of the homing of CD34+ hematopoietic progenitors.

BACKGROUND: Treatment with natalizumab, an antibody blocking the α4-integrin, is associated with increased numbers of circulating CD34+ cells in the peripheral blood of patients with multiple sclerosis. OBJECTIVE: To determine whether natalizumab mobilizes CD34+ cells from or inhibits homing to the bone marrow (BM). DESIGN: Fifty-two patients with relapsing-remitting multiple sclerosis treated with natalizumab were included. Flow cytometric analyses; polymerase chain reaction assays for JC (John Cunningham) virus DNA detection; and adhesion, migration, and apoptosis assays of immunomagnetically enriched peripheral blood and BM CD34+ cells were conducted. A comparison was made with CD34+ cells from granulocyte colony-stimulating factor-mobilized peripheral blood or steady-state BM of age- and sex-matched healthy donors. RESULTS: We found adhesion and migration of peripheral blood-derived CD34+ cells to be reduced. In BM aspirates from natalizumab-treated patients, the cellularity, the proportion, and the adhesive capacity of CD34+ cells were normal. The JC virus was undetectable. CONCLUSIONS: Natalizumab mediates an increase in circulating CD34+ cells by interfering with homing to the BM. Thus, CD34+ cells appear unlikely to represent a source mobilizing JC virus out of the BM in patients treated with natalizumab.

Higher proportions of circulating FOXP3+ and CTLA-4+ regulatory T cells are associated with lower fractions of memory CD4+ T cells in infants.

In adults, a majority of FOXP3(+) T(regs) expresses CTLA-4, and this costimulatory molecule is essential to control the expansion of other T cells. However, it remains to be investigated whether FOXP3(+) and/or CTLA-4(+) T(regs) are associated with the expression of memory markers and homing receptors on CD4(+) T cells. Thus, in a prospective newborn-infant cohort study, we examined the proportions of FOXP3(+) and CTLA-4(+) T(regs) within the CD4(+)CD25(+) T cell population and the fractions of CD4(+) T cells that expressed CD45RA, CD45RO, HLA-DR, α(4)ß(7), CD62L, and CCR4 at several time-points during the first 3 years of life using flow cytometry. With the use of multivariate factor analysis, we found that a high proportion of FOXP3(+) or CTLA-4(+) T(regs) during the first 18 months of life was associated positively with the fraction of T cells that expressed a naïve phenotype (CD45RA and α(4)ß(7)) and inversely related to the fraction of T cells that expressed a memory phenotype (CD45RO and CCR4) later in childhood. In conclusion, FOXP3(+) or CTLA-4(+) T(regs) may modulate CD4(+) T cell activation and homing receptor expression in children.

Dynamic development of homing receptor expression and memory cell differentiation of infant CD4+CD25high regulatory T cells.

Migration of CD4+CD25+FOXP3+ regulatory T cells (Treg) is important for suppressing immune responses in different tissues. Previous studies show that the majority of Treg at birth express gut homing receptor alpha(4)beta(7) and that only few express CCR4, while the reverse pattern is found in adults. The age at which homing receptor switch occurs in vivo is not known. In this study, we show, in a prospective study of human infants from birth to 3 years of age, that homing receptor switch from alpha(4)beta(7) to CCR4 commences between 1 1/2 and 3 years of age and that Treg at that age also had started their switch to a memory phenotype. The majority of naive Treg express alpha(4)beta(7) in infants but not in adults, while the majority of memory Treg express CCR4 both infants and adults. The homing receptor expression on Treg corresponds to their actual migration properties, because Treg from cord blood migrate foremost toward the gut-associated chemokine CCL25. CD4+FOXP3+ T cell numbers increase rapidly in the circulation during the first days of life indicating conversion to suppressive Treg from CD25(high) Treg precursors. These findings suggest that the gut is the primary site of Treg stimulation to exogenous Ags during the first 18 mo of life and that homing receptor switch toward a more extra-intestinal phenotype occurs thereafter.

Alternatively activated macrophages regulate extracellular levels of the hormone placental lactogen via receptor-mediated uptake and transcytosis.

