Experimental acute Babesia caballi infections: II. Response of platelets and fibrinogen

Ponies were acutely infected with Babesia caballi by inoculation with infected red blood cells (RBCs) containing 1 × 10⁸ and 1 × 10⁹ piroplasms. A series of blood samples taken before and after inoculation were analyzed for platelets and fibrinogen, and the results compared with similar analyses made on challenged, premunized ponies and on equids inoculated with uninfected RBCs. In acutely infected animals there were immediate decreases in platelet counts that persisted at least through Day 18 after inoculation (AI). Concomitantly, plasma fibrinogen levels rose, reaching peak values between Days 6–17. Clot retractions in vitro were impaired in these ponies during Days 9–16. No large diminutions in platelet counts or elevations of fibrinogen levels were observed in the challenged, premunized ponies or the group transfused with uninfected RBCs. In fact, the effect of challenge was to maintain or increase platelet counts. Our results plainly indicate that B. caballi can elicit alterations in clotting factor levels in its hosts during acute infections.     

Effects of Malaria Parasite Density on Blood Cell Parameters

Changes in blood cell parameters are already a well-known feature of malarial infections. To add to this information, the objective of this study was to investigate the varying effects that different levels of parasite density have on blood cell parameters. Patients diagnosed with malaria at Phobphra Hospital, Tak Province, Thailand between January 1^st 2009 and January 1^st 2012 were recruited as subjects for data collection. Blood cell parameters of 2,024 malaria-infected patients were evaluated and statistically analyzed. Neutrophil and platelet counts were significantly higher, however, RBC count was significantly lower in patients with P. falciparum infection compared to those with P. vivax infection (p<0.0001). Leukocyte counts were also significantly higher in patients with high parasitemia compared to those with low and moderate parasitemia. In terms of differential leukocyte count, neutrophil count was significantly higher in patients with high parasitemia compared to those with low and moderate parasitemia (p<0.0001). On the other hand, both lymphocyte and monocyte counts were significantly lower in patients with high parasitemia (p<0.0001). RBC count and Hb concentration, as well as platelet count were also significantly reduced (p<0.05) and (p<0.0001), respectively. To summarize, patients infected with different malaria parasites exhibited important distinctive hematological parameters, with neutrophil and eosinophil counts being the two hematological parameters most affected. In addition, patients infected with different malarial densities also exhibited important changes in leukocyte count, platelet count and hemoglobin concentration during the infection. These findings offer the opportunity to recognize and diagnose malaria related anemia, help support the treatment thereof, as well as relieve symptoms of severe malaria in endemic regions.     

Plasmodium berghei: correlation of in vitro erythrophagocytosis with the dynamics of early-onset anemia and reticulocytosis in mice.

Infection of BALB/c mice with Plasmodium berghei results in an anemia which is excessive to that which can be accounted for solely by direct destruction of infected erythrocytes by the mature schizonts at the time of merozoite release. Mice infected with 10⁴ infected erythrocytes exhibited a progressive anemia beginning on Day 7. Significant reticulocytosis was first observed on Day 9 and parasitemia tended to parallel reticulocytosis with a lag of about 1 day. In studies of erythrophagocytosis, washed erythrocytes from randomly selected mice infected with 10⁵ infected red blood cells were phagocytized by peritoneal macrophages in vitro to a significantly greater extent on Days 3–5 postinfection than were erythrocytes taken from normal controls. The degree of erythrophagocytosis reached a peak on Day 4 and returned to control levels on Days 6 and 7. Erythrocytes taken from infected animals on Day 7 and incubated in normal plasma were phagocytized to a significantly greater extent than were normal erythrocytes incubated in normal plasma or erythrocytes from infected mice incubated in plasma from infected animals. The enhanced in vitro erythrophagocytosis observed on Days 3–5, which preceded and coincided with the beginning of the early-onset anemia on Day 5, may correlate with in vivo phenomena which may contribute to the developing anemia. Furthermore, the restoration of enhanced erythrophagocytosis by normal plasma seems to indicate that some component(s) of normal plasma may be depleted during the early stages of P. berghei infection.     

