Cytoadherence in human falciparum malaria as a cause of respiratory distress

The ultrastructure of three cases of fatal human falciparum malaria was studied in order to identify the cytoadherence of the endothelial cells in relation to parasitized red blood cells and septal interstitial changes which could be related to respiratory distress. Two cases showed marked endothelial oedema narrowing the capillary lumen with areas of adherence preferentially related to knobs, accompanied by septal interstitial oedema. One case showed no endothelial cells oedema, no knobs in parasitized red blood cells with no cytoadherence, no septal interstitial oedema and no respiratory distress. Cytoadherence seems to be the mechanism responsible for the septal pulmonary changes in severe falciparum malaria.     

A primate model for human cerebral malaria: Plasmodium coatneyi-infected rhesus monkeys.

A major factor in the pathogenesis of human cerebral malaria is blockage of cerebral microvessels by the sequestration of parasitized human red blood cells (PRBC). In vitro studies indicate that sequestration of PRBC in the microvessels is mediated by the attachment of knobs on PRBC to receptors on the endothelial cell surface such as CD36, thrombospondin (TSP), and intercellular adhesion molecule-1 (ICAM-1). However, it is difficult to test this theory in vivo because fresh human brain tissues from cerebral malarial autopsy cases are not easy to obtain. Although several animal models for human cerebral malaria have been proposed, none have shown pathologic findings that are similar to those seen in humans. In order to develop an animal model for human cerebral malaria, we studied brains of rhesus monkeys infected with the primate malaria parasite, Plasmodium coatneyi. Our study demonstrated PRBC sequestration and cytoadherence of knobs on PRBC to endothelial cells in the cerebral microvessels of these monkeys. Cerebral microvessels with sequestered PRBC were shown by immunohistochemical analysis to possess CD36, TSP, and ICAM-1. These proteins were not evident in the cerebral microvessels of uninfected control monkeys. Thus, our study indicates, for the first time, that rhesus monkeys infected with P. coatneyi can be used as a primate model to study human cerebral malaria. By using this animal model, we may be able to evaluate strategies for the development of vaccines to prevent human cerebral malaria.     

Ultrastructure of the bone marrow in HIV infection: evidence of dyshaemopoiesis and stromal cell damage.

The ultrastructure of bone marrow cells was studied in nine patients infected with the human immunodeficiency virus (HIV). Two of these (cases 1 and 3) were thrombocytopenic, had never suffered from opportunistic infections and had not received any drugs prior to the time of study. A number of ultrastructural abnormalities were found in a variable proportion of the affected cell types in all nine patients. These were: (a) an increased prevalence of multivesicular bodies within several cell types and of abnormalities of the nuclear membrane in neutrophil granulocytes, (b) an increase in the size of the Golgi apparatus and in the quantity of endoplasmic reticulum in neutrophil granulocytes, (c) dysplastic features, including multiple long intranuclear clefts and large cytoplasmic vacuoles in some erythroblasts and (d) vacuolation of the plasma cells. Other abnormalities seen in a proportion of the patients were: (a) cylindrical confronting cisternae (CCC) in some of the lymphocytes, macrophages (phagocytic reticular cells), non-phagocytic reticular cells (including adventitial cells) and endothelial cells of marrow sinusoids, (b) tubuloreticular structures (TRS) in some lymphocytes, plasma cells, monocytes and endothelial cells and (c) precipitates of protein within occasional erythroblasts and marrow reticulocytes. There was also a striking and hitherto undescribed abnormality of the structure of the nucleus in intersinusoidal and perisinusoidal non-phagocytic reticular cells. This was seen in six patients, including case 3, and was characterized by the extensive detachment of masses of abnormally electron-dense heterochromatin from the nuclear membrane, the presence of a uniformly thin layer of electron-dense material at the inner surface of the areas of nuclear membrane denuded of heterochromatin masses and an abnormal electron lucency of areas containing euchromatin. The CCC and TRS were found in the six patients with the lowest number of circulating CD4-positive T cells. The precipitation of protein within erythroid cells may have been caused by the oxidant effect of dapsone or high doses of co-trimoxazole. The abnormalities in the stromal cells and in particular the nuclear changes seen in the non-phagocytic reticular cells support the possibility that one of the mechanisms underlying the cytopenia in patients infected with HIV may be a disturbance of the microenvironmental regulation of haemopoiesis.     

