Glut1 glucose transporter in the primate choroid plexus endothelium.

The objective of the present study was to define the cellular location of the Glut1 glucose transporter in the primate choroid plexus. Immunogold electron microscopy indicated that Glut1 epitopes were associated primarily with choroid plexus endothelial cells. Digitized analyses of electron microscopic images provided quantitative estimates of the relative number of Glut1 glucose transporter epitopes on luminal and abluminal endothelial cell membranes within the choroid plexuses. We recorded a high density of Glut1 in the microvascular endothelium of primate choroid plexus, which was consistent in vervet monkeys (5-10 Glut1 gold particles per micrometer of endothelial cell plasma membrane), as well as in baboons (5-20 Glut1 gold particles per micrometer of capillary plasma membrane). In the baboon choroid plexus, we observed that perivascular cells (presumed to be pericytes) were also Glut1-positive, but with substantially reduced activity compared with endothelial cells. Occasional Glut1-immunogold particles were also seen in the basolateral membranes of the choroid plexus cuboidal cells. Light microscopic immunocytochemistry confirmed the abundance of Glut1 immunoreactivity in choroid plexus endothelial cells of vervet monkeys and baboons. A similar pattern was observed in surgically resected human choroid plexus, suggesting differences between primates, including humans and laboratory animals. The only difference was that erythrocytes within the human choroid plexus exhibited a florid Glut1-positive response, but were weakly immunoreactive in nonhuman primates. The observation of high glucose transporter densities in choroid plexus endothelial cells is consistent with the suggestion that choroidal epithelia and capillaries provide a metabolic work capability for maintaining ionic gradients and secretory functions across the blood-CSF barriers.

A single glucose transporter configuration in normal primate brain endothelium: comparison with resected human brain.

Cellular distribution of the Glut1 glucose transporter in normal primate brains was analyzed by immunogold electron microscopy. Two configurations of endothelial Glut1 glucose transporter (high and low density capillaries) have been found in resections of traumatically injured and epileptogenic human brain; the objective of the present study was to ascertain whether these same 2 capillary populations, expressing high and low glucose transporter densities, were the common configuration in normal brain. The relative numbers of Glut1 glucose transporter-associated gold particles on luminal and abluminal endothelial cell membranes were determined within the cerebral cortex of several normal, nonhuman primates. Low Glut1 densities were seen in brain endothelia of both the rhesus and squirrel monkey cortex, with slightly greater quantities of Glut1 in vervet monkey cortices. The Glut1 transporter was most highly expressed in the baboon cortex, approaching the concentrations seen in human brains. In the rhesus, squirrel, and vervet monkeys, Glut1 concentrations were greater on the abluminal than luminal capillary membranes. In contrast, mean luminal membrane Glut1 concentrations were greater in baboons, resembling the distribution seen in the human brain. Brain regional differences in transporter concentration were seen in comparing membrane densities in the baboon cortex (approximately 15 Glut1-gold particles per micrometer), hippocampus (approximately 12 Glut1 gold particles per micrometer), cerebellum (approximately 6 Glut1-gold particles per micrometer), and retinal microvasculature (approximately 20 Glut1-gold particles per micrometer). We conclude that a single, uniform Glut1 distribution characterizes brain capillaries of normal nonhuman primates, and hypothesize that the presence of high and low density glucose transporter endothelial cells (seen in human traumatic injury and seizure resections) represents a pathologic response to brain insult.

Micro-system for the primary immunization of baboon (Papio cynocephalus) peripheral blood mononuclear cells with sheep erythrocytes in vitro.

A micro-culture system for the stimulation of baboon peripheral blood mononuclear cells (PBMC) with sheep erythrocytes (SRBC) in vitro was established. PBMC cultures in 96-well microtiter plates were maintained in 300 microliter of culture medium containing a murine mixed lymphocyte reaction (MLR) supernatant and SRBC. Cultures of 7.5 x 10(5) PBMC and 6 x 10(6) SRBC resulted in the highest anti-SRBC plaque-forming cell (PFC) response. It was also determined that the presence of 50 microliter of murine MLR supernatant was required for optimal PFC generation and that the cultures required supplementation with nutrient cocktail 3 days post-immunization. Although PFC were detectable on days 4-8, the maximum expression of PFC occurred on day 7.

