Michael J. Dascombe

Nitric oxide and reactive nitrogen intermediates during lethal and nonlethal strains of murine malaria

The virulence of Plasmodia depends partly on the strain of parasite and partly on the host. In this study, Plasmodium berghei N/13/1A/4/203 caused the death of mice, whereas Plasmodium chabaudi chabaudi AS was not lethal. Current opinion is that nitric oxide (NO) and other reactive nitrogen intermediates (RNI) are produced in several host organs during malaria to resist infection or produce tissue damage. NO and RNI production in blood or plasma, brain, liver and spleen in MF1 mice was investigated during P. berghei and P. c. chabaudi infection, in order to help determine whether changes in NO production are beneficial or detrimental to the host in vivo. NO production was measured both directly and indirectly as nitrites and nitrates, to represent RNI. No changes in blood NO were detected in P. berghei infected mice, but increases were observed in brain, liver and spleen. In P. c. chabaudi infected mice, rises in NO concentration were observed in blood and spleen, whereas a decline in liver NO was seen, but there were no changes in brain. Liver contained the highest concentration of RNI, but increasing concentrations were seen in both plasma and spleen in both P. berghei and P. c. chabaudi infected mice. These results show that NO and RNI production alters during murine malaria. The changes depend upon the tissue, the day of infection, the degree of parasitaemia, the strain of Plasmodia and the method of measuring NO biosynthesis. Lethal P. berghei induced NO production in the mid and late stages of infection in mice when parasitaemia was high, whereas in nonlethal P. c. chabaudi infection, NO production was increased in the early and late stages when parasitaemia was low. These data are consistent with a role for NO in the protection of the MF1 mouse against Plasmodia. Failure to clear the parasite is associated with evidence of increased NO production in brain and liver, which may contribute to the pathology of malaria, but this hypothesis requires confirmation from other experimental approaches.

Expression of inducible nitric oxide synthase (iNOS) mRNA in target organs of lethal and non-lethal strains of murine malaria

Nitric oxide (NO) is a putative mediator of the immunological and/or pathological responses to malaria, consequently it is a potential target for novel drug therapy. Numerous cell types increase expression of inducible nitric oxide synthase (iNOS) under inflammatory conditions, the most relevant stimuli being cytokines and endotoxins. In this study the expression of iNOS mRNA in several target organs (brain, liver, spleen) of malaria have been investigated in MF1 mice during lethal Plasmodium (P.) berghei and non‐lethal P. c. chabaudi infection. In P. berghei malaria, iNOS mRNA decreased in liver and was unchanged in spleen during the period of rising parasitaemia, but increased in both organs late in the infection, when parasitaemia was high and death imminent. In mice infected with P. c. chabaudi, spleen iNOS mRNA increased progressively throughout the early, peak and recovery periods of parasitaemia, but decreased in liver. Brain iNOS mRNA decreased in samples collected throughout the time courses of both infections. Hence it is evident that changes in iNOS mRNA in murine malaria depend upon the tissue, day of infection, degree of parasitaemia and strain of Plasmodium. These data indicate induction of iNOS mRNA in the spleen has a role in combating these strains of Plasmodium in MF1 mice. Failure to clear lethal P. berghei parasitaemia was associated with increased iNOS mRNA expression in the liver, which may contribute to the pathology of this malaria.

Pharmacological assessment of the role of nitric oxide in mice infected with lethal and nonlethal species of malaria

This pharmacological investigation sought to determine whether nitric oxide (NO) had an antiparasitic effect and/or mediated pathology in mice infected with nonlethal P. chabaudi or lethal P. berghei. Nitric oxide synthase (NOS) inhibitors were evaluated for their ability to inhibit the rise in reactive nitrogen intermediates (RNI) induced by bacterial lipopolysaccharide (LPS) in mice. The more effective compound, aminoguanidine (AG) inhibited the rise in RNI induced by P. chabaudi and increased mortality, but had no effect on parasitaemia. Inducers and donors of NO were screened for their ability to increase RNI and the most effective agents evaluated for their ability to modify P. berghei infection. S‐Nitrosoglutathione had little effect, but LPS decreased parasitaemia and mortality. An inconsistent relationship is evident between the abilities of these agents to modify NO activity and their effects on malaria in mice. Increased mortality in mice with P. chabaudi treated with AG indicates a reduction in resistance. The absence of an effect on parasitaemia by a NOS inhibitor or NO donor indicates either RNI have insignificant antimalarial action in vivo or the efficacy of the compounds is inadequade. Resistance to P. berghei in LPS‐treated mice demonstrates an antiparasitic effect, but this may be attributable to factors other than NO.

