An immune control model for viral replication in the CNS during presymptomatic HIV infection.

The brain is targeted by human immunodeficiency virus type 1 (HIV-1) during the course of untreated infection, leading to cognitive impairment, neurological damage and HIV encephalitis (HIVE). To study early dynamics of HIV entry into the brain, we examined a unique autopsy series of samples obtained from 15 untreated individuals who died in the presymptomatic stages of infection from non-HIV causes. HIV was detected and quantified by limiting dilution PCR and genetically characterized in the V3 region of env. Limiting dilution was shown to be essential for correct estimation of genetic partitioning between brain- and lymphoid-associated HIV populations. While no actively expressing HIV-infected cells were detected by immunohistochemistry, variable and generally extremely low levels of proviral DNA were detected in presymptomatic brain samples. V3 region sequences were frequently genetically distinct from lymphoid-associated HIV variants, with association index (AI) values similar to those observed in cases of HIVE. Infiltration of CD8 lymphocytes in the brain was strongly associated with expression of activation markers (MHCII; R = 0.619; P < 0.05), the presence of HIV-infected cells (proviral load; R = 0.608; P < 0.05) and genetic segregation of brain variants from populations in lymphoid tissue (AI value, R = -0.528; P approximately 0.05). CD8 lymphocytes may thus limit replication of HIV seeded into the brain in early stages of infection. Neurological complications in AIDS occur when this control breaks down, due to systemic immunosuppression from HIV that destroys CD8 lymphocyte function and/or through the evolution of more aggressive neuropathogenic variants.

The trans Golgi network is lost from cells infected with African swine fever virus.

The cellular secretory pathway is important during the assembly and envelopment of viruses and also controls the transport of host proteins, such as cytokines and major histocompatibility proteins, that function during the elimination of viruses by the immune system. African swine fever virus (ASFV) encodes at least 26 proteins with stretches of hydrophobic amino acids suggesting entry into the secretory pathway (R. J. Yanez, J. M. Rodriguez, M. L. Nogal, L. Yuste, C. Enriquez, J. F. Rodriguez, and E. Vinuela, Virology 208:249-278, 1995). To predict how and where these potential membrane proteins function, we have studied the integrity of the secretory pathway in cells infected with ASFV. Remarkably, ASFV caused complete loss of immunofluorescence signal for the trans Golgi network (TGN) marker protein TGN46 and dispersed the AP1 TGN adapter complex. Loss of TGN46 signal was not due to degradation of TGN46, suggesting redistribution of TGN46 to other membrane compartments. ASFV markedly slowed transport of cathepsin D to lysosomes, demonstrating that loss of TGN structure correlated with loss of TGN function. ASFV shows a tropism for macrophages, and it is possible that ASFV compromises TGN function to augment the activity of viral membrane proteins or to suppress the function of host immunoregulatory proteins.

Demonstration of polysaccharide capsule in Campylobacter jejuni using electron microscopy.

Recently, we reported that Campylobacter jejuni, an important gastrointestinal pathogen, has the genetic determinants to produce a capsular polysaccharide (Karlyshev et al., Mol. Microbiol. 35:529-541, 2000). Despite these data, the presence of a capsule in these bacteria has remained controversial. In this study we stain C. jejuni cells with the cationic dye Alcian blue and demonstrate for the first time by electron microscopy that C. jejuni cells produce a polysaccharide capsule that is retained in the coccoid form but is absent in a kpsM mutant.

Schistosoma mansoni phosphofructokinase: immunolocalization in the tegument and immunogenicity.

