Bone marrow origin of Ia-positive cells in the medulla rat thymus.

Irradiated rats were reconstituted with bone marrow from F1 hybrids. Ia antigen of donor-bone marrow origin was detected by an immunoperoxidase technique on cryostat sections and found predominantly in the medulla of rat thymus 2 wk after reconstitution. These Ia-bearing cells increased in number with time after reconstitution, but the Ia on the cortical epithelial cells remained of host origin. The nature of the bone marrow-derived cells and their implication for major histocompatibility complex restriction are discussed.

Ia antigens in rat kidney, with special reference to their expression in tubular epithelium.

The distribution of Ia antigens in rat kidneys has been investigated by an immunoperoxidase technique, using monoclonal antibodies directed at framework determinants on molecules equivalent to gene products of the I-A and I-E/C regions of the mouse major histocompatibility complex (MHC). Antigens mapping to both loci were detected in proximal convoluted tubule epithelium and on scattered dendritic cells in the interstitial connective tissues, glomeruli, and mucosa of the renal pelvis. All other structures were negative. Genetic studies indicate that the levels of expression of Ia molecules from both genetic loci are controlled by non-MHC genes in epithelium, but not on lymphocytes. The subcellular distribution of Ia molecules has been investigated in tubular epithelium and is discussed in relation to their possible functions in epithelia.

[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.

Identification of a broad-specificity nucleoside transporter with affinity for the sugar moiety in Giardia intestinalis trophozoites.

A broad-specificity nucleoside transporter has been identified in Giardia intestinalis trophozoites, using a rapid sampling assay to measure influx of [3H]deoxycytidine, [3H]adenosine and [3H]guanosine at 0 degrees C. The influx of each labelled nucleoside was inhibited strongly by all common, naturally-occurring nucleosides but only poorly or not at all by nucleobases, indicating that the transporter recognizes structural features on the furanosyl moiety of ribo- and 2'-deoxyribonucleosides. Both 2'- and 5'-deoxyadenosine were potent inhibitors of influx (greater than 95% inhibition at 2 mM), whereas 3'-deoxyadenosine was significantly less effective (approx. 70% inhibition), and 2',3'-dideoxycytidine and cytosine arabinoside were virtually inactive (0-20% inhibition). The data reveal that the 2'- and 5'-hydroxyl groups are not necessary for the recognition of nucleosides by this transporter. However, the 3'-hydroxyl appears to be important. Michaelis-Menten constants (Km) were calculated for the influx at 0 degrees C of deoxycytidine (220 +/- 116 microM) and adenosine (45 +/- 24 microM), with respective Vmax values of 13 +/- 4 and 11 +/- 2 pmol min-1 (10(6) cells)-1. Only 12-26% of [3H]thymidine influx occurred through this transporter, the remainder entering the cells through a thymine/uracil-specific transporter described previously. Thymidine exhibited a Ki of 205 +/- 90 microM against [3H]deoxycytidine influx.

Two genes encoding homologous 70-kDa surface proteins are present within individual trophozoites of the binucleate protozoan parasite Giardia intestinalis.

A 0.52-kb DNA sequence encoding part of a major surface antigen of Giardia intestinalis trophozoites has been amplified by the polymerase chain reaction (PCR) using primers specific for nucleotide sequences that are conserved between two apparently related genes, tsp11 (cloned previously from the Australian G. intestinalis isolate, Ad-1) and tsa417 (cloned from the Afghanistani isolate, WB). Restriction analysis revealed that the DNA amplified from each of seven axenic isolates of G. intestinalis was not homogeneous, even though the template DNA had been purified from cultures that had been established from single trophozoites. Every isolate yielded two PCR products, whose respective cleavage fragments corresponded to tsp11 (HindIII+, PstI-, KpnI-) and to tsa417 (HindIII-, PstI+, KpnI+). This was confirmed by cloning individual amplification products into the plasmid vector, pGEM-7Zf(+). The sequence of one cloned fragment (1-P4), derived from the Ad-1 isolate, but possessing restriction sites characteristic of tsa417, exhibited 98.6% identity over 425 bp with tsa417 and only 63.8% identity with the corresponding region of tsp11. The data indicate that individual trophozoites of all the isolates examined contain a copy of each of these homologous genes.

A method for specifically detecting internal immunoglobulin by immunofluorescence.

The detection of specifically internal immunoglobulin by staining of fixed preparations of lymphoid cells with appropriate fluorescein-conjugated antisera can be hampered by binding of the conjugates to membrane-bound immunoglobulin. Without preventing staining through membrane-bound immunoglobulin, it is impossible to be sure whether lightly stained cells contain only small amounts of immunoglobulin or wehter the staining is entirely due to the membrane-bound material. Surface immunoglobulin can be stripped from tonsil cells prior to making preparation, and this can be achieved without loss of viable cells. As compared with untreated preparations, pronase-stripped smears contain little non-cellular debris and greatly reduced numbers of small lymphocytes faintly stained by polyvalent anti-human immunoglobulin. The remaining stained cells can be said to specifically contain internal immunoglobulin and are easily scored.

