CD44+ cytokeratin-positive tumor cells in blood and bone marrow are associated with poor prognosis of patients with gastric cancer.

BACKGROUND: The phenotypic heterogeneity of circulating tumor cells (CTC) in peripheral blood and disseminated tumor cells (DTC) in bone marrow is an important constraint for clinical decision making. Here, we investigated the implications of two different subpopulations of these cells in gastric cancer (GC). METHODS: GC patients (n = 228) who underwent elective gastric resections were prospectively examined for CTC/DTC. The cells obtained from peripheral blood and bone marrow aspirates were sorted by flow cytometry and CD45- cells expressing cytokeratins (8, 18, and 19) and CD44 were identified by immunofluorescent double staining. RESULTS: Ninety-three (41%) patients had cytokeratin-positive tumor cells in either blood or bone marrow, while cells expressing CD44 were found in 22 (10%) cases. CK+CD44+ cells were significantly more common among patients with distant metastases (50 vs 19%, P = 0.001), while no such correlations were demonstrated for CK+CD44- cells. Detection of CK+CD44+ cells, but not CK+CD44-, was associated with significantly shortened survival. Moreover, the Cox proportional hazards model identified CK+CD44+ cells as a negative prognostic factor with an odds ratio of 2.38 (95% CI 1.28-4.41, P = 0.006). CONCLUSION: CD44+ phenotype of cytokeratin-positive cells in blood and bone marrow is an independent prognostic factor in patients with gastric cancer.

Interactions of tumour-derived micro(nano)vesicles with human gastric cancer cells.

BACKGROUND: Tumour cells release membrane micro(nano)fragments called tumour-derived microvesicles (TMV) that are believed to play an important role in cancer progression. TMV suppress/modify antitumour response of the host, but there is also some evidence for their direct interaction with cancer cells. In cancer patients TMV are present in body fluid and tumour microenvironment. The present study aimed at characterization of whole types/subpopulations, but not only exosomes, of TMV from newly established gastric cancer cell line (called GC1415) and to define their interactions with autologous cells. METHODS: TMV were isolated from cell cultures supernatants by centrifugation at 50,000×g and their phenotype was determined by flow cytometry. The size of TMV was analysed by dynamic light scattering and nanoparticle tracking analysis, while morphology by transmission electron microscopy and atomic force microscopy. Interactions of TMV with cancer cells were visualized using fluorescence-activated cell sorter, confocal and atomic force microscopy, biological effects by xenografts in NOD SCID mice. RESULTS: Isolated TMV showed expression of CD44H, CD44v6 (hyaluronian receptors), CCR6 (chemokine receptor) and HER-2/neu molecules, exhibited different shapes and sizes (range 60-900 nm, highest frequency of particles with size range of 80-120 nm). TMV attached to autologous cancer cells within 2 h and then were internalized by them at 24 h. CD44H, CD44v6 and CCR6 molecules may play a role in attachment of TMV to cancer cells, while HER-2 associated with CD24 be involved in promoting cancer cells growth. Pre-exposure of cancer cells to TMV resulted in enhancement of tumour growth and cancer cell-induced angiogenesis in NOD SCID mice model. CONCLUSIONS: TMV interact directly with cancer cells serving as macro-messengers and molecular cargo transfer between gastric cancer cells resulting in enhancement of tumour growth. TMV should be considered in future as target of anticancer therapy.

Properties of monocytes generated from haematopoietic CD34(+) stem cells from bone marrow of colon cancer patients.

