Subtle ecosystem effects of microplastic exposure in marine mesocosms including fish.

For two months, communities in 5.8 m outdoor marine mesocosms were exposed to 700 μm sphere-shaped polystyrene (PS) beads in dosages between 0.08 and 80 g/m.

Malabsorption and nutritional balance in the ICU: fecal weight as a biomarker: a prospective observational pilot study.

Introduction: Malabsorption, which is frequently underdiagnosed in critically ill patients, is clinically relevant with regard to nutritional balance and nutritional management. We aimed to validate the diagnostic accuracy of fecal weight as a biomarker for fecal loss and additionally to assess fecal macronutrient contents and intestinal absorption capacity in ICU patients.

Methods: This was an observational pilot study in a tertiary mixed medical-surgical ICU in hemodynamically stable adult ICU patients, without clinically evident gastrointestinal malfunction.

Assessment of small bowel function in critical illness: potential role of citrulline metabolism.

Small intestinal function in critically ill patients should ideally be assessed in order to determine the preferred feeding route, timing, and composition of enteral nutrition. Additionally, evaluation of small bowel function may lead to new insights aimed to maintain enterocyte integrity. Critically ill patients are likely to have impaired enterocyte function mainly as a consequence of diminished splanchnic blood flow associated with mucosal hyperpermeability and bacterial translocation, a pathological state believed to be pivotal in the development of sepsis and multiple organ dysfunction syndrome (MODS).

Diagnosing malabsorption in the intensive care unit.

Malabsorption as a result of decreased intestinal function is a frequently occurring problem in intensive care units. Small bowel dysfunction may lead to malnutrition and may predispose patients to infectious complications (sepsis) and may be linked to increased hospitalization duration, morbidity and mortality. There are several small bowel function tests, such as faecal fat excretion and sugar absorption tests, but data specifically applicable to the intensive care setting are limited.

A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle.

Green fluorescent protein (GFP) is a well-established reporter protein for the examination of biological processes. This report describes a recombinant Plasmodium berghei, PbGFPCON, that constitutively expresses GFP in a growth responsive manner in its cytoplasm from a transgene that is integrated into the genome and controlled by the strong promoter from a P. berghei elongation factor-1alpha gene.

Pregnancy induces minor histocompatibility antigen-specific cytotoxic T cells: implications for stem cell transplantation and immunotherapy.

Recipients of HLA-identical stem cell transplants have a poorer transplant outcome if the donor is female rather than male. We analyzed whether pregnancy primes for minor histocompatibility (H) antigens. Peripheral blood mononuclear cells (PBMCs) from healthy multiparous female blood donors were depleted for CD4+, CD14+, CD16+, and CD19+ cells, stained with minor H antigen-specific HLA-A2 tetramers, sorted by fluorescence-activated cell sorting, and tested for cytotoxic activity.

Intrinsic genetic instability of normal human lymphocytes and its implication for loss of heterozygosity.

A combination of flow cytometry and microsatellite analysis was used to investigate loss of expression of HLA-A and/or HLA-B alleles and concurrent LOH at polymorphic chromosome 6 loci both in freshly isolated lymphocytes (in vivo mutations) and in lymphocytes cultured ex vivo. The fraction of in vivo mutants that showed LOH at 6p appeared to vary from 0%-49% for various donors. During culturing ex vivo, HLA-A(-) cells arose at a high rate and showed simultaneous loss of expression at the linked HLA-B locus.

Molecular methods for the detection of mutations.

We report the results of a collaborative study aimed at developing reliable, direct assays for mutation in human cells. The project used common lymphoblastoid cell lines, both with and without mutagen treatment, as a shared resource to validate the development of new molecular methods for the detection of low-level mutations in the presence of a large excess of normal alleles. As the "gold standard, " hprt mutation frequencies were also measured on the same samples.

Autoreactive and immunoregulatory T-cell subsets in insulin-dependent diabetes mellitus.

Aims/hypothesis: Type I (insulin-dependent) diabetes mellitus is a T-cell mediated autoimmune disease. Several subsets of T-cells, in particular CD4+ and in vivo activate CD45RA+RO+ T-cells, have been shown to be increased at disease onset. The functional implications of these relative increases in CD4 T-cells were investigated.

CD11b and L-selectin expression on eosinophils and neutrophils in blood and induced sputum of patients with asthma compared with normal subjects.

Background: Patients with asthma show altered surface expression of the adhesion molecules CD11b and L-selectin on airway granulocytes compared with blood granulocytes.