Characterization of the high mannose asparagine-linked oligosaccharides synthesized by Schistosoma mansoni adult male worms.

This report describes the structures of the high-mannose-type N-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni adult male worms. Adult male schistosomes were incubated in vitro in media containing either [2-3H]mannose, [6-3H]glucosamine or [6-3H]galactose to allow metabolic radiolabeling of the oligosaccharide moieties of newly synthesized glycoproteins. Glycopeptides were prepared from the radiolabeled glycoproteins by digestion with Pronase and fractionation by chromatography on concanavalin A-Sepharose. Eleven percent of [3H]mannose incorporated into the schistosome glycopeptides was recovered in high mannose-type Asn-linked oligosaccharides which bound to the immobilized lectin. Upon treatment of [3H]mannose-labeled glycopeptide with endo-beta-N-acetylglucosaminidase H, the high mannose-type chains were released and their structures were determined by high performance liquid chromatography, methylation analysis, acetolysis and exoglycosidase digestion. The major species of high mannose-type chains synthesized by S. mansoni adult males have the composition Man7GlcNAc2, Man8GlcNac2 and Man9GlcNA2. Structural analyses indicate that these oligosaccharides are similar to high mannose-type chains synthesized by mammalian cells.

Inhibition of p-450 aromatase prevents feminisation and induces protection during cysticercosis.

Cysticercotic male mice undergo an impressive feminisation process, characterised by 200 times increased serum 17beta-estradiol levels while testosterone and dihydrotestosterone are 90% reduced, which results in elevated parasite burden. Administration of Fadrozole (an aromatase inhibitor) in male and female mice suppressed the production of 17beta-estradiol, accompanied with a 70% reduction in parasite burden. This protective effect was associated in male mice with a recovery of the specific cellular immune response. Interleukin-6 (IL-6) serum levels, and its production by splenocytes, was augmented by 80%, together with a 10-fold increase in its expression in testes of infected male mice. Fadrozole treatment returned these levels to baseline values. Aromatase expression in the testes of infected male mice was not affected by Fadrozole. These results suggest that aromatase and IL-6 are key molecules in the production of the feminisation undergone by infected male mice and to Fadrozole treatment as a possible new therapeutic approach to cysticercosis.

Development of a molecular probe to baboon interleukin-10 mRNA for in situ hybridization during experimental schistosomiasis.

A nucleic acid probe complementary to baboon interleukin 10 (IL-10) mRNA was developed for in situ hybridization. Highly conserved IL-10 protein sequences from several mammals were aligned to design oligonucleotide primers flanking a 270-bp sequence of the target cDNA. RNA was isolated from stimulated peripheral blood mononuclear cells (PBMC). IL-10 cDNA was reverse-transcribed from the total PBMC RNA and amplified with the polymerase chain reaction (PCR). Cloning and sequencing of the PCR product confirmed it to be of baboon IL-10 origin, with 97.8% identity to human and 100% identity to macaque mRNA sequences. The baboon IL-10 DNA probe hybridized in Southern blots to a 7.9-Kbp or 8.6-Kbp band after digestion of genomic baboon DNA with Bam H1 or Eco R1, respectively. Preliminary results with an antisense riboprobe derived from this sequence showed the presence of IL-10 mRNA in sections of granulomatous tissues.

Voltage- and GABA-evoked currents from Müller glial cells of the baboon retina.

The electrophysiological features of isolated baboon Müller cells was investigated using the whole-cell voltage-clamp technique. Application of depolarizing voltage steps evoked transient inward and delayed outward currents. The transient currents disappeared when extracellular Na+ was replaced by choline+ and were substantially decreased by application of tetrodotoxin (1 microM). The outward currents were strongly diminished by extracellular Ba2+ (1 mM), and the hyperpolarization-generated inward currents disappeared following application of Ba2+. The recently described gamma-aminobutyric acid A (GABAA) receptor currents were increased by flunitrazepam, nordiazepam, pentobarbital and Zn2+, as well as by the inverse agonist DMCM. These results suggest that the baboon Müller cells possess the same voltage-dependent current pattern as those from other species, e.g. humans, whereas their GABAA receptors react in an uncharacteristic manner to DMCM and Zn2+, when compared with neuronal GABAA receptors.

