T1/ST2-deficient mice demonstrate the importance of T1/ST2 in developing primary T helper cell type 2 responses.

We have generated mice with a deficiency in T1/ST2 expression to clarify the roles of T1/ST2 in T helper cell type 2 (Th2) responses. Using immunological challenges normally characterized by a Th2-like response, we have compared the responses of T1/ST2-deficient mice with those generated by wild-type mice. Using a primary pulmonary granuloma model, induced with Schistosoma mansoni eggs, we demonstrate that granuloma formation, characterized by eosinophil infiltration, is abrogated in T1/ST2-deficient mice. Furthermore, we clearly demonstrate that in the absence of T1/ST2 expression, the levels of Th2 cytokine production are severely impaired after immunization. Thus, in a secondary pulmonary granuloma model, draining lymph node cells from the T1/ST2-deficient animals produced significantly reduced levels of IL-4 and IL-5, despite developing granulomas of a magnitude similar to those of wild-type mice and comparable antigen-specific immunoglobulin isotype production. These data clearly demonstrate that T1/ST2 expression plays a role in the development of Th2-like cytokine responses and indicate that effector functions are inhibited in its absence.

Prevention of lysosomal storage in Tay-Sachs mice treated with N-butyldeoxynojirimycin.

The glycosphingolipid (GSL) lysosomal storage diseases result from the inheritance of defects in the genes encoding the enzymes required for catabolism of GSLs within lysosomes. A strategy for the treatment of these diseases, based on an inhibitor of GSL biosynthesis N-butyldeoxynojirimycin, was evaluated in a mouse model of Tay-Sachs disease. When Tay-Sachs mice were treated with N-butyldeoxynojirimycin, the accumulation of GM2 in the brain was prevented, with the number of storage neurons and the quantity of ganglioside stored per cell markedly reduced. Thus, limiting the biosynthesis of the substrate (GM2) for the defective enzyme (beta-hexosaminidase A) prevents GSL accumulation and the neuropathology associated with its lysosomal storage.

Migration of noble gas tracers at the site of an underground nuclear explosion at the Nevada National Security Site.

As part of an underground gas migration study, two radioactive noble gases (37Ar and 127Xe) and two stable tracer gases (SF6 and PFDMCH) were injected into a historic nuclear explosion test chimney and allowed to migrate naturally. The purpose of this experiment was to provide a bounding case (natural transport) for the flow of radioactive noble gases following an underground nuclear explosion. To accomplish this, soil gas samples were collected from a series of boreholes and a range of depths from the shallow subsurface (3 m) to deeper levels (~160 m) over a period of eleven months. These samples have provided insights into the development and evolution of the subsurface plume and constrained the relative migration rates of the radioactive and stable gas species in the case when the driving pressure from the cavity is low. Analysis of the samples concluded that the stable tracer SF6 was consistently enriched in the subsurface samples relative to the radiotracer 127Xe, but the ratios of SF6 and 37Ar remained similar throughout the samples.

The Ro60 autoantigen binds endogenous retroelements and regulates inflammatory gene expression.

Autoantibodies target the RNA binding protein Ro60 in systemic lupus erythematosus (SLE) and Sjögren's syndrome. However, it is unclear whether Ro60 and its associated RNAs contribute to disease pathogenesis. We catalogued the Ro60-associated RNAs in human cell lines and found that among other RNAs, Ro60 bound an RNA motif derived from endogenous Alu retroelements. Alu transcripts were induced by type I interferon and stimulated proinflammatory cytokine secretion by human peripheral blood cells. Ro60 deletion resulted in enhanced expression of Alu RNAs and interferon-regulated genes. Anti-Ro60-positive SLE immune complexes contained Alu RNAs, and Alu transcripts were up-regulated in SLE whole blood samples relative to controls. These findings establish a link among the lupus autoantigen Ro60, Alu retroelements, and type I interferon.

Immune mechanisms associated with the rejection of fetal murine proislet allografts and pig proislet xenografts: comparison of intragraft cytokine mRNA profiles.