Objective: To investigate whether this modulation is related to disease activity or due to transendothelial migration, we compared the CD11b and L-selectin expression on blood and induced sputum eosinophils and neutrophils between patients with asthma and normal subjects.

Methods: Eleven normal subjects (21-43 years), nine patients (21-34 years) with mild atopic asthma and 10 patients (20-47 years) with moderate to severe atopic asthma on regular treatment with inhaled steroids underwent sputum induction by inhalation of nebulized hypertonic saline (4.

Generation of dendritic cells expressing bcr-abl from CD34-positive chronic myeloid leukemia precursor cells.

Patients with a relapse of chronic myeloid leukemia (CML) after allogeneic bone marrow transplantation can be successfully treated with blood mononuclear cells from the original bone marrow donor. However, the antileukemic effect of this treatment is often accompanied by graft-versus-host disease (GVHD). Treatment with cytotoxic T-lymphocyte (CTL) lines or clones that are specifically generated against leukemic antigen-presenting cells from the patient, may separate antileukemic effects from GVHD.

Functional expression of minor histocompatibility antigens on human peripheral blood dendritic cells and epidermal Langerhans cells.

Adequate presentation and cell surface expression of foreign minor histocompatibility antigens (mHag) to allogeneic T cells can lead to graft versus-host disease (GvHD) after HLA matched bone marrow transplantation (BMT). Cells of the dendritic cell (DC) lineage, including epidermal Langerhans cells (LC), are the most potent inducers of primary alloreactive T cell responses in vivo and in vitro. To explore the possible role of peripheral blood DC and of skin derived LC in the induction of alloimmune responses against mHag, we analysed the functional expression of mHag on these professional antigen-presenting cells (APC).

Evaluation of various methods to quantify endothelial cells attached to vascular prostheses: comparison with a new "gold standard" FACS method.

For in vitro evaluation of functional properties of endothelial cells seeded on synthetic vascular prostheses accurate and reproducible quantification of cells is mandatory. Comparison of these properties with those resulting from other studies requires correlation of the functional parameters to reliably counted cell numbers. The accuracy of methods of quantification currently being used is unknown due to the lack of a "gold standard" method to which these methods can be compared.

Antigen capture and major histocompatibility class II compartments of freshly isolated and cultured human blood dendritic cells.

Dendritic cells (DC) represent potent antigen-presenting cells for the induction of T cell-dependent immune responses. Previous work on antigen uptake and presentation by human DC is based largely on studies of blood DC that have been cultured for various periods of time before analysis. These cultured cells may therefore have undergone a maturation process from precursors that have different capacities for antigen capture and presentation.

Computer software for testing drug susceptibility of malaria parasites.

A computer program is described for the automated analysis of data obtained by flow cytometry for in vitro antimalarial drug susceptibility testing. Samples of malaria-infected red blood cells (RBC), which were cultured in the presence of different concentrations of antimalarial drugs, were stained with Hoechst. The Hoechst fluorescence intensity of infected RBC corresponds to DNA content of the parasites and to their stage of development.

Characterization of exocytosis in electropermeabilized neutrophils by flow cytometric analysis: difference in sensitivity to calcium and guanosine-5'-[gamma-thio]triphosphate.

When rabbit neutrophils were subjected to two electrical discharges of 4.75 kV/cm, the cells became permeable to propidium iodide. Measurement of propidium iodide fluorescence using flow cytometry showed that all cells in the suspension were permeabilized.

Usage of TCRAV and TCRBV gene families in human fetal and adult TCR rearrangements.

We have investigated fetal and adult T-cell receptor (TCR) A and B V-gene repertoires both by fluorescence-activated cell sorter (FACS) analysis with the available TCR V region-specific mAbs and by the polymerase chain reaction (PCR) with TCR V gene family-specific oligonucleotides. Among the low number of CD3+ T cells, most of the TCR V regions tested for could be detected by FACS analysis in liver, bone marrow, and spleen derived from a 14-week-old fetus and two 15-week-old fetuses. Similarly, the PCR analysis showed that the majority of the TCRAV and TCRBV families were expressed in the peripheral organs of the 13-week-old fetus, although an apparent absence of particular TCR V families was found in liver and bone marrow.

Selection of defined cell types by flow-cytometric cell sorting.