Alternatively activated (M2) macrophages regulate immune responses and tissue remodelling. In many tissues including placenta, M2 express stabilin-1, a multidomain protein that exerts a dual role as a scavenger receptor for acetylated low density lipoprotein (acLDL) and SPARC (secreted protein acidic and rich in cysteine) and as an intracellular cargo carrier for SI-CLP. Using yeast two-hybrid screening, we identified the developmental hormone placental lactogen (PL) as a novel ligand of stabilin-1. In Chinese hamster ovary-stabilin-1 cells and M2, FACS and confocal microscopy demonstrated that stabilin-1 mediates internalization and endosomal sorting of PL. In M2 macrophages, PL was partially degraded in lysosomes; part of PL escaped degradation and was delivered to novel PL+ storage vesicles lacking endosomal/lysosomal markers. During formation, PL+ vesicles underwent transient interaction with the trans-Golgi network (TGN). Upon placement of PL-loaded M2 into PL-free medium, PL was secreted into the supernatant. Leupeptin, an inhibitor of lysosomal hydrolases, reduced PL degradation, enhanced sorting of PL into the TGN/storage vesicle pathway and increased PL secretion. Thus, processing of PL in M2 macrophages occurs either by the classical lysosomal pathway or by a novel TGN-associated trans-secretory pathway. Macrophages isolated from human placental villi efficiently endocytosed PL-FITC and transported it to the storage vesicles. Our data show that extracellular PL levels are determined by uptake, degradation, storage, and release in M2. During pregnancy PL concentration reaches 10 microg/ml in maternal circulation and stays below 0.5 microg/ml in fetal circulation. We propose that stabilin-1-positive macrophages determine the difference in PL levels between maternal and fetal circulation.

Regulation of trafficking receptor expression in human forkhead box P3+ regulatory T cells.

Forkhead Box P3(+) (FOXP3(+)) T cells are regulatory cells important for maintaining immune tolerance. While chemokine- and other homing-receptors are important for T cell migration, it has been unclear how they are regulated in FOXP3(+) T cells. We thoroughly investigated, ex vivo and in vitro, the regulation of chemokine receptor expression on human FOXP3(+) T cells in neonatal cord blood, adult peripheral blood, and tonsils. We found that human FOXP3(+) T cells undergo changes in trafficking receptors according to their stages of activation and differentiation. FOXP3(+) T cells are divided into CD45RA(+) (naive type) and CD45RO(+) (memory type) FOXP3(+) T cells in neonatal blood, adult blood, and tonsils. CD45RA(+)FOXP3(+) T cells mainly express lymphoid tissue homing receptors (CD62L, CCR7, and CXCR4), while CD45RO(+)FOXP3(+) T cells highly express both Th1 and Th2-associated trafficking receptors along with the lymphoid tissue homing receptors at reduced frequencies. Up-regulation of Th1/Th2-associated trafficking receptors begins with activation of CD45RA(+)FOXP3(+) T cells and is completed after their differentiation to CD45RO(+) cells. Some chemokine receptors such as CXCR5 and CXCR6 are preferentially expressed by many FOXP3(+) cells at a specific stage (CD69(+)CD45RO(+)) in tonsils. Our in vitro differentiation study demonstrated that CD45RA(+)FOXP3(+) T cells indeed undergo chemokine receptor switch from CD45RA(+) (secondary lymphoid tissue homing) to CD45RO(+) type (lymphoid and nonlymphoid tissue homing). The orderly regulation of trafficking receptors in FOXP3(+) T cells according to stages of differentiation and activation is potentially important for their tissue-specific migration and regulation of immune responses in humans.

Detailed analysis of intrahepatic CD8 T cells in the normal and hepatitis C-infected liver reveals differences in specific populations of memory cells with distinct homing phenotypes.