Schistosoma mansoni depends for its survival on glycolysis. Two glycolytic enzymes, glyceraldehyde-3P-dehydrogenase and triose-phosphate dehydrogenase, found in both the adult and schistosomular tegument, have been reported to confer partial protection against cercarial infection. This paper describes the immunogenic properties of phosphofructokinase (PFK), a rate-limiting enzyme of glycolysis, and its localization in the tegument and adjacent tissues. Recombinant schistosome PFK was used as antigen. A polyclonal antibody against purified PFK from Fasciola hepatica was affinity purified using recombinant PFK and used in combination with immunogold labelling to identify PFK by transmission electron microscopy in cryosections. In both adult worms and in schistosomula most immunogold label localized in the cytoplasmic syncytial region with less being found in the tegument. There was no significant PFK localization within or external to the outer membrane. Sera from mice immunized with recombinant S. mansoni PFK with Freund's adjuvant or alum plus rIL-12 demonstrated high titres of anti-PFK IgG, but no protection against cercarial infection. Sera from mice that were acutely or chronically infected or multiply exposed to irradiated cercariae did not recognize recombinant schistosome PFK in either Western blotting or ELISA. Similarly, sera from humans infected with S. mansoni did not recognize PFK. We conclude that in spite of the high immunogenicity of rPFK in mice, it is not a significant immunogen during the course of infection and does not confer protection from schistosomiasis. One main difference between PFK and the other 2 glycolytic enzymes seems to be the inaccessibility of PFK to the outside surface of the tegument.

Immunogenicity of plasmid DNA encoding the 62 kDa fragment of Schistosoma japonicum myosin.

The recombinant Schistosoma mansoni 62 kDa myosin fragment, rIrV-5, is highly protective in experimental animals, however, vaccination of mice and rats with the recombinant Schistosoma japonicum homologue, rSj62, did not induce significant resistance against S. japonicum infection. To explore alternative ways of presenting this antigen, we further constructed a plasmid (VRSj62) which encodes Sj62 using the VR1020 vector and tested it in vaccination experiments. Four immunisations with 10 microg VRSj62 DNA alone were sufficient to induce high and progressively increasing levels of IgG antibodies against rSj62 with increasing numbers of injections in CBA/Ca mice (IgG titre > or =1:25000), and three injections with 50 microg VRSj62 DNA alone induced significant IgG responses in C57Bl/6 mice (IgG titre, 1:1600). However, vaccination with plasmid DNA entrapped in cationic liposomes or together with pUC19 DNA as a source of CpG motifs, both of which have been reported to enhance immune responses, did not enhance specific antibody production. In spite of the stimulation of specific antibodies against rSj62 with the naked DNA construct no resistance to challenge was demonstrated.

Molecular cloning and characterization of a novel Schistosoma japonicum "irradiated vaccine-specific" antigen, Sj14-3-3.

A Schistosoma japonicum cDNA coding for a full length S. japonicum 14-3-3 protein was obtained by antibody screening of an adult worm cDNA library using sera taken from mice vaccinated with UV-attenuated cercariae, which are capable of transferring high levels of passive immunity to this parasite. The deduced amino acid sequence consists of 254 amino acids and is highly homologous with 14-3-3 family of proteins from a variety of species (55-69% identity). The recombinant S. japonicun 14-3-3 protein (rSj14-3-3) was expressed and purified in pGEX/E. coli, and in Western blotting was strongly recognised by sera from mice, rats and bovines vaccinated with irradiated S. japonicum cercariae. Analysis of mRNA showed that Sj14-3-3 is expressed in sporocysts and adult worms, but not in cercariae, however mouse antisera against rSj14-3-3 recognised a 29 kDa native antigen in antigen preparations made from eggs, cercariae, schistosomula and adult worms of S. japonicum indicating that this antigen is present in all life-cycle stages. The presence of the native antigen in detergent extracts of intact schistosomula suggests that it is also present in the schistosomular tegument which is the most vulnerable target for immune attack. However, antisera against rSj14-3-3 did not recognise a similar band in S. mansoni or S. haematobium antigens, indicating that, like the UV-attenuated vaccines, this protein induced species-specific immune responses. Southern blot analysis suggested that there may exist more than one gene copy and/or polymorphism for Sj14-3-3. Immunoelectron microscopy confirmed that the native antigen is present throughout the body of adult worms including the tegument, but is less abundant in the muscles. The potential of rSj14-3-3 as a vaccine is now under further investigation.

An ultrastructural study of the phagocytosis of Burkholderia pseudomallei.

Burkholderia pseudomallei causes melioidosis and is believed to be an intracellular pathogen in human and animal disease. The uptake of B. pseudomallei by mouse peritoneal macrophages and cells in tissue culture was examined by electron microscopy. In all the systems studied B. pseudomallei were phagocytosed and apparently inhibited the normal processes of intracellular killing. Destruction of the phagosome membrane occurred and the bacteria escaped into the cytoplasm.

