Biomedical subjects

M Aikawa

Publications and source records attributed to M Aikawa.

At least 181 records · Page 10Linked to original sources

PubMed1992

Localization of a 230-kD parasitophorous vacuole membrane antigen of Plasmodium berghei exoerythrocytic schizonts (LSA-2) by immunoelectron and confocal laser scanning microscopy.

Using antiserum to a 230-kD parasitophorous vacuole membrane (PVM) antigen of Plasmodium berghei exoerythrocytic schizonts as a specific probe for the PVM, we studied the three-dimensional structure of this membrane within infected host cells by immunoelectron microscopy and confocal laser scanning microscopy at 3, 4, and 50 hr after sporozoite invasion. Fluorescent label was not detected at 3 hr, but was associated with the cytoplasm of 24-hr-old exoerythrocytic parasites. Specific labeling of the PVM was not observed by immunoelectron microscopy until 50 hr, when numerous vesicles and finger-like projections of the PVM were found in the cytoplasm of infected host cells. Labeled vesicles were often isolated and located at the periphery of the infected hepatocyte. Confocal microscopy demonstrated that these vesicles formed discontinuous chains that extended from 3-10 microns away from the parasite. These structures appear to be similar to the membranous clefts of Plasmodium-infected erythrocytes, and may be important in the movement of host or parasite proteins within infected hepatocytes.

Animals↗

PubMed1992

Pathology of falciparum malaria in Vietnam.

Autopsy samples from the brains of 20 patients who died of falciparum malaria were examined by light microscopy and by an immunohistologic method. Particular attention was paid to a comparison of the pathologic features of the white matter and the cortex. In the high-sequestration (greater than 50%) group (n = 8), the mean +/- SD percentage of cerebral microvessels that showed parasitized red blood cell (PRBC) sequestration was 71.2 +/- 8.1% in the cortex and 84.0 +/- 6.7% in the white matter. The difference in the PRBC sequestration rate between cortex and white matter was statistically significant (P less than 0.01). Perivascular and ring hemorrhages were seen more frequently in the white matter than in the cortex. Deposition of IgG and Plasmodium falciparum antigen in the cerebral microvessels was more highly significant in the white matter than in the cortex (P less than 0.01). Our study demonstrated that the localized concentration of PRBC sequestration in the brain correlated with the marked immunohistologic differences in the microvessels of cortex and white matter.

Adult↗

PubMed1992

Renal pathology in owl monkeys vaccinated with Plasmodium falciparum asexual blood-stage synthetic peptide antigens.

Renal specimens from Aotus monkeys were studied by light microscopy and immunohistochemistry to examine pathologic changes following vaccination with synthetic peptides corresponding to the 35-kD, 55-kD, and 83-kD asexual blood stage antigens of Plasmodium falciparum. The monkeys were vaccinated and later challenged with P. falciparum. In the monkeys vaccinated with Centers for Disease Control peptides (group I), specimens from four of six postvaccinated animals had mild to severe mesangial proliferation and two had diffuse interstitial nephritis. Specimens from three monkeys vaccinated with Colombia peptides (group II) had mild to severe mesangial proliferation and one had interstitial nephritis. In the hybrid polymer-vaccinated monkeys (group III), specimens from three animals had mild to moderate mesangial proliferation and one had severe interstitial nephritis. On the other hand, the control group immunized with bovine serum albumin (group IV) showed that specimens from three animals had mild to severe mesangial proliferation and two had severe interstitial nephritis. In the nonimmunized group (group V), specimens from three animals had moderate to severe mesangial proliferation and two had severe and mild interstitial nephritis. Immunohistochemical analysis using the peroxidase-antiperoxidase method revealed mesangial deposits of P. falciparum antigens in 11 of 14 vaccinated monkeys and in five of 10 unvaccinated controls. These results show that treatment of monkeys with prospective malaria vaccines does not increase the frequency of occurrence or of the severity of renal lesions. These data thus provide a baseline for assessing the safety of synthetic malarial vaccines in the future.