Host control of malaria infections: constraints on immune and erythropoeitic response kinetics

The two main agents of human malaria, Plasmodium vivax and Plasmodium falciparum, can induce severe anemia and provoke strong, complex immune reactions. Which dynamical behaviors of host immune and erythropoietic responses would foster control of infection, and which would lead to runaway parasitemia and/or severe anemia? To answer these questions, we developed differential equation models of interacting parasite and red blood cell (RBC) populations modulated by host immune and erythropoietic responses. The model immune responses incorporate both a rapidly responding innate component and a slower-responding, long-term antibody component, with several parasite developmental stages considered as targets for each type of immune response. We found that simulated infections with the highest parasitemia tended to be those with ineffective innate immunity even if antibodies were present. We also compared infections with dyserythropoiesis (reduced RBC production during infection) to those with compensatory erythropoiesis (boosted RBC production) or a fixed basal RBC production rate. Dyserythropoiesis tended to reduce parasitemia slightly but at a cost to the host of aggravating anemia. On the other hand, compensatory erythropoiesis tended to reduce the severity of anemia but with enhanced parasitemia if the innate response was ineffective. For both parasite species, sharp transitions between the schizont and the merozoite stages of development (i.e., with standard deviation in intra-RBC development time ≤2.4 h) were associated with lower parasitemia and less severe anemia. Thus tight synchronization in asexual parasite development might help control parasitemia. Finally, our simulations suggest that P. vivax can induce severe anemia as readily as P. falciparum for the same type of immune response, though P. vivax attacks a much smaller subset of RBCs. Since most P. vivax infections are nonlethal (if debilitating) clinically, this suggests that P. falciparum adaptations for countering or evading immune responses are more effective than those of P. vivax.     

Plasmodium chabaudi AS: erythropoietic responses during infection in resistant and susceptible mice.

The course of anemia and the erythropoietic response in the bone marrow, spleen, and blood were studied during Plasmodium chabaudi AS infection in resistant C57BL/6 (B6) and susceptible A/J (A) mice. Infections in B6 mice were characterized by moderate levels of both parasitemia and anemia and survival. In contrast, A mice experienced high parasitemia, severe anemia, and high mortality rates. During the period of anemia, erythropoiesis, as measured by in vivo 59Fe incorporation, was significantly more depressed in bone marrow and more increased in the spleen in resistant B6 mice. The increase in splenic 59Fe incorporation was a function of the size of the spleen. Bone marrow CFU-E were decreased to 50% of control in both strains, while splenic CFU-E were increased twofold greater in B6 mice compared to those in A mice. However, the absolute numbers of CFU-E per spleen in the two strains were not significantly different during peak parasitemia. Bone marrow BFU-E were transiently increased before peak parasitemia whereas splenic BFU-E peaked during peak parasitemia. A mice had significantly lower numbers of BFU-E per spleen on all days except at peak parasitemia. The frequency of blood-borne BFU-E and plasma erythropoietin titers was increased earlier and to a greater extent in A mice. These results suggest that an impaired amplification of late-stage splenic erythropoiesis may be an important determinant in the severity of anemia and lethality of infection with P. chabaudi AS in A mice. Moreover, these results demonstrate that the defective amplification of splenic erythropoiesis in A mice is neither caused by a defect in the mobilization of BFU-E from the bone marrow to the spleen nor caused by a defect in erythropoietin production.     

The influence of oxidative bursts of phagocytes on red blood cell oxidation in anemic cattle infected with Theileria sergenti

The primary clinical symptom of Japanese bovine theileriosis, caused by the intraerythrocytic protozoan Theileria sergenti, is anemia, but the underlying mechanism of this anemia remains unknown. To elucidate the pathogenesis of anemia developing in bovine theileriosis, we investigated the relationship between oxidative bursts of peripheral blood phagocytes (neutrophils and monocytes) and the oxidation of red blood cells (RBC) to the development of anemia in cattle experimentally infected with T. sergenti. The levels of methemoglobin (MetHb) and malondialdehyde (MDA), as a parameter of intracellular and membrane oxidative damage in RBC and of production of hydrogen peroxide (H₂O₂) in phagocytes, were low before the onset of anemia; these parameters began to increase remarkably with decreasing packed cell volume and increasing parasitemia during the course of the anemia, which returned to initial levels during convalescence from anemia. A positive correlation between H₂O₂ production of phagocytes and each of the oxidative indices of MetHb and MDA was also noted during the onset of anemia. The levels of antioxidants, namely reduced glutathione and glucose-6-phosphate dehydrogenase, in RBC also decreased during the progression of anemia. These results suggest that oxidative damage of RBC has a close relationship with the onset of anemia in bovine theileriosis, and that oxidative bursts of phagocytes may play a part in the pathogenesis of anemia in infected cattle.     

