[Urolithiasis in the Long-Term GnRH Agonist Treatment of Patients with Paraphilia: 3 Case Studies]. / Urolithiasis bei der Langzeitherapie mit GnRH-Agonisten bei Paraphilien ­ 3 Fallberichte.

The outpatient forensic aftercare department of the Charité Berlin treated 32 paraphilic sex offenders with GnRH analogues within the past 5 years. Out of those patients, three men suffered from urolithiasis and were in need of treatment. All 3 patients had previously developed osteopenia/osteoporosis while on antiandrogen treatment.This article describes these 3 cases and suggests an intense consideration of the possible occurrence of urolithiasis in sex offenders on antiandrogen treatment.

Characterization of thoracic duct cells that transfer polyarthritis.

Polyarthritis may result from the haematogenous distribution of arthritogenic effector lymphocytes that emerge in the efferent lymph and pass through the thoracic duct (TD) to the circulation. We therefore examined whether TD cells collected from rats in the late prodrome of adjuvant-induced arthritis (AA) could transfer polyarthritis adoptively and whether these cells included a subpopulation of arthritogenic cells that could be identified phenotypically. Unfractionated TD cells collected from donor rats 9 days after adjuvant inoculation were injected intravenously into normal syngeneic recipients in numbers equivalent to the overnight harvest from a single donor. TD cell subpopulations, equivalent in number to proportions in the same inoculum, were prepared by negative selection. Unfractionated TD cells transferred polyarthritis without in vitro stimulation or conditioning of recipient animals. Abrogation of arthritogenicity by depletion of alpha/beta TCR(+) cells showed that the polyarthritis was transferred by T cells. Negatively selected CD4(+) but not CD8(+) TD cells transferred AA. An arthritogenic subpopulation of CD4(+) T cells, enriched by either negative or positive selection, expressed the activation markers CD25 (IL-2 receptor alpha), CD71 (transferrin receptor), CD134 (OX40 antigen) and MHC class II. Cells expressing these markers were more numerous in TD lymph from arthritic rats than in lymph from normal rats and they included the majority of large CD4(+) T cells. Thus, arthritogenic effector T cells bearing activation markers are released into the central efferent lymph in the late prodrome of AA. Recruitment of these arthritogenic cells to synovium probably determines the polyarticular pattern of AA.

Recruitment of mononuclear leucocytes to osteoarthritic human synovial xenografts in the ears of SCID mice.

A system has been established to assess the recruitment of 99mTc-hexamethylpropylene amine oxamine (99mTc-HMPAO)-labelled PBMC and [125I]iododeoxyuridine-labelled Con A stimulated lymphoblasts to allogeneic human synovial xenografts in the ears of SCID mice. Successful engraftment of osteoarthritic synovium was achieved in approximately 90% of cases and a connection between the human microvasculature of the xenograft and the circulation of the mouse was shown. Cells were delivered to the xenograft by a system of regional vascular perfusion, thus avoiding the major murine vascular beds. The accumulation of 99mTc-HMPAO-labelled PBMC in mouse ears was monitored in real time. Direct injection of xenograft-bearing ears with recombinant human TNF-alpha, 7 h prior to perfusion, increased the accumulation of both PBMC and lymphoblasts in cytokine-injected ears compared to contralateral control-injected ears. Autoradiography revealed the presence of [125I]iododeoxyuridine-labelled lymphoblasts associated with human microvasculature within the xenograft. However, the increased accumulation of lymphoblasts in cytokine-injected ears occurred in the tissues surrounding the xenograft, where lymphoblasts were associated more often with murine than human vessels. Although the system described offers advantages over similar models, the propensity for mouse endothelium to interact with human leucocytes is likely to be a generic disadvantage for models of human leucocyte recruitment to xenografts in immunodeficient mice.

Effects of tumor necrosis factor-alpha, interleukin 1beta, and activated peripheral blood mononuclear cells on the expression of adhesion molecules and recruitment of leukocytes in rheumatoid synovial xenografts in SCID mice.