Animals↗

PubMed1992

Placental pathology in Plasmodium berghei-infected rats.

The pathologic changes in placentae of pregnant rats infected with Plasmodium berghei at different stages of gestation were studied using light and electron microscopy and immunohistochemistry. The major changes observed were thickening and duplication of the trophoblastic basement membrane, and accumulation of parasitized erythrocytes and occasional mononuclear cells in the maternal blood space. Immunohistochemical examination of nine placentae revealed that six stained positively for IgG, two for IgM, and four for P. berghei antigen. No C3 deposition was detected. The findings in this study indicate that the variable parasitologic-clinical course from benign to fatal of P. berghei infection in pregnant rats makes it a potentially valuable model of human gestational malaria infection.

Animals↗

PubMed1991

Phagocytosis of liposomes by macrophages: intracellular fate of liposomal malaria antigen.

Liposomes containing a synthetic recombinant protein were phagocytosed by macrophages, and the internalized protein was recycled to the cell surfaces where it was detected by enzyme-linked immunosorbent assay. The transit time of the liposome-encapsulated protein from initial phagocytosis of liposomes to appearance of protein on the surfaces of macrophages was determined by pulse-chase experiments. The macrophages were pulsed with liposomes containing protein and chased with empty liposomes, and vice versa. The amount and rate of protein antigen expression at the cell surfaces depended on the quantity of encapsulated protein ingested by the macrophages. Although liposomes were rapidly taken up by macrophages, the liposome-encapsulated protein was antigenically expressed for a prolonged period (at least 24 h) on the cell surface. Liposomes were visualized inside vacuoles in the macrophages by immunogold electron microscopy. The liposomes accumulated along the peripheries of the vacuoles and many of them apparently remained intact for a long time (greater than 6 h). However, nonliposomal free protein was also detected in the cytoplasm surrounding these vacuoles, and it was concluded that the free protein in the cytoplasm was probably en route to the macrophage surface. Exposure of the cells to ammonium chloride did not inhibit the appearance of liposomal antigenic epitopes on the cell surface, and this suggests that expression of the liposomal antigenic epitopes at the surface was not a pH-sensitive phenomenon. There was no significant effect of a liposomal adjuvant, lipid A, on the rate or extent of surface expression of the liposomal protein.

Amino Acid Sequence↗

PubMed1991

Characterization and localization of Plasmodium falciparum surface antigens on infected erythrocytes from west African patients.

The malaria-induced surface antigens on Plasmodium falciparum-infected erythrocytes from West African patients were characterized by agglutination of infected cells by human sera, surface immunofluorescence of live infected cells, inhibition of cytoadherence to C32 melanoma cells by human sera, immunoelectron microscopy (immunoEM), and immunoprecipitation. In a nonimmune individual, serum antibody reactivity to surface antigens of infected cells was acquired during convalescence, as tested by all five methods, and was generally parasite isolate-specific. By contrast, adult hyperimmune West African sera reacted with many isolates, including isolates from geographically distinct regions. A quantitative correlation was established between agglutination and surface immunofluorescence assay titers, and between surface immunofluorescence assay and immunoEM reactivity, suggesting that a single antigen or a set of coexpressed antigens is being detected. Surface iodination of infected cells identified trypsin-sensitive high M, antigens in the sodium dodecyl sulfate extract. All sera tested that agglutinated infected cells also immunoprecipitated these antigens. The same surface antigens were immunoprecipitated by the homologous convalescent serum as by adult sera. By immunoEM these antigens were localized exclusively at the knob-like protrusions of infected cells, where they may participate in adherence to vascular endothelium.

Africa, Western↗

PubMed1991

Modification of host cell membrane lipid composition by the intra-erythrocytic human malaria parasite Plasmodium falciparum.

