Tumor necrosis factor alpha and the anemia associated with murine malaria.

The anemia associated with malaria is complex, and multiple factors contribute to its severity. An increased destruction and a decreased production of erythrocytes are involved; however, the mechanisms responsible remain unclear. Tumor necrosis factor alpha (TNF-alpha), released by macrophages in response to infection, is thought to play a role through its ability to inhibit erythropoiesis. In these studies we have examined erythropoiesis in mice infected with Plasmodium berghei and in mice infused with recombinant TNF-alpha via implanted osmotic pumps. In both groups of mice there was (i) a reduction of pluripotent stem cells in the bone marrow and a concomitant increase in the spleen, (ii) a reduction of erythroid progenitor cells, and (iii) a reduced incorporation of 59Fe into erythrocytes. When P. berghei-infected mice were given antiserum against recombinant murine TNF, erythropoiesis was partially restored. There was a significant increase in bone marrow stem cells, erythroid progenitor cells, and 59Fe incorporation into erythrocytes in P. berghei-infected mice that had been treated with anti-TNF. How TNF may act, directly or indirectly, to inhibit erythropoiesis is not yet clear. These results demonstrate that TNF mediates, in part, the anemia associated with malaria.

Inhibition of erythropoiesis by a soluble factor in murine malaria.

To study the cellular mechanisms involved in the ineffective erythropoiesis associated with malaria, an in vitro proliferative assay was used to measure the response to erythropoietin (Epo) of erythroid progenitor cells from malaria-infected mice. In this assay, spleen (SP) cells from phenylhydrazine (PHZ)-treated mice (PHZ-SP), enriched for erythroid progenitor cells, respond to Epo in a dose-dependent manner. Despite a similar degree of anemia, SP and bone marrow (BM) cells from Plasmodium berghei- or P. vinckei-infected mice did not show a significant response to Epo in this assay. When SP or BM cells from malaria-infected mice were added to cultures of SP or BM cells from PHZ-treated mice the response to Epo of these cells was significantly inhibited. Removal of parasitized red blood cells (pRBC) from SP cells of P. berghei-infected mice had no effect on the ability of the cells to inhibit the response to Epo. Adherent SP cells and SP cells positive for the Mac-1 antigen, from malaria-infected mice, were shown to be enriched for cells that could inhibit the response to Epo. Cell-free conditioned media (CM) prepared from SP cells of P. berghei- or P. vinckei-infected mice or from normal SP cells incubated with pRBC were also able to inhibit the response to Epo of SP cells from PHZ-treated mice. These investigations have shown that during the course of malaria infection, cells appear in the SP and BM capable of inhibiting, via soluble mediators, the response to Epo of erythroid progenitor cells. The cells responsible are probably macrophages. The nature of the factor(s) and its mechanism of action are not known. Through the ability to inhibit erythropoiesis, soluble factors may, in part, mediate the anemia associated with malaria.

Murine malaria decreases hemopoietic stem cells.

The causes of anemia and immunosuppression, major outcomes of malaria, are not well established. This study was undertaken to investigate whether erythropoietin (EP) production is adequate and whether the hemopoietic stem cells (CFU-S) were affected during the course of infection. Groups of female Balb/c mice infected with Plasmodium vinckei vinckei, Plasmodium berghei, or Plasmodium chabaudi adami were exposed to five hours of simulated altitude equivalent to 22,000 ft. Plasma samples were collected for EP bioassay and radioimmunoassay (RIA). Using radioiron incorporation as an index of erythropoiesis, differences in response to infection with different species of plasmodia were observed. In general, decreases in erythropoietic activity were observed in bone marrow and spleen as the infection progressed and continued to be depressed after apparent resolution of a nonlethal infection with P. chabaudi adami. Marrow from infected and control femurs were tested for CFU-S content using the spleen colony assay. The cellularity and CFU-S content of the femoral marrow decrease as the parasitemia increases. All three species of plasmodia stimulate EP production during peak parasitemias, indicating that adequate amounts of EP are available to the erythron during malarial infection. Depletion of CFU-S and probable lack of compensatory turnover of CFU-S may contribute to the disease characteristics of malaria.

Recent advances in applied malaria immunology.

Our present knowledge of cellular and humoral factors which are involved in immunity to plasmodial infections are discussed. Immunization against plasmodial infection has been achieved in birds, rodents, simians, and humans. Avian hosts have been immunized against gametocytes which resulted in inhibition of gametocytes within the mosquito vector. Immunization of humans against plasmodial gametocytes would indirectly protect them against malaria by blocking mosquito transmission to other susceptible individuals. Immunization by sporozoites provides short-lived protection against sporozoite challenge, but gives no protection against erythrocytic forms. Some success has been obtained in immunizing avian and mammalian hosts with exoerythrocytic forms obtained from cultured avian cells. The most significant advances have occurred in immunizing simian hosts against simian or human malaria by vaccinating with fresh erythrocytic merozoites or a nonviable lyophilized antigen obtained from intraerythrocytic forms. The development of an antigen preparation suitable for use as a human malaria vaccine is dependent upon prior development of an in vitro system which would provide adequate amounts of parasite material. Efforts to cultivate the sporogonic, exoerythrocytic, and erythrocytic, and erythrocytic phases of plasmodia as well as the feasibility of using these forms for vaccination are discussed.