Schistosomiasis mansoni in baboons. VI. Plastic-adherent-cell suppression of the primary in vitro antibody response of peripheral blood mononuclear cells to a heterologous antigen (SRBC) during chronic infection.

Peripheral blood mononuclear cells (PBMC) from five of six baboons (Papio cynocephalus) with chronic schistosomiasis mansoni showed a marked reduction in the ability to generate anti-sheep erythrocyte (SRBC) plaque-forming cells (PFC) after primary in vitro immunization as compared to the PFC responses of PBMC from normal (non-infected) baboons. Removal of the plastic-adherent (PLAD) cells from the PBMC of these animals results in a population of cells capable of responding to in vitro immunization with SRBC at a level equal to or higher than their normal counterparts. Reconstitution of plastic-non-adherent cells with PLAD cells re-establishes suppression. In contrast, the single apparently non-suppressed infected baboon showed reduced responses after PLAD cell removal, but, upon reconstitution with PLAD cells, responses were higher than those obtained before cell separation. No evidence of PLAD suppressor cells in normal animals was found, and indeed the data suggest that PBMC from normal animals may require a PLAD accessory cell for full responsiveness.

Experimental Schistosoma mansoni infection in a small New World monkey, the saddle-back tamarin (Saguinus fuscicollis).

Twelve young adult captivity-born tamarin monkeys (Saguinus fuscicollis) were each exposed to 150 cercariae of Schistosoma mansoni (KEB strain): 6 by the percutaneous (pc) and 6 by the subcutaneous (sc) route. The prepatent period, as determined by eggs in the feces, was 34-39 days for both groups. Weekly quantitative fecal examinations revealed that although both groups actively passed eggs for as long as the duration of the experiment (18 months), the sc group excreted a significantly higher number of eggs/gram/day in the feces than did the pc group. Eggs recovered from the feces of both groups were viable: they hatched and the miracidia invaded snails which subsequently yielded infective cercariae. A significantly greater number of worms was recovered from tamarins infected by the sc route as compared to those with pc infections, and large numbers of eggs were found in affected organs of the sc group. Chronically infected tamarins with high tissue egg loads developed focal granulomatous nodules along the serosal walls of the small intestines, which in some cases, apparently had budded off to lie free in the abdominal cavity. Hepatic granulomas from animals with acute infections were significantly larger than those from chronically infected monkeys. Our results strongly suggest the presence of a skin barrier to infection in the tamarin monkey, and that if this barrier is bypassed, the tamarin can serve as a permissive host for S. mansoni. The small body size of this monkey (less than 400 g) is an obvious reason for the establishment of the tamarin monkey as a laboratory nonhuman primate model for human schistosomiasis mansoni.

Immunizing efficacy of a Kenyan strain of Schistosoma mansoni in rhesus monkeys (Macaca mulatta).

The efficacy as an immunizing agent of the Kenyan strain of Schistosoma mansoni against challenge infection in rhesus monkeys was demonstrated. An initial exposure of 200 cercariae immunized monkeys against a challenge dose of 2,000 cercariae administered 16 weeks later. The penetration rate in rhesus monkeys was 99%, the same as in baboons. The prolongation of the time for immunity to develop in baboons, compared to rhesus monkeys, shown with this strain of S. mansoni is therefore not due to a reduced potential for immunization by this strain in the rhesus monkey.

Coagulation factor XIIa (activated Hageman factor) inhibitor from adult Schistosoma mansoni.

An inhibitory activity for the contact phase of the intrinsic coagulation pathway was demonstrated in an extract of adult Schistosoma mansoni. Inhibition is apparently specific for the enzymatic activation of Factor XI (pre-PTA) by Factor XIIa (activated Hageman factor). This phenomenon offers an explanation for the schistosomal evasive mechanism of the host contact hemostatic defense system.

Schistosoma mansoni: anti-SMw32 proteinase response in vaccinated and challenged baboons.