Endotoxin fever in granulocytopenic rats: evidence that brain cyclooxygenase‐2 is more important than circulating prostaglandin E2

PGE2 is a recognized mediator of many fevers, and cyclooxygenase (COX) is the major therapeutic target for antipyretic therapy. The source, as well as the site of action of PGE2, as an endogenous pyrogen, is widely accepted as being central, but PGE2 in the circulation, possibly from leukocytes, may also contribute to the development of fever. However, bacterial infections are important causes of high fever in patients receiving myelosuppressive chemotherapy, and such fevers persist despite the use of COX inhibitors. In the study reported here, the febrile response to bacterial LPS was measured in rats made leukopenic by cyclophosphamide. A striking increase in LPS fever occurred in these granulocytopenic rats when compared with febrile responses in normal animals. Unlike LPS fever in normal rats, fever in granulocytopenic rats was neither accompanied by an increase in blood PGE2 nor inhibited by ibuprofen. Both leukopenic and normal rats showed LPS‐induced COX‐2‐immunoreactivity in cells associated with brain blood vessels. Furthermore, LPS induced an increase of PGE2 in cerebrospinal fluid. Induction of COX‐2‐expression and PGE2 production was inhibited by ibuprofen in normal but not in leukopenic rats. Although the results presented are, in part, confirmatory, they add new information to this field and open a number of important questions as yet unresolved. Overall, the present results indicate that, in contrast to immunocompetent rats, leukocytes and/or other mechanisms other than PGE2 are implicated in the mechanisms restricting and reducing the enhanced febrile response to endotoxin in immunosuppressed hosts.

Nitroglycerine reduces neutrophil activation and acute damage in latissimus dorsi muscle grafts.

BACKGROUNDnDamage to the latissimus dorsi muscle (LDM) may jeopardize a successful outcome to dynamic cardiomyoplasty. We and others have demonstrated muscle damage in LDM in various species including humans. Ischemia is now recognized to be an important contributory factor. We postulated that glyceryl trinitrate, a nitric oxide donor, might protect against ischemic endothelial dysfunction and so reduce resultant muscle damage.nnnMETHODSnIn 20 adult rats the left LDM was mobilized on its thoracodorsal neurovascular pedicle and maintained as an orthotopic graft. Half of the animals received glycerol trinitrate intraoperatively and postoperatively for 24 hours. The other half served as untreated controls. Each group was further subdivided into two groups (n = 5 in each): animals in which the LDM was excised after 4 hours for myeloperoxidase studies, and animals in which the LDM was excised at 24 hours for analysis of muscle damage by histology and enzyme macrohistochemistry. Blood samples were taken at 24 hours for assay of plasma nitrite and nitrate as nitric oxide metabolites.nnnRESULTSnGlycerol trinitrate-treated animals had higher plasma nitric oxide metabolite levels after 24 hours (after nitrate reductase treatment, total nitrite, 78.3+/-11.8 nmol/mL, mean +/- SEM) than controls (42.1+/-3.7 nmol/ mL, p = 0.008). The proportion of viable LDM in glycerol trinitrate-treated animals was greater than in untreated animals, mainly in the middle and distal regions of the graft (middle region, 96.3%+/-0.5% versus 75.7%+/-4.1%, p<0.001; distal region, 94.4%+/-0.8% versus 40.9%+/-3.1%, p<0.001). Macrohistochemical findings correlated well with the histologic findings. Myeloperoxidase activity (U/g) was markedly lower in glycerol trinitrate-treated LDMs, mainly in the distal part of the graft (glycerol trinitrate versus control, 20.5+/-2.1 versus 40.9+/-3.1 U/g, p<0.001).nnnCONCLUSIONSnGlycerol trinitrate significantly reduced acute damage to the distal two-thirds of the mobilized LDM, possibly by modifying leukocyte activation and endothelial dysfunction associated with ischemic injury.

