Plasma-derived proteins in airway defence, disease and repair of epithelial injury

One significant characteristic of the airway mucosa in vivo, that cannot easily be mimicked in vitro, is its microcirculation, which generates a highly dynamic, biologically active milieu of plasma-derived molecules that may pass to the airway lumen in vivo. New data on the mechanisms of airway mucosal exudation indicate that the protein systems of circulating plasma may contribute significantly to the biology and immunology of the lamina propria, its surface epithelium and the luminal surface, not only in injured airways, but also in airways that are activated but display no sign of oedema, epithelial disruption, or increased absorption capacity. We suggest that present knowledge of the mechanisms of plasma exudation, together with rapidly emerging information (not detailed herein) on receptors, target cells and cellular responses to the plasma-derived molecules, must be considered in any realistic model that investigates "immuno-inflammatory" mechanisms of the airway mucosa.     

Cytolysis and piecemeal degranulation as distinct modes of activation of airway mucosal eosinophils

In contrast to the protective effect of chronic caloric restriction on tumor development, we have shown that fasting sustained tumor initiation in rat liver by a noninitiating dose of diethylnitrosamine. Here we investigated whether fasting had a similar favorable effect on initiation in the colorectal mucosa in 80 male F344 rats. Animals fasted for 4 days were given a single s.c. dose of azoxymethane (AOM) (20 mg/kg) on the first day of re-feeding, and rates of kinetic proliferative parameters, and development of the pre-neoplastic lesions such as aberrant crypt foci (ACF), were evaluated. Starvation before AOM treatment enhanced the growth of ACF, as shown by the significantly higher crypt multiplicity of fasted/re-fed rats as compared with fully fed rats (3.97 +/- 0.50 vs. 2.64 +/- 0.20, p < or = 0.025). This difference was associated with perturbations in cell death and cell proliferation. Fasting induced apoptosis and depressed cell division, while re-feeding had opposite effects, resulting in a higher percentage of S-phase cells at the time of AOM injection and 2 days thereafter. Starvation-induced apoptosis may represent the mitogenic stimulus to an increase in the number of cells susceptible to AOM damage, and may favor its fixation, leading to enhanced growth of ACF. Our data therefore suggest that fasting/re-feeding enhances colon cancer.     

Xenoislet transplantation: experimental and clinical aspects

Ten diabetic renal transplant patients had porcine fetal islet-like cell clusters (ICC) injected intraportally or placed under the kidney capsule. In some patients, temporary graft survival was achieved, as evidenced by the urinary excretion of small amounts of porcine C-peptide (4 patients) and the identification of some intact insulin-staining cells in a biopsy specimen (1 patient). Glucose metabolism remained unaffected. To improve the results, better islets and better immunosuppressive protocols are required. We found that, while fetal porcine ICC produced insulin only after several weeks, adult islets gave immediate insulin production. The search for an optimal immunosuppression was conducted in the pig-to-rat islet transplant model. A clear inhibitory effect on the xenograft rejection was observed when using some of the new drugs. The best results were achieved with a triple drug regimen consisting of cyclosporine, mycophenolate mofetil and leflunomide.     

Characterization of mixed syngeneic-allogeneic and syngeneic-xenogeneic islet-graft rejections in mice. Evidence of functional impairment of the remaining syngeneic islets in xenograft rejections

Allogeneic mouse islets or xenogeneic rat islets, or fetal porcine islets were implanted under the renal capsule of C57BL/6 mice either alone or carefully mixed with syngeneic islets. With this experimental model the syngeneic islets, although not rejected themselves, are exposed to cytokines and inflammatory mediators released during either allograft or xenograft rejection. No differences in insulin content could be observed between mixed islet grafts and pure syngeneic islet grafts 6 wk after transplantation. Neither was there any morphological evidence of a non-specific destruction of syngeneic islets. These findings suggest that the mechanisms of both allograft and xenograft rejections are highly specific. The hormone release from the mixed syngeneic-allogeneic grafts was similar to that from pure syngeneic islet grafts. In contrast, a pronounced impairment of both the first and second phases of insulin release was observed 2 wk after implantation in mixed syngeneic-xenogeneic islet grafts. When perfusing the mixed islet graft after completed rejection of the concordant xenogeneic rat islets (6 wk after implantation), the insulin release from the remaining syngeneic mouse islets was identical to that of control grafts. However, syngeneic mouse islets exposed to the rejection mechanism of the discordant xenogenic pig islet-like cell clusters did not attain a complete functional recovery.     

Natural killer cells determine development of allergen-induced eosinophilic airway inflammation in mice

The earliest contact between antigen and the innate immune system is thought to direct the subsequent antigen-specific T cell response. We hypothesized that cells of the innate immune system, such as natural killer (NK) cells, NK1.1(+) T cells (NKT cells), and gamma/delta T cells, may regulate the development of allergic airway disease. We demonstrate here that depletion of NK1.1(+) cells (NK cells and NKT cells) before immunization inhibits pulmonary eosinophil and CD3(+) T cell infiltration as well as increased levels of interleukin (IL)-4, IL-5, and IL-12 in bronchoalveolar lavage fluid in a murine model of allergic asthma. Moreover, systemic allergen-specific immunoglobulin (Ig)E and IgG2a levels and the number of IL-4 and interferon gamma-producing splenic cells were diminished in mice depleted of NK1.1(+) cells before the priming regime. Depletion of NK1.1(+) cells during the challenge period only did not influence pulmonary eosinophilic inflammation. CD1d1 mutant mice, deficient in NKT cells but with normal NK cells, developed lung tissue eosinophilia and allergen-specific IgE levels not different from those observed in wild-type mice. Mice deficient in gamma/delta T cells showed a mild attenuation of lung tissue eosinophilia in this model. Taken together, these findings suggest a critical role of NK cells, but not of NKT cells, for the development of allergen-induced airway inflammation, and that this effect of NK cells is exerted during the immunization. If translatable to humans, these data suggest that NK cells may be critically important for deciding whether allergic eosinophilic airway disease will develop. These observations are also compatible with a pathogenic role for the increased NK cell activity observed in human asthma.     