Antibodies to a cysteinyl proteinase of the trematode Schistosoma mansoni have been detected in serum from infected mice and humans. We have evaluated antiproteinase responses in infected baboons and in baboons vaccinated with irradiated, cryopreserved schistosomules prior to challenge. Prechallenge sera and normal, uninfected control sera were nonreactive by ELISA and immunoblots. Serum antibodies were first detectable by ELISA at two months post-challenge in both challenged (C) and vaccinated-challenged (V-C) baboons (serum dilution 1:200). By four months post-challenge, ELISA absorbance values for subgroup C baboons were significantly higher than for V-C counterparts. The immunoblot technique provided a more sensitive means of detecting antibody early in the infection. One month post-challenge, 7 of 12 C and V-C sera (diluted 1:100) contained measurable anti-proteinase antibody. By month two, 12 of 12 were immunoblot-positive. Baboons vaccinated but not challenged (subgroup V) remained negative. The presence of the anti-proteinase antibody appears to be a sensitive and early marker for infection by S. mansoni.

Schistosoma mansoni in baboons. III. The course and characteristics of infection, with additional observations on immunity.

Parasitological, clinical, and histopathological observations on 54 baboons infected with Schistosoma mansoni are presented. The baboon and S. mansoni constitute a compatible host-parasite system, evidence by the infectivity of cercariae (98% penetration, 42% adult worm recovery), and the long, fertile life of the worms. Baboons tolerated the infection well, with clinical illness a rarity in moderately infected baboons. Pathological findings were generally unremarkable. An acute "toxemic" phase occurred 66 days or less following a large cercarial exposure in three baboons. Worm burdens were not significantly reduced during the course of prolonged infection, but prolonged infections resulted in decreased oviposition by the worms and in an anterior shift in egg deposition from the colon to the small intestine. Concomitant immunity was also a feature of baboon infections. Decreased oviposition and the anterior shift are probably manifestations of a second phase of immunity, distinct from concomitant immunity. The baboon is similar to man and the grivet monkey in that in all three species immunity is slow to develop.

GABAA receptor currents recorded from Müeller glial cells of the baboon (Papio cynocephalus) retina.

The effect of gamma-aminobutyric acid (GABA) application on acutely isolated, non-cultivated Muller glial cells from the baboon retina was studied using the whole-cell voltage-clamp technique. Application of GABA (0.1 mM) generated inward currents at a holding potential of -80 mV as well as an increase in current noise. The GABA-activated current had a reversal potential of 18.6 mV and was therefore supposed to be a Cl- current (ECl = 5 mV). The GABAA receptor agonist muscimol (0.1 mM) elicited an inward current and bicucullin (0.5 mM), a blocker of the GABAA receptor, diminished the GABA responses in our experiments completely. Baclofen (0.1 mM), a GABAB agonist, neither had an effect when applied under conditions where the dominant Muller cell K+ currents were unblocked, nor when the K+ currents were blocked by application of Ba2+ (1 mM). Glycine (0.1 mM) was ineffective as well. From these results we conclude that the baboon retinal Muller cells possess GABAA receptors. However, these have recently been discovered on skate Muller cells whereas GABAA receptors could not be found on Muller cells of guinea pig, pig, mouse, rat and rabbit.

Mitogenic responses of baboon (Papio cynocephalus) peripheral blood mononuclear cells. A possible role for monocytes.

Removal of carbonyl iron-adherent/phagocytic cells from baboon peripheral blood mononuclear cells generally resulted in a depressed blastogenic response to both pokeweed mitogen and concanavalin A. In the majority of cases, no alteration in dose requirement nor shift in kinetics was apparent. Staining for peroxidase activity indicated a reduced proportion of monocytes in the population of cells treated with iron. Therefore, the results of this study strongly suggest a potentiating role for monocytes in lectin stimulation of baboon lymphocytes.

Immunochemical analysis of baboon (Papio cynocephalus) IgG subclasses.

Few reports are presently available on the existence of IgG subclasses in nonhuman primates. Papain and trypsin digestion of baboon (Papio cynocephalus cynocephalus and P. cynocephalus hamadryas) IgG proteins, in the absence of cysteine, revealed the occurrence of two different protein populations, one being protease-resistant, the other being protease-sensitive. The papain-resistant population is rendered sensitive to digestion upon addition of cysteine. Ion exchange chromatography of the papain-resistant IgG population subsequently demonstrated that it is composed of two different subpopulations varying in their ionic binding affinities. Peptide maps of the Fc fragments of the papain sensitive population and of the Fc fragments of the two papain-resistant subpopulations differing in their binding affinities for ion exchange resins, were different from each other. The biochemical identification of the baboon IgG proteins presented here strongly suggests that they are composed of at least three different subclasses.