The phospholipid and fatty acid compositions of the host infected erythrocyte plasma membrane (IEPM) have been determined for erythrocytes infected with the human malaria parasite Plasmodium falciparum. IEPM were prepared by selective lysis of the host erythrocyte (but not of the parasite membranes) with 0.1% saponin, followed by differential centrifugation. The purity of the IEPM was determined by measuring the membrane-specific enzyme markers acetylcholinesterase, glutamate dehydrogenase and lactate dehydrogenase, and by immunoelectron microscopy using monoclonal antibodies specific for human erythrocyte glycophorin A (4E7) and for a 195 kDa parasite membrane glycoprotein (Pf6 3B10.1). Both approaches demonstrated that the host erythrocyte plasma membrane preparation was free from contamination by parasite membranes. During intra-erythrocytic development of the parasite, the phospholipid composition of the erythrocyte membrane was strikingly altered. IEPM contained more phosphatidylcholine (38.7% versus 31.7%) and phosphatidylinositol (2.1% versus 0.8%) and less sphingomyelin (14.6% versus 28.0%) than normal uninfected erythrocytes. Similar alterations in phospholipid composition were determined for erythrocyte membranes of parasitized cells isolated by an alternative method utilizing polycationic polyacrylamide microbeads (Affigel 731). The total fatty acid compositions of the major phospholipids in IEPM were determined by g.l.c. The percentage of polyunsaturated fatty acids in normal erythrocyte phospholipids (39.4%) was much higher than in phospholipids from purified parasites (23.3%) or IEPM (24.0%). The unsaturation index of phospholipids in IEPM was considerably lower than in uninfected erythrocytes (107.5 versus 161.0) and was very similar to that in purified parasites (107.5 versus 98.5). Large increases in palmitic acid (C16:0) (from 21.88% to 31.21%) and in oleic acid (C18:1) (from 14.64% to 24.60%), and major decreases in arachidonic acid (C20:4) (from 17.36% to 7.85%) and in docosahexaenoic acid (C22:6) (from 4.34% to 1.8%) occurred as a result of infection. The fatty acid profiles of individual phospholipid classes from IEPM resembled in many instances the fatty acid profiles of parasite phospholipids rather than those of uninfected erythrocytes. Analysis of IEPM from P. falciparum-infected erythrocytes (trophozoite stage) revealed that, during intra-erythrocytic maturation of the parasite, the host erythrocyte phospholipid composition was markedly refashioned. These alterations were not dependent on the method used to isolate the IEPM, with similar results obtained using either a saponin-lysis method or binding to Affigel beads. Since mature erythrocytes have negligible lipid synthesis and metabolism, these alterations must occur as a result of parasite-directed metabolism of erythrocyte lipids and/or trafficking of lipids between the parasite and erythrocyte membranes.

Acetylcholinesterase↗

PubMed1991

Plasmodium cynomolgi: immunization of a rhesus monkey with exoerythrocytic stages cultured in autologous hepatocytes.

To investigate the immune response to exoerythrocytic stages of malaria parasites, a rhesus monkey was immunized with autologous primary hepatocyte cultures infected with 7-day-old liver stage parasites of Plasmodium cynomolgi. A primary antibody response against EE stage antigens was obtained, and boosted after injection of homologous viable sporozoites. Antibodies directed against sporozoites and blood stages were also detected. The polyvalent immune response observed demonstrates the antigenicity of the liver stages and suggests their involvement in the general immune response against malaria.

Animals↗

PubMed1991

The peritrophic membrane as a barrier: its penetration by Plasmodium gallinaceum and the effect of a monoclonal antibody to ookinetes.