The influence of oxidative bursts of phagocytes on red blood cell oxidation in anemic cattle infected with Theileria sergenti

The primary clinical symptom of Japanese bovine theileriosis, caused by the intraerythrocytic protozoan Theileria sergenti, is anemia, but the underlying mechanism of this anemia remains unknown. To elucidate the pathogenesis of anemia developing in bovine theileriosis, we investigated the relationship between oxidative bursts of peripheral blood phagocytes (neutrophils and monocytes) and the oxidation of red blood cells (RBC) to the development of anemia in cattle experimentally infected with T. sergenti. The levels of methemoglobin (MetHb) and malondialdehyde (MDA), as a parameter of intracellular and membrane oxidative damage in RBC and of production of hydrogen peroxide (H2O2) in phagocytes, were low before the onset of anemia; these parameters began to increase remarkably with decreasing packed cell volume and increasing parasitemia during the course of the anemia, which returned to initial levels during convalescence from anemia. A positive correlation between H2O2 production of phagocytes and each of the oxidative indices of MetHb and MDA was also noted during the onset of anemia. The levels of antioxidants, namely reduced glutathione and glucose-6-phosphate dehydrogenase, in RBC also decreased during the progression of anemia. These results suggest that oxidative damage of RBC has a close relationship with the onset of anemia in bovine theileriosis, and that oxidative bursts of phagocytes may play a part in the pathogenesis of anemia in infected cattle.     

Transfer of immunity to Babesia microti of human origin using T lymphocytes in mice

Lymph node and spleen cells from mice infected with Babesia microti of human origin developed the ability to transfer adoptive immunity to naive mice within 25 days after infection. This protective activity was greater in cells obtained at 32 days than in cells obtained at 25 days postinfection and remained stable up to 52 days postinfection. Recipients of lymph node cells and spleen cells displayed similar peak parasitemias although 2 days after peak parasitemia, immune spleen cell recipients had significantly lower parasitemias than immune lymph node cell recipients. Strong protective activity was demonstrated when cells were transferred 1 day postinfection, while equal numbers of cells, transferred 3 days postinfection did not confer significant protection over nonimmune cells. There was also a suggestion that the number of immune spleen cells necessary for significant protection was directly related to the number of parasites inoculated. The subpopulation of lymphocytes responsible for the transfer of adoptive immunity to B. microti of human origin was then studied in BALB/c mice depleted of T lymphocytes by thymectomy and lethal irradiation. One day after infection with B. microti, T-cell-depleted mice were given complement-treated immune spleen cells, anti-θ serum-treated immune spleen cells, nonimmune spleen cells, or no cells. Similar experiments were performed comparing the effects of anti-immunoglobulin serum-treated and unfractionated immune spleen cells on B. microti parasitemia. Treatment with anti-θ serum abrogated the protective activity of immune spleen cells while anti-immunoglobulin serum treatment had no effect. These results suggest that immunologic memory of B. microti in BALB/c mice is modulated by T rather than B lymphocytes.     

Dexamethasone suppresses the generation of phenylhydrazine-induced anemia in the rat.

The immunoactive steroid dexamethasone (DXM) was administered to rats injected with a dose of phenylhydrazine (PHZ) known to induce anemia. PHZ treatment alone resulted in a hemolytic anemia that was most pronounced on Days 1-7 after injection. This anemia was accompanied by a leukocytosis that was greatest on Days 2-7 following PHZ treatment. Lymphocytes accounted for greater than 75% of this incremental increase. In contrast, rats treated with PHZ as well as with DXM displayed erythrocyte counts and hematocrits within the normal range. Although the reticulocyte counts of these rats were higher than those of controls, they were significantly lower than those of animals receiving PHZ alone. In addition, DXM suppressed the leukocytosis and splenomegaly resulting from PHZ administration and inhibited the rise in plasma IgG titers induced by PHZ exposure. DXM also altered the ratio of peripheral blood T and B lymphocytes of PHZ-treated rats. DXM suppression of PHZ-induced anemia is further confirmation that this anemia is associated with immune activation.     