Serial laparoscopic biopsies of liver and spleen from Schistosoma-infected baboons (Papio spp.).

PURPOSE: To obtain large, serial biopsy samples from the liver and spleen by using laparoscopy. Large samples were needed for measurement of inflammatory mediators during various stages of schistosomiasis. METHODS: Each of the seven female baboons (Papio sp.) underwent as many as three laparoscopies, for a total of 19 laparoscopic procedures. This process permitted sampling of the liver, spleen, and mesenteric lymph nodes before and at 6 and 9 weeks after infection with Schistosoma mansoni. All surgery was performed through three trocar sites. Postoperative care included preemptive analgesia. After surgery, we monitored the animals' appetite and measured the core body temperature and activity by using implanted radiofrequency transmitters. RESULTS: We obtained samples of the liver and splenic biopsies during all 19 laparoscopic procedures. The mean weight of the liver biopsies was 3.7 g and that of the spleen samples was 5.3 g. We encountered small adhesions during 5 of the 12 reoperations. Eating and activity rapidly returned after surgery. CONCLUSIONS: Laparoscopy permitted collection of large, serial biopsies with apparently limited stress to the animals. Laparoscopy can be used for biopsies in studies to characterize disease response, confirm normal organ histology prior to drug toxicity studies, determine target-organ drug concentrations in pharmacokinetic studies, and measure drug residues. This refinement likely will reduce required animal numbers by decreasing the effect of surgery compared to that of the experimental conditions, enhance animal well-being, and permit repeated measurements in an animal that serves as its own control.

Serum factors from infected baboons inhibit oviposition and cause unpairing of Schistosoma mansoni in vitro.

A reliable in vitro fecundity assay for Schistosoma mansoni was established. The main features that reduced variability in in vitro oviposition were pre-selection and randomization of worm pairs producing moderate numbers of eggs in initial 2-day culture, and short pre-incubation in serumless medium prior to addition of test sera to the cultures. In 4 of 6 total experiments testing the effects of serum from chronically infected baboons, significant (P less than or equal to 0.025) fecundity reduction ranging from 29 to 82% was found. Chronically infected baboon serum also caused consistently higher unpairing than normal serum. These results demonstrate the existence of serum factors which inhibit egg production and maintenance of the paired status of Schistosoma mansoni in vitro.

Haptenation of adult Schistosoma mansoni and assessment of humorally mediated damage in vitro.

Adult Schistosoma mansoni were haptenated with DNP under mild conditions using DNP-liposomes, as devised by Levi-Schaffer et al. (1983, Am. J. Trop. Med. Hyg. 32: 343) for haptenating schistosomula. The effects of complement, alone or with anti-DNP antibodies, on tegumental morphology of DNP-haptenated adult worms were assessed by both scanning and transmission electron microscopy, as a simple model system for possible in vitro effects of complement and anti-schistosomal antibodies on normal adult worms. Complement-mediated cytotoxicity, as measured by tegumental changes in both normal and haptenated worms, was observed and indicates a possible role for complement in humorally mediated damage of adult schistosomes. The damage to haptenated adults in the presence of both complement and anti-DNP was greater than that to nonhaptenated worms similarly treated. However, anti-DNP plus complement caused less damage to normal worms than did complement alone, perhaps the result of blocking by antibodies bound to the tegument via Fc receptors. Evidence herein presented implicates both classical and alternative complement activation by the adult worm's tegument. Tegumental tubercle smoothing was one manifestation of damage, as assessed by SEM. Smoothing appears to progress from spine blunting to disappearance, arguing for resorption rather than shedding as the mechanism for spine loss. Because the spines are mostly actin bundles, we suggest that complement-mediated intrategumental calcium ion flux could lead to spine resorption resulting from actin filament depolymerization and/or calcium-induced severing of actin filaments and dissociation of actin filament cross-linking proteins. This process could have adaptive value to the adult worm, in that the spines may be a repository of readily mobilizable actin molecules for tegumental maintenance and repair.

Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula.

This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.