We studied the point at which a monoclonal antibody (mAb C5) to a surface protein (Pgs25) on Plasmodium gallinaceum ookinetes blocked the infection of Aedes aegypti mosquitoes. The antibody did not block the development of zygotes to ookinetes in vitro. Development of ookinetes to oocysts in the mosquito was blocked to the same extent whether zygotes grew to ookinetes in the presence of mAb C5 or the antibody was added after the ookinetes had reached full development. When ookinetes developed in vitro in the presence of mAb C5, antibody remained on the surface of the parasite for the next 50 hr and did not block attachment to the peritrophic membrane. When ookinetes were fed to mosquitoes, two subpopulations of mosquitoes were observed (high numbers of oocysts per midgut and low numbers of oocysts per midgut). mAb C5 reduced the number of oocysts per midgut in the subpopulation that had low numbers of oocysts. The subpopulation that had high numbers of oocysts was unaffected by antibody, indicating that the antibody did not block invasion of the midgut epithelium. When mAb C5 was fed with gametocytes, the parasites invaded the epithelium at the same time (between 30 and 35 hr after the blood meal) as in controls, although at a markedly reduced rate. The ultrastructural observations were consistent with a block of parasites within the peritrophic membrane and not with a block at the epithelium, as parasites were not seen to accumulate within the space between the peritrophic membrane and the epithelium. The mechanism by which mAb C5 to Pgs25 of P. gallinaceum blocks the penetration of the peritrophic membrane remains undefined. We present evidence that the parasite modifies the peritrophic membrane during penetration, an observation first made for Babesia microti during penetration of the peritrophic membrane in Ixodes ticks. Ookinetes in the absence of antibodies appeared to disrupt the layers of the peritrophic membrane, suggesting an enzymatic mechanism for penetration.

Aedes↗

PubMed1991

Selective association of a fragment of the knob protein with spectrin, actin and the red cell membrane.

The knob protein of Plasmodium falciparum is essential for the formation of knob-like protrusions on the host erythrocyte membrane. A functional domain of the knob protein was identified. This peptide formed stable complexes with the two major red cell skeletal proteins, spectrin and actin. When introduced into resealed normal erythrocytes, the peptide associated selectively with the cytoplasmic surface of the membrane and formed knob-like electron dense deposits. Knobs are thought to play an important role in the immunopathology of P. falciparum infections. Our findings provide a first step towards understanding the molecular basis for selective membrane changes at knobs.

Actins↗

PubMed1991

Primary structure and localization of a conserved immunogenic Plasmodium falciparum glutamate rich protein (GLURP) expressed in both the preerythrocytic and erythrocytic stages of the vertebrate life cycle.

A gene coding for a 220-kDa glutamate rich protein (GLURP), an exoantigen of Plasmodium falciparum, was isolated and its nucleotide sequence was determined. The deduced amino acid sequence contains 2 repeat regions. The sequence of one of these was shown to be conserved among geographically dispersed isolates, and a fusion protein containing that sequence was able to stimulate B- and T-cells. Antibodies against GLURP stained erythrocytic stages of the parasite as well as the hepatic stage as detected by electron microscopy.

Amino Acid Sequence↗

PubMed1991

Induction and localization of Plasmodium falciparum stress proteins related to the heat shock protein 70 family.

Induction of heat shock-related stress proteins Pfhsp and Pfgrp, similar in sequence to hsp70 (heat shock protein) and grp78 (glucose-regulated protein), respectively, was studied in culture-derived parasite Plasmodium falciparum. Elevation in temperature from 26 degrees C to 37 degrees C and higher caused significant induction of Pfhsp with a moderate effect on the synthesis of Pfgrp also. Synthesis of Pfgrp, however, was not induced by partial glucose deprivation. On the contrary, lack of glucose in the medium resulted in cessation of protein synthesis in the parasites. Other known inducers of grp synthesis in mammalian cells, i.e., calcium ionophore A23187 and inhibitors of glycosylation (tunicamycin, 2-deoxy glucose) were also without any apparent effect on the synthesis of Pfgrp. Heat shock-induced responses were transient in nature: removal of stress caused repression of these responses. The effect of glucose deprivation was only partially reversible with better recovery if parasites were subjected to glucose starvation at 26 degrees C than at 37 degrees C. Northern blot analysis and in vitro translation of mRNA revealed a parallel increase in the levels of mRNA for Pfhsp upon heat shock. Immuno-gold electron microscopy with cultured parasites revealed nuclear location of Pfhsp and primarily cytoplasmic (probably endoplasmic reticulum) location of Pfgrp. These findings suggest that SDEL (carboxy terminal sequence of Pfgrp) might play a similar role in the cellular localization of Pfgrp as does the sequence KDEL in mammalian cells and HDEL in yeast.