Monocytes exhibit direct and indirect antitumour activities and may be potentially useful for various forms of adoptive cellular immunotherapy of cancer. However, blood is a limited source of them. This study explored whether monocytes can be obtained from bone marrow haematopoietic CD34(+) stem cells of colon cancer patients, using previously described protocol of expansion and differentiation to monocytes of cord blood-derived CD34(+) haematopoietic progenitors. Data show that in two-step cultures, the yield of cells was increased approximately 200-fold, and among these cells, up to 60 % of CD14(+) monocytes were found. They consisted of two subpopulations: CD14(++)CD16(+) and CD14(+)CD16(-), at approximately 1:1 ratio, that differed in HLA-DR expression, being higher on the former. No differences in expression of costimulatory molecules were observed, as CD80 was not detected, while CD86 expression was comparable. These CD14(+) monocytes showed the ability to present recall antigens (PPD, Candida albicans) and neoantigens expressed on tumour cells and tumour-derived microvesicles (TMV) to autologous CD3(+) T cells isolated from the peripheral blood. Monocytes also efficiently presented the immunodominant HER-2/neu369-377 peptide (KIFGSLAFL), resulting in the generation of specific cytotoxic CD8(+) T lymphocytes (CTL). The CD14(++)CD16(+) subset exhibited enhanced cytotoxicity, though nonsignificant, towards tumour cells in vitro. These observations indicate that generation of monocytes from CD34(+) stem cells of cancer patients is feasible. To our knowledge, it is the first demonstration of such approach that may open a way to obtain autologous monocytes for alternative forms of adaptive and adoptive cellular immunotherapy of cancer.

Interactions of human monocytes with TMVs (tumour-derived microvesicles).

The tumour microenvironment represents a dynamic complex milieu, which includes tumour cells, cells of the immune system and other (cellular and non-cellular) components. The role of these particular 'puzzle pieces' may change substantially due to their mutual interactions. The present review concerns different opinions on interactions that occur between monocytes, tumour cells and TMVs (tumour-derived microvesicles).

Nomenclature of monocytes and dendritic cells in blood.

Monocytes and cells of the dendritic cell lineage circulate in blood and eventually migrate into tissue where they further mature and serve various functions, most notably in immune defense. Over recent years these cells have been characterized in detail with the use of cell surface markers and flow cytometry, and subpopulations have been described. The present document proposes a nomenclature for these cells and defines 3 types of monocytes (classical, intermediate, and nonclassical monocytes) and 3 types of dendritic cells (plasmacytoid and 2 types of myeloid dendritic cells) in human and in mouse blood. This classification has been approved by the Nomenclature Committee of the International Union of Immunological Societies, and we are convinced that it will facilitate communication among experts and in the wider scientific community.

Preparations of intravenous immunoglobulins diminish the number and proinflammatory response of CD14+CD16++ monocytes in common variable immunodeficiency (CVID) patients.

We have studied the effect of intravenous immunoglobulins (IVIG) on monocyte subpopulations and cytokine production in patients with CVID. The absolute number of CD14(+)CD16(++) monocytes decreased on average 2.5-fold 4h after IVIG and after 20h returned to the baseline. The cytokine level in the supernatants of peripheral blood mononuclear cells (PBMC) after ex vivo LPS stimulation demonstrated the >2-fold decrease in TNF production 4h after IVIG. The TNF expression, which is higher in the CD14(+)CD16(++) monocytes, was decreased in these cells by IVIG in 4/7 CVID cases. In vitro exposure of the healthy individuals' monocytes to the IVIG preparation resulted in reduced TNF production, which was overcome by blockade of the FcγRIIB in the CD14(+)CD16(++) CD32B(high) monocytes. Our data suggest that reduction in the number of CD14(+)CD16(++) monocytes and the blockade of their cytokine production via triggering CD32B can contribute to the anti-inflammatory action of IVIG.

Allogeneic haematopoietic stem cell transplantation as therapy for chronic granulomatous disease--single centre experience.

Chronic granulomatous disease (CGD) is phagocytic cell metabolic disorder resulting in recurrent infections and granuloma formation. This paper reports the favourable outcome of allogeneic transplantation in six high-risk CGD patients. The following donors were used: HLA-matched, related (two) and unrelated (three), and HLA-mismatched, unrelated (one). One patient was transplanted twice using the same sibling donor because of graft rejection at 6 months after reduced-intensity conditioning transplant (fludarabine and melphalan). Myeloablative conditioning regimen consisted of busulphan and cyclophosphamide. Stem cell source was unmanipulated bone marrow containing: 5.2 (2.6-6.5) × 10(8) nucleated cells, 3.8 (2.0-8.0) × 10(6) CD34+ cells and 45 (27-64) × 10(6) CD3+ cells per kilogramme. Graft-versus-host disease prophylaxis consisted of cyclosporine A and, for unrelated donors, short course of methotrexate and anti-T-lymphocyte globulin. Mean neutrophile and platelet engraftments were observed at day 22 (20-23) and day 20 (16-29), respectively. Pre-existing infections and inflammatory granulomas resolved. With the follow-up of 4-35 months (mean, 20 months), all patients are alive and well with full donor chimerism and normalized superoxide production.

