Number Of Cells In Peripheral Blood Research Articles

Seasonal changes in suppressive capacity of CD4+ CD25+ T cells from patients with hayfever are allergen‐specific and may result in part from expansion of effector T cells among the CD25+ population

Suppression of allergen-stimulated peripheral blood CD4(+) CD25(-) effector T cells by CD4(+) CD25(+) regulatory T cells obtained from subjects with allergic rhinoconjunctivitis is reduced during the pollen season when compared with out of season. We examined possible explanations for this effect of seasonal pollen exposure on suppression of allergen responses. CD4(+) CD25(-) and CD4(+) CD25(+) T cells were isolated from blood obtained from 44 volunteers with allergic rhinoconjunctivitis during and out of the UK grass pollen season. Co-cultures were performed with grass pollen extract and house dust mite (HDM) to examine allergen specificity. The frequency of IL-5 and IL-10 producing cells was determined by ELISPOT and the expression of T cell activation markers and the CD25(+) regulatory T cell-associated transcription factor Foxp3 were examined. Lactic acid stripping of IgE was used to determine IgE dependence of T cell responses. The seasonal reduction in suppression by CD4(+) CD25(+) T cells was confirmed and was shown to be allergen specific because suppression of HDM-stimulated cultures was not affected significantly. The CD4(+) CD25(+) population contained IL-5 and IL-10 producing cells but increases in their frequencies with seasonal pollen exposure were not significant. Both activation marker and Foxp3 expression increased during the pollen season. IgE stripping reduced CD4(+) and CD4(+) CD25(-) T cell responses to allergen, but had no effect on suppression by CD4(+) CD25(+) T cells. The seasonal reduction in suppression of grass pollen-stimulated effector T cells by CD4(+) CD25(+) T cells is allergen specific and cannot be explained by increased IgE-facilitated allergen presentation. We suggest that changes in the proportion of effector to regulatory T cells among the CD25(+) population isolated may partially explain these findings, and that trafficking to the site of allergic disease may reduce allergen-specific regulatory T cell numbers in peripheral blood.

Natural killer cell number and phenotype in bovine peripheral blood is influenced by age

Natural killer (NK) cells are critical to the innate defence against intracellular infection. High NK cell frequencies have been detected in human neonates, which may compensate for the relative immaturity of the specific immune response. Additionally, phenotypic subsets of NK cells have been identified in humans with different functional properties. In this study, we examined the age distribution and phenotype of NK populations in bovine peripheral blood, including neonatal animals. We found that the NK cell populations defined by the phenotypes CD3 −CD2 + and NKp46 + largely overlapped, so that the majority of NK cells in bovine peripheral blood were CD3 −CD2 +NKp46 +. The remainder of the NK-like cells comprised two minor populations, CD3 −CD2 +NKp46 − and CD3 −CD2 −NKp46 +; the relative proportions of these varied with age. The lowest frequency of NK cells was recorded in 1-day-old calves, with the highest frequency in day 0 calves. The phenotypic characteristics of CD3 −CD2 + and NKp46 + NK populations were similar; both populations expressed CD45RO, CD45RB, CD11b, CC84, CD8αα and CD8αβ and did not express CD21, WC1, CD14 or γδ TCR. Age-related phenotypic differences were apparent. The phenotypic characteristics of three NK subpopulations were described; a significantly greater proportion of the CD3 −CD2 −NKp46 + population expressed CD8α compared to CD3 −CD2 +NKp46 + cells. Furthermore, a significantly greater proportion of the CD3 −CD2 +NKp46 − population expressed CD8 compared to total CD3 −CD2 + cells. Adult cattle had a significantly higher proportion of perforin + cells compared to calves aged ≤6 weeks. In this age group, the majority of perforin + cells expressed NKp46, while in adults the majority of perforin + cells were NKp46 −. However, the proportion of NKp46 + and CD3 −CD2 + cells that expressed perforin was not significantly different in any age group tested.