On the mechanism of phenylhydrazine-induced hemolytic anemia

The thiobarbituric acid (TBA)-reactants have been measured in the circulating red blood cells (RBC) and RBC trapped in the spleens of normal and phenylhydrazine-treated rats. There was significant TBA-reactivity in the circulating RBC of phenylhydrazine-treated rats which was increased 3-fold in RBC obtained from the spleen. Since lipid peroxidation accompanies formation of TBA-reactive malonyldialdehyde, it is suggested that phenylhydrazine induces anemia as a consequence of peroxidation of RBC membrane lipids and this effect may be a result of the autoxidation of the drug and the interaction of oxygen radicals with membrane lipids.     

Subchronic Toxicological Study of Two Artemisinin Derivatives in Dogs

The objective of our study was to profile and compare the systematic changes between orally administered artesunate and intramuscularly injected artemether at a low dose over a 3-month period (92 consecutive days) in dogs. Intramuscular administration of 6 mg kg^-1 artemether induced a decreased red blood cell (RBC) count (anemia), concurrent extramedullary hematopoiesis in the spleen and inhibition of erythropoiesis in the bone marrow. We also observed a prolonged QT interval and neuropathic changes in the central nervous system, which demonstrated the cortex and motor neuron vulnerability, but no behavioral changes. Following treatment with artesunate, we observed a decreased heart rate, which was most likely due to cardiac conduction system damage, as well as a deceased RBC count, extramedullary hematopoiesis in the spleen and inhibition of erythropoiesis in the bone marrow. However, in contrast to treatment with artemether, neurotoxicity was not observed following treatment with artesunate. In addition, ultra-structural examination by transmission electron microscopy showed mitochondrial damage following treatment with artesunate. These findings demonstrated the spectrum of toxic changes that result upon treatment with artesunate and artemether and show that the prolonged administration of low doses of these derivatives result in diverse toxicity profiles.     

Nippostrongylus brasiliensis: mast cells and histamine levels in tissues of infected and normal rats.

The role of the spleen during Babesia microti and B. hylomysci infection was investigated by examining the course of infection in both intact and splenectomized mice. The presence of the spleen was critical during the early stages of infection to control excessive multiplication of either parasite, a role taken over by other lymphoid sites as the infection progressed. Mice splenectomized prior to or within 1 week of B. microti inoculation developed extended infections with some deaths, and others were unable to check their parasitemias. All intact mice, and those splenectomized 1 week after infection with B. microti, recovered completely with subsequent development of sterile immunity. Mice splenectomized prior to or within 1 week of B. hylomysci inoculation succumbed to hyperacute infections: Some of the intact mice, and those splenectomized 12 days after infection, recovered but continued to harbor a low-grade infection with periodical recrudescences. Erythrophagocytosis of infected and uninfected erythrocytes was detected in saline preparations and impression smears of spleen and bone marrow and rarely in blood smears of infected mice. This coincided with anemia, splenomegaly, and relatively high levels of opsonizing antibodies, especially during B. microti infection. The colloidal carbon clearance method was used to investigate the phagocytic activity of the reticuloendothelial system. Carbon clearance rates increased rapidly during both infections, but peak B. hylomysci parasitemia coincided with reticuloendothelial phagocytic depression and death of the host. Babesia microti stimulated a consistently higher reticuloendothelial phagocytic activity with higher erythrophagocytosis both in the spleen and bone marrow than did B. hylomysci.     

Comparison of the effects of IL-1 alpha and TNF-alpha on phagocyte accumulation and murine antibacterial immunity

IL-1 and TNF both are reported to increase host antibacterial resistance. To directly compare their effects on tissue phagocyte accumulation and antibacterial activity, we infused recombinant human IL-1 alpha and TNF-alpha into C3H/HeJ mice. Although IL-1, at a dose of 1 microgram/day, did not significantly elevate blood neutrophil concentrations, it increased the number of PMNs within the spleen three to fourfold within 2 days. Similar neutrophil accumulation also occurred in the lungs, bone marrow, and liver of treated animals without detectable changes in macrophage numbers. IL-1 also increased myelopoiesis in the spleen by Days 3-4 of infusions. The capacity of splenocytes from IL-1-treated animals to kill Listeria monocytogenes in vitro and to suppress listeria proliferation in vivo after the intravenous infusion of bacteria both rose in parallel with PMN accumulation. Comparable doses of TNF also enhanced listeria killing in vivo but in contrast to IL-1, it significantly depressed peripheral blood neutrophil counts, and inhibited splenic neutrophil accumulation and in vitro listericidal activity in listeria-infected mice. Our results suggest that IL-1 enhances host resistance to infection by increasing tissue neutrophil accumulation while TNF protects by a different mechanism, despite a net inhibitory effect on neutrophil accumulation.     