Intrauterine exposure to lead may enhance sensitization to common inhalant allergens in early childhood: a prospective prebirth cohort study.

BACKGROUND: Several in vivo and in vitro studies have shown that metal-rich particles may enhance allergic responses to house dust mites and induce an increased release of allergy-related cytokines. OBJECTIVES: The main goal of this analysis is to define the possible association of intrauterine exposure to lead and mercury with the occurrence of skin sensitization to common aeroallergens in early childhood. MATERIAL AND METHODS: The present study refers to a sample of 224 women in the second trimester of pregnancy recruited from Krakow inner city area who had full term pregnancies and whose children underwent skin prick testing (SPT) at the age of 5. Lead and mercury levels were assessed in cord blood and retested in children at age of 5 years. Aeroallergen concentrations in house dust were measured at the age of 3 years. The main health outcome (atopic status) was defined as the positive SPT to at least one common aeroallergen (Der f1, Der p1, Can f1 and Fel d1) at the age of 5 years. In the statistical analysis of the association between atopic status of children and exposure to metals, the study considered a set of covariates such as maternal characteristics (age, education, atopy), child's gender, number of older siblings, prenatal (measured via cord blood cotinine) and postnatal environmental tobacco smoke together with exposure to polycyclic aromatic hydrocarbons (PAH) as measured by PAH-DNA adducts. RESULTS AND CONCLUSION: In the binary regression analysis, which controlled for the confounders, the risk ratio (RR) estimate for atopic sensitization was significantly associated with the lead exposure (RR=2.25, 95%CI: 1.21-4.19). In conclusion, the data suggest that even very low-level of prenatal lead exposure may be implicated in enhancing sensitization to common aeroallergens in early childhood.

Circulating tumour-derived microvesicles in plasma of gastric cancer patients.

Cell membrane microfragments called microvesicles (MV) originating from different cells are circulating in the blood of healthy subjects and their elevated numbers are found in different diseases, including cancer. This study was designed to characterise MV present in plasma of gastric cancer patients. Since majority of MV in blood are platelets-derived (PMV), plasma samples deprived of PMV were used. In comparison to control, the number of MV in patients was significantly elevated in all stages, higher in more advanced disease. Patients' MV showed an increased membrane expression of CCR6 and HER-2/neu. The proportion of MV carrying some leucocyte determinants was low and similar in patients and control. Transmission electron microscopy showed their substantial heterogeneity in size and shape. The size determined by dynamic light scattering analysis confirmed this heterogeneity. The MV size distribution in patients was broader within the range of 10-800 nm, while in control MV showed 3-mode distribution within the range of 10-400 nm. Atomic force microscopy confirmed MV size heterogeneity with implication that larger objects represented aggregates of smaller microparticles. Patients' MV exhibited increased absolute values of zeta potential, indicating a higher surface charge. Tumour markers HER-2/neu, MAGE-1, c-MET and EMMPRIN were detected both in control and patients' samples with stronger expression in the latter. Significantly higher expression of MAGE-1 and HER-2/neu mRNA was observed in individual patients. All together, it suggests that at least some MV in plasma of gastric cancer patients are tumour-derived. However, their role in cancer requires further studies.

The B-cell compartment in the peripheral blood of children with different types of primary humoral immunodeficiency.