Increase in primitive stem cell numbers in equine peripheral blood following stress

Stout et al. (Amer Surg. 73:1106‐1110, 2007) reported base‐line levels of primitive stem cells circulating in adult porcine peripheral blood, which increased dramatically after just 90 minutes of trauma. Ongoing studies have shown similar stem cells in adult equine blood and that base‐line levels varied according to breed of the horse. The current study was undertaken to determine whether these stem cells would increase in number in the peripheral blood following stress in the same horse. Stress was defined as moderate exercise, i.e., 10 minutes of cantering. Blood withdrawal followed the guidelines of FVSU‐IACUC. Adult horses had 8 ml of blood withdrawn by venipuncture immediately prior to and immediately after exercising. After gravity separation, the plasma was withdrawn and 15 microliters of plasma from each sample was mixed with 15 microliters of 0.4% Trypan blue. The resultant solution was mixed, placed onto a hemocytometer and photographed: blastomere‐like stem cells (BLSCs) were Trypan blue positive and < 2.0 microns in size; transitional‐BLSCs (Tr‐BLSCs) were both Trypan blue positive & negative and 2‐5 microns in size; and epiblast‐like stem cells (ELSCs) were Trypan blue negative and 6‐8 microns in size (Young and Black, Minerva Biotech 17:55‐63, 2005). This study demonstrates that moderate exercise will increase the level of these primitive pluripotent stem cells in the blood of adult equines.

Impaired immune function in Gulf War Illness

BackgroundGulf War Illness (GWI) remains a serious health consequence for at least 11,000 veterans of the first Gulf War in the early 1990s. Our understanding of the health consequences that resulted remains inadequate, and this is of great concern with another deployment to the same theater of operations occurring now. Chronic immune cell dysfunction and activation have been demonstrated in patients with GWI, although the literature is not uniform. We exposed GWI patients and matched controls to an exercise challenge to explore differences in immune cell function measured by classic immune assays and gene expression profiling.MethodsThis pilot study enrolled 9 GWI cases identified from the Department of Veterans Affairs GWI registry, and 11 sedentary control veterans who had not been deployed to the Persian Gulf and were matched to cases by sex, body mass index (BMI) and age. We measured peripheral blood cell numbers, NK cytotoxicity, cytokines and expression levels of 20,000 genes immediately before, immediately after and 4 hours following a standard bicycle ergometer exercise challenge.ResultsA repeated-measures analysis of variance revealed statistically significant differences for three NK cell subsets and NK cytotoxicity between cases and controls (p < 0.05). Linear regression analysis correlating NK cell numbers to the gene expression profiles showed high correlation of genes associated with NK cell function, serving as a biologic validation of both the in vitro assays and the microarray platform. Intracellular perforin levels in NK and CD8 T-cells trended lower and showed a flatter profile in GWI cases than controls, as did the expression levels of the perforin gene PRF1. Genes distinguishing cases from controls were associated with the glucocorticoid signaling pathway.ConclusionGWI patients demonstrated impaired immune function as demonstrated by decreased NK cytotoxicity and altered gene expression associated with NK cell function. Pro-inflammatory cytokines, T-cell ratios, and dysregulated mediators of the stress response (including salivary cortisol) were also altered in GWI cases compared to control subjects. An interesting and potentially important observation was that the exercise challenge augments these differences, with the most significant effects observed immediately after the stressor, possibly implicating some block in the NK and CD8 T-cells ability to respond to "stress-mediated activation". This has positive implications for the development of laboratory diagnostic tests for this syndrome and provides a paradigm for exploration of the immuno-physiological mechanisms that are operating in GWI, and similar complex syndromes. Our results do not necessarily elucidate the cause of GWI, but they do reveal a role for immune cell dysfunction in sustaining illness.

Increases In CD25 + T Cell Numbers In Peripheral Blood Of Children With Allergic Asthma Correlate With Measures Of Airway Obstruction, But Not Serum IgE Levels

Increases In CD25 + T Cell Numbers In Peripheral Blood Of Children With Allergic Asthma Correlate With Measures Of Airway Obstruction, But Not Serum IgE Levels

Tumor Necrosis Factor-α Blockade Leads to Decreased Peripheral T Cell Reactivity and Increased Dendritic Cell Number in Peripheral Blood of Patients with Ankylosing Spondylitis