OBJECTIVE: To examine expression of adhesion molecules within human synovial xenografts in vivo after injection of human cytokines or peripheral blood mononuclear cells (PBMC). METHODS: Rheumatoid synovium was transplanted into subcutaneous pouches in the ears of severe combined immunodeficient (SCID) mice. Tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), or PBMC were injected into the xenografts. The grafts were assessed by immunohistochemistry and quantitative video image analysis. RESULTS: TNF-alpha, IL-1beta, and concanavalin A activated PBMC increased the expression of vascular cellular adhesion molecule 1 and intercellular adhesion molecule 1 significantly on vessels and nonvascular synovial cells compared with injection of medium only. Vascular expression of E-selectin, but not of MHC Class II, was also increased. These stimuli also induced the migration of murine leukocytes into the xenografts. CONCLUSION: Rheumatoid synovial xenografts in SCID mice respond by upregulation of adhesion molecule expression when challenged in vivo with proinflammatory cytokines or activated PBMC.

Trapping and immobilization of Nippostrongylus brasiliensis larvae at the site of inoculation in primary infections of interleukin-5 transgenic mice.

Interleukin-5 (IL-5) transgenic mice are highly resistant to primary infections with the intestinal nematode Nippostrongylus brasiliensis; few parasites are found in the intestines of infected animals, and egg production is minimal. While adult worms may be damaged in the intestine, larval migration, development, and viability may also be impaired in other tissues. This study addresses the migration of N. brasiliensis larvae through the skin and lungs and associated cellular responses in primary infections of IL-5 transgenic mice. Although some larvae may have been trapped and killed in the lungs of IL-5 transgenic mice, most apparently failed to reach this site. Two or more hours after infection of IL-5 transgenic mice, eosinophils were a major component of the cellular infiltrate at the subcutaneous site of injection, and localized eosinophil degranulation was extensive. Seventy-five to ninety-five percent of the larvae injected into subcutaneous air pouches in IL-5 transgenic mice were retained there for at least 24 h. In contrast, in nontransgenic mice, less than 20% of larvae could be recovered from the skin 2 or more h postinjection, and eosinophil activity was modest at all times. The data strongly suggest that eosinophils can restrict the movement of N. brasiliensis larvae in the first few hours of a primary infection and that this has profound effects on later stages of parasite development. Preexisting eosinophilia, due either to allergy or to infection with tissue-invasive helminth species, may therefore confer some degree of immediate and nonspecific resistance in primary infections with parasitic worms.

Molecular systematics of the parasitic protozoan Giardia intestinalis.

The long-standing controversy regarding whether Giardia intestinalis is a single species prevalent in both human and animal hosts or a species complex consisting of morphologically similar organisms that differ in host range and other biotypic characteristics is an issue with important medical, veterinary, and environmental management implications. In the past decade, highly distinct genotypes (some apparently confined to particular host groups) have been identified by genetic analysis of samples isolated from different host species. The aim of this study was to undertake a phylogenetic analysis of G. intestinalis that were representative of all known major genetic groups and compare them with other Giardia species, viz. G. ardeae, G. muris, and G. microti. Segments from four "housekeeping" genes (specifying glutamate dehydrogenase, triose phosphate isomerase, elongation factor 1 alpha, and 18S ribosomal RNA) were examined by analysis of 0.48-0.69-kb nucleotide sequences determined from DNA amplified in polymerase chain reactions from each locus. In addition, isolates were compared by allozymic analysis of electrophoretic data obtained for 21 enzymes representing 23 gene loci. The results obtained from these independent techniques and different loci were essentially congruous. Analyses using G. ardeae and/or G. muris as outgroups supported the monophyly of G. intestinalis and also showed that this species includes genotypes that represent at least seven deeply rooted lineages, herein designated assemblages A-G. Inclusion of G. microti in the analysis of 18S rRNA sequence data demonstrated the monophyly of Giardia with the same median body morphology but did not support the monophyly of G. intestinalis, instead placing G. microti within G. intestinalis. The findings support the hypothesis that G. intestinalis is a species complex and suggest that G. microti is a member of this complex.

Large-scale synthesis of hematoregulatory nonapeptide SK&F 107647 by fragment coupling.