The aim of this study was to evaluate the B-cell compartment in the peripheral blood of children with different types of hypogammaglobulinemia: common variable immunodeficiency (CVID), transient hypogammaglobulinemia of infancy (THI), and selective IgA deficiency (SIgAD). We analyzed by flow cytometry the changes in the B-cell subsets with age and showed that children with an early-onset CVID develop similar pattern of B-cell subsets as adult patients with CVID with age, as the levels of memory B cells (CD19/CD27) and class-switched memory B cells (CD19/CD27/IgD/IgM), in contrast to age-matched control group, did not increase with age. Children with SIgAD displayed similar changes as patients with CVID only within the class-switched memory B-cell subpopulation. No significant differences in the level of memory B cells and class-switched memory B cells in children with THI in comparison to age-matched control group were observed. There were no differences in the percentage of immature B cells (CD19/CD21) among all studied groups. As B-cell subsets in children with THI were normal during entire period of hypogammaglobulinemia, the persistence of low levels of memory B-cell subsets in some children may facilitate the diagnosis of CVID.

Rapid full engraftment and successful immune reconstitution after allogeneic hematopoietic stem cell transplantation with reduced intensity conditioning in Omenn syndrome.

OS is a variant of SCID characterized by generalized erythroderma, alopecia, eosinophilia, and elevated IgE levels. It is fatal unless treated with allogeneic HSCT, which is the only curative approach. However, treatment related complications and graft rejection are major obstacles to the success of treatment. In this report, we describe a patient with OS, complicated by prolonged cytomegalovirus infection, successfully treated by reduced intensity conditioning allogeneic HSCT from sibling donor.

Detection of isolated tumour cells in the blood and bone marrow of patients with gastric cancer by combined sorting, isolation and determination of MAGE-1, -2 mRNA expression.

The detection of isolated (circulating or disseminated) tumour cells (ITC) in patients with cancer requires very sensitive methods, as such cells are very rare. In the present study, the method that combines the negative isolation of CD45- leukocytes from the blood and bone marrow of patients with gastric cancer by flow cytometry, followed by the positive isolation of single cytokeratin-positive (CK+) cells by a Laser Capture Microdissection System for the determination of MAGE-1, -2 mRNA expression was used to detect ITC. This study shows that this method is highly sensitive as it allows to determine beta-actin-mRNA expression in a single CK+ cell. Using > or =5 CK+ cells as a cut-off level, the MAGE-1 mRNA expression was detected in 100% of CK+ cells in the peripheral blood and in 75% of bone marrow samples of patients with gastric cancer. The MAGE-2 mRNA expression was observed in 40 and 58% of samples, respectively. Furthermore, an analysis of primary tumours and locoreginal lymph nodes with respect to the mRNA expression of the two genes showed that MAGE-1 mRNA expression was detected in 88% of the primary tumours and in 67% of the lymph node samples, whereas the MAGE-2 mRNA expression was observed in 72 and 67% of the cases, respectively. Thus, the method described here allows the precise and sensitive determination of tumour-associated gene expression in single ITC present in the blood and bone marrow of patients with gastric cancer.

Human monocytes both enhance and inhibit the growth of human pancreatic cancer in SCID mice.

BACKGROUND: Monocytes/macrophages exhibit antitumour potential, but clinicopathological evidence suggests that they may both inhibit and enhance tumour growth. The purpose of this study was to determine the effect of monocytes on the growth of human pancreatic cancer (HPC-4) in severe combined immunodeficient (SCID) mice. MATERIALS AND METHODS: Freshly isolated human monocytes or CD14+ cells from cocultures with tumour cells were coengrafted, at various ratios, with HPC-4 cells into SCID mice. The tumour size and angiogenesis were determined. RESULTS: At a high ratio of monocytes to cancer cells the enhancement and, at a low ratio, the inhibition of tumour growth was observed. Multiple intratumoral applications of monocytes in large numbers also enhanced tumour growth. Deactivation of monocytes by a short pre-exposure to tumour cells in vitro before engraftment led to increased tumour growth. Monocytes used in large numbers and deactivated monocytes in low doses enhanced tumour-induced angiogenesis. CONCLUSION: Monocytes may both facilitate and suppress the growth of human tumours in SCID mice and both the number of monocytes, as well as the state of monocyte deactivation are critical for the final outcome of monocyte-tumour interactions.

Expansion and differentiation of CD14+CD16(-) and CD14+ +CD16+ human monocyte subsets from cord blood CD34+ hematopoietic progenitors.