Studies on immunity to Babesia gibsoni in dogs immunostimulation by Bordetella bronchiseptica

Four species of bacteria, Corynebacterium anaerobium 578, Actinobacillus pleuropneumoniae G-4, Mycobacterium bovis BCG, and Bordetella bronchiseptica A-2, were injected intravenously into mice (5 weeks old, ICR-SPF). The clearance of carbon from the blood stream and the weights of the spleen and liver were determined as indicators of RES stimulation. Mouse footpad reaction was assessed as an indicator of delayed-type hypersensitivity to each species of bacteria. The immuno-stimulative activity of each species of bacteria against bovine serum albumin was monitored by passive hemagglutination assay and the macrophage migration-inhibition test in guinea pigs. Based on the results of the experiments described above, B. bronchiseptica was selected as an immunostimulator (Ims) for immunization trials of the hemo-protozoan parasite, Babesia gibsoni, with inactivated merozoites of B. gibsoni (BgK). Twelve dogs, pointers about 6 months old, were divided into four groups of three dogs each. Group 1 dogs were initially injected with Ims, and later injected with BgK and Ims (BgK+Ims) after a 3-week interval. Group 2 and Group 3 dogs were injected twice, at a 3-week interval, with BgK+Ims and BgK, respectively, and Group 4 served as a control. As the results, the serum antibody titres of Group 1 and 2 were several times higher than that of Group 3, and the cell-mediated immunity to parasites was noticeably stimulated by immunization with BgK+Ims. The peak level of parasitemia following the challenge were over 10% for Group 4 and 4.5% for Group 3, while levels for Group 1 and 2 were 2.5% and less than 1%, respectively. No such major clinical signs of babesiosis as jaundice and anemia were observed in Group 1 or 2.     

The parasitemia of cloned Trypanoplasma borreli Laveran and Mesnil, 1901, in laboratory-infected common carp (Cyprinus carpio L.)

The course of parasitemia of cloned Trypanoplasma borreli in laboratory-infected common carp was investigated. In 25-42-g carp kept at 20 C, the prepatent period was 8 days; after a phase of exponential growth, the parasitemia peaked at day 39 postinjection (PI) at a level of about 10(3) T. borreli/microliters blood. This maximum was followed by a chronic phase of about 6 wk with large numbers of T. borreli. At 20 wk PI, T. borreli was absent in infected carp. In 2.2-g carp kept at 20 C, the prepatent period was 4 days only, and the parasitemia peaked at day 23 PI. At 30 C, T. borreli was present in the blood only for 12 wk, and the number of T. borreli did not exceed 162 trypanoplasms/microliters blood. Carp kept at 8 and 15 C showed retarded development of parasitemia. The prepatent period lasted longer and the generation time was increased, but the level of parasitemia was not affected. Carp, inoculated at 8 C and then warmed to 20 C on days 27 and 55 PI, developed a parasitemia of 10(4) flagellates/microliters blood and showed high mortalities. During the prepatent period, T. borreli was found in the muscle tissue of the inoculation area but in no other tissue. In the kidney, T. borreli was found 27 hr PI, whereas in the circulating blood it was manifest at day 3 PI. At the same time it was manifest in the liver and spleen.     