In hepatitis C virus (HCV) infection the immune response is ineffective, leading to chronic hepatitis and liver damage. Primed CD8 T cells are critical for antiviral immunity and subsets of circulating CD8 T cells have been defined in blood but these do not necessarily reflect the clonality or differentiation of cells within tissue. Current models divide primed CD8 T cells into effector and memory cells, further subdivided into central memory (CCR7+, L-selectin+), recirculating through lymphoid tissues and effector memory (CCR7-, L-selectin-) mediating immune response in peripheral organs. We characterized CD8 T cells derived from organ donors and patients with end-stage HCV infection to show that: 1) all liver-infiltrating CD8 T cells express high levels of CD11a, indicating the effective absence of naive CD8 T cells in the liver. 2) The liver contains distinct subsets of primed CD8+ T cells including a population of CCR7+ L-selectin- cells, which does not reflect current paradigms. The expression of CCR7 by these cells may be induced by the hepatic microenvironment to facilitate recirculation. 3) The CCR7 ligands CCL19 and CCL21 are present on lymphatic, vascular, and sinusoidal endothelium in normal liver and in patients with HCV infection. We suggest that the recirculation of CCR7+/L-selectin- intrahepatic CD8 T cells to regional lymphoid tissue will be facilitated by CCL19 and CCL21 on hepatic sinusoids and lymphatics. This centripetal pathway of migration would allow restimulation in lymph nodes, thereby promoting immune surveillance in normal liver and renewal of effector responses in chronic viral infection.

Adhesion molecule and homing receptor expression on blood and milk polymorphonuclear leukocytes during the periparturient period of dairy cattle.

Adhesion molecule and homing receptor expression on blood and milk polymorphonuclear leukocytes (PMN) from periparturient dairy cattle was studied. Both percentages and the mean fluorescence intensity (MFI) of PMN expressing CD11a, CD44, CD62L, and LPAM-1 (alpha4 beta7) were evaluated at seven time points during the twenty-one day period post calving. CD11a and CD62L were expressed on 94-100% of PMN in both blood and milk and there were no significant differences in these percentages at any time point. LPAM-1 was expressed on 3-10% of the PMN in the blood and 13-45% in the milk and the percentage of cells expressing LPAM-1 in milk was significantly (P<0.05) greater than in blood at 0, 4, 10, 14, 18 and 21 days after calving. CD44 was expressed on 11-39% of the PMN in blood and 33-69% in the milk and the percentage of cells expressing CD44 in milk was significantly (P<0.05) greater than in blood at all time points. The MFI of CD11a on milk PMN was consistently higher than that of blood PMN throughout the study period and significantly (P<0.05) higher at days 4, 10 and 18 after calving.

In vivo evaluation of 111In-labeled T-lymphocyte homing in experimental colitis.

UNLABELLED: Blockade of lymphocyte recruitment to the intestinal mucosa is considered a useful therapy for inflammatory bowel disease (IBD) and anti-alpha4 antibodies have clinical benefit in patients with active Crohn's disease. The aim of this study was to evaluate a scintigraphic technique to assess lymphocyte homing to the colon in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced experimental colitis (TNBS colitis) in vivo. METHODS: TNBS-sensitized and nonsensitized murine total lymphocytes or CD4+ lymphocytes were radiolabeled with 111In-oxinate. Cells were injected into control mice (n = 5) or mice with TNBS colitis (n = 5). Specific abdominal radioactive uptake was determined by SPECT using a dedicated pinhole system 48 h after cell transfer. Radioactive colon uptake was correlated with histology and colon weight as parameters of inflammation. RESULTS: The radioactive colon uptake was most evident in mice with TNBS colitis that received sensitized lymphocytes (uptake ratio [mean +/- SEM], 0.51 +/- 0.03 vs. 0.22 +/- 0.04; P = 0.004). The sensitized 111In-labeled lymphocytes exacerbated colitis compared with nonsensitized lymphocytes. The colon uptake correlated well with both colon weight and histologic score (R2 = 0.836 and 0.933, respectively). The use of purified 111In labeled CD4+ lymphocytes resulted in a similar scintigraphic pattern. Administration of an anti-alpha4 antibody decreased radioactivity colon uptake of the (111)In-labeled cells compared with the control antibody in mice with TNBS colitis (uptake ratio, 0.72 +/- 0.14 to 0.33 +/- 0.03; P = 0.012). CONCLUSION: Animal pinhole SPECT can be applied for temporal and spatial analysis of the lymphocyte homing process in experimental colitis. This technique makes possible the in vivo evaluation of therapeutic efficacy of new drugs that interfere with lymphocyte migration. Moreover, colon uptake of radioactivity can be used as a parameter of disease activity in experimental colitis.

