Secondary bacterial infection in plasma endotoxin levels and the acute-phase response of mice infected with Trypanosoma brucei brucei.

Murine Trypanosoma brucei brucei infection leads to elevated plasma endotoxin-like activity levels not related to parasitaemia levels accompanied by the development of acute-phase response and increased plasma levels of serum amyloid P (SAP) and haptoglobin (Hp). To determine the source of the endotoxin-like activity and role of secondary bacterial infection in the pathogenesis of trypanosomosis, infected mice were treated with the antibiotic ciprofloxacin. Plasma endotoxin-like activity levels, irrespective of treatment, were elevated three- to fourfold, beginning 7 days after infection. Plasma protein concentrations increased markedly following infection from 7 days after infection (DAI). Peak Hp and SAP concentrations in ciprofloxacin-treated and -untreated infected mice were attained 7 and 14 DAI, respectively. Thereafter, both protein levels gradually declined until the end of the experiment, but Hp levels for non-treated mice declined up to 21 DAI and thereafter significantly increased on 28 and 35 DAI. Whole-trypanosome lysate and the membrane-enriched fraction demonstrated endotoxin-like activity, with the former having higher levels. The results suggest that the endotoxin-like activity in trypanosome fractions and plasma of infected mice is due to the trypanosome. Further elevation of haptoglobin during the late stages of infection in non-treated mice suggests the involvement of secondary bacterial infection.

Lipopolysaccharide binding protein in the acute phase response of experimental murine Trypanosoma brucei brucei infection.

Cellular responses to lipopolysaccharide (LPS) are enhanced by LPS-binding protein (LBP). The present study investigated the acute phase response of LBP during Trypanosoma brucei brucei infection in mice. Mean plasma concentrations of LBP increased two-fold by the seventh day following infection, but decreased to intermediate levels by the 14th day. There were no significant differences in LBP concentrations of infected/antibiotic-treated and infected/untreated mice. At 35 days post-infection, the infected mice were treated with the anti-trypanosomal diminazine aceturate (Berenil). LBP levels of the mice then decreased to pre-infection levels within one-week. This demonstrated that LBP is an acute phase protein during murine trypanosomosis. Furthermore, opportunistic secondary bacterial infection during trypanosomosis did not seem to play an important role in the changes in plasma LBP levels. We speculate that the marked concomitant increases in plasma LBP and endotoxin-like activity following murine trypanosome infection might play an important role in the pathogenesis of trypanosomosis.

Acute phase response in mice experimentally infected with Trypanosoma congolense: a molecular gauge of parasite-host interaction.

Mice infected with Trypanosoma congolense developed a severe anaemia 1 week after infection, which persisted till treatment with diminazine aceturate when the packed cell volume (PCV) recovered to pre-infection levels. This was accompanied by a marked increase in the plasma levels of the acute phase proteins (APP), serum amyloid P-component (SAP) and haptoglobin (Hp). The initial peak levels of Hp and SAP were attained 7 and 12 days post-infection (DPI), respectively. Thereafter SAP levels decreased significantly to near pre-infection levels, but later increased even after treatment to give a second peak 34 DPI after which there was a decline till the study was terminated. The Hp levels on the other hand decreased to an intermediate level after the initial peak increasing to a second peak 22 DPI. Thereafter Hp decreased significantly following diminazine aceturate treatment to reach pre-infection levels within 5 days post-treatment. This indicates that T. congolense-infected mice develop severe anaemia accompanied by an acute phase response leading to an increase in SAP and Hp but that following treatment divergent responses occurred indicating differences in the pathways for stimulation of the APP. Haptoglobin was shown to be an earlier indicator of infection and a better marker in monitoring the response to treatment.

The role of the polyamine inhibitor eflornithine in the neuropathogenesis of experimental murine African trypanosomiasis.

The treatment of late-stage human African trypanosomiasis is complicated by a post-treatment reactive encephalopathy, also referred to as a 'reactive arsenical encephalopathy', that may be fatal. This study used a well established experimental mouse system to assess the use of the trypanostatic drug, eflornithine, in the management of this post-treatment reaction. Female CD-1 mice infected with an eflornithine-resistant trypanosome stabilate and treated with the trypanocidal compound diminazene aceturate on or after day 21 post-infection develop a reactive encephalopathy and relapsing parasitaemia. If these animals are re-treated with diminazene aceturate, a severe encephalopathy develops histologically comparable with that of human cases and characterized by a severe meningoencephalitis and astrogliosis. Histopathological and immunocytochemical examination shows that administration of eflornithine before or after the development of this reactive encephalopathy prevented or ameliorated the inflammatory reaction. Since an eflornithine resistant stabilate was used, this effect appears to be independent of the drug's trypanostatic action and illustrates an important, previously unrecognized, pharmacological property of eflornithine. Consideration can now be given to the use of eflornithine for the management of human trypanosomiasis cases, even where trypanosome resistance to eflornithine exists.

A substance P antagonist, RP-67,580, ameliorates a mouse meningoencephalitic response to Trypanosoma brucei brucei.

Mice infected with the protozoan parasite Trypanosoma brucei brucei and treated subcuratively with the trypanocidal drug diminazene aceturate develop an acute inflammatory meningoencephalitis with associated astrocytic proliferation. This reaction is very similar to that seen in the fatal posttreatment reactive encephalopathies that can occur in human African trypanosomiasis. The 11-amino acid neuropeptide substance P (SP) has recently been identified as a mediator in many inflammatory responses, and the development of potent, highly specific, nonpeptide SP antagonists has provided a new opportunity to investigate the possible involvement of SP in a variety of pathological conditions. We therefore postulated that SP may play a role in the development of the posttreatment inflammatory encephalopathy found in this experimental mouse model of African trypanosomiasis. In the present study RP-67,580, a SP antagonist that binds specifically to NK-1 receptors, was given intraperitoneally at a dose of 2 mg/kg twice daily to mice in which a severe meningoencephalitis had been produced. A significant reduction in both the severity of the inflammatory response (P = 0.0001) as well as the degree of astrocyte activation (P < 0.001) was found in the brains of these animals as compared with control mice that had not received RP-67,580. An inactive enantiomer of this SP antagonist, RP-68,651, had no effect on the central nervous system inflammatory reaction. We conclude from these findings that the neuropeptide SP plays a key role in the development of the severe central nervous system inflammatory response associated with African trypanosomiasis.

Topical chemotherapy for experimental murine African CNS-trypanosomiasis: the successful use of the arsenical, melarsoprol, combined with the 5-nitroimidazoles, fexinidazole or MK-436.