Ultrastructure of the bone marrow in HIV infection: evidence of dyshaemopoiesis and stromal cell damage

The ultrastructure of bone marrow cells was studied in nine patients infected with the human immunodeficiency virus (HIV). Two of these (cases 1 and 3) were thrombocytopenic, had never suffered from opportunistic infections and had not received any drugs prior to the time of study. A number of ultrastructural abnormalities were found in a variable proportion of the affected cell types in all nine patients. These were: (a) an increased prevalence of multivesicular bodies within several cell types and of abnormalities of the nuclear membrane in neutrophil granulocytes, (b) an increase in the size of the Golgi apparatus and in the quantity of endoplasmic reticulum in neutrophil granulocytes, (c) dysplastic features, including multiple long intranuclear clefts and large cytoplasmic vacuoles in some erythroblasts and (d) vacuolation of the plasma cells. Other abnormalities seen in a proportion of the patients were: (a) cylindrical confronting cisternae (CCC) in some of the lymphocytes, macrophages (phagocytic reticular cells), non-phagocytic reticular cells (including adventitial cells) and endothelial cells of marrow sinusoids, (b) tubuloreticular structures (TRS) in some lymphocytes, plasma cells, monocytes and endothelial cells and (c) precipitates of protein within occasional erythroblasts and marrow reticulocytes. There was also a striking and hitherto undescribed abnormality of the structure of the nucleus in intersinusoidal and perisinusoidal non-phagocytic reticular cells. This was seen in six patients, including case 3, and was characterized by the extensive detachment of masscs of abnormally electrondense heterochromatin from the nuclear membrane, the presence of a uniformly thin layer of electron-dense material at the inner surface of the areas of nuclear membrane denuded of heterochromatin masses and an abnormal electron lucency of areas containing euchromatin. The CCC and TRS were found in the six patients with the lowest number of circulating CD4-positive T cells. The precipitation of protein within erythroid cells may have been caused by the oxidant effect of dapsone or high doses of co-trimoxazole. The abnormalities in the stromal cells and in particular the nuclear changes seen in the non-phagocytic reticular cells support the possibility that one of the mechanisms underlying the cytopenia in patients infected with HIV may be a disturbance of the microenvironmental regulation of haemopoiesis.     

An Investigation into the Development and Fate of Neutrophil Giant Metamyelocytes using the Techniques of Electron Microscopy and High Resolution Autoradiography

The neutrophil giant metamyelocytes present in vitamin B₁₂- and folate-deficient patients were studied using the techniques of electron microscopy and electron microscope autoradiography. The ultrastructural features of the cytoplasm of a proportion of these cells resembled those of promyelocytes and myelocytes, particularly with respect to the types of neutrophil granules present. This finding suggests that the giant metamyelocytes result from an abnormal type of development in promyelocytes and myelocytes which have been arrested or retarded in their progress through the cell cycle. The hypothesis that giant metamyelocytes eventually die within the marrow was supported by the observations that a significant proportion of these cells contain intracytoplasmic autophagic vacuoles, that some giant metamyelocytes suffer from a marked depression of RNA and protein synthesis and that degenerating giant metamyelocytes can be recognized within the cytoplasm of some bone marrow macrophages.     

Dyserythropoiesis and ineffective erythropoiesis in Plasmodium vivax malaria

Summary. Nine Thai adults with P. vivax malaria were investigated. Light and electron microscope studies of marrow aspirates revealed morphological evidence of dyserythro-poiesis in six of them, Dyserythropoiesis was most marked in the four most anaemic patients. In these four patients the electron microscope also revealed the presence of erythro-blasts at various stages of degradation within the cytoplasm of macrophages. Neither the dyserythropoiesis nor the ineffective erythropoiesis could be attributed to a deficiency of vitamin B₁₂, folate or iron. The abnormalities of erythropoiesis seemed to result from the P. vivax infection itself. Other bone marrow reactions seen in this infection included macrophage hyperplasia, plasmacytosis and increased eosinophil granulocytopoiesis. Unlike in severe P. falciparum malaria, the microvasculature of the marrow was not obstructed by parasitized red cells.     

