Edward S. Rector

Isolation and characterization of an α-satellite repeated sequence from human chromosome 22

We constructed a library in λIL47.1 with DNA isolated from flow-sorted human chromosome 22. Over 50% of the recombinants contained the same highly repetitive sequence. When this sequence was used to probe Southern blots of EcoRI-digested genomic DNA, a ladder of bands with increments of about 170 bp was observed. This sequence comigrates with satellite III in Ag+/Cs2SO4 gradients and may account for at least part of the 170 bp Hae III ladder seen in isolated satellite III DNA. Partial sequence analysis revealed homology to the 171 bp monomeric repeat unit of α-R1-DNA and the X specific α-satellite consensus sequence. After low stringency in situ hybridization, silver grains were found over the centromeres of a number of chromosomes. Under high stringency conditions, however, the labeling was concentrated over the centromeric region of chromosome 22. This localization was confirmed using DNA from a panel of human/hamster cell lines which showed that the homologous 2.1 and 2.8 kb EcoR1 restriction fragments were chromosome 22 specific. These clones therefore contain chromosome 22 derived α-satellite sequences analogous to other chromosome-specific satellite sequences described previously.

A comparison of biologically variable ventilation to recruitment manoeuvres in a porcine model of acute lung injury

BackgroundBiologically variable ventilation (return of physiological variability in rate and tidal volume using a computer-controller) was compared to control mode ventilation with and without a recruitment manoeuvre – 40 cm H2O for 40 sec performed hourly; in a porcine oleic acid acute lung injury model.MethodsWe compared gas exchange, respiratory mechanics, and measured bronchoalveolar fluid for inflammatory cytokines, cell counts and surfactant function. Lung injury was scored by light microscopy. Pigs received mechanical ventilation (FIO2 = 0.3; PEEP 5 cm H2O) in control mode until PaO2 decreased to 60 mm Hg with oleic acid infusion (PaO2/FIO2 <200 mm Hg). Additional PEEP to 10 cm H2O was added after injury. Animals were randomized to one of the 3 modes of ventilation and followed for 5 hr after injury.ResultsPaO2 and respiratory system compliance was significantly greater with biologically variable ventilation compared to the other 2 groups. Mean and mean peak airway pressures were also lower. There were no differences in cell counts in bronchoalveolar fluid by flow cytometry, or interleukin-8 and -10 levels between groups. Lung injury scoring revealed no difference between groups in the regions examined. No differences in surfactant function were seen between groups by capillary surfactometry.ConclusionsIn this porcine model of acute lung injury, various indices to measure injury or inflammation did not differ between the 3 approaches to ventilation. However, when using a low tidal volume strategy with moderate levels of PEEP, sustained improvements in arterial oxygen tension and respiratory system compliance were only seen with BVV when compared to CMV or CMV with a recruitment manoeuvre.

A method for the preparation of protein-protein conjugates of predetermined composition

A novel procedure for the synthesis of well-defined protein-protein conjugates is described using ovalbumin (OA) and IgG as test proteins. This procedure involves the highly selective and rapid reaction of alkyl halide and sulfhydryl groups, which have been grafted, respectively, onto the proteins to be conjugated. Accordingly, iodoacetylated IgG, (ICH2CO)nIgG, was prepared by the reaction of the epsilon-amino groups of IgG with the N-hydroxysuccinimide ester of iodoacetic acid (NHIA), the degree of iodoacetylation (n) being proportional to the concentration of NHIA. OA was reacted with S-acetylmercaptosuccinic anhydride (SAMSA) under conditions yielding, on the average, a monosubstituted derivative. Following removal of the protective S-acetyl group, the resulting -SH derivative of OA was reacted with (ICH2CO)nIgG. The OAx-IgG conjugates so produced were characterized by gel filtration, specific radioactivity (using tritiated OA) and immunodiffusion. It was found that the average number of OA molecules coupled per IgG molecule could be controlled by varying the degree of iodoacetylation of IgG.