Cytostatic and cytotoxic effects of activated macrophages and nitric oxide donors on Brugia malayi.

The susceptibility of Brugia malayi microfilariae and adults to injury by the murine macrophage cell line J774 activated with gamma interferon and bacterial lipopolysaccharide has been examined in vitro. Parasites of both stages showed a decline in viability over 48 h of coculture with activated macrophages, assessed by their capacity to reduce the tetrazolium salt 3-[4,5-diethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), although adult parasites were more resistant than microfilariae. Removal of parasites to cell-free medium following exposure to activated macrophages for up to 48 h resulted in partial recovery of their capacity to reduce MTT, suggesting that the effects were primarily cytostatic. However, prolonged exposure to activated J774 cells for 72 h resulted in parasite death. Addition of the nitric oxide synthase inhibitor L-NMMA (N(G)-monomethyl-L-arginine monoacetate) indicated that nitric oxide derivatives were responsible for cytostasis and ultimate toxicity. The toxicity of nitric oxide derivatives was confirmed by coincubation of parasites with chemical donors, although far higher concentrations were required than those generated by activated J774 cells, implying additional complexity in macrophage-mediated cytotoxicity. These experiments further suggested that peroxynitrite or its by-products were more potently damaging to filariae than nitric oxide per se. Examination of ultrastructural changes on exposure of parasites to activated macrophages or donors of nitric oxide indicated that hypodermal mitochondria were highly vacuolated, with less prominent cristae. The data are discussed with reference to immunity to lymphatic filariae and their mechanisms of energy generation.

The gene B protein localises to the surface of Leishmania major parasites in the absence of metacyclic stage lipophosphoglycan.

The stage specific Gene B protein (GBP) of Leishmania major localises to the surface of infective metacyclic parasites, where it associates with the developmentally regulated surface glycoconjugate, lipophosphoglycan (LPG). This association has been proposed to aid maintenance of GBP on the parasite surface. In this paper, we show that the abundance of GBP on the extracellular metacyclic cell surface is in the order of 100,000 copies per cell. This level of expression is comparable to that seen in the intracellular amastigote stage, in which GBP is also localised to the surface, despite the lack of metacyclic stage specific LPG. Furthermore GBP expressed from an episome in avirulent parasites, which are unable to synthesise metacyclic LPG or endogenous GBP, also localises to the parasite surface. These data demonstrate that GBP can maintain a surface localisation in the absence of metacyclic LPG, suggesting that it is able to associate with other glycoconjugates on the surface of infective parasites.

Brugia malayi: localisation and differential expression of extracellular and cytoplasmic CuZn superoxide dismutases in adults and microfilariae.

We have determined the levels of superoxide dismutase (SOD) in different stages of the lymphatic filarial nematode parasite of man, Brugia malayi. Adult male worm extracts showed the highest levels of enzyme activity at 34.5 U mg-1, and there was no significant difference in the overall levels of SOD in extracts of adult female worms and microfilariae (27.1 and 26.7 U mg-1, respectively). SOD activity was detected in the culture medium of parasites maintained in vitro, with particularly high levels of specific activity in media in which males and females were maintained (357 and 339 U mg-1, respectively), indicative of active secretion. In all cases, this was accounted for predominantly by CuZn SOD, assessed by potassium cyanide inhibition. Northern blots with cDNA probes specific for cytoplasmic and extracellular CuZn SODs indicated that levels of mRNA for the cytoplasmic form were similar between adults and microfilariae, whereas expression of the extracellular form was 10x higher in adult worms. Western blots with an antibody to recombinant CuZn SOD demonstrated that higher levels of the extracellular protein were present in adult male worms, whereas the cytoplasmic form was present in roughly equivalent amounts in males, females, and microfilaria. Iodination and immunoprecipitation experiments indicated that the extracellular enzyme was accessible to surface labeling of both male and female adult worms, but not microfilaria. Immuno-electron microscopy showed that CuZn SOD was localised predominantly in the hypodermis of adult parasites, with an asymmetric distribution in the intercordal regions suggestive of compartmentalisation into several distinct syncytia. No labeling was evident in the cuticle, and thus the accessibility of the extracellular enzyme to extrinsic iodination in adult worms remains unclear. No binding of antibody was demonstrable in the glandular region of the oesophagus or the uterus of females, presumed to be major sites of synthesis for secreted proteins. Dense labeling was observed in the seminal fluid surrounding spermatazoa in the vas deferens of male parasites. These data also suggest that, as observed in mammals, nematode spermatazoa are particularly susceptible to oxidative damage and are protected during storage by secreted anti-oxidant enzymes.