Human cerebral malaria

Possible factors contributing to the development of cerebral malaria were discussed based on pathological changes in Burmese patients who died of cerebral malaria. Blockage of cerebral capillaries by Plasmodium falciparum infected erythrocytes appeared to be the principal cause of cerebral malaria. From electron microscopic results, it was concluded that knobs on infected erythrocytes acted as focal junctions which mediated adhesion to endothelial cells. The knobs are, therefore, important contributors to the blockage of the capillary lumen and ensuing pathological changes in cerebral tissues. Host cell molecules such as OKM5 and thrombospondin may function as endothelial cell surface receptors for the attachment of knobs of P. falciparum infected erythrocytes. Immunological events might also play a role in the pathogenesis of cerebral malaria. This was suggested by the presence of IgG, IgM, P. falciparum antigens, and knob proteins in the cerebral capillaries of the people with cerebral malaria. It will be important to assess the candidate malaria vaccines now in development not only for their efficacy in reducing parasitemia but for effects they may have on the sequestration of infected erythrocytes in the brain.     

The Mr 95,000 gelatin-binding protein in differentiated human macrophages and granulocytes

Cultured adherent human macrophages and a promonocytic cell line, U 937, were previously shown to produce a Mr 95,000 gelatin-binding protein. The protein has no immunologic cross-reactivity with the well- characterized gelatin-binding protein fibronectin and the Mr 70,000 gelatin-binding protein produced by a variety of mesenchymal or epithelial cell types (T. Vartio et al, J Biol Chem 257:8862, 1982). In the present study the Mr 95,000 protein was found in Triton X-100 extracts of granulocytes purified from human blood buffy coat. The protein, as isolated by gelatin-agarose, was immunologically cross- reactive with the corresponding macrophage protein in immunoblotting assay. When peripheral blood and bone marrow cells were examined for the presence of the Mr 95,000 protein by indirect immunofluorescence, positive staining was detected only in differentiated granulocytes but not to any significant extent in metamyelocytes, myelocytes, promyelocytes, or in normal or leukemic blasts. In granulocytes the protein had a granular cytoplasmic distribution. In freshly prepared monocyte cultures, the Mr 95,000 protein was detected in low amounts in the cytoplasm, while along with differentiation of the cells into macrophages, the immunofluorescence increased in a reticular and vesicular cytoplasmic pattern and in a juxtanuclear cap, probably representing the Golgi complex. In conclusion, the Mr 95,000 gelatin- binding protein was specifically detected in macrophages and granulocytes and may thus serve as a differentiation marker for these phagocytic cells.     

Neutrophil Marrow Profiles in Patients with Rheumatoid Arthritis and Neutropenia

Neutrophil marrow cellularity was determined in 14 neutropenic patients with rheumatoid arthritis (RA) from measurements of neutrophil-normoblast ratios in marrow biopsies and ferrokinetic estimates of marrow normoblasts. A marrow profile was developed for each patient comprising the numbers of promyelocytes and myelocytes, of metamyelocytes and bands, and of segmented neutrophils in whole marrow. In each case a maturation ratio was calculated by dividing the number of metamyelocytes and bands by the number of promyelocytes and myelocytes. The physiologic marrow response to loss of neutrophils from circulation was assumed to be an increase in promyelocytes and myelocytes due to proliferation and influx, a reduction in segmented cells due to early release, and a normal maturation ratio. The results were interpreted in the light of the 95% confidence limits for data previously obtained from 13 normal subjects: in patients with neutropenia reduced or basal numbers of promyelocytes and myelocytes were interpreted as absence of the anticipated proliferative response; increased numbers of marrow segmented cells were attributed to failure of release; a low maturation ratio was assessed to reflect intramedullary cell loss. The pattern in two patients with Felty's syndrome was consistent with a physiological response to neutrophil destruction. The other 12 patients had neutrophil marrow abnormalities. Seven patients with Felty's syndrome and four patients without splenomegaly had absolute or relative hypoplasia of neutrophil marrow or low maturation ratios. One patient with a normal spleen size had an increased number of marrow segmented cells yet failed to mobilize cells normally in response to dialysis coil-activation of C3. Abnormalities of neutrophil marrow may contribute to neutropenia in RA irrespective of the presence of splenomegaly. Recognition of neutrophil marrow abnormalities in these patients may be of value in prognosis and management.     