Cell sorting by flow cytometry usually involves preliminary staining with fluorescent dyes or reagents (antibodies or probes) that interact specifically with cellular constituents. Passage through a focused beam of light enables cells to be sorted on the basis of their light-scattering or fluorescence characteristics. Integration with other techniques and refinement of labelling specificity is enabling high-speed sorting to be developed for an expanding range of both analytical and preparative applications--including isolation of specific cells for PCR amplification, establishing high-expressing cell clones and chromosome sorting.

Flow cytometric screening of blood samples for malaria parasites.

An automated method for the detection and estimation of malaria parasites in blood samples using flow cytometry is presented. In a single-step procedure 50 microliters of blood sample was collected in 1 ml of lysis solution containing formaldehyde, causing red blood cells to lyse while parasites and white blood cells are preserved. Thus prepared, samples could be transported and remained stored in lysis solution until flow cytometric analysis was performed.

Clonal involvement of granulocytes and monocytes, but not of T and B lymphocytes and natural killer cells in patients with myelodysplasia: analysis by X-linked restriction fragment length polymorphisms and polymerase chain reaction of the phosphoglycerate kinase gene.

To determine the clonal nature of hematopoiesis and to assess lineage involvement in patients with myelodysplastic syndromes (MDS), we used restriction fragment length polymorphisms of the X-linked genes phosphoglycerate kinase (PGK1) and hypoxanthine phosphoribosyltransferase (HPRT) and the X-linked probe M27 beta. Eleven female MDS patients heterozygous for at least one of these probes were studied: 3 with refractory anemia (RA), 2 with RA with ringed sideroblasts (RARS), 2 with chronic myelomonocytic leukemia (CMML), and 4 with RA with excess of blasts in transformation (RAEB-t). All exhibited clonal hematopoiesis as determined by Southern analysis of DNA prepared from peripheral blood (PB) and/or bone marrow (BM) cells.

Detection of mixed chimaerism in flow-sorted cell subpopulations by PCR-amplified VNTR markers after allogeneic bone marrow transplantation.

The amplification of Variable Number of Tandem Repeats (VNTR) by the polymerase chain reaction (PCR) was used to determine the extent of chimaerism in flow sorted lymphoid and myeloid cell populations following allogeneic bone marrow transplantation (BMT). Pre-BMT screening with a set of five VNTR revealed that at least one marker was maximally informative in 95% of donor-recipient pairs. Mixing reconstruction experiments indicated that detection of 1-5% of the minor cell population in a sample of 5 x 10(3) nucleated cells is feasible.

Flow cytometric double labeling technique for screening of multidrug resistance.

We investigated the capabilities of flow cytometry in the analysis of a multidrug resistant (MDR) human ovarian cancer cell line 2780AD and its drug sensitive parental A2780. A functional assay using daunorubicin (DNR) as a fluorescent probe was combined with an immunofluorescence assay of P-glycoprotein (P-gp) using the monoclonal antibody MRK-16. Functionally MDR could be demonstrated by the lower DNR-content of MDR cells compared to DNR-content of drug sensitive cells.

Automated flow cytometric analysis of drug susceptibility of malaria parasites.

A method is described for the fully automated reading of Plasmodium falciparum drug susceptibility tests. Cultured material was fixed and could be stored for greater than or equal to 6 months until analysis. The parasites were stained for DNA with the fluorescent dye Hoechst 33258 and analyzed by flow cytometry.

Gating of the so-called 'lymphocytic' cell population for the quantification of natural killer cells (CD16+) by flow cytometry causes loss of CD16 positive cells.

Natural killer cells can phenotypically be identified as CD16 positive with a specific monoclonal antibody (B73.1 = Leu-11c) by either immunofluorescence microscopy or by flow cytometry. The standard procedure in flow cytometry is to set a window or gate around the so called lymphocytic population, based on scatter characteristics.

T-cell phenotypes after stimulation of human mononuclear cells by pokeweed mitogen or pokeweed mitogen bound to erythrocytes.

Stimulation of human peripheral blood mononuclear cells (PBMC) by pokeweed mitogen bound to erythrocytes (E-PWM) has been found to result in an increased blast cell formation, lymphocyte proliferation, and enhanced immunoglobulin production, compared to stimulation of PBMC by PWM. Using flow cytometric analysis we compared the T helper/inducer (CD4+) to T suppressor/cytotoxic (CD8+) cell ratios of PBMC after stimulation by PWM and by E-PWM. E-PWM was found to induce significantly lower CD4+:CD8+ ratios on days 6 and 9 of the culture than PWM did.