Localization of parasite antigens in Cryptosporidium parvum-infected epithelial cells using monoclonal antibodies.

An immunogold ultrastructural study was made of Cryptosporidium parvum-infected intestinal cells from SCID mice to locate parasite antigens recognized by monoclonal antibodies raised against sporozoite or oocyst wall antigens. The results suggested that these antigens were present in more than one life-cycle stage and demonstrated that the intracellular parasite modified the parasitophorous vacuole membrane and villous membrane surrounding the parasite. In an immunofluorescence antibody test monoclonal antibody (MAb) 1B5 reacted with the oocyst wall, MAb 2C3 with the whole sporozoite and MAb 2B2 with the sporozoite surface. Western and dot-blot studies demonstrated that different carbohydrate epitopes were recognized by the respective sporozoite-reactive antibodies. In the ultrastructural examination MAb 1B5 reacted with macro- and microgametocytes as well as the oocyst wall. In the macrogametocyte MAb 1B5 recognized the large electron-dense bodies characteristic of this stage and, in some parasites, the parasitophorous vacuole and the parasite pellicle. The sporozoite-reactive MAbs were able to bind to all developmental stages. These antibodies recognized the parasite cytoplasm and, additionally, MAb 2B2 produced substantial labelling of the parasite membrane. Significantly, both these antibodies also detected antigen in the parasitophorous vacuole membrane and, to a lesser extent, the villous membrane surrounding the parasite.

Subcellular localization of Pfs16, a Plasmodium falciparum gametocyte antigen.

We have used immunoelectron microscopy to investigate the subcellular location of Pfs16 in Plasmodium falciparum. It was detected in the outer membrane region of gametocytes and more specifically on the parasitophorous vacuole membrane (pvm), since, during gametogenesis when the pvm disintegrates, the majority of the antigen was detected on the remains of this membrane in multilaminated whorls and not on the gamete plasma membrane. The antigen was also present on other gametocyte cellular structures, including those which we believe to be Garnham bodies, present in the host cell cytoplasm of some gametocytes. The antigen was present too on the membrane surrounding cytosomes and the resulting food vacuoles in the parasite cytoplasm.

Antigens targeted to the Leishmania phagolysosome are processed for CD4+ T cell recognition.

Processing of antigen for recognition by class II-restricted CD4+ T cells occurs within acidic compartments of the antigen-presenting cell. The exact nature of this compartment has yet to be precisely defined, however, but may vary depending upon the cell type studied and the antigen used. The acidic compartments of macrophages are also responsible for the degradation of ingested micro-organisms and play host to others which are adapted to an intracellular existence. To determine whether the phagolysosome (PL) formed in activated macrophages after ingestion of Leishmania parasites is also a site for entry of antigen into the class II presentation pathway, we have used the approach of genetic transformation. Hence, Leishmania were transfected with the genes for the protein antigens ovalbumin (OVA) and beta-galactosidase (beta-gal) and after infection were able to deliver these antigens specifically into the PL. Delivery of antigen to this site resulted in the ability of infected macrophages to present these antigens to antigen-specific CD4+ T cells. After taking into account the absolute levels of antigen uptake by macrophages, a 4-h processing period for OVA delivered by this or a soluble route led to equivalent levels of T cell activation. Unlike macrophages pulsed with soluble OVA, those with PL-targeted OVA still retained the ability to stimulate T cells after a 24-h processing period. This enhanced lifespan of antigen in macrophages corresponded to the kinetics of degradation of the parasite, suggesting slow release of antigen into the processing pathway. beta-gal presentation from the PL was tenfold less efficient under the same conditions. In addition to providing the first information on antigen processing in a protozoan PL, these studies highlight the usefulness of genetically transformed parasites for these types of studies.