Quantitative expression of the major homing integrins alphaL and alpha4 on human cord blood hematopoietic progenitor and stem cells.

An increased traffic of hematopoietic progenitor cells (HPC) between bone marrow and peripheral organs is a peculiar feature of the allergic inflammation. It has been recently reported that the sublingual form of specific immunotherapy (SLIT) is capable of reducing such an increased HPC traffic. The House Dust Mite major antigen Der p1 has been proved to up-regulate the expression of the ICAM-1 and VCAM-1 endothelial addressins, supporting the view of an inflammatory cell recruiting at the site of allergen extract administration. In the present work we have investigated, by flow-cytometric techniques, the expression of the two major integrins CD11a (LFA-1) and CD49d (VLA-4) that are the homing receptor cognate for ICAM-1 and VCAM-1 on human cord blood CD34 hematopoietic progenitor and stem cells. Even if both the investigated molecules resulted detectable on CD34+ HPC surfaces, being the system redundant, the density of the cellular expression was significantly higher for CD49d (median value: 158) than CD11a (median value: 20.5), suggesting a preferential usage of the homing axis VLA-4/VCAM-1. Results consistency with outcomes of clinical trials that relate SLIT efficacy to allergen dosage is discussed.

Monocyte and neutrophil adhesion molecule expression during acute hyperglycemia and after antioxidant treatment in type 2 diabetes and control patients.

OBJECTIVE: We hypothesized that acute hyperglycemia (an independent cardiovascular risk factor) increases the expression of proatherogenic leukocyte adhesion molecule in type 2 diabetes and controls and that the expression of these adhesion molecules would be antioxidant sensitive. METHODS AND RESULTS: Twenty-three type 2 diabetes patients and 13 control patients underwent two oral glucose tolerance tests 14 days apart and took placebo or 800 IU daily of oral alpha tocopherol between tests. Monocyte and neutrophil expression of adhesion molecules Mac-1, LFA-1 and 3, ICAM-1, and VLA-4 were measured at 0, 120, and 240 minutes by using laser flow cytometry. Baseline adhesion molecule expression did not differ between groups, but there was a rapid, highly significant increase (P<0.0001) in the intensity of monocyte Mac-1 expression after a glucose load in both groups. Alpha-tocopherol supplementation reduced only Mac-1 expression in the diabetes group (P=0.03). CONCLUSIONS: Acute glycemic excursions of any degree cause highly significant, rapid increases in monocyte Mac-1 expression in type 2 diabetes patients and controls. Mac-1 mediates leukocyte vascular infiltration and is prothrombotic. These data suggest a mechanism for the link between glycemic excursions and increased vascular event rates.

Comparison of processing four and five times the patients' blood volume during peripheral blood stem cell collection and analysis of CD34+38--and CD34+49d+ subsets during apheresis.

PURPOSE: We investigated whether increasing the patients' processed blood volume (BV) during peripheral blood stem cell collection (PBSCC) from four to five times leads to a greater yield of CD34+ cells. We also studied the kinetics of CD34+38- and CD34+49d+ subsets and compared the amount of transfused cells with engraftment. METHODS: All patients ( n=20) received chemotherapy followed by G-CSF for PBSC mobilization. Samples from the patients' peripheral blood and the PBSC harvests were taken after processing 1-, 4-, and 5 times the patients' calculated BV. RESULTS: The mean total yields of CD34+, CD34+38-, and CD34+49d+ cells were 15.69-, 1.13- and 4.17 x 10(6)/kg body weight, respectively. The mean increase for these subsets between 4- and 5 BV was 10%, 8%, and 21%, respectively. Based on the mean number of 2.25 (range 2-3) planned courses of high-dose chemotherapy (HDC) per patient, the mean yield of CD34+ cells per kg body weight and intended course of HDC after 4- and 5 BV was 6.31- and 6.97 x 10(6) ( P=0.014). Twenty HDC were evaluable for engraftment. There was some correlation between the number of transfused CD34+ and CD34+38- cells and WBC engraftment ( r = -0.66 and --0.69; P<0.01) and CD34+ cells and platelet engraftment ( r = -0.56; P= 0.013). No toxicity occurred during PBSCC, although the mean platelet count dropped by 50% which must be kept in mind regarding the additional application of anti-coagulants and the fact that most patients had large indwelling catheters. CONCLUSION: Processing 4 BV is sufficient to collect >5 x 10(6) CD34+ cells/kg body weight and intended course of HDC in most patients, although extension to 5 BV further increases the total yield of CD34+ cells.