Linear and convergent routes for the large-scale preparation of the hematoregulatory nonapeptide (Glp-Glu-Asp)2-DAS-(Lys)2 (2, SK&F 107647) were investigated. A convergent approach ('3 + 2'-route employing Boc-and benzyl ester protecting groups) was selected for the preparation of multihundred-gram quantities of 2. Key steps were the preparation and the coupling of tripeptide hydrochloride (HCl.H)2-DAS-(Lys(Z)-OBn)2 (6, DAS-2,7-L,L-diaminosuberic acid) and tripeptide Glp-Glu(OBn)-Asp(OBn)-OH (26). Several coupling reagents were investigated in order to reduce the amount of epimerization of this fragment coupling. TDBTU [O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl-1,1,3,3-tetrameth yluronium tetrafluoroborate] was identified as the condensation reagent of choice. Using this synthetic route > 97% pure final product in an overall yield of 35% calculated on di-Boc protected 2,7-L,L-diaminosuberic acid was prepared.

Accessible xenografts of human synovium in the subcutaneous tissues of the ears of SCID mice.

This work was undertaken to examine whether human synovium could be engrafted into subcutaneous pouches in the ears of severe combined immunodeficient (SCID) mice. Synovium was transplanted into surgically constructed ear pouches. The grafts were examined by histological and immunohistochemical methods after varying periods after engraftment, or after percutaneous injection of TNF-alpha. Normal, osteo-arthritic and rheumatoid synovium was engrafted successfully in subcutaneous ear pouches. The general morphology and cellular compositions of xenografts were retained including human endothelial cells. In rheumatoid xenografts, macrophages, fibroblasts and lymphocytes persisted for at least 4 weeks. Vascular expression of intercellular adhesion molecule-1 (ICAM-1) was maintained but expression of vascular adhesion molecule-1 (VCAM-1), E-selectin and MHC class II diminished with time. Percutaneous injection of TNF-alpha induced up-regulation of VCAM-1. Human synovium can be engrafted into subcutaneous ear pouches in SCID mice. The xenografts are accessible and respond to injection of a pro-inflammatory cytokine.

A new locus (vsp417-4) belonging to the tsa417-like subfamily of variant-specific surface protein genes in Giardia intestinalis.

A new variant-specific surface protein gene locus (vsp417-4) of Giardia intestinalis is described. Vsp417-4 represents the fourth member of a gene subfamily that is based on a previously described gene, tsa417 ( =vsp417-1). The new locus was detected by characterising DNA amplified in polymerase chain reactions from the 3' ends of divergent homologues (vsp417-4(A-I), vsp417-4(A-II)) found respectively in isolates belonging to the genetic Assemblage A/Group I ('A-I') and Assemblage A/Group II ('A-II') subtypes of G. intestinalis. The complete vsp417-4(A-I) gene was isolated on a 6.2-kb HindIII fragment by screening a genomic DNA library prepared from a type A-I isolate, Ad-1/C7. The deduced polypeptide (VSP417-4(A-I); 709 amino acids, Mr 72662) has properties characterising it as a Giardia variant-specific surface protein, namely a high cysteine content (11.85 mol%), 29 copies of the four amino-acid 'CXXC' motif, and conserved N-terminal signal peptide and C-terminal hydrophobic (membrane-spanning) segments--the latter terminating with the invariant, hydrophilic motif '-CRGKA'. An extended polyadenylation signal sequence (CTTAGRTAGTAAAY), which appears to be a characteristic feature of VSP genes in Giardia, is situated immediately beyond the stop codon. VSP417-4(A-I) shares 87% sequence identity with VSP417-4(A-II) over its C-terminal 235 amino acids, but only 57-58% identity with VSP417-1, VSP417-2 and VSP417-3 which are encoded by other vsp417 family genes identified in these genotypes. Southern hybridisations, using probes derived from the 5' segment of vsp417-4(A-I), indicated the presence of at least five to six closely related loci in both type A-I and type A-II isolates.

Interleukin-5 transgenic mice show enhanced resistance to primary infections with Nippostrongylus brasiliensis but not primary infections with Toxocara canis.