To study the effect of tumor necrosis factor-alpha (TNF-alpha) antagonist (etanercept) treatment on the peripheral T cell reactivity of patients with ankylosing spondylitis (AS). Peripheral blood mononuclear cells were collected from 40 patients with AS at baseline, after 2 and 6 weeks of etanercept treatment or placebo treatment, and from healthy controls. The number of cells secreting various cytokines was detected by enzyme linked immunospot. Serum soluble interleukin 2 (IL-2) receptor level was measured by ELISA. T cell proliferation was assayed with the WST-1 live cell-staining method. The myeloid dendritic cell (mDC) and regulatory T cell (Treg) levels were analyzed by fluorescence activated cell sorting. . After 2 and 6 weeks of etanercept treatment, the number of TNF-alpha-secreting monocytes decreased. Although the T cell proliferation rate remained stable, the number of T cells secreting IL-2 and interferon-gamma under anti-CD3/anti-CD28 stimulation was significantly decreased. The level of serum soluble IL-2R (sIL-2R), a T cell activation marker, also declined. The changes in T cell reactivity were correlated with a significant increase in MHC Class II-positive mDC cells in circulation. An increase in Treg cell numbers was also observed. . The anti-TNF-alpha therapy blockaded MHC Class II-positive mDC maturation, enhanced regulatory T cell levels, and suppressed the functions of effector T cells. The reduced T cell reactivity could contribute to the efficacy of the TNF-alpha antagonist therapy in patients with AS.

Epstein-Barr virus and HIV play no direct role in persistent generalized lymphadenopathy syndrome.

Persistent generalized lymphadenopathy (PGL) and polyclonal B cell activation are features of infection with HIV. Epstein-Barr virus (EBV) and HIV are known to activate B cells in vitro, but whether they are important B cell activators in patients infected with HIV is less clear. In this study, lymph node tissue was obtained from 10 patients with PGL and assessed for evidence of EBV and HIV gene sequences. DNA was extracted and specific viral gene sequences identified using the polymerase chain reaction (PCR). EBV sequences were difficult to detect in the PGL tissue, with a signal intensity similar to that of other benign and malignant lymphoid conditions not associated with EBV. HIV sequences were also rare in the PGL tissue, consistent with HIV infection of the small number of peripheral blood cells and nodal T cells likely to be present in such a sample. These findings suggest that the polyclonal B cell activation typical of HIV is not driven by direct EBV or HIV infection of B cells.

Impaired response to GM-CSF and G-CSF, and enhanced apoptosis in C/EBPβ-deficient hematopoietic cells

AbstractTranscription factors known as CCAAT enhancer binding proteins (C/EBPs) are involved in hematopoietic differentiation, including myelopoiesis and granulopoiesis. C/EBPβ-deficient mice develop normally; however, they exhibit defective macrophage function, resulting in increased susceptibility to infection. Little is known about the role of C/EBPβ in granulopoiesis; therefore, we examined granulopoiesis in C/EBPβ-deficient mice. Morphology, the number of peripheral blood and bone marrow cells, and the expression of genes specific for the myeloid lineage were normal in C/EBPβ-deficient mice. Interestingly, the hematopoietic progenitor cells of C/EBPβ-deficient mice did not respond normally to granulocyte/macrophage-colony stimulating factor and granulocyte colony stimulating factor. In addition, C/EBPβ-deficient neutrophils displayed enhanced apoptosis compared with wild-type neutrophils. Our present results indicate that C/EBPβ helps regulate survival of neutrophils, downstream of the granulocyte colony stimulating factor receptor.

‘Iron‐saturated’ lactoferrin is a potent natural adjuvant for augmenting cancer chemotherapy