Effects of methylxanthine drugs on pyrogen-induced hyperthermia.

The hyperthermic response to pyrogen was not potentiated by caffeine or theophylline administered i.p. into cats or rabbit. Injection of these drugs into the anterior hypothalamus or into the third cerebral ventricle in cats was also without effect on pyrexia. These results support the hypothesis that cyclic AMP in the anterior hypothalamus does not mediate pyrogen-induced hyperthermia in cats.

Fever induced by macrophage inflammatory protein-1β in the rat is independent of hypothalamic interleukin-1β or interleukin-6

Abstract 1. It has been proposed that macrophage inflammatory protein-1β (MIP-1β) induces fever via a cyclooxygenase-independent pathway. The roles of interleukin-1β (IL-1β) and interleukin-6 (IL-6) in MIP-1β-induced fever were investigated in the rat, by using specific antibodies against these cytokines. 2. The intrahypothalamic injection of anti-MIP-1β, 15xa0min before the injection at the same site of MIP-1β significantly blocked the fever induced by this cytokine. 3. The pre-treatment with antibodies against IL-1β or IL-6 receptors, did not alter MIP-1β-induced fever. 4. These data support the hypothesis that MIP-1β produces fever via a parallel pathway, independent of the classical prostaglandin-dependent pathway adduced in the development of fever for IL-1β and IL-6.

Ketanserin effects on thermoregulation in malarial and normal rats

Malaria, which is caused by parasitic protozoa of the genus Plasmodium, continues to be a major global problem due to the development of resistance to antimalarial drugs, the failure to eradicate the insect vector and the present lack of effective vaccines. Laboratory investigations of malaria and antimalarial drugs commonly employ mice or rats inoculated with the rodent-specific parasite, Plasmodium berghei (P. be~-ghei) .~.~ However, malarial infection causes a fall in core temperature in both the mouse and the rat, not pyrexia as seen in human^.^,^ The mechanisms underlying the development of hypothermia in malarial rodents are not known, but it is conceivable that both peripheral and central homeostatic mechanisms are disturbed by the disease. We have observed that the turnover of serotonin (5-hydroxytryptamine, 5-HT), a putative neurotransmitter reducing both body temperature and food intake, is increased in the brains of rats infected by P. berghei.6-8 The data presented here are the results of a pharmacological study with the 5-HT, antagonist ketanserin to test the hypothesis that 5-HT2 receptors mediate the hypothermia andlor hypophagia associated with malaria in the rat.

Behavioural thermoregulatory responses to malaria infection and interleukin-1β in rats.

Abstract 1. Behavioural thermoregulation was assessed by the ambient temperatures selected by rats moving freely around an enclosed temperature gradient circuit (15 to 32°C). 2. Interleukin-1β (10xa0μg/kg i.p.) increased both colonic and selected ambient temperatures, but malaria ( Plasmodium berghei ) had no effect on either temperature, although malarial rats responded to interleukin-1β with pyrexia. 3. Immune status of lymph and spleen cells appeared unaltered by malaria in rats. 4. These data indicate that malarial rats do not have a pyrogenic drive to develop fever.

Expression of NADPH-diaphorase in nucleus tractus solitarius after peripheral injection of E. coli endotoxin in rats

The possibility that peripheral exogenous pyrogens can activate brainstem nuclei by abdominal viscera aAerents was studied using the NADPH-diaphorase method in E. coli lipopolysaccharide treated rats with and without ibu- profen pre-treatment. 2. NADPH-d staining revealed: (i) a significant increase in the number of NADPH-d labeled neurons in the subpos- tremal area of the nucleus tractus solitarius lipopolysaccharide treated rats compared with control animals (p < 0.05), and (ii) an almost similar number of NADPH-d positive neurons in both control and ibuprofen pre- treated rats (p = 0.091). 3. These data indicate that peripheral administration of an exogenous pyrogen stimulates the synthesis of nitric oxide in the nucleus tractus solitarius, and are consistent with the hypothesis of a direct neural link between the periphery and the hypothalamus.