Characteristics of thymidine transport in Giardia intestinalis trophozoites.

The transport of thymidine by the protozoan parasite Giardia intestinalis was examined at 0 degrees C. This temperature prevented attachment of the cells to vessel walls, so that a rapid sampling technique could be used. Thymidine influx (distinguished from gross uptake) was readily measurable at 0 degrees C and was specific and saturable. The transporter appears to be a facilitative carrier, exhibiting a high affinity for thymidine (Km = 50 microM). Thymine and uracil were the most effective inhibitors (Ki = 30 microM and 45 microM, respectively), followed by thymidine, deoxyuridine and uridine (Ki = 64-96 microM). Cytosine, cytidine and deoxycytidine were not inhibitory, even at high concentrations. The data indicate that the oxygen at position 4 of the pyrimidine ring is essential for recognition by the transporter, whereas the 5-methyl group of thymine is unimportant. The furanose ring appears not to be recognized, since D-ribose was non-inhibitory and uridine and deoxyuridine were equally inhibitory but less so than uracil and thymine. This carrier probably mediates the transport of uracil, as well as uridine and thymidine, although influx of the base remains to be measured.

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.

A gene encoding a 69-kilodalton major surface protein of Giardia intestinalis trophozoites.

A gene encoding a 68.5-kDa trophozoite surface protein (TSP11) of the Australian Giardia intestinalis (syn. G. lamblia) isolate, Ad-1, has been cloned from a genomic expression library screened with an antiserum specific for 3 major surface antigens. Sequence analysis of two overlapping genomic fragments identified a single open reading frame that contained no introns and predicted a cysteine-rich, 667-residue polypeptide with features common to other trophozoite surface proteins. These include the presence of 27 copies of the 4-amino acid Cys-X-X-Cys motif, an N-terminal signal sequence and a highly conserved, hydrophobic C-terminal segment. Transcripts from the tsp11 gene were detected as a single band on Northern blots using total RNA extracted from Ad-1 trophozoites. Primer extension analysis indicated that the mRNA has a 5' untranslated region of only 5 nt, similar to the very short (1-6 nt) leader sequences reported for other Giardia mRNAs. A large portion of the promoter distal segment of tsp11 has homology with tsa417, a gene encoding a 72.5-kDa trophozoite surface antigen of the Afghanistan-derived G. intestinalis isolate, WB [13].

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.

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.

Giardia intestinalis: detection of major genotypes by restriction analysis of gene amplification products.

The polymerase chain reaction (PCR) has been used to amplify a 0.52 kb segment of Giardia intestinalis DNA, using primers specific for nucleotide sequences conserved within two genes (tsp11 and tsa417) that encode homologous, cysteine-rich trophozoite surface proteins. Using products amplified from axenic isolates belonging to genetic groups I and II (defined on the basis of allozyme electrophoresis data), restriction endonuclease analysis revealed both tsp11-like and tsa417-like fragments within all samples. The study also identified among the amplification products of group II organisms an additional fragment, containing a novel PstI site, that is not detected in the reaction products of group I isolates. The recovery of three distinct PCR products from each group II isolate was verified by cloning the fragments into the plasmid vector pGEM-7. Fragments containing the new PstI site possess the ClaI site common to both tsp11 and tsa417-like fragments, but they lack the HindIII site which characterizes tsp11-like fragments and also lack the PstI and KpnI sites which characterize tsa417-like fragments. Spot-blot analyses using cloned fragments of all three types as probes showed strong homologous hybridization but weak heterologous hybridization, indicating that each type differs substantially in nucleotide sequence from the others. Because the samples of Giardia DNA used in the PCR were purified from cultures that had been established from single trophozoites, the data indicate that individual trophozoites belonging to genetic group II possess three homologous genes defined by these related fragments. The presence of a PstI site in the amplified segment of the newly-discovered third gene of group II organisms provides a simple diagnostic means of differentiating group I and II isolates.

Giardia intestinalis: electrophoretic evidence for a species complex.

The technique of allozyme electrophoresis was applied to 29 Australasian stocks and 48 clones of Giardia intestinalis from humans as a means of increasing the number of genetic markers currently available for identification and classification. Fifty different enzymes were examined and of these 26 loci were found to be suitable for use as genetic markers. The data indicate the presence of four discrete genetic groups within the sample of G. intestinalis examined. The groups had fixed genetic differences at 23-69% of loci established. The evidence suggests that G. intestinalis is a species complex. The results have important implications for the systematics of human isolates of Giardia, as well as for studies on the epidemiology and demography of giardiasis in Australia and elsewhere.

Changes in allozyme pattern of the protozoan parasite Giardia intestinalis.