In this study, interleukin-5 (IL-5) transgenic mice with lifelong eosinophilia were assessed for resistance to primary infections with two tissue-invading nematodes, Nippostrongylus brasiliensis and Toxocara canis. Relative to nontransgenic littermates, three lines of IL-5 transgenic mice with varying degrees of eosinophilia all displayed enhanced resistance to N. brasiliensis. Although the timing of final worm expulsion was similar in transgenic and nontransgenic hosts, intestinal worms in transgenic mice were fewer in number throughout infection, failed to increase in size over the course of the infection, and were much less fecund. In contrast, T. canis larvae were recovered in similar numbers from tissues of transgenic mice with "low" or "high" eosinophilia and from nontransgenic mice. These results and other data suggest that eosinophils can contribute to host resistance to some parasite species. Parasite transit time through the host may correlate with relative sensitivity to eosinophils.

Comparison of tsa417-like variant-specific surface protein (VSP) genes in Giardia intestinalis and identification of a novel locus in genetic group II isolates.

A gene (vsp417-3A-II) encoding a new member of the variant-specific surface protein (VSP) family of Giardia is described. Vsp417-3A-II is detected exclusively in isolates belonging to the genetic Assemblage A/Group II sublineage of G. intestinalis, and it is closely related to a previously characterized VSP gene (tsp11, herein renamed vsp417-2A-I) found in Group I isolates of G. intestinalis. Analysis of DNA amplified from the genomes of Group II isolates using consensus primers also revealed products corresponding to the vsp417-2 locus (vsp417-2A-II allele), indicating that vsp417-2A-II and vsp417-3A-II represent separate loci in these organisms. Southern hybridizations revealed a single genomic copy of vsp417-3A-II in Group II isolates, but efforts to detect this locus in Group I organisms were unsuccessful. Together with the vsp417-1 (tsa417) locus that was first identified in the genetic Group I isolate WB and which has been detected since in all tested genetic Group I isolates (as vsp417-1A-I) and in genetic Group II isolates (as vsp417-1A-II), vsp417-3A-II represents a third member of a stable subset of tsa417-like VSP genes in genetic Group II G. intestinalis. The encoded polypeptide, VSP417-3A-II (667 residues, predicted M(r) 69120) possesses a high cysteine content (12.1 mol%), 28 copies of the metal-binding -CXXC- motif, and a highly conserved C-terminal hydrophobic domain--similar to all other characterized Giardia VSP. A revised nomenclature for Giardia VSP genes is proposed, prompted by the need to describe the homologues found in the various major genotypes of the species complex, G. intestinalis.

Lymphocyte-filled villi: comparison with other lymphoid aggregations in the mucosa of the human small intestine.

BACKGROUND & AIMS: Solitary lymphoid structures that may be sites of primary extrathymic T-cell differentiation have been described recently in murine (cryptopatches) and rat (lymphocyte-filled villi) small intestine. This study tests the hypothesis that similar structures occur in human small intestine. METHODS: Normal small intestine was obtained during surgery. Fixed tissue was examined histologically, and frozen sections were examined by an indirect immunoperoxidase technique using a panel of mouse monoclonal antibodies. RESULTS: A new isolated lymphoid structure, with epithelium resembling follicle-associated epithelium of Peyer's patches, is described as a lymphocyte-filled villus. These structures contain major histocompatibility complex (MHC) class II-positive dendritic cells, a majority of memory T cells, a variable B-cell component, and no evidence of immature lymphocytes that express either c-kit or CD1a. Two previously described lymphoid aggregations (isolated lymphoid follicles and submucosal lymphoid aggregations) are components of a single structure. The complete structure contains a B-cell follicle, T cells with mainly memory (CD45RO-positive) phenotype, high endothelial venules, and no detectable population of immature lymphocytes. CONCLUSIONS: A new solitary lymphoid structure is described in the human small intestine. Neither these structures nor isolated lymphoid follicles appear to be similar to solitary primary lymphoid structures in rodent intestine.

Comparison of the levels of intra-specific genetic variation within Giardia muris and Giardia intestinalis.