CD34(+) cord blood cells expressing cutaneous lymphocyte-associated antigen are enriched in granulocyte-macrophage progenitors and support extensive amplification of dendritic cell progenitors.

OBJECTIVE: We evaluated the frequency of hematopoietic progenitor cells (HPC) in CD34(+)CLA(+) (cutaneous lymphocyte-associated antigen) and CD34(+)CLA(-) cord blood cells, and followed cellular growth and HPC production during cultures in Flt3 ligand, thrombopoietin, and stem cell factor (FTS). MATERIALS AND METHODS: Immunomagnetic bead-purified CD34(+) cells were sorted into CD34(+)CLA(+) or CD34(+)CLA(-) cells. HPC frequency was assessed by clonal assays in methylcellulose either ex vivo or after, 7, 14, or 21 days of culture with FTS. Dendritic cell (DC) progenitors were evaluated after induction of FTS-amplified cells into DC using secondary cultures containing granulocyte-macrophage colony-stimulating factor and interleukin-4. RESULTS: Ex vivo, granulocyte-macrophage progenitors were more frequent and erythroid progenitors were less frequent in the CLA(+) fraction. In FTS culture, CD34(+)CLA(+) cells produced greater absolute numbers of CD34(+) cells, granulocyte-macrophage-, erythroid-, and DC (including Langerhans cell-related) progenitors compared to CD34(+)CLA(-) cells. In CD34(+)CLA(+) cultures, CLA(+) cells steadily decreased with time, and CD34(+)CLA(-) cells appeared. In CD34(+)CLA(-) cultures, CLA(+) cells were generated, increased up to day 7, and decreased thereafter. CLA was expressed only on CD34(-) cells in these cultures. Ex vivo, CD34(+)CLA(+) cells could be subdivided further into CD38(low) and CD38(high) cells. Cord blood and growth factor-mobilized CD34(+) cells contained more CLA(+)CD38(low) cells than nonmobilized peripheral blood CD34(+) cells and proliferated more extensively with FTS than the latter cells. CONCLUSIONS: CD34(+)CLA(+) cells contain a rather immature progenitor capable of high proliferation and extensive amplification of HPC in vitro. This progenitor may be localized in the CD34(+)CLA(+)CD38(low) fraction. In addition, cultures of CD34(+)CLA(+) cells from cord blood produced CD34(+)CLA(-) cells, suggesting that these cells may derive directly from CD34(+)CLA(+) cells in vivo.

Granulocyte colony-stimulating factor and lineage-independent modulation of VLA-4 expression on circulating CD34+ cells.

Although the use of allogeneic transplants of peripheral blood stem/progenitor cells (PBSCs) is increasing, the precise mechanism of PBSC mobilization has not yet been fully clarified. We examined the expression of some adhesion molecules on CD34+ cells from steady-state bone marrow (BM), granulocyte colony-stimulating factor (G-CSF)-mobilized PBSCs, and cytotoxic drugs plus G-CSF-mobilized PBSCs. Irrespective of mobilization method, very late antigen (VLA)-4 expression on circulating CD34+ cells was significantly lower than on steady-state BM CD34+ cells. To elucidate the influence of lineage commitment on VLA-4 expression of circulating CD34+ cells, we analyzed VLA-4 expression on different subsets of CD34+ cells with or without CD33, CD38, CD5, or CD10 antigens, or Glycophorin A in G-CSF-mobilized PBSCs and steady-state BM from related donors, using 3-color flow cytometry. VLA-4 on circulating CD34+ subsets was less expressed than on each corresponding subset of steady-state BM CD34+ cells. Furthermore, VLA-4 positive rates showed no significant difference among the CD34+ subsets. Finally, the data comparing CD34+ cells from steady-state and G-CSF-mobilized PBSCs revealed no differences in terms of VLA-4 expression. These data suggest that reduced expression of VLA-4 may be a result of peripheralization of CD34+ cells from bone marrow, which occurs in a G-CSF- and lineage-independent fashion.