Expression of a mitochondrial stress protein in the protozoan parasite Leishmania major.

The DNA sequence has been determined of a gene from Leishmania major that shares sequence identity with members of the eukaryotic heat shock protein (hsp) 70 gene family. The deduced open reading frame for translation shares a number of features common to hsp70 stress proteins, including conserved amino acids implicated in ATP binding and a putative calmodulin-binding site. In addition, the protein has an N-terminal sequence characteristic of a mitochondrial targeting signal. Specific antibodies to this protein, generated by the use of recombinant fusion peptides, recognise a 65 kDa molecule of pI 6.7. This molecule is constitutively expressed and localises to the mitochondrion in all stages of the parasite life cycle. These features suggest a role for this protein as a molecular chaperone in Leishmania.

The case for perfusion fixation of large tissue samples for ultrastructural pathology.

We present the case for the perfusion fixation of large, freshly isolated tissue samples of liver and lung from dog, rat, and mouse. Individual lobes of liver and lung were fixed by vascular perfusion using a technique that is simple and quick to perform and results in a reproducibly high standard of ultrastructural preservation compared to immersion fixation. A major advantage to the use of this technique lies in the ability to provide tissue samples for diagnostic ultrastructural pathology and toxicological pathology while avoiding the need for whole-organ or whole-body perfusion fixation. This advantage therefore permits the use of fresh tissue from the same organ for other investigative purposes (e.g., drug metabolism studies and pharmacokinetics), thereby allowing correlation of structure and function. The advantages of perfusion fixation compared to immersion fixation are discussed, and potential applications for tissue preparation of postmortem specimens and also for scanning electron microscopy are indicated.

Gastric neuroendocrine cell hyperplasia after treatment with the long-acting, potent H2-receptor antagonist SK&F 93479.

The time course and dose response of the neuroendocrine cell hyperplasia in the oxyntic mucosa of the rat was examined after treatment with the potent, long-acting H2-receptor antagonist SK&F 93479 at doses of 0 and 1000 mg/kg orally for 1, 3, 7, and 14 days and at doses of 0, 40, 200, and 1000 mg/kg orally for 1 and 6 months. The number of oxyntic neuroendocrine cells (chromogranin-positive) increased after 7 days of treatment. In the 1- and 6-month studies with doses of 1000 mg/kg, the grading for the number of oxyntic chromogranin-positive cells was 2.5 to 3 times the control levels, and they were distributed mostly throughout the mucosa, whereas at lower doses, which did not produce carcinoid tumours at 2 years, the neuroendocrine cells were distributed in the lower half of the mucosa with 1.5- to 2-fold increases in grades for cell numbers. Increases in cell numbers and cell distribution may be useful factors in the evaluation of the neuroendocrine cell hyperplasia found in, for example, the Zollinger-Ellison syndrome and chronic atrophic gastritis, in which hypergastrinaemia and fundic neuroendocrine cell hyperplasia are present.

Study of foveal tritanopia.

This paper discusses the possibility there is a difference between men and women in foveal tritanopia. The discussion is based on a study carried out by Cobb and McCrossan in 1973 in which they measured the luminosity curves of the fovea in five women and five men. The instrument used was a Wright colorimeter which measured the luminosity curves with a 2 degree 12' field and a 0 degree 12.5' field. Comparison shows a loss of sensitivity to blue for the curve obtained with the 0 degrees 12.5 relative to the curve obtained with the 2 degree 12' field. Male subjects obtained two maxima with the 0 degree 12.5' field, usually at 555 nm and 595 nm, whereas for females on maximum, usually at 555 nm and from 555 nm to the long wavelength end of the spectrum, curves followed loosely the curve obtained with the 2 degree 12' field. Thus, a significant difference was found between the males and the females in their response to the longer wave-lengths when the 0 degree 12.5' (0 degree 12.5') field was used. In addition to this there were also large individual differences in the matching points obtained by the males while the differences among the females were much smaller.