Bovine lactoferrin (bLf), an iron-containing natural defence protein found in bodily secretions, has been reported to inhibit carcinogenesis and the growth of tumours. Here, we investigated whether natural bLf and iron-saturated forms of bLf differ in their ability to augment cancer chemotherapy. bLf was supplemented into the diet of C57BL/6 mice that were subsequently challenged subcutaneously with tumour cells, and treated by chemotherapy. Chemotherapy eradicated large (0.6 cm diameter) EL-4 lymphomas in mice that had been fed iron-saturated bLf (here designated Lf(+)) for 6 weeks prior to chemotherapy, but surprisingly not in mice that were fed lesser iron-saturated forms of bLf, including apo-bLf (4% iron saturated), natural bLf (approximately 15% iron saturated) and 50% iron-saturated bLf. Lf(+)-fed mice bearing either EL-4, Lewis lung carcinoma or B16 melanoma tumours completely rejected their tumours within 3 weeks following a single injection of either paclitaxel, doxorubicin, epirubicin or fluorouracil, whereas mice fed the control diet were resistant to chemotherapy. Lf(+) had to be fed to mice for more than 2 weeks prior to chemotherapy to be wholly effective in eradicating tumours from all mice, suggesting that it acts as a competence factor. It significantly reduced tumour vascularity and blood flow, and increased antitumour cytotoxicity, tumour apoptosis and the infiltration of tumours by leukocytes. Lf(+) bound to the intestinal epithelium and was preferentially taken up within Peyer's patches. It increased the production of Th1 and Th2 cytokines within the intestine and tumour, including TNF, IFN-gamma, as well as nitric oxide that have been reported to sensitize tumours to chemotherapy. Importantly, it restored both red and white peripheral blood cell numbers depleted by chemotherapy, potentially fortifying the mice against cancer. In summary, bLf is a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy, but needs to be saturated with iron to be effective.

Elevation in Peripheral Blood Circulating Tumor Cell Number Correlates with Macroscopic Progression in UICC Stage IV Colorectal Cancer Patients

Aims: Cytokeratin(CK) based real-time RT-PCR assays (QRT-PCR) are now available for peripheral blood circulating tumor cell (CTC) evaluations in colorectal cancer(CRC) patients. Results are non-existent for the application of these techniques to the determination of progression and therapy response in Dukes stage D CRC patients. Patients and methods: Each month 30 ml peripheral blood of 30 Dukes D patients (17 with progression) were drawn. CEA, CA19-9, CA72-A and TPA-M determinations were made. CK20, thymidilate synthase(TS) QRT-PCR was performed, as well. Buffy coat was used for immunmagnetic cancer cell isolation and CTC counting. Correlation between elevated CTC and macroscopic progression within 3 months was determined by Chi2 test. Results: Microscopic CTC single cell, doublet, cluster number were found in correlation with CK20 QRT-PCR results (p < 0.01). There was a significant increase in microscopic CTC number, CK20 and decrease in TS QRT-PCR levels (p < 0.05) in the peripheral blood of the non-responder as compared to responder patients. Elevation of the CTC was in significant correlation with macroscopic progression of the disease in 3 months (p < 0.01). Conclusions: CTC number reflects the chemotherapeutic sensitivity of CRC patients. Elevation of circulating tumor cell number in peripheral blood is in correlation with macroscopic progression.

Effect of whole body proton irradiation on hematological and gastrointestinal damages

With increasing the chance of cancer radiotherapy or spaceflight with mission or simple trip, a considerable concern about the absorbed amount of radiation and its deleterious effect on physiological system has been increased. Many efforts have been contributed to estimate the risk, however, there is little known about the spectrum of radiations during the flight as well as the effects of low-dose radiation. Here, we aimed to investigate the effect of whole body proton irradiation at a recommended dose limit of occupational (20mGy per year) and the standardized radio-therapeutic fraction dose (2Gy) on gastro-intestinal damages, and hematological variables compared with gamma irradiation. Exposure to low dose radiation has not shown any distinctive changes on hematological (the number of bone marrow cells and peripheral blood cells) and gastrointestinal damages whatever the source of radiation is proton or gamma ray. However, the therapeutic dose of radiation significantly decreased the number of spleen cells and white blood cells about 1.5- and 4-fold, respectively. Especially the number of lymphocytes was only 20% of the untreated control mice. The frequency of micronuclei in bone marrow cells was not affected in proton irradiated mice in contrast to that of gamma irradiation. The apoptosis of intestinal crypts confirmed by TUNEL assay was markedly increased by the therapeutic dose of radiation, and the magnitude of the apoptosis in gamma irradiated mice was severer than that of proton. The degree of damages with gamma irradiation seemed to be more profound than proton even the same total dose of radiation, suggesting that the physiological effects were dependent on the characteristic of the source of radiation.