BACKGROUND: Previous in vivo depletion studies of CD4 and CD8 T cells indicated that different rejection mechanisms operate for proislet allografts and xenografts. The cellular and molecular mechanisms of acute proislet allograft and xenograft rejection have therefore been characterized and directly compared. METHODS: The intragraft cytokine mRNA profile in rejecting BALB/c (H-2d) proislet allografts was analyzed in control, CD4 T cell-depleted, and CD8 T cell-depleted CBA/H (H-2k) recipient mice using semi-quantitative reverse transcriptase-assisted polymerase chain reaction (RT-PCR). The cytokine profiles for proislet allografts and pig proislet xenografts at 3-10 days posttransplant were directly compared and correlated with graft histopathology. RESULTS: Allograft rejection was protracted (2-3 weeks), characterized by infiltrating CD8 T cells and CD4 T cells (no eosinophils) and was associated with a Th1-type CD4 T cell response (IL-2, IFN-gamma, and IL-3 mRNA) and a CD8 T cell-dependent spectrum of cytokine gene expression (IL-2, IFN-gamma, IL-3, and IL-10 mRNA). Xenograft rejection was rapid (6-8 days), involved predominantly CD4 T cells and eosinophils, and in contrast to allografts, exhibited intragraft mRNA expression for the Th2 cytokines IL-4 and IL-5. CONCLUSIONS: Proislet allograft and xenograft rejection differ in the tempo of destruction, phenotype of the cellular response and intragraft profile of cytokine mRNA. The recruitment of eosinophils only to the site of xenorejection correlates with IL4 and IL-5 mRNA expression. These findings suggest that different anti-rejection strategies may need to be developed to optimally target the allograft and the xenograft response.

Matrix metalloproteinase expression in an experimentally-induced DTH model of multiple sclerosis in the rat CNS.

In an experimentally-induced DTH model of MS, we examined mRNA and protein expression of a range of MMPs and of TNFalpha to establish the contribution that individual MMPs might make to the pathogenesis. In control rat brain, mRNA for all of the MMPs examined was detectable. However, by immunohistochemistry, only MMP-2 could be detected. In the DTH lesions, significant increases in the level of mRNA expression were observed for MMP-7, MMP-8, MMP-12, and TNFalpha. Where expression of MMP mRNA was increased, there was a corresponding increase in protein expression detected by immunohistochemistry. To determine whether the upregulated MMPs could invoke destructive events in the CNS, highly purified activated MMP-7, MMP-8, and MMP-9 were stereotaxically injected into the brain parenchyma. All provoked recruitment of leukocytes and BBB breakdown. In addition, MMPs 7 and 9 induced loss of myelin staining. In conclusion, specific MMPs are upregulated in DTH lesions; for the most part, measurement of mRNA was a predictor of increased protein expression. From our injections of MMPs, it is clear that the upregulated MMPs in the DTH lesions could participate in the disruption of the BBB, leukocyte recruitment, and tissue damage.

Ultrastructural studies of an immune-mediated inflammatory response in the CNS parenchyma directed against a non-CNS antigen.

We have shown previously that heat-killed bacillus Calmette-Guerin injected into the brain parenchyma becomes sequestered behind the blood brain barrier for months undetected by the immune system. However, independent peripheral sensitization of the immune system to bacillus Calmette-Guérin results in recognition of bacillus Calmette-Guérin in the brain and the induction of focal chronic lesions [Matyszak M. K. and Perry V. H. (1995) Neuroscience 64, 967 977]. We carried out ultrastructural studies of these lesions. Prior to subcutaneous challenge we used immunohistochemistry to detect bacillus Calmette-Guérin which was found in cells with the morphology of macrophages/microglia and in perivascular macrophages. Eight to 14 days after subcutaneous challenge there was a conspicuous leucocyte infiltration at the site of bacillus Calmette-Guérin deposits within the brain parenchyma. The majority of these cells were macrophages and lymphocytes, with some lymphocytes showing characteristic blast morphology. Dendritic cells in close contact with lymphocytes were prominent. Inflammatory cells were found in perivascular cuffs and within the brain parenchyma. The tissue was oedematous and some axons were undergoing Wallerian degeneration with associated myelin degeneration. Throughout the lesions, but more commonly at the edges, we detected macrophages containing myelin in their cytoplasm close to intact axons and axons with evidence of remyelinating sheaths, suggestive of primary demyelination. In older lesions, two to three months after the peripheral challenge, the oedema was less pronounced and there was little evidence of Wallerian degeneration. There were still many macrophages. lymphocytes and dendritic cells, although the number of these cells was lower than in earlier lesions. Late lesions also contained many plasma cells which were not present in early lesions. In these late lesions there were bundles of axons with no myelin or a few axons with thin myelin sheaths, suggestive of persistent or ongoing demyelination or remyelination. These observations show that, during a delayed-type hypersensitivity lesion in the CNS, the leucocyte populations change with time, and suggest that the mechanisms and type of tissue damage are different in the early and late stages of the lesion.