The extent of intra-specific genetic variation between isolates of Giardia muris was assessed by allozyme electrophoresis. Additionally, the levels of allozymic variation detected within G. muris were compared with those observed between members of the two major assemblages of the morphologically distinct species Giardia intestinalis. Four isolates of G. muris were analysed. Three (Ad-120, -150, -151) were isolated from mice in Australia, while the fourth (R-T) was isolated from a golden hamster in North America. The 11 isolates of G. intestinalis (Ad-1, -12, -2, -62, representing genetic Groups I and II of Assemblage A and BAH-12, BRIS/87/HEPU/694, Ad-19, -22, -28, -45, -52, representing genetic Groups III and IV of Assemblage B) were from humans in Australia. Intra-specific genetic variation was detected between G. muris isolates at four of the 23 enzyme loci examined. Similar levels of variation were found within the genetic groups that comprise Assemblages A and B of G. intestinalis. These levels of intra-specific variation are similar to those observed within other morphologically-distinct species of protozoan parasites. We suggest that the magnitude of the genetic differences detected within G. muris provides an indication of the range of genetic variation within other species of Giardia and that this can be used as a model to delineate morphologically similar but genetically distinct (cryptic) species within this genus.

Novel lineages of Giardia intestinalis identified by genetic analysis of organisms isolated from dogs in Australia.

Infection of suckling mice with Giardia trophozoites recovered from the intestines of 11 dogs autopsied in Central and Southern Australia in each case produced an established isolate. In contrast, only 1 isolate was obtained by inoculation of faecal cysts. The organisms grew poorly in comparison with isolates from humans or non-canine animal hosts. Light microscopy revealed that the trophozoites had median bodies with the 'claw hammer' appearance typical of G. intestinalis (syn. G. duodenalis, G. lamblia) but that they differed in shape and nuclear morphology from axenic isolates of human or canine origin. Allozymic analysis of electrophoretic data representing 26 loci and phylogenetic analysis of nucleotide sequences obtained from DNA amplified from the glutamate dehydrogenase locus showed that the 11 isolates examined from Australian dogs were genetically distinct from all isolates of G. intestinalis that have been established previously from humans and animals, and also from G. muris. Both analytical methods placed 10 of the Australian canine isolates into a unique genetic lineage (designated Assemblage C) and the eleventh into a deep-rooted second branch (designated Assemblage D), each well separated from the 2 lineages (Assemblages A and B) of G. intestinalis that encompass all the genotypes known to infect humans. In contrast, 4 axenic isolates derived from dogs in Canada and Europe (the only other isolates to have been established from dogs) have genotypes characteristic of genetic Assemblages A or B. The findings indicate that the novel Giardia identified in these rural Australian dogs have a restricted host range, possibly confined to canine species. The poor success rate in establishing Giardia from dogs in vitro suggests, further, that similar genotypes may predominate as canine parasites world-wide. The absence of such organisms among isolates of Giardia that have been established from humans by propagation in suckling mice indicates that they are unlikely to infect humans. However, infection of humans by those dog-derived genotypes that grow in vitro cannot be excluded.

Immune responses of IL-5 transgenic mice to parasites and aeroallergens

Eosinophils have long been thought to be effectors of immunity to helminth but have also been implicated in the pathogenesis of asthma. Patterns of cytokine production in the host may influence the pathogenesis of these diseases by regulating the activities of eosinophils and other components of the immune response. Mice which constitutively over-express IL-5 have profound and life-long eosinophilia in a restricted number of tissues. Although eosinophils from IL-5 transgenics are funtionally competent for a number of parameters considered to be important in inflammation, untreated animals are overtly normal and free of disease. In addition, the responses of these animals when exposed to aeroallergens and helminth present a number of apparent paradoxes. Eosinophil accumulation in tissue adjacent to major airways is rapid and extensive in transgenics exposed to the aeroallergen, but even after treatment with antigen over many months these mice show no evidence of respiratory distress or pathology. Helminth-infected IL-5 transgenics and their non-transgenic littermates develop similar inflammatory responses at mucosal sites and are comparable for a number of T cell and antibody responses, but they differ considerably in their ability to clear some parasite species. The life-cycle of Nippostrongylus brasilensis is significantly inhibited in IL-5 transgenics, but that of Toxocara canis is not. Our results suggest that eosinophilia and/or over-expression of IL-5 may actually impair host resistance to Schistosoma mansoni and Trichinella spiralis. The pathogenesis of diseases in which eosinophils are involved may therefore be more complex than previously thought.

Localization of the transmembrane 4 superfamily (TM4SF) member PETA-3 (CD151) in normal human tissues: comparison with CD9, CD63, and alpha5beta1 integrin.