Origin and significance of a thermostable granulocyte antigen

In 1972 the thermostabile antigen of granulocytes was for the first time isolated by Thoss and Abendroth from punctates of the joint of patients with rheumatoid arthritis. Its origin from mature neutrophil granulocytes was ascertained by fluorescence-microscopic investigations. In the present paper the existence of thermostabile antigen of granulocytes in neutrophil granulocytes could be confirmed. The fluorescence pattern of neutrophil granulocytes of healthy persons did not show any differences in comparison to patients with inflammatory or myeloproliferative diseases as well as granulocytes from punctates of the joint or sternal marrow. With the help of punctates of the lymph-nodes the presence of thermostabile antigens of granulocytes in cells of the lymphatic system could be excluded. In smears of the sternal marrow positive fluorescence could be proved in the myelopoesis in neutrophil metamyelocytes. Quantitative investigations in inflammatory and myeloproliferative diseases as well as in granulocytopenias showed that the TSGA -serum values of the numbers of granulocytes go parallel. In punctates of the joint of patients with rheumatoid arthritis we found concentrations of thermostabile antigens of granulocytes which up to 50-fold were above the normal values of the serum. There was a close correlation to the number of granulocytes in the synovial fluid and to the cytological local activity. The TSGA -level can be regarded as indicator of granulocytic activation.     

Observations on the ultrastructure of sinusoids and reticular cells in human bone marrow

Summary The ultrastructural characteristics of sinusoids in human bone marrow were generally similar to those previously reported in the rat. However, human sinusoids differed from rat sinusoids in displaying frequent tight junctions between adjacent overlapping or interdigitating endothelial cells. In both species, large areas of the sinusoidal walls were devoid of much subendothelial connective tissue and of an outer adventitial cell layer. The marrow sinusoids of humans resembled those of rabbits and rats in that processes of macrophage cytoplasm protruded through endothelial cells into the sinusoidal lumen; thin veils derived from such processes were apposed over the inner surfaces of some endothelial cells. Features observed in pathological human marrow not noted in rodent marrow are the phagocytosis of extruded erythroblast nuclei and of abnormal erythroblasts by perisinusoidal adventitial cells (reticular cells) and the presence of large secondary lysosomes and siderosomes in sinusoidal endothelial cells. When compared with mouse bone marrow, human bone marrow contained very few non-phagocytic reticular cells that were unassociated with sinusoids and other blood vessels. There were no absolute ultrastructural differences between perisinusoidal adventitial cells, intersinusoidal non-phagocytic reticular cells and macrophages lacking phagosomes or containing only a few small phagosomes. Consequently, on some occasions, these cell types could not be reliably identified from the study of a single thin section. Extracellular reticulin fibres were found adjacent both to non-phagocytic reticular cells and to macrophages. Mitosis was observed in macrophages, albeit very rarely.     

Thrombospondin mediates the cytoadherence of Plasmodium falciparum- infected red cells to vascular endothelium in shear flow conditions

Cerebral malaria is thought to involve specific attachment of Plasmodium falciparum-infected knobby red cells to venular endothelium. The nature of surface ligands on host endothelial cells that may mediate cytoadherence is poorly understood. We have investigated the effects of soluble thrombospondin, rabbit antiserum raised against thrombospondin, and human immune serum on cytoadherence of parasitized erythrocytes in ex vivo mesocecum vasculature. Preincubation of infected red cells with soluble thrombospondin or human immune serum inhibits binding of infected red cells to rat venular endothelium. Infusion of the microcirculatory preparation with rabbit antithrombospondin antibodies before perfusion of parasitized erythrocytes also resulted in decreased cytoadherence. In addition, incubation of infected cells with human immune sera obtained from malaria patients significantly inhibited the observed cytoadherence. Our results indicate that thrombospondin mediates binding of infected red cells to venular endothelium and may thus be involved in the pathogenesis of cerebral malaria.     