Airway smooth muscle cell proliferation: characterization of subpopulations by sensitivity to heparin inhibition

Growth and maturation state of airway smooth muscle cells (SMCs) are determinants of asthma pathophysiology. Heparin reduces airway SMC proliferation and arterial SMC replication and phenotypic modulation. Distinct arterial SMC subtypes, differing in heparin sensitivity, have been characterized. We assessed the cellular mechanisms underlying the growth and phenotype of heparin-treated canine tracheal myocytes in primary culture. Heparin reduced replication by 40%. Immunoblot assay of myosin, actin, and myosin light chain kinase revealed heparin had no effect on rapid spontaneous phenotypic modulation after the cells were plated. Heparin increased cellular protein and vimentin contents in confluent cultures, suggesting that it may induce hypertrophic growth. Cell cycle analysis revealed that heparin decreased serum-stimulated replicating myocyte number by 40%. Also, G2-M transit was 20% slower for the set of SMCs that proceeded past G1 in the presence of heparin. These data indicate that heparin does not inhibit airway SMC replication by blocking modulation from the contractile state. Moreover, airway smooth muscle is composed of distinct SMC populations differing in mitogen and antiproliferative mediator responsiveness. Identification of functionally divergent subgroups suggests that distinct sets of SMCs may contribute differentially to airway physiology and pathophysiology.Growth and maturation state of airway smooth muscle cells (SMCs) are determinants of asthma pathophysiology. Heparin reduces airway SMC proliferation and arterial SMC replication and phenotypic modulation. Distinct arterial SMC subtypes, differing in heparin sensitivity, have been characterized. We assessed the cellular mechanisms underlying the growth and phenotype of heparin-treated canine tracheal myocytes in primary culture. Heparin reduced replication by 40%. Immunoblot assay of myosin, actin, and myosin light chain kinase revealed heparin had no effect on rapid spontaneous phenotypic modulation after the cells were plated. Heparin increased cellular protein and vimentin contents in confluent cultures, suggesting that it may induce hypertrophic growth. Cell cycle analysis revealed that heparin decreased serum-stimulated replicating myocyte number by 40%. Also, G2-M transit was 20% slower for the set of SMCs that proceeded past G1 in the presence of heparin. These data indicate that heparin does not inhibit airway SMC replication by blocking modulation from the contractile state. Moreover, airway smooth muscle is composed of distinct SMC populations differing in mitogen and antiproliferative mediator responsiveness. Identification of functionally divergent subgroups suggests that distinct sets of SMCs may contribute differentially to airway physiology and pathophysiology.

Heterogeneity of polyclonal IgE characterized by differential charge, affinity to protein A, and antigenicity

Functional and physical heterogeneity of polyclonal IgE has been reported. Extremely low serum concentrations of IgE have limited the study of these important differences. We have purified polyclonal dog IgE and developed polyclonal and monoclonal (mAb C2) anti-dog IgE antibodies. In this study chromatofocusing of dog IgE revealed two biologically active IgE fractions: IgE1 eluted at pH 5.0, and IgE2 eluted at pH 4.7. The two IgE subforms (IgEs) exhibited typical IgE characteristics: positive in the 48-hour passive cutaneous anaphylaxis response, heat-labile, identical molecular weight, and reactive to polyclonal anti-dog IgE. However, the two IgEs were found to be significantly heterogeneous. IgE1 bound to protein A and did not react with mAb C2 in ELISA and isoelectric focusing-immunoblotting, whereas IgE2 did not bind to protein A and reacted with mAb C2. Further, in sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting, IgE2, but not IgE1, reacted with seven well-defined mAb anti-human IgE antibodies and an mAb anti-mouse IgE antibody, even though both IgE1 and IgE2 reacted with polyclonal anti-human and anti-mouse IgE. Neuraminidase or endoglycosidase treatment did not abolish the differential antigenicity and charge of IgE1 and IgE2, although the antigenicity of IgE2 was significantly reduced after incubation with endoglycosidase. These data suggest that carbohydrate moieties are not involved in the observed differences in antigenicity and charge and that the two IgE molecules represent distinct isotypes. In studies with seven purified IgE fractions obtained from different ragweed-allergic dogs, the distribution of ragweed IgE2 varied 200-fold, whereas ragweed total IgE levels varied only fourfold. This raises the possibility of a relationship between different IgEs and the allergic response.