Differences in the contribution of CD4+ T cells to proislet and islet allograft rejection correlate with constitutive class II MHC alloantigen expression.

Allografts of BALB/c (H-2d) fetal proislets facilitated long-term (> 100 days) reversal of streptozotocin-induced diabetes in CBA/H (H-2k) mice treated with a combination of anti-CD4 and anti CD8 mAbs. Anti-CD8 monotherapy was partially effective in restoring normoglycemia but anti-CD4 mAb treatment of host animals failed to promote allograft function. In contrast, allografts of BALB/c adult islets demonstrated indefinite reversal of diabetes in recipient mice treated only with anti-CD8 mAb. Anti-CD4 monotherapy resulted in only transient restoration of normoglycemia. These findings clearly demonstrate (1) a critical role for CD8 T cells in the acute rejection of pancreatic islet tissue allografts and (2) tissue-specific differences in the participation of CD4 T cells as primary effectors in the rejection reaction. Immunohistochemical studies showed that the capacity for CD4 T cells to initiate the rejection of proislet but not adult islet allografts correlates with the presence/absence, respectively, of graft parenchymal cells that constitutively express Class II MHC alloantigens. Proislet grafts, unlike transplants of purified adult islets, contain heterogeneous tissue components including Class II MHC+ve duct epithelium. Thus, the participation of CD8 and CD4 T cells as primary effectors of graft rejection depends on which class or classes of MHC antigens are constitutively expressed on graft parenchymal cells and are available for recognition. Islet tissue in both rejecting proislet and islet allografts showed de novo induction of Class II MHC alloantigens only after severe disruption to islet architecture had been achieved by infiltrating mononuclear cells. Thus, at this stage of advanced allograft injury, CD4 T cells have the potential to act as secondary effectors, possibly by amplifying the inflammatory reaction and thus accelerating graft destruction. The capacity for antirejection mAb therapy to establish transplant tolerance was facilitated in the islet allograft model where it was necessary to target only the CD8 T cell subpopulation.

Analysis of the Th1/Th2 paradigm in transplantation: interferon-gamma deficiency converts Th1-type proislet allograft rejection to a Th2-type xenograft-like response.

The rejection mechanisms for fetal proislet allografts and pig proislet xenografts in mice are characterized by different intragraft cytokine mRNA profiles and cellular responses. Allograft rejection is predominantly CD8 T-cell-dependent and is associated with a Th1-type cytokine pattern (i.e., IFN-gamma, IL-2 but no IL-4 or IL-5 mRNA). In contrast, xenograft rejection is CD4 T-cell-dependent and is accompanied by a strong Th2-type response (i.e., enhanced expression of IL-4 and IL-5 mRNA) and by marked eosinophil accumulation at the graft site. We have now examined and compared the regulatory role of IFN-gamma in both proislet allograft and xenograft rejection processes. The histopathology and intragraft cytokine mRNA profile of BALB/c (H-2d) proislet allografts were examined in IFN-gamma-deficient and wild-type C57BL/6J recipient mice. The survival of pig proislet xenografts was also assessed in IFN-gamma -/- and wild-type hosts. Both proislet allografts and xenografts were acutely rejected in IFN-gamma -/- and wild-type mice. Unlike the conventional allograft reaction, which lacks eosinophil infiltration, the rejection of proislet allografts in IFN-gamma-deficient hosts correlated with intragraft expression of IL-4 and IL-5 mRNA (i.e., a Th2-type response) and eosinophil recruitment. The rejection of proislet allografts and xenografts can therefore occur by IFN-gamma-independent pathways; IFN-gamma, however, regulates the pathology of the allograft reaction but not the xenograft response. The immune destruction of proislet allografts is not prevented by Th2 cytokine gene expression; instead, the latter correlated with the recruitment of unconventional inflammatory cells (eosinophils), which may play an accessory role in effecting graft injury. Significantly, the Th1-to-Th2-like switch resulted in the novel conversion of an allograft rejection reaction into a xenograft-like rejection process.