Spleen in falciparum malaria: ultrastructural study

An ultrastructural study was undertaken of the spleen of 13 year-old-boy who died of falciparum malaria. The spleen revealed the following: both parasitized and non-parasitized erythrocytes are phagocytosed in large numbers by macrophages, littoral and reticular cells. Blood congestion and trapping of parasitized erythrocytes are commonly seen in splenic sinusoids and cords. Erythrocytes forming rosette structure around immuno-presenting cells is observed. The results of this study provide evidence that the mechanisms underlying splenic host defence in malaria include both immunological and non-immunological interaction with erythrocytes. Splenic trapping of parasitized erythrocytes is an important defence mechanism and the phagocytosis of erythrocytes probably accounts for anaemia.     

Observations on the ultrastructure of sinusoids and reticular cells in human bone marrow.

The ultrastructural characteristics of sinusoids in human bone marrow were generally similar to those previously reported in the rat. However, human sinusoids differed from rat sinusoids in displaying frequent tight junctions between adjacent overlapping or interdigitating endothelial cells. In both species, large areas of the sinusoidal walls were devoid of much subendothelial connective tissue and of an outer adventitial cell layer. The marrow sinusoids of humans resembled those of rabbits and rats in that processes of macrophage cytoplasm protruded through endothelial cells into the sinusoidal lumen; thin veils derived from such processes were apposed over the inner surfaces of some endothelial cells. Features observed in pathological human marrow not noted in rodent marrow are the phagocytosis of extruded erythroblast nuclei and of abnormal erythroblasts by perisinusoidal adventitial cells (reticular cells) and the presence of large secondary lysosomes and siderosomes in sinusoidal endothelial cells. When compared with mouse bone marrow, human bone marrow contained very few non-phagocytic reticular cells that were unassociated with sinusoids and other blood vessels. There were no absolute ultrastructural differences between perisinusoidal adventitial cells, intersinusoidal non-phagocytic reticular cells and macrophages lacking phagosomes or containing only a few small phagosomes. Consequently, on some occasions, these cell types could not be reliably identified from the study of a single thin section. Extracellular reticulin fibres were found adjacent both to non-phagocytic reticular cells and to macrophages. Mitosis was observed in macrophages, albeit very rarely.     

Knob antigen deposition in cerebral malaria

Plasmodium falciparum-infected erythrocytes attach to the endothelial cells via electron-dense knobs and this attachment has been suggested as one of the contributing factors in the development of cerebral malaria. Monoclonal antibodies against an 80-95 Kd knob protein were prepared and applied to brain tissue from cerebral malaria patients. The deposition of the 80-95 Kd knob protein antibodies was observed in the basement membrane of cerebral capillaries by the peroxidase anti-peroxidase method. This result indicates involvement of knob protein deposition in the pathogenesis of cerebral malaria.     

Erythrophagocytosis in the Human Bone Marrow

Erythrocyte destruction by erythrophagocytosis and red blood cell disintegration has been studied in methacrylate embedded human bone marrow. Phagocytosis of erythrocytes is demonstrated in macrophages within the bone marrow sinusoids, in the bone marrow reticulum cells and in the nursing cells of the erythroclastic islands. Red cell destruction takes place both in normal and in pathologically altered bone marrow, but is often markedly increased in hypoplastic bone marrow disease. The bone marrow is considered representing the main erythroblastic organ in normal individuals, removing aged red cells from the circulation by erythrophagocytosis within bone marrow sinusoids. Further disintegration of the erythrocytes in the bone marrow reticulum cells provides directly components for rebuilding of new erythrocytes in the erythroblastic islands.     