Novel IgE peptide‐based vaccine prevents the increase of IgE and down‐regulates elevated IgE in rodents

Background Immunotherapy with anti‐IgE antibodies for treatment of allergy is promising but a short half‐life and extremely high cost limit its application.

Dietary repletion can replenish reduced T cell subset numbers and lymphoid organ weight in zinc-deficient and energy-restricted rats

The objective of the present study was to investigate the time course for recovery of lymphoid tissue and T cell subset numbers when Zn-deficient (ZD) or energy-restricted (ER) rats were repleted with control diet; in a second experiment, the link between the stress axis and lymphoid organs was explored. During the deficiency phase, rats were fed a ZD (<1 mg Zn/kg) or control diet (30 mg Zn/kg, nutritionally complete) either as pair-fed controls (ER) or ad libitum-fed controls (CTL) for 3 weeks. During the repletion phase, all rats were fed control diet ad libitum for 3, 7 or 23 d. After the deficiency phase, ZD and ER had lower T cell subset numbers in the thymus compared with CTL, and ZD had reduced T cell subset numbers in the spleen compared with both ER and CTL. T cell subset numbers and lymphoid organ weights recovered from dietary Zn deficiency and energy restriction by 7 d of repletion (except 23 d for thymus weight in ZD), while body weight required more than 23 d for recovery. At the end of the deficiency phase, ZD and ER had higher circulating corticosterone concentrations compared with CTL; plasma TNFalpha was not detectable and there were no differences in plasma haptoglobin, an acute-phase protein. In conclusion, Zn deficiency and energy restriction elevated circulating corticosterone and reduced T cell subset numbers in the thymus and spleen of growing rats. Repletion with a nutritionally complete diet allowed recovery of T cell subset numbers and lymphoid organ weight.

Regulation of B16F1 melanoma cell metastasis by inducible functions of the hepatic microvasculature.

We have previously shown that circulating intravascular cells generally arrest by mechanical restriction in the hepatic sinusoids, causing rapid release of nitric oxide (NO) which is cytotoxic to these cells and inhibits their growth into metastatic tumours. Here, we present evidence that these NO-dependent cytotoxic mechanisms are susceptible to upregulation by lipopolysaccharide (LPS). Five x 10(5) fluorescently labelled melanoma cells were injected into the mesenteric vein of C57BL/6 mice to effect their localisation in the hepatic microvasculature. Test mice were then given 1 mg/kg LPS intraperitoneally (i.p.) to activate the microvascular cells. By electron paramagnetic resonance (EPR) spectroscopy, the expression of NO in the liver was significantly increased by 8 h in the LPS-treated mice. The non-selective NO synthase inhibitor L-NAME inhibited the induction of NO by LPS, while its inactive enantiomer D-NAME had no significant effect. Using immunohistochemistry (IHC), iNOS-positive microvascular cells were detected in the terminal portal venule (TPV) region of the liver 8 h after LPS stimulation. LPS treatment also increased the retention of melanoma cells in the liver between 8 and 24 h, especially in the TPV region. Eight hours after cell injection, local expression of VCAM-1 and ICAM-1 was detected by double-label immunohistochemistry at the sites of tumour cell arrest. Expression of these adhesion molecules was enhanced in mice treated with LPS. Using flow cytometry, 98% of the B16F1 melanoma cells expressed VLA-4, the counter receptor of VCAM-1, and approximately 1.5% expressed LFA-1, the counter receptor of ICAM-1. LPS did not significantly alter the expression of either counter receptor on melanoma cells in vitro or in vivo. By DNA end-labelling, the rates of melanoma cell apoptosis were significantly increased from 8 to 24 h in the TPV region (but not in the sinusoids) of LPS-treated mice. Fourteen days after tumour cell injection, the LPS-treated mice had a significantly smaller hepatic metastatic tumour burden than the control mice. These data suggest that LPS can inhibit the metastasis of melanoma cells in the liver by inducing the expression of NO and adhesion molecules by the hepatic endothelium. The induction of iNOS and the inducible cytotoxic effect of LPS appear to be primarily located within the TPV region of the liver acinus.