To determine whether monocytes can be generated from CD34+ hematopoietic progenitors in large numbers, cord blood CD34+ cells were first expanded for 3-10 days in X-VIVO 10 medium supplemented with FCS, stem cell factor (SCF), thrombopoietin (TPO), and Flt-3 Ligand (Flt-3L), and then differentiated in IMDM medium supplemented with FCS, SCF, Flt-3L, IL-3 and M-CSF for 7-14 days. These two step cultures resulted in up to a 600-fold mean increase of total CD14+ cells. Using this approach, two subpopulations of monocytes were obtained: CD14+CD16(-) and CD14++CD16+ occurring at 2:1 ratio. 1.25(OH)2 Vitamin D3 added to the differentiation medium altered this ratio by decreasing proportion of CD14++CD16+ monocytes. In comparison to CD14+CD16(-), the CD14++CD16+ cells showed different morphology and an enhanced expression of CD11b, CD33, CD40, CD64, CD86, CD163, HLA-DR, and CCR5. Both subpopulations secreted TNF and IL-12p40 but little or no IL-10. CD14++CD16+ monocytes released significantly more IL-12p40, were better stimulators of MLR but showed less S. aureus phagocytosis. These subpopulations are clearly different from those present in the blood and may be novel monocyte subsets that represent different stages in monocyte differentiation with distinct biological function.

Tumour-derived microvesicles modulate biological activity of human monocytes.

Tumour cells are shedding membrane fragments (tumour-derived microvesicles, TMV) that may interact with cells of immune system. Our previous observations indicated that TMV carry several surface determinants and mRNA of tumour cells and transfer some of them to monocytes. This study determined the effect of TMV on biological activity of human monocytes as the precursors of tumour infiltrating macrophages (TIM). It was found that TMV activated monocytes as shown by an increased HLA-DR expression, induced production of ROI (reactive oxygen intermediates) and of tumour necrosis factor (TNF), interleukin (IL)-10, IL-12p40 accumulation of mRNA and their secretion. Induction of TNF synthesis was CD44 dependent as blocking of CD44 on monocytes abolished its secretion. TMV-treated monocytes showed an increased antitumour activity as judged by enhanced cytotoxicity/cytostasis against tumour cells in vitro. Taken together, these results indicate that TMV significantly modulate biological activity of monocytes and thus mimic the effect of tumour cells on them. This may suggest that tumour cells interact with TIM not only via direct contact, soluble factors, but also TMV.

Circulating tumour cells and survival of patients with gastric cancer.

BACKGROUND: The prognostic significance of the presence of tumour cells in the blood of gastric cancer patients remains unclear. Their occurrence and its association with the stage of disease and long-term survival was determined. PATIENTS AND METHODS: Fifty-seven patients with stage I-IV gastric cancer were divided into two groups: these with and these without circulating tumour cells that were identified as cytokeratin positive (CK+) cells among CD45- cells (obtained by sorting of CD45+ leukocytes). RESULTS: Tumour cells were detected prior surgery in the peripheral blood of 54.4% patients but no clear association with the stage of disease was observed. After gastrectomy detection rate was 21.1%. There was no significant difference in the 5-year survival of patients, with or without CK+ in the blood. CONCLUSION: The presence of circulating tumour cells is of no prognostic value in patients with resectable gastric cancer.

Immunophenotype of isolated tumour cells in the blood, bone marrow and lymph nodes of patients with gastric cancer.

Immunophenotype of isolated (disseminated or circulating) tumour cells (ITC) in the blood, bone marrow and lymph nodes were studied in patients with gastric cancer. Coexpression of metalloproteinases inducer (EMMPRIN), chemokine receptors (CCR6, CXCR4) and adhesion molecules (Ep-CAM, CD44) was determined on cytokeratin positive (CK+) cells in CD45- cell population sorted out from the blood and/or bone marrow. Eight cytospin samples of blood and 69 samples of bone marrow containing CK+ cells from patients with gastric cancer were included into study. Expression of EMMPRIN and CCR6 were noted in a half of CK+ samples (of blood/bone marrow) whereas the expression of CXCR4 and Ep-CAM was much lower. Analysis of paired data of these determinants expression on CK+ cells showed no association between them. Expression of EMMPRIN, Ep-CAM, CCR6, CCR7, CXCR1, and CXCR4 on ITC in lymph nodes was determined by flow cytometry. In 18 lymph nodes (out of 36 assayed) CK+ cells were found. The expression of CCR6 and Ep-CAM on CK+ cells was observed in almost all studied lymph nodes, CXCR1--in half of them. The expression of EMMPRIN and CCR7 cells was lower. These results suggest that ITC of gastric cancer express variably several molecules that may be involved in metastasis formation.