The present study compares the allelic profiles of Giardia intestinalis grown in vivo and in vitro. Three clinical isolates of G. intestinalis were established in suckling mice and subsequently adapted to in vitro culture to test the null hypothesis that samples of the same clinical isolate grown in different culture conditions have identical allelic profiles. For each isolate, a mouse-derived and an axenically cultured sample were analysed electrophoretically at 11 enzyme loci. In each case, the axenically cultured sample of each isolate showed marked allelic differences from its corresponding in vivo sample. These data suggest that there may be either regulated expression of alternative genes encoding distinct isozymes (i.e. gene switching) or selection by different growth conditions of specific genotypes from a mixture present within the original clinical isolate. Although these hypotheses are not tested in this study, the data highlight the importance of confirming that allozymes (or isozymes) are stable genetic characters for the identification and characterization of protozoan taxa.

Measurement of virulence of aeromonads using a suckling mouse model of infection.

Virulent and avirulent strains of Aeromonas spp. were identified and virulence quantified using an animal model. Virulence was measured by determining a 50% lethal dose (LD50) 43 h after oral administration of live bacteria. The LD50 of virulent Aeromonas isolates ranged from log10 7.53 (mean) organisms to log10 8.88 (mean). Some isolates were avirulent in this model. Detection of cytotoxic activity in culture supernatants correlated with virulence (Fisher exact test, P = 0.0029). There was no correlation between LD50 and the source of the isolate, beta-haemolysis or lipopolysaccharide (LPS) banding profile on SDS-PAGE. In this animal model, virulence was multifactorial in that: (i) bacterial multiplication in the gut was associated with fatal infection; (ii) the increase in bacterial numbers in the gut of mice administered a lethal dose of bacteria was accompanied by accumulation of fluid; and (iii) there was evidence of extraintestinal spread of infection. Protection of suckling mice by rabbit antiserum to Aeromonas cell envelopes was observed.

[AIDS of the central nervous system]. / AIDS im ZNS.

The central nervous system of nearly every HIV-positive patient becomes affected by the AIDS virus itself or by one of the associated diseases during the course of the illness. Early diagnosis of lesions which demand therapeutic consequences is of the most importance concerning prolongation of life and improvement in its quality. In spite of the frequent underestimation of cerebral involvement by imaging methods and their unspecific findings they are often the only diagnostic means which permit-timely diagnosis and, at least in some diseases, therapeutic monitoring. Indications for cranial computed tomography (CCT) or magnetic resonance tomography (MRT) are already present with mild or transient neurological or psychiatric symptoms or the extracerebral manifestation of neurotropic organisms or tumours which metastasize to the brain, even in patients without subjective complaints.

Peyer's patch organogenesis--cytokines rule, OK?

Targeted inactivation of genes in the tumor necrosis factor (TNF)/lymphotoxin (LT) ligand and receptor system has recently revealed essential roles forthese molecules in lymphoid tissue development and organization. Lymphotoxin-alpha beta (LT alpha beta)/lymphotoxin-beta receptor (LT beta-R) signaling is critical for the organogenesis of lymph nodes and Peyer's patches and for the structural compartmentalization of the splenic white pulp into distinct B and T cell areas and marginal zones. Moreover, an essential role has been demonstrated for TNF/p55 tumor necrosis factor receptor (p55TNF-R) signaling in the formation of splenic B lymphocyte follicles, follicular dendritic cell networks, and germinal centers. In contrast to a previously described essential role for the p55TNF-R in Peyer's patch organogenesis, we show in this report that Peyer's patches are present in both TNF and p55TNF-R knockout mice, demonstrating that these molecules are not essential for the organogenesis of this lymphoid organ. Furthermore, we show that in the absence of TNF/p55TNF-R signaling, lymphocytes segregate normally into T and B cell areas and a normal content and localization of dendritic cells is observed in both lymph nodes and Peyer's patches. However, although B cells are found to home normally within Peyer's patches and in the outer cortex area of lymph nodes, organized follicular structures and follicular dendritic cell networks fail to form. These results show that in contrast to LT alpha beta signaling, TNF signaling through the p55TNF-R is not essential for lymphoid organogenesis but rather for interactions that determine the cellular and structural organization of B cell follicles in all secondary lymphoid tissues.

Thymus-dependent and thymus-independent subpopulations of intestinal intraepithelial lymphocytes: a granular subpopulation of probable bone marrow origin and relationship to mucosal mast cells.

Intraepithelial lymphocytes have been examined histochemically in the small intestines of normal rats and mice and in thymus-deficient animals (B rats and nude mice). It is concluded that these cells are heterogeneous, consisting of at least two subpopulations. One population contains granules, is thymus-independent, and is probably bone-marrow-derived. The other population does not contain granules and appears to be thymus-dependent. It is suggested that the granular cell, which may be a precursor of mucosal mast cells, is more properly a previously unrecognized nonlymphoid leukocyte of bone marrow origin.