Xenograft rejection of porcine islet-like cell clusters in normal, interferon-gamma, and interferon-gamma receptor deficient mice

BACKGROUND: An increase in serum alpha-glutathione S-transferase concentration (GST) has been shown to be a more sensitive and specific marker of hepatocellular damage than equivalent increases in transaminase activities. A randomized clinical trial of 60 liver transplants in 49 patients was carried out to assess the clinical benefits of GST monitoring as a supplementary test to routine liver function tests during the first 3 postoperative months after liver transplantation. METHODS: Mortality and morbidity were compared in graft recipients who had their GST reported daily to the ward (reporting group) and graft recipients who did not. RESULTS: The 3-month survival rate was significantly greater in the reporting group (P=0.033) and the risk of graft loss was halved (relative hazard ratio=0.50; P=0.29). The reporting group also had significantly more patients who spent less than 3 weeks in the hospital throughout the follow-up period (P=0.036). In addition, the reporting group experienced a lower frequency of biopsies per graft (P=0.038), less severe rejection (P=0.015), and a lower incidence of infection episodes per graft (P=0.03). GST increased by >50% above the upper limit of the reference range at a median of 1 day before the equivalent change in alanine transaminase in association with allograft rejection in the combined groups (95% confidence interval=1 to 2 days) but was lower on the day of diagnosis of rejection in the reporting group (P=0.02). This is compatible with the earlier diagnosis of rejection in the reporting group. CONCLUSIONS: We conclude that the monitoring of GST may improve patient care, reducing both mortality and morbidity.     

Xenograft rejection of porcine islet-like cell clusters in immunoglobulin- or Fc-receptor gamma-deficient mice

The aim of the present study was to evaluate the role of xenoreactive antibodies in islet-like cell cluster (ICC) xenograft rejection. For this purpose, normal mice, mice with a targeted disruption of the Fc-receptor (FcR) gamma-chain, or the membrane exon of the immunoglobulin mu-chain gene, were transplanted with fetal porcine ICC under the kidney capsule. Mice lacking the FcR gamma have no functional FcR for IgG or IgE. Mice with disruption of the immunoglobulin mu-chain cannot produce antibodies, because B cell development is arrested at the stage of preB cells. All animals, irrespective of recipient group, readily rejected the ICC xenograft. Analyses of the pattern of cellular infiltration revealed only minor dissimilarities between the different experimental groups. Xenograft destruction was evident on day 6 after transplantation, and a large number of mononuclear cells were found to be evenly distributed throughout the ICC graft. The majority of the infiltrating cells were large, macrophage-like cells expressing the macrophage-specific phenotype marker F4/80. CD3-positive T lymphocytes were found to be mainly accumulated in the peripheral parts of the ICC xenograft. This study has demonstrated that xenoreactive antibodies are not crucial to ICC xenograft rejection in the pig-to-mouse model.     

A Novel Particle Trap Impactor for Use with the Gas-Quenching Probe Sampling System

A novel particle trap impactor has been developed for use with the gas-quenching probe in order to exclude solid particles from entering into the probe during sampling of gaseous metallic species in fluidized bed combustion conditions. The impactor must be small in size (Ø_impactor ≤ Ø_probe = 45 mm) but capable of collecting a relatively large amount of particles at elevated temperatures. As the first step, the impactor was designed, constructed, and tested at room temperature for KCI aerosol particles. The impactor with a nozzle of 0.95 mm in diameter, in combination with the orifice-to-jet diameter ratio of 1.5 and the ratio of the jet-to-plate spacing to jet diameter at 1.4 yielded a sharp cutoff curve with a maximum collection efficiency of about 0.9 and a √Stk₅₀ value of about 0.22. In addition, the collection efficiency of the impactor was compared with the particle removal efficiency of a filter of the same type as the filter previously used with the gas-quenching probe. The difference from the comparison is very small, indicating that the impactor can be used to replace the filter to prevent fly ash particles from entering the gas-quenching probe in fluidized bed combustion conditions.     

Evidence of a negative feedback system regulating the total beta-cell volume in nondiabetic rats that received pancreas transplants

BACKGROUND: The aim of the present study was to investigate the long-term regulation of pancreatic beta-cell volume after pancreas transplantation into adult rats. METHODS: A syngeneic pancreaticoduodenal transplantation was made in normoglycemic Wistar-Furth rats. By this means, the recipients doubled their pancreatic islet volume. Nine months after transplantation, the total beta-cell volume was measured in serial pancreatic sections immunostained for insulin from both the native and transplanted pancreata, and in the native pancreas of age-matched Wistar-Furth rats that did not receive transplants. RESULTS: No changes in the volume of individual beta-cells were seen. A 50% decrease in total beta-cell volume was observed in both the native and transplanted pancreas when compared with that of age-matched controls. However, the combined beta-cell volumes of the native and transplanted pancreas in the rats that received transplants were similar to those of the native pancreas in control animals. No signs of increased apoptosis in any of the glands could be seen during the first postoperative week or after 9 months. CONCLUSIONS: These findings provide evidence of a negative feedback system, which regulates the total beta-cell volume to the levels seen in age-matched rats that did not receive transplants. The underlying mechanisms for the decreased beta-cell volume are unknown, but may involve a diminished replicatory rate of the beta-cells.