Cellular and Some Biochemical Changes in Blood and Peritoneal Fluid Constituents in Awassi Lambs Following Elective Castration

No reports could be cited in the recent literature concerning the effects of surgical castration on blood and peritoneal fluid constituents in lambs. The purpose of this study was to evaluate the effects of surgical castration on cellular and some biochemical parameters of blood and peritoneal fluid in Awassi lambs. Eight, clinically healthy, 5-month-old, Awassi lambs were surgically castrated. Peritoneal fluid and blood samples were collected prior to castration (day 0) and on days 1, 3 and 7 post castration. Total nucleated cell count (TNCC) was determined using an electric cell counter. Total protein (TP), albumin, fibrinogen, creatinine, urea, glucose, alkaline phosphatase (ALP), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) concentrations were analyzed using commercially available kits. Following castration, the number of peripheral fluid white blood cells (WBC) and the percentage of neutrophils significantly increased (p 0.05) in the percentage of lymphocytes in the peritoneal fluid. The concentration of peritoneal fluid TP, urea, creatinine, glucose, ALP and AST significantly increased (p<0.05) while LDH concentration did not change. Results of this study show that changes in the blood and peritoneal fluid cellular and biochemical components are likely to follow elective surgical castration in lambs. These changes must be taken in consideration when peritoneal fluid samples from lambs are evaluated for the diagnosis of abdominal diseases.

Nilotinib treatment in mouse models of P190 Bcr/Abl lymphoblastic leukemia

BackgroundPh-positive leukemias are caused by the aberrant fusion of the BCR and ABL genes. Nilotinib is a selective Bcr/Abl tyrosine kinase inhibitor related to imatinib, which is widely used to treat chronic myelogenous leukemia. Because Ph-positive acute lymphoblastic leukemia only responds transiently to imatinib therapy, we have used mouse models to test the efficacy of nilotinib against lymphoblastic leukemia caused by the P190 form of Bcr/Abl.ResultsAfter transplant of 10,000 highly malignant leukemic cells into compatible recipients, untreated mice succumbed to leukemia within 21 days, whereas mice treated with 75 mg/kg nilotinib survived significantly longer. We examined cells from mice that developed leukemia while under treatment for Bcr/Abl kinase domain point mutations but these were not detected. In addition, culture of such cells ex vivo showed that they were as sensitive as the parental cell line to nilotinib but that the presence of stromal support allowed resistant cells to grow out. Nilotinib also exhibited impressive anti-leukemia activity in P190 Bcr/Abl transgenic mice that had developed overt leukemia/lymphoma masses and that otherwise would have been expected to die within 7 days. Visible lymphoma masses disappeared within six days of treatment and leukemic cell numbers in peripheral blood were significantly reduced. Treated mice survived more than 30 days.ConclusionThese results show that nilotinib has very impressive anti-leukemia activity but that lymphoblastic leukemia cells can become unresponsive to it both in vitro and in vivo through mechanisms that appear to be Bcr/Abl independent.

Evaluation of stem cell administration in a model of kidney ischemia-reperfusion injury

Ischemia-reperfusion injury is a common early event in kidney transplantation and contributes to a delay in organ function. Acute tubular necrosis, impaired kidney function and organ leukocyte infiltration are the major findings. The therapeutic potential of stem cells has been the focus of recent research as these cells possess capabilities such as self-renewal, multipotent differentiation and aid in regeneration after organ injury. FTY720 is a new synthetic compound that has been associated with preferential migration of blood lymphocytes to peripheral lymph nodes instead of inflammatory sites. Bone marrow stem cells (BMSC) and/or FTY720 were used as therapy to promote recovery of tubule cells and avoid inflammation at the renal site, respectively. Mice were submitted to renal ischemia-reperfusion injury and were either treated with two doses of FTY720, 10×106 BMSC, or both in order to compare the therapeutic effect with non-treated and control animals. Renal function and structure were investigated as were cell numbers in peripheral blood and spleen. Activation and apoptosis markers were also evaluated in splenocytes using flow cytometry. We found that the combined therapy (FTY720+BMSC) was associated with more significant changes in renal function and structure after ischemia-reperfusion injury when compared with the other groups. Also a decrease at cell numbers and prevention of spleen cells activation and apoptosis was observed. In conclusion, in our model it was not possible to demonstrate the potential of stem cells alone or in combination with FTY720 to promote early kidney recovery after ischemia-reperfusion injury.