Computational modeling of biomechanics and biorheology of heated red blood cells

Because of their compromised deformability, heat denatured erythrocytes have been used as labeled probes to visualize spleen tissue or to assess the ability of the spleen to retain stiff red blood cells (RBCs) for over three decades, e.g., see Looareesuwan et al. N. Engl. J. Med. (1987). Despite their good accessibility, it is still an open question how heated RBCs compare to certain diseased RBCs in terms of their biomechanical and biorheological responses, which may undermine their effective usage and even lead to misleading experimental observations. To help answering this question, we perform a systematic computational study of the hemorheological properties of heated RBCs with several physiologically relevant static and hemodynamic settings, including optical-tweezers test, relaxation of prestretched RBCs, RBC traversal through a capillary-like channel and a spleen-like slit, and a viscometric rheology test. We show that our in silico RBC models agree well with existing experiments. Moreover, under static tests, heated RBCs exhibit deformability deterioration comparable to certain disease-impaired RBCs such as those in malaria. For RBC traversal under confinement (through microchannel or slit), heated RBCs show prolonged transit time or retention depending on the level of confinement and heating procedure, suggesting that carefully heat-treated RBCs may be useful for studying splenic- or vaso-occlusion in vascular pathologies. For the rheology test, we expand the existing bulk viscosity data of heated RBCs to a wider range of shear rates (1–1000 s⁻¹) to represent most pathophysiological conditions in macro- or microcirculation. Although heated RBC suspension shows elevated viscosity comparable to certain diseased RBC suspensions under relatively high shear rates (100–1000 s⁻¹), they underestimate the elevated viscosity (e.g., in sickle cell anemia) at low shear rates (<10 s⁻¹). Our work provides mechanistic rationale for selective usage of heated RBC as a potentially useful model for studying the abnormal traversal dynamics and hemorheology in certain blood disorders.     

Effects of selenium supplementation on virus-induced inflammatory heart disease

The effects of 10 wk of selenium (Se) supplementation (5 ppm) in drinking water on immune responses and resistance to a myocarditic Coxsackie virus B3 (CB3) infection were studied in female Balb/c mice. Se supplementation reduced CB3-induced mortality: at day 14 postinoculation, survival was 58% in the Se-treated group as compared to 25% in the untreated group. Whole-blood glutathione peroxidase (GSH-Px) activity was elevated by 68% (p < 0.001) and Se content in the liver by 24% (p < 0.001). Red (RBC) and white blood cell (WBC) counts, as well as the number of cells in the spleen and thymus, were unaffected. The cellular counts of T-lymphocytes (CD4⁺, CD8⁺) and natural killer (NK⁺) cells in the blood were not affected. However, the CD4⁺/CD8⁺ ratio (5.2) tended to increase after Se supplementation (5.9). The spleen lymphoproliferative response to T and B-cell mitogens were increased by 9 and 43%, respectively (ns), in the Se-supplemented group. The total NK cell activity in blood and spleen showed minor increases, but when the activity in the blood was expressed per cell, the increase amounted to 35% (ns) with Se supplementation. The inflammatory and necrotic lesions in the ventricular myocardium at 7 and 14 d postinoculation were not significantly reduced by Se treatment, probably owing to the increased survival with Se even of mice with the most pronounced heart damage; comparable untreated mice were estimated to have died at day 14. Results indicate that modest doses of Se can improve immune function, which may increase the general resistance to this viral infection.     

Induction of Antibody Synthesis by Purified Immunogenic RNA <Emphasis Type="Italic">in vitro</Emphasis>

WE have described an RNA fraction derived from phenol-extracted livers of immunized rabbits, which induced specific antibody production when mixed with normal rabbit spleen cells in vitro^1,2. Similar fractions have been described by others using spleen, lymph node or peritoneal exudate cells of mice, rats or rabbits^3–12 as sources of the RNA fraction. In all cases it has been assumed that the RNA-donor cell type was a macrophage. Considerable controversy has been generated by these experiments and data have been published to show that (a) the RNA is neither specific¹³ nor newly synthesized¹⁴ and (b) the RNA fraction contains antigen or fragments thereof^15–18. Here we show that the data obtained with the rabbit-DNP system² extend to another laboratory model, the mouse-sheep red blood cell (RBC) system. Our earlier work^1,2 suggested that the immunogenic RNA is produced in the macrophage cell, that it is specific and that it is confined to a discrete fraction of the extractable RNA. For these reasons we thought it desirable (a) to compare directly the capacities of both liver and spleen tissue RNA extracts to induce antibody-plaque-forming cells in vitro, (b) to compare the effects of RNAase and pronase on the immunogenic capacity of the RNA fraction and (c) to investigate the distribution of the immunogenic RNA fraction relative to the total RNA fractions.