Long-term studies on rhesus monkeys (Macaca mulatta) immunized against Plasmodium knowlesi.

Studies carried out on four rhesus monkeys (Macaca mulatta) that had been vaccinated against Plasmodium knowlesi show that the immunized animals were protected against a challenge with a heterologous strain of P. knowlesi. This protection was shown to be present even 4 years after the immunization schedule has been completed. The effect could not be attributed toprevious infections with the parasite, since four control rhesus monkeys that had recovered from one to four challenges with P. knowlesi died when exposed to the heterologous strain. Data obtained from the lymphocyte transformation test and the radioimmunoassay are also presented.

Cultivation of the erythrocytic stages of Plasmodium berghei in Leydig cell tumor cultures.

Twelve different established cell-lines were used in attempts to cultivate the erythrocytic stages of Plasmodium berghei, P. vinckei vinckei, P. coatneyi or P. knowlesi. Intracellular parasites were seen in only mouse Leydig cell testicular tumor (LCT) cultures inoculated with red cells infected with P. berghei. Intracellular parasites were present at 15 to 96 h after inoculation, being most numerous at 36 h. Most intracellular stages were rings, trophozoites, schizonts and merozoites; gametocytes were few in number and present only at 36 and 48 h. Intracellular parasites were normal in general morphology and staining characteristics at 15 to 48 h, but were abnormal after 72 h. Infected host cells exhibited progressive nuclear and cytoplasmic degenerative changes, which ultimately resulted in death of the cell. Uninfected cells appeared normal. The ability of parasites in LCT cultures to produce infections upon injection into mice was similar to that obtained with control cultures without LCT cells.

Cultivation of the erythrocytic stages of Plasmodium berghei in primary bone marrow cells.

Cultures of primary bone marrow cells, obtained from the tibiae and femora of hamsters (HBM), mice (MBM), or rats (RBM) were inoculated with red cells infected with Plasmodium berghei, P. vinckei vinckei, or P. knowlesi. Merozoites, rings, trophozoites, schizonts and a few gametocytes were seen in HBM and MBM cells inoculated with P. berghe-infected red cells. At 1 to 2 or 3 days after inoculation in HBM and MBM, the number of intracellular asexual stages decreased slightly or remained the same, whereas at 3 to 7 or 8 days a 4 to 7-fold increase occurred. Parasites then decreased in number from days 8 to 11 and no parasites were seen on day 12. Intracellular parasites were most numerous at 7 and 8 days in MBM and HBM cultures, respectively. Mice injected with supernatant or cells from control cultures (without cultured cells) or primary cultures inoculated 1 and 2 days earlier with P. berghei- or P. vinckei vinckei-infected red cells always became infected. Mice injected with material from 3-day-old control and experimental cultures usually became infected, and those injected with 4- to 12-day-old cultures never became infected.

Ultrastructural study of Plasmodium knowlesi antigen used in vaccination of rhesus monkeys.

Material from various steps obtained in the French pressure cell technic of preparing antigen from Plasmodium knowlesi-infected red cells, was examined by elctron microscopy. A positively charged colloidal iron solution was used to differentiate between membranes of host red cells and parasites. Red cell membranes take the stain, wheras parasite membranes do not. This antigen which has been used previously to protect monkeys against P. knowlesi appears to consist almost entirely of membrane-bounded vesicles. Some of these vesicles contain a fine granular material, whereas others appear empty. The antigen failed to stain with the positively charged iron solution, which suggests that it is free of contamination by host cell membrane.

Delayed dermal hypersensitivity in rhesus monkeys (Macaca mulatta) immunized against Plasmodium knowlesi.

Eighteen Rhesus monkeys were immunized with a fresh or lyophilized antigen preparation obtained from erythrocytes infected with Plasmodium knowlesi. Sixteen of these monkeys showed a positive delayed hypersensitivity response when subsequently skin tested with the immunizing antigen. Ten of the 16 positive reactors survived a P, knowlesi challenge, the remaining 6 showed several indications of protection. None of the 18 control monkeys gave a positive skin reaction or survived the challenging inoculum. Macro- and microscopical evidence suggests that the dermal reaction is of a delayed type. Preliminary biochemical analysis of the antigen used in this experiment is included and its significance is discussed. Hematological data is also presented.