Membrane Channel Connexin 32 Maintains Lin−/c-kit+ Hematopoietic Progenitor Cell Compartment: Analysis of the Cell Cycle

Membrane channel connexin (Cx) forms gap junctions that are implicated in the homeostatic regulation of multicellular systems; thus, hematopoietic cells were assumed not to express Cxs. However, hematopoietic progenitors organize a multicellular system during the primitive stage; thus, the aim of the present study was to determine whether Cx32, a member of the Cx family, may function during the primitive steady-state hematopoiesis in the bone marrow (BM). First, the numbers of mononuclear cells in the peripheral blood and various hematopoietic progenitor compartments in the BM decreased in Cx32-knockout (KO) mice. Second, on the contrary, the number of primitive hematopoietic progenitor cells, specifically the Lin(-)/c-kit(+)/Scal(+ )fraction, the KSL progenitor cell compartment, also increased in Cx32-KO mice. Third, expression of Cx32 was detected in Lin(-)/c-kit(+) hematopoietic progenitor cells of wild-type mice (0.27% in the BM), whereas it was not detected in unfractionated wild-type BM cells. Furthermore, cell-cycle analysis of the fractionated KSL compartment from Cx32-KO BM showed a higher ratio in the G(2)/M fraction. Taken together, all these results imply that Cx32 is expressed solely in the primitive stem cell compartment, which maintains the stemness of the cells, i.e., being quiescent and noncycling; and once Cx32 is knocked out, these progenitor cells are expected to enter the cell cycle, followed by proliferation and differentiation for maintaining the number of peripheral blood cells.

Abnormal hematopoiesis in Gab2 mutant mice

Gab2 is an important adapter molecule for cytokine signaling. Despite its major role in signaling by receptors associated with hematopoiesis, the role of Gab2 in hematopoiesis has not been addressed. We report that despite normal numbers of peripheral blood cells, bone marrow cells, and c-Kit(+)Lin(-)Sca-1(+) (KLS) cells, Gab2-deficient hematopoietic cells are deficient in cytokine responsiveness. Significant reductions in the number of colony-forming units in culture (CFU-C) in the presence of limiting cytokine concentrations were observed, and these defects could be completely corrected by retroviral complementation. In earlier hematopoiesis, Gab2-deficient KLS cells isolated in vitro responded poorly to hematopoietic growth factors, resulting in an up to 11-fold reduction in response to a cocktail of stem cell factor, flt3 ligand, and thrombopoietin. Gab2-deficient c-Kit(+)Lin(-) cells also demonstrate impaired activation of extracellular signal-regulated kinase (ERK) and S6 in response to IL-3, which supports defects in activating the phosphatidylinositol-3 kinase (PI-3K) and mitogen-associated protein kinase (MAPK) signaling cascades. Associated with the early defects in cytokine response, competitive transplantation of Gab2(-/-) bone marrow cells resulted in defective long-term multilineage repopulation. Therefore, we demonstrate that Gab2 adapter function is intrinsically required for hematopoietic cell response to early-acting cytokines, resulting in defective hematopoiesis in Gab2-deficient mice.

Abnormal surface markers expression on bone marrow CD34+ cells and correlation with disease activity in patients with systemic lupus erythematosus