Depletion of natural killer cells from the graft reduces interferon-gamma levels and lipopolysaccharide-induced tumor necrosis factor-alpha release in F1 hybrid mice with acute graft-versus-host disease.

BACKGROUND We wished to determine whether removal of NK1.1+ cells from the graft provides protection against acute graft-versus-host disease (GVHD) by obviating the Th1 immune response that underlies the development of this disease. METHODS Graft-versus-host (GVH) reactions were induced in two groups of (C57BL/6 x DBA/2)F1 hybrid mice. The first received grafts harvested from polyinosinic:polycytidylic acid-stimulated, C57BL/6 donors and depleted in vitro of NK1.1+ cells. This treatment provides protection against GVHD-associated mortality and cachexia. The second received unmodified grafts. We compared interferon-gamma and interleukin-10 production as well as the levels of engraftment in these two groups. Lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-alpha) release was also compared since TNF-alpha levels in GVH mice following injection of a sublethal dose of endotoxin provide an index of macrophage priming by Th1 cytokines. RESULTS Interferon-gamma production was absent in recipients of NK1.1-depleted grafts at the time when high levels were seen in recipients of unmodified grafts. Following lipopolysaccharide injection, high levels of TNF-alpha were observed in recipients of unmodified grafts, whereas negligible amounts were present in recipients of NK1.1-depleted grafts. The use of NK1.1-depleted grafts did not result in a reduced level of engraftment of CD4+ or CD8+ cells. CONCLUSIONS These results suggest that NK1.1 depletion of the graft confers protection against mortality by interfering with an immunoregulatory mechanism that results in the development of a Th1 response in GVH mice, and does not result in abortion of the graft. Because macrophage priming is prevented, recipients are also protected from the exaggerated sensitivity to endotoxin seen in mice with acute GVHD.

Characterization of suppressor T cell clones derived from a mouse tolerized with conjugates of ovalbumin and monomethoxypolyethylene glycol.

The induction of antigen-specific tolerance in mice by conjugates of ovalbumin (OVA) and monomethoxypolyethylene glycol (mPEG) previously had been shown to be associated with the generation of antigen-specific suppressor T (Ts) cells. For the elucidation of the nature of these Ts cells, five nonhybridized OVA-specific Ts cell clones were generated from the spleen cells of a BDF1 mouse which had been immunosuppressed by the tolerogenic conjugate, OVA(mPEG)12. The cloned Ts cells were maintained in vitro by periodic stimulation with OVA and feeder cells and were able to suppress the in vitro antibody production in an OVA-specific and MHC class I (H-2Kd or H-2Dd)-restricted manner. All these Ts cell clones were shown to be Thy1.2+, CD4-, CD5-, CD8+, and to express CD3 and the alpha beta heterodimer of the T cell receptor. The cell-free extracts of these cells contained soluble suppressor factors which could mimic in vitro the suppressive activity of the intact cells. In contrast to cytotoxic T lymphocytes (CTL), none of the cloned Ts cells were endowed with cytolytic activity as revealed in the perforin-mediated microhemolysis and in the 18-hr51Cr release assays. These results demonstrate that (i) OVA-specific Ts cell clones can be generated from mice pretreated with OVA(mPEG)12 by employing conventional T cell culture techniques, and (ii) these Ts cells are functionally different from conventional CD8+ CTL.