Amino Acid Sequence↗

PubMed1991

In vitro effects of berberine sulphate on the growth and structure of Entamoeba histolytica, Giardia lamblia and Trichomonas vaginalis.

The plant alkaloid, berberine sulphate, inhibited the growth of Entamoeba histolytica, Giardia lamblia and Trichomonas vaginalis in BI-S-33 medium, and induced morphological changes in the parasites. Exposure of E. histolytica to berberine caused a clumping of chromatin in the nucleus, and the formation of autophagic vacuoles and aggregates of small vacuoles in the cytoplasm. In berberine-treated G. lamblia, an irregularly-shaped vacuole appeared in the cytoplasm and gradually enlarged during culture. The trophozoites became swollen and deposits of glycogen were seen in the cytoplasm. Trichomonas vaginalis was also affected by the berberine; autophagic vacuoles increased in number soon after exposure, and one large vacuole, which was characteristic of treated cells, appeared. These observations demonstrate that, in vitro, this drug was effective against E. histolytica, G. lamblia and T. vaginalis. Another alkaloid, coptisine, was inactive against the three parasites.

Animals↗

PubMed1991

Asexual development of Cryptosporidium parvum within a differentiated human enterocyte cell line.

Unremitting diarrhea with malabsorption is associated with Cryptosporidium parvum infection of the small intestine in patients with AIDS. The lack of a well-defined in vitro model of C. parvum infection has severely hampered research into the biology of cryptosporidial invasion of the host epithelial cell and development of new pharmacologic and immunologic therapies. The adherent human intestinal epithelial cell line HT29 when grown in glucose-free medium develops morphologic and functional characteristics of the small intestine enterocyte and was used to develop an in vitro model of infection. Cryptosporidium oocysts obtained from AIDS patients were applied to a monolayer of cloned, differentiated HT29.74 cells. Cells were fixed and stained to estimate the degree of parasite infection. Schizonts were easily distinguished from the host cell by light microscopy. Twenty-four hours after 10(5) oocysts were added to approximately 10(6) HT29.74 cells, Cryptosporidium infection rates varied from 50 to 120 schizonts per 1,000 cells. Among 14 different experiments, the mean infection rate was 91 (+/- 18) schizonts per 1,000 cells. Electron microscopy at 6 and 24 h confirmed intracellular localization and development of schizonts. The morphologic features of the cryptosporidial schizonts within HT29.74 cells, which included the presence of a dense band and feeder layer, were identical to those described during cryptosporidial infection of human enterocytes in patients with AIDS. Fewer schizonts were observed at 5 days and beyond. Infection of differentiated HT29.74 cells (62 and 65 schizonts per 1,000 cells at 24 and 72 h, respectively) was over five times more efficient than infection of undifferentiated HT29.74 cells (9 and 5 schizonts per 1,000 cells at 24 and 72 h, respectively). In vitro infection of differentiated HT29.74 cells will allow a better understanding of the mechanisms by which C. parvum infects the small intestinal epithelium and will allow a systematic evaluation of new therapeutic agents.

Animals↗

PubMed1991

Microvascular sequestration of parasitized erythrocytes in human falciparum malaria: a pathological study.

Thirty-nine falciparum malaria autopsy cases from the Hospital for Tropical Diseases, Mahidol University, Bangkok, Thailand were divided into two groups that had had either cerebral malaria (CM) or non-cerebral malaria (NCM). We then studied significant pathological differences between these groups in order to investigate the correlation between parasitized erythrocyte (PRBC) sequestration in small blood vessels in the brain, heart, lungs and small intestines. We found that the percentage of PRBC sequestration in the organs which we studied was higher in the CM patients than in the NCM patients. The difference of PRBC sequestration among the organs of two groups was significant (P less than 0.05). In the CM group, the average percentage of PRBC sequestration in the brain was significantly higher than in the heart, lungs and small intestines (P less than 0.05). No statistically significant difference was found between PRBC sequestration in the brains, hearts, lungs and small intestines in the NCM group (P greater than 0.05). Our study indicates that severity of malaria in the CM patients depends on PRBC sequestration, especially in the brain. A combination of functional disturbances of the other organs, in addition to the cerebral pathology, may augment the severity of the disease.