Defects of hematopoietic stem cells (HSCs) have been suggested to contribute to the development of systemic lupus erythematosus (SLE). The aim of this study was to investigate the phenotypic characteristics of bone marrow (BM) CD34(+) cells in patients with SLE and its relationship with SLE disease activity. Ten SLE patients and 10 healthy subjects were recruited and their BM CD34(+) cells were analyzed by flow cytometric analysis with CD45/SSC gating for the expression of CD90, CD95, CD117, CD123, CD164, CD166, FAS-L, and HLA-DR. The percentage of BM CD34(+) cells was significantly decreased in active SLE patients (1.48 +/- 0.41%, n = 7) compared to the healthy controls (2.31 +/- 0.75%, n = 10, p < 0.01), but no significant difference was found between the inactive patients (2.04 +/- 0.44%, n = 3) and the controls. The expression of CD95, CD123, and CD166 on BM CD34(+) cells were significantly increased in SLE patients (48.31 +/- 10.59%, 44.9 +/- 21.5%, 30.9 +/- 19.54%, respectively, n = 10) when compared with the control subjects (24.33 +/- 11.1%, 19.5 +/- 4.4%, 10.7 +/- 5.5%, respectively, n = 10, p < 0.05). The increased CD123 expression was negatively correlated with the number of peripheral white blood cells (r = -0.700, p < 0.05, n = 10). The percentage of CD166 expression was found significantly correlated with the index of SLE disease activity (r = 0.472, p < 0.05, n = 10) and 24 h proteinuria (r = 0.558, p < 0.05, n = 10), but negatively correlated with serum C3 level (r = -0.712, p < 0.01, n = 10). Our study found that the surface marker expression of CD95, CD123, and CD166 on BM CD34(+) cells were significantly increased in patients. This supports the hypothesis that there are abnormalities of the HSC in SLE. Since CD166 and CD123 correlated with the overall lupus activity, their role as a biomarker of inflammatory disease activity also requires further study.

Comparative Study of Circadian Variation in Numbers of Peripheral Blood Cells among Mouse Strains: Unique Feature of C3H/HeN Mice

We examined strain differences in numbers of blood cells and their circadian rhythms in male Jcl:ICR, BALB/cA, C57BL/6J and C3H/HeN mice. The total numbers of circulating white blood cells (WBCs) were increased during subjective day and night, and the peaks in the active period were common to all strains. However, the number of WBCs in C3H/HeN mice remained lower and plasma levels of corticosterone (CS) were slightly higher throughout the day compared with the other strains. The numbers of circulating red blood cells (RBC) also differed according to strain. The numbers of RBCs, hematocrit (HCT) and hemoglobin (HGB) were considerably lower in C3H/HeN mice compared with the other strains, although mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) were highest among the tested strains. We found that serum erythropoietin (EPO) levels were considerably higher in C3H/HeN mice than in the other three strains. The high EPO level might be related to the unique features of RBCs in C3H/HeN mice. The present observations provide basic information about the numbers of peripheral blood cells and their circadian rhythm in mouse models and also demonstrate a unique feature of C3H/HeN mice.

Multifunctional Role of Caspase-3 in Regulating Hematopoietic Stem Cells.

The processes of cell cycle control, differentiation and apoptosis are closely intertwined in controlling cell fate during development and in adult homeostasis. Molecular pathways connecting these events in stem cells are poorly defined and we were particularly interested in the cysteine-aspartic acid protease, Caspase-3, an ‘executioner' caspase also implicated in the regulation of the cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1. These latter proteins are known to participate in primitive hematopoietic cell cycling and self-renewal. We demonstrated high levels of Caspase-3 mRNA and protein in immunophenotypically defined mouse hematopoietic stem cells (HSC). Using mice engineered to be deficient in Caspase-3, we observed a consistent reduction of lymphocytes in peripheral blood counts and a slight reduction in bone marrow cellularity. Notably, knockout animals had an increase in the stem cell enriched Lin−cKit+Sca1+Flk2low (LKSFlk2lo) cell fraction. The apoptotic rates of LKS cells under homeostatic conditions as assayed by the Annexin V assay were not significantly different from controls. However, in-vitro analysis of sorted LKS cells revealed a reduced sensitivity to apoptotic cell death in absence of Caspase-3 under conditions of stress (cytokine withdrawal or gamma irradiation). Primitive hematopoietic cells displayed a higher proliferation rate as demonstrated by BrdU incorporation and a significant reduction in the percentage of cells in the quiescent stage of the cell cycle assessed by the Pyronin-Y/Hoechst staining. Upon transplantation, Caspase-3−/− stem cells demonstrated marked differentiation abnormalities with significantly reduced ability to differentiate into multiple hematopoietic lineages while maintaining an increased number of primitive cells. In a competitive bone marrow transplant using congenic mouse stains Capase-3 deficient HSC out-competed WT cells at the stem cell level, while giving rise to comparable number of peripheral blood cells as the WT controls. Transplant of WT BM cells into Caspase-3 deficient mice revealed no difference in reconstitution ability, suggesting negligible effect of the Caspase-3−/− niche microenvironment to stem cell function. These data indicate that Caspase-3 is involved in the regulation of differentiation and proliferation of HSC as a cell autonomous process. The molecular bases for these effects remain to be determined, but the multi-faceted nature of the changes seen suggest that Caspase-3 is central to multiple regulatory pathways in the stem cell compartment.

