Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes

The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10(4) physical particles of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hybridization stringency, 32P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus-containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments.     

Simple apparatus for collecting estuarine sediments and suspended solids to detect solids-associated virus

Laboratory trials of a new sampler for collection of estuarine sediment-associated virus resulted in a recovery effectiveness averaging 30% for two enteroviruses and rotavirus SA11. A minimal recovery potential of 54% was calculated when losses caused by virus concentration procedure inadequacies were excluded. Both sediment-associated and suspended solids-associated viruses were collected with the sampler. Recoveries of 61 and 60% poliovirus and rotavirus, respectively, were obtained from salt water-suspended, solids-associated virus. The unique advantage of the sampler for selective collection of virus-associated top layers of sediment, plus collection over extensive areas, resulted in recovery of more virus than was obtained with a commonly used dredge-type sampler.     

Interleukin-4 is chemotactic for mouse macrophages.

An important component of the cell-mediated immune response often is the migration of macrophages to the site of immune activity. Although much evidence suggests that macrophage migration is regulated by antigen-specific T cells, the influence of T cell-derived cytokines on macrophage chemotaxis has not been well studied. Here we present evidence that interleukin-4 (IL-4), a cytokine derived from T helper 2 (Th 2) cells, is chemotactic for mouse peritoneal macrophages. In an in vitro chemotaxis assay using Boyden chambers, recombinant IL-4 was chemotactic for mouse peritoneal exudate macrophages. This response was inhibited in a dose-dependent manner by the anti-IL-4 antibody, 11B11. As shown here and previously, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), cytokines derived from T helper 1 cells, are not chemotactic for mouse macrophages.     

Rheological properties of cross-linked hyaluronan-gelatin hydrogels for tissue engineering

Hydrogels that mimic the natural extracellular matrix (ECM) are used in three-dimensional cell culture, cell therapy, and tissue engineering. A semi-synthetic ECM based on cross-linked hyaluronana offers experimental control of both composition and gel stiffness. The mechanical properties of the ECM in part determine the ultimate cell phenotype. We now describe a rheological study of synthetic ECM hydrogels with storage shear moduli that span three orders of magnitude, from 11 to 3 500 Pa, a range important for engineering of soft tissues. The concentration of the chemically modified HA and the cross-linking density were the main determinants of gel stiffness. Increase in the ratio of thiol-modified gelatin reduced gel stiffness by diluting the effective concentration of the HA component.     

Binding of iodinated multipotential colony-stimulating factor (interleukin-3) to murine bone marrow cells

Multipotential colony-stimulating factor (Multi-CSF or interleukin-3) was radioiodinated to high specific radioactivity (1-4 X 10(5) cpm/ng) with no detectable loss of biological activity and its binding to murine bone marrow cells and factor-dependent cell lines studied. Both the native glycosylated molecule purified from a cloned T-cell line (LB-3) and the purified non-glycosylated recombinant molecule produced by E. coli could be radioiodinated. Comparative binding studies with these derivatives demonstrated equal binding affinities and equal numbers of binding sites on various cell types indicating that carbohydrate moieties are not involved in the binding interactions. Binding of 125I-Multi-CSF to several factor-dependent continuous hemopoietic cell lines showed the presence of specific receptors on all cell lines, the receptor number per cell varying from 700 to 13,000 and the apparent dissociation constant from 400 pM to 1 nM. Specific binding of 125I-Multi-CSF was also observed to normal murine hemopoietic cells and the binding to murine bone marrow cells was studied in detail. Bone marrow cells showed 117-130 receptors per cell on average and an apparent dissociation constant of 126-233 pM. However, quantitative autoradiographic analysis indicated that receptors for 125I-Multi-CSF were not distributed randomly on bone marrow cells--nucleated erythroid and lymphoid cells were not labeled while essentially all neutrophilic granulocyte, eosinophilic granulocyte and monocytic cells were labeled. Moreover, in each of the labeled cell lineages grain counts (reflecting receptor number) decreased with increasing maturation and a small subpopulation of marrow cells (0.4-1.5% and including blast cells, monocytes, promyelocytes, and myelocytes) exhibited very high grain counts. The existence of such a subset of marrow cells raises the possibility of functional heterogeneity among marrow cells in their response to Multi-CSF.