Laryngeal manifestations of gout.

Gout is a disorder of purine metabolism characterized by hyperuricemia with rare involvement of the head and neck. We present a 72-year-old woman with a known history of gout who presented with hoarseness and a lesion suspicious for carcinoma of the larynx. Endoscopic biopsy revealed a tophus of the true vocal cord with characteristic birefringent crystalline deposits and giant cell granuloma. There have been limited reports of gouty involvement of the larynx, more commonly involving cricoarytenoid arthritis. Tophi of the laryngeal soft tissues are exceedingly rare. In this paper we will discuss the pathophysiology and management of this interesting clinical entity.

Lentivirus capture directly from cell culture with Q-functionalised microcapillary film chromatography.

A new disposable adsorbent material for fast anion-exchange capture of nano-complexes without prefiltering, clarification or pre-processing of samples was developed based on plastic microcapillary films (MCFs). An MCF containing 19 parallel microcapillaries, each with a mean internal diameter of 142 ± 10 µm, was prepared using a melt extrusion process from an ethylene-vinyl alcohol copolymer (EVOH). The MCF internal surfaces were functionalised using branched chain chemistries to attach quaternary amine groups producing an anion-exchange adsorbent. The purification of nano-complexes using this newly fabricated MCF-EVOH-Q was successfully demonstrated with the capture of lentivirus from pre-filtered culture harvest. This 5m chromatographic substrate was found to bind and elute ∼40% of bound lentivirus or 2.5 × 10(6)infectious units (ifu). The unique properties of this chromatographic substrate that allow the passage of large particulates was further demonstrated with the capture of lentiviral particles from unfiltered un-processed culture media containing cells and cell debris. Using this approach, 56% or 1 × 10(7)ifu of captured lentivirus was eluted. A device based on this new material might be used at an early stage in clinical lentiviral production to harvest lentiviral particles, directly from bioreactors.

Eosinophils are not required for the rejection of neovascularized fetal pig proislet xenografts in mice.

The rejection of neovascularized pig proislet (islet precursor) xenografts in mice is a CD4 T cell-dependent process involving invasion of the graft site mainly by host CD4 T cells and eosinophils. We previously identified CD4 T cell-dependent enhancement of intragraft IL-3, IL-4, and IL-5 mRNA expression during acute xeno-rejection in CBA/H recipient mice. In the present study we investigated the role of each cytokine and the involvement of eosinophils in the rejection of pig proislet xenografts using cytokine gene knockout mice (IL-4 -/- and IL-5 -/-) and the treatment of transplant recipients with anti-IL-3 mAb. In IL-4 -/- mice, IL-5 -/- recipient animals, and anti-IL-3 mAb-treated CBA/H mice, eosinophil accumulation at the transplant site was inhibited or ablated, but the kinetics of xenograft rejection was unaltered. Prolonged xenograft survival was only achieved in anti-CD4 mAb-treated mice and consistently correlated with the absence of intragraft IL-3, IL-4, and IL-5 mRNA enhancement. Together these findings indicate that neither IL-3, nor IL-4, nor IL-5 individually plays an obligatory role in the rejection process. The cytokine mRNA profile correlating with the lack of eosinophil recruitment was variable; the data suggest that IL-4 regulates eosinophil involvement in the xeno-rejection reaction indirectly via effects on IL-5 and IL-3 transcript expression. There is also suggestive evidence that IL-5 may influence IL-3 and IL-4 mRNA expression via feedback inhibition. Eosinophils, therefore, do not play an essential role in the rejection of neovascularized pig proislet xenografts in mice.