脑型疟发生的免疫病理机制

脑型疟(cerebral malaria)是疟疾感染的严重并发症,近年来其发生的免疫病理学机制受到极大关注。早期的研究认为,脑型疟的发生主要与感染疟原虫的红细胞和脑血管内皮细胞黏附,导致脑血管阻塞有关。然而,越来越多的证据表明,脑型疟的发生主要由疟原虫感染后引起的免疫病理反应所导致,与炎症因子的过量释放和免疫细胞在脑血管的浸润密切相关。本文就近年来脑型疟发生的免疫病理机制的研究进展作一综述。     

Neutrophil marrow release

Summary Neutrophil marrow egress is governed by several processes. The most important are cell maturation, functional behavior of marrow sinusoids and humoral or neurovascular factors. Neutrophil release cannot be observed directly but is reflected in the size, cellular composition and kinetics of the nonproliferating pool of granulocytopoiesis in bone marrow and of blood neutrophil pool. These experimentally determined parameters were used as the basis of a mathematical model study.The model describes two catenated compartments, the nonproliferating pool of granulocytopoiesis in marrow and the total blood granulocyte pool. Cell transit from one pool to the other was assumed to be age-dependent. It was expressed by a positive sloping sigmoidal function that defines the egress potential of the cells that increases with cell maturation.During maturation granulocytopoietic cells develop intense motility which determines the morphology of the cells on smears. Relationship between cell motility and its morphology was defined by functions determining the age-dependent probabilities of cell fixation as metamyelocytes, band- and segmented forms, respectively.The parameters of this model could be so adjusted that all experimental data were matched within experimental errors. Thus, qualitative and quantitative information on neutrophil marrow egress was obtained for normal and pathological states of granulocytopoiesis.This work was supported by Swiss National Research Fund grant No. 3.9002.72.     

A Quantitative Assessment of Neutrophil Marrow in Seven Patients with Chronic Granulocytic Leukaemia

Neutrophil marrow cellularity was determined in seven patients with chronic granulocytic leukaemia (CGL). The size of the mitotic pool (promyelocytes and myelocytes) and the number of metamyelocytes and bands and of segmented neutrophils in the postmitotic pool were determined from measurements of neutrophil--erythroid ratios in marrow biopsy sections and ferrokinetic estimates of marrow normoblasts. A section mitotic index was calculated in each patient from the numbers of mitotic figures and mitotic pool cells counted on marrow sections. Basal values previously established in normal subjects for the mitotic pool, for metamyelocytes and bands, and for segmented neutrophils, were 2.11 +/- 0.36 x 10(9) cells/kg, 3.33 +/- 0.61 x 10(9) cells/kg, and 2.26 +/- 0.42 x 10(9) cells/kg, respectively (+/- 1 SD, n = 13). The basal section mitotic index was 0.07 +/- 0.01 (+/- 1 SD, n = 13). In the seven patients with CGL the mitotic pool comprised 3.71--25.70 x 10(9) cells/kg, metamyelocytes and bands 7.70--51.02 x 10(9) cells/kg, and segmented neutrophils 3.45--28.81 x 10(9) cells/kg. Mitotic indices ranged from 0.04 to 0.10. No relationship was found between marrow cellularity and blood neutrophil count. A negative correlation existed between mitotic pool cellularity and mitotic index (r = -0.76, n = 7 pairs). The results provide quantitative affirmation of neutrophil marrow hyperplasia and of increased neutrophil production by the marrow in CGL.     

Primary structure and subcellular localization of the knob-associated histidine-rich protein of Plasmodium falciparum.

Plasmodium falciparum-infected erythrocytes bind to venular endothelial cells by means of electron-dense deformations (knobs) on the parasitized erythrocyte surface. The primary structure of a parasite-derived histidine-rich protein associated with the knob structure was deduced from cDNA sequence analysis. The 634 amino acid sequence is rich in lysine and histidine and contains three distinct, tandemly repeated domains. Indirect immunofluorescence, using affinity-purified monospecific antibodies directed against recombinant protein synthesized in Escherichia coli, localized the knob-associated histidine-rich protein to the membrane of knobby infected erythrocytes. Immunoelectron microscopy established that the protein is clustered on the cytoplasmic side of the erythrocyte membrane and is associated with the electron-dense knobs. A role for this histidine-rich protein in knob structure and cytoadherence is suggested based upon these data.