The Extramedullar Dissemination of Acute Myeloid Leukemic Cells Depends on CD31 (PECAM-1) and CD38 Coexpression Level.

The number of peripheral white blood cells (WBC) shows a wide range in acute myeloblastic leukemia (AML) patients. A high WBC count constitutes an adverse prognosis factor when at presentation and (myelo)monocytic subtypes were more frequently associated with extramedullar tumor sites. Marrow endothelial cells express CD31 and CD31/CD31 interaction is known to promote the migration in physiological mechanisms, as for example transendothelial migration of neutrophiles and monocytes in diapedesis. CD31 is a specific receptor of CD38 which is associated with numerous molecules on the surface of blood cells. Moreover, CD38 could interact with hyaluronate, a component of the extracellular matrix, 2 hyaluronate-binding sites having been reported in its extracellular domain. These elements lead us to postulate that CD31 and CD38 are colocalized on AML cells and that an excess of CD31 promotes the egress of the cells from the marrow compartment, while an excess of CD38 favors their anchorage to marrow microenvironment.The CD38/CD31 colocalization was demonstrated using FRET and cocapping strategies. FRET experiments were performed with a 488 nm laser flow cytometer, Cy3-conjugated anti-CD38 mAb and FITC-conjugated mAb against CD31. For cocapping experiments, we induced capping of CD31 with anti-CD31 mAb at 37°C; the cells were then fixed and labeled with anti-CD38 mAb. Transendothelial migration has been studied using anti-CD31 mAb and anti-CD38 blocking mAbs in Transwell® experiments performed with TrHBMEC cell line. To study the CD38/hyaluronate interaction, cells were treated or not with all-trans retinoic acid to induce CD38 overexpression just before plating the cells onto hyaluronate-coated dish. The in vivo influence of CD31 and CD38 coexpression level was evaluated by studying the phenotype of marrow leukemic cells (S/N ratio of the MFI) and the WBC count of 78 consecutive patients with newly diagnosed de novo AML. In all experiments, P value <0.05 has been considered as statistically significant.FITC-conjugated anti-CD31 induced increase in MFI of Cy-3 by 108.4% and we observed a CD31/CD38 cocapping. The inhibition of migration by anti-CD31 blocking mAb ranged from 2% to 66% of control and was correlated to the CD31 expression level (R=0.9236; P<0.0001), while migration in presence of anti-CD38 blocking mAb was about 80% of the control without correlation with CD38 expression level, suggesting a lesser role in the transendothelial migration process. The blasts exposed to ATRA exhibited a 1.3±0.1-fold increase in the CD38 expression (S/N of MFI) and a 40±10 % increase in adhesion to hyaluronate. The expression of CD31 and CD38 on the membrane of marrow leukemic cells of the AML patients were correlated (R=0.6621; P<0.0001). Moreover, peripheral WBC count was correlated with the CD31/CD38 ratio (R=0.5286; P=0.0129) and an excess of CD31 antigen was associated with an increased peripheral WBC count when comparing patients with ratio >1 to those with ratio <1 (P<0.0001). Interestingly, CD31/CD38 ratio was higher in AML (myelo)monocytic subtypes when comparing M4/M5 to all other FAB subtypes (1.2±0.1 and 0.7±0.1, respectively; P<0.0001).These findings suggest that extramedullar dissemination of AML cells depends on CD31 and CD38 coexpression level and give new insights into novel strategies to reduce the number of extramedullar tumor sites, notably in AML (myelo)monocytic subtypes.