It has recently been shown that several members of the tetraspan superfamily, including CD9 and CD63, associate with each other and with beta1 integrins. In this study, we examined the distribution of a recently identified tetraspan, PETA-3 (CD151), and of CD9, CD63, alpha5beta1, and the integrin beta1 chain in normal human tissues by the indirect immunoperoxidase and alkaline phosphatase-anti-alkaline phosphatase techniques. PETA-3 showed a broad distribution and was expressed by endothelium, epithelium, Schwann cells, and dendritic cells and by skeletal, smooth, and cardiac muscle. Expression in skin was mostly restricted to the basal cells of the epidermis and was downregulated on differentiation. In the small intestine, PETA-3 was expressed by crypt and villous enterocytes with a mostly basolateral distribution, but was not detectable on the brush border. CD9 was expressed on the plasma membrane of enterocytes in crypts and at the bases of the villi whereas CD63 demonstrated a unique granular appearance concentrated in the apical cytoplasm below the brush border. The findings of this study show co-localization of PETA-3 with CD9, CD63, alpha5beta1, and beta1 in particular tissues, demonstrating that tetraspan/integrin complexes may occur. However, the lack of co-localization of these antigens in other tissues also implies distinct roles for these molecules.

Deregulated inflammatory response in mice lacking the STK/RON receptor tyrosine kinase.

Immune and inflammatory responses must be rightly regulated to maintain a homoeostatic balance between an effective immune response and tissue damage to the host. NO is a principal mediator of many of the cytokine-inducible macrophage activities during a normal cell-mediated immune response. STK, the murine homologue of the human RON receptor tyrosine kinase, is expressed on murine resident peritoneal macrophages. The ligand for STK, macrophage-stimulating protein (MSP), is a serum protein that is activated by members of the coagulation cascade in response to tissue damage. In addition to its potential to induce chemotaxis and phagocytosis of C3bi-coated erythrocytes, MSP has an inhibitory effect on the production of NO by activated peritoneal macrophages in vitro. Here we demonstrate that peritoneal macrophages from mice lacking STK produce elevated levels of NO in response to interferon (IFN)-gamma in a dose-dependent manner, without the need for a co-stimulus. However, production of pro-inflammatory cytokines by activated macrophages from stk -/- mice is unaltered. In vivo, stk -/- mice exhibit increased inflammation in an IFN-gamma-mediated delayed-type hypersensitivity reaction and increased susceptibility to lipopolysaccharide (LPS)-induced endotoxic shock. Furthermore, the levels of NO in the serum of mice injected with LPS are significantly higher than those in control littermates. Nevertheless, the serum levels of IFN-gamma and the intermediate cytokines generated by the inflammatory response, which have previously been shown to play a role in septicaemic shock, do not differ significantly from controls. These data suggest that the STK receptor suppresses NO production, therefore ameliorating the potentially tissue-damaging effects of a cell-mediated immune response, through negative regulation of the IFN-gamma signalling pathway.

Immune responses of IL-5 transgenic mice to parasites and aeroallergens.

Eosinophils have long been thought to be effectors of immunity to helminths but have also been implicated in the pathogenesis of asthma. Patterns of cytokine production in the host may influence the pathogenesis of these diseases by regulating the activities of eosinophils and other components of the immune response. Mice which constitutively over-express IL-5 have profound and life-long eosinophilia in a restricted number of tissues. Although eosinophils from IL-5 transgenics are functionally competent for a number of parameters considered to be important in inflammation, untreated animals are overtly normal and free of disease. In addition, the responses of these animals when exposed to aeroallergens and helminths present a number of apparent paradoxes. Eosinophil accumulation in tissues adjacent to major airways is rapid and extensive in transgenics exposed to the aeroallergen, but even after treatment with antigen over many months these mice show no evidence of respiratory distress or pathology. Helminth-infected IL-5 transgenics and their non-transgenic littermates develop similar inflammatory responses at mucosal sites and are comparable for a number of T cell and antibody responses, but they differ considerably in their ability to clear some parasite species. The life-cycle of Nippostrongylus brasiliensis is significantly inhibited in IL-5 transgenics, but that of Toxocara canis is not. Our results also suggest that eosinophilia and/or over-expression of IL-5 may actually impair host resistance to Schistosoma mansoni and Trichinella spiralis. The pathogenesis of diseases in which eosinophils are involved may therefore be more complex than previously thought.