Risk of wheezing associated with house-dust mite allergens and indoor air quality among three-year-old children. Kraków inner city study.

OBJECTIVES: The aim of the study was to describe the distribution of house-dust mite (HDM) allergens in homes of three-year-old children and to test the hypothesis whether the content of HDM allergens exceeding 2 microg/g of dust may be regarded as a risk level possibly affecting respiratory health in early childhood. MATERIALS AND METHODS: House-dust samples were collected in 275 dwellings from mattresses, children's bedrooms and kitchen floors. In the laboratory, dust samples were analyzed for Der f 1 and Der p 1 using monoclonal antibody enzyme-linked immunosorbent assays (ELISA). At the time of the house-dust collection, mothers were interviewed on the household characteristics and their children's respiratory health. Respiratory outcome variables included wheezing or whistling in the chest irrespective of respiratory infections. The number of the wheezing episodes and their duration in days over the last 6 months were recorded in the questionnaire. In the multivariate Poisson regression analysis on the association between the occurrence of wheezing and exposure, a set of potential confounders, such as child's gender, maternal education, maternal allergy, older siblings, presence of moulds, house dampness, and environmental tobacco smoke (ETS) was taken into account. RESULTS: The adjusted incidence rate ratios (IRR) of wheezing ascribed to a higher HDM level (> 2.0 microg/g dust) were 1.84 (95% CI: 1.45-2.34) for duration of wheezing and 1.56 (95% CI: 0.88-2.75) for episodes. Of the confounders taken into consideration, the presence of moulds had the strongest impact on the risk of wheezing (IRR = 4.24; 95% CI: 3.08-5.84). CONCLUSION: The data support the view that exposure to a higher level of HDM allergens increases the burden of respiratory diseases in the early childhood and the effect is independent of maternal atopy, ETS, and moulds in homes.

Hyaluronan carried by tumor-derived microvesicles induces IL-10 production in classical (CD14++CD16-) monocytes via PI3K/Akt/mTOR-dependent signalling pathway.

Tumor-derived microvesicles (TMV) can mimic effects of tumor cells leading to an increased anti-inflammatory cytokine production, such as interleukin 10 (IL-10), by tumor-infiltrating monocytes and macrophages. Yet, the mechanism of IL-10 induction by TMV in monocytes remains unclear. The co-incubation of TMV derived from the human pancreas carcinoma cell line (HPC-4) with human monocytes resulted in a nearly 30-fold increase in IL-10 protein production. This effect operates at the level of transcription since monocytes transduced with an adenovirus containing IL-10-promoter luciferase reporter gene showed a 5-fold induction of luciferase activity after treatment with TMV. Since tumor cells can express hyaluronan (HA), which participates in tumor invasion and metastases, we have tested its effect on IL-10 expression. We showed that HA at the concentration of 100µg/ml induces IL-10 protein expression and the IL-10 promoter activation in monocytes. Moreover, hyaluronidase treatment of TMV reduced IL-10 protein production by 50% and promoter activity by 40%. Inhibitors of the PI3K/Akt/mTOR pathway reduced both, TMV-induced IL-10 promoter activity and protein production, and the same was observed in monocytes when stimulated by HPC-4 cells or HA. Inhibition of PI3K activity down-regulated phosphorylation of the Akt and (to a lesser extent) mTOR proteins in monocytes following TMV or HA stimulation. When comparing monocyte subsets, TMV induced IL-10 protein and mRNA synthesis only in classical CD14++CD16- but not in CD16-positive monocytes. Our data show that TMV induce IL-10 synthesis in human classical monocytes via HA, which, in turn, activates the PI3K/Akt/mTOR pathway.