Animals↗

PubMed1991

Renal pathology in Saimiri monkeys during a vaccine trial using the recombinant circumsporozoite protein of Plasmodium vivax.

Renal specimens of squirrel monkeys (Saimiri sciureus boliviensis) were studied by light microscopy and immunohistochemistry to examine the pathologic changes during vaccine trials with four recombinant circumsporozoite (CS) proteins (rPvCS-1, rPvCS-2, rPvCS-3, NS1(81) V20) of Plasmodium vivax. The monkeys were vaccinated and later challenged with P. vivax sporozoites. Among the 33 posttrial biopsies, 17 had mild to moderate mesangial proliferation and nine had interstitial nephritis. Immunohistochemistry by the peroxidase-antiperoxidase (PAP) method revealed IgG deposits in only three of 24 specimens and failed to demonstrate C3 deposits and P. vivax antigens in their glomeruli. There was no relationship between the severity of nephropathy and intensity of parasitemia. The intensity of parasitemia was the same in the vaccinated and control groups. Vaccinated monkeys from the groups (rPvCS-1, rPvCs-2, rPvCS-3) had no differences in renal pathology from the unvaccinated controls, but one group vaccinated with NS1(81) V20 did not develop renal changes.

Amino Acid Sequence↗

PubMed1991

Ultrastructural localization of a protective 68,000 molecular weight antigen in Schistosoma mansoni.

Vaccination with SMW 68, an Mr 68,000 glycoprotein of Schistosoma mansoni, induces significant protection in mice against challenge schistosome infection. This resistance occurs without the use of adjuvants and without sensitizing animals to granuloma formation. Likewise, passive transfer of monoclonal antibody (MAb) 31-3B6 against SMW 68 confers partial protection against challenge infection. As a first step in understanding how the immune response to this molecule leads to resistance, SMW 68 was localized in three developmental stages of the parasite by immunoelectron microscopy using MAb 31-3B6 and polyclonal antisera raised against purified SMW 68. In cercariae and schistosomula, MAb 31-3B6 bound electron-dense granules within the head gland and similar granules in the preacetabular glands. In adult worms, SMW 68 or related antigens were found to be widely distributed in tissues. Binding of specific antisera was most pronounced in the gut and tegument of male worms, but less so in subtegumental muscles. We conclude that SMW 68 is presented to the immune system in various ways during parasite development. The protective protein or epitope is excreted, and presented on the surface and in the cytoplasm at various stages of the life cycle. The relationship of the location of this protein to its role in protective immunity is discussed.

Animals↗

PubMed1991

Strain specificity in the liver-stage development of Plasmodium falciparum in primary cultures of new world monkey hepatocytes.

Primary cultures of Aotus and Saimiri monkey hepatocytes were infected with sporozoites of the Plasmodium falciparum NF 54 strain from mosquitoes fed on gametocyte cultures, and with sporozoites of the P. falciparum Santa Lucia strain from mosquitoes fed on an infected Aotus monkey. After 4-8 days, one exoerythrocytic (EE) parasite per 30,000 sporozoites was detected in one of three experiments performed with the P. falciparum NF54 strain. However, numerous EE parasites were detected in Aotus and Saimiri cells infected with sporozoites of the P. falciparum Santa Lucia strain. At day 6, most of the parasites contained several hundred nuclei, and were morphologically similar to those previously described in vivo using light or electron microscopy. A monoclonal antibody directed against the repeat region of the circumsporozoite protein of P. falciparum labeled the plasma and parasitophorous vacuole membrane of five-day-old EE parasites by immunoelectron microscopy, thus supporting previous observations by immunofluorescence indicating that the CS protein persists throughout the EE development of P. falciparum. These results demonstrate that liver stages of P. falciparum can be obtained in Aotus and Saimiri monkey cells, they also suggest a parasite strain specificity for hepatocytes.

Animals↗