Adhesion molecules in multiple sclerosis: relation to subtypes of disease and methylprednisolone therapy.

OBJECTIVES: To determine levels of adhesion molecules in blood and cerebrospinal fluid (CSF) samples from patients with different subtypes and activities of multiple sclerosis (MS) and to assess the effect of intravenous methylprednisolone sodium succinate treatment on the levels of soluble adhesion molecules. DESIGN: The expressions of very late activation antigen 4 (VLA-4), lymphocyte function associated antigen 1 (LFA-1), vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) were determined immunocytochemically, and levels of soluble VCAM-1, ICAM-1, and E-selectin, by means of enzyme immunoassay technique. The volumes of T2- and T1-weighted MS plaques and brain atrophy were determined by means of the semiautomatic magnetic resonance imaging (MRI) segmentation technique. SETTING: A university hospital in Finland. PATIENTS: One hundred subjects (71 patients with MS and 29 healthy control subjects). The subtypes of MS were relapsing-remitting (RRMS [n = 26]), secondary progressive (SPMS [n = 20]), and primary progressive (PPMS [n = 25]). RESULTS: In patients with RRMS and SPMS, the expressions of VLA-4 and LFA-1 on immune cells from blood were at least 1.5- to 3-fold higher than in controls (RRMS, P = .002 and P<.001, respectively; SPMS, P = .03 and P =.001, respectively). In RRMS, LFA-1 and ICAM-1 expression in blood was more up-regulated than in SPMS (P = .03 and P = .01, respectively). The expressions of adhesion molecules on CSF lymphocytes in RRMS and SPMS were of similar magnitude, but the proportions of CSF VLA-4- and LFA-1-expressing lymphocytes were 3- to 4-fold higher than in controls (P = .04 and P = .008, respectively). The levels of serum soluble VCAM-1 were higher in SPMS than in RRMS (P = .005) or PPMS (P = .04). Intravenous methylprednisolone treatment of patients with RRMS in exacerbation caused a significant reduction in the serum levels of soluble VCAM-1 and E-selectin (P<.001). In SPMS, the volumes of T2-weighted plaques correlated with the serum level of soluble ICAM-1 (r = 0.64; P = .03). CONCLUSIONS: Up-regulated adhesion molecules in blood and CSF indicate sustained potential for inflammation in the CNS throughout the clinical spectrum of MS. Therapies interfering with cell adhesion may be of key importance in suppressing MS.

Alpha 4 beta 1 integrin/VCAM-1 interaction activates alpha L beta 2 integrin-mediated adhesion to ICAM-1 in human T cells.

Modulation of integrin affinity and/or avidity provides a regulatory mechanism by which leukocyte adhesion to endothelium is strengthened or weakened at different stages of emigration. In this study, we demonstrate that binding of high-affinity alpha 4 beta 1 integrins to VCAM-1 strengthens alpha L beta 2 integrin-mediated adhesion. The strength of adhesion of Jurkat cells, a human leukemia T cell line, or MnCl2-treated peripheral blood T cells to immobilized chimeric human VCAM-1/Fc, ICAM-1/Fc, or both was quantified using parallel plate flow chamber leukocyte detachment assays in which shear stress was increased incrementally (0.5-30 dynes/cm2). The strength of adhesion to VCAM-1 plus ICAM-1, or to a 40-kDa fragment of fibronectin containing the CS-1 exon plus ICAM-1, was greater than the sum of adhesion to each molecule alone. Treatment of Jurkat or blood T cells with soluble cross-linked VCAM-1/Fc or HP2/1, a mAb to alpha 4, significantly increased adhesion to ICAM-1. These treatments induced clustering of alpha L beta 2 integrins, but not the high-affinity beta 2 integrin epitope recognized by mAb 24. Up-regulated adhesion to ICAM-1 was abolished by cytochalasin D, an inhibitor of cytoskeletal rearrangement. Taken together, our data suggest that the binding of VCAM-1 or fibronectin to alpha 4 beta 1 integrins initiates a signaling pathway that increases beta 2 integrin avidity but not affinity. A role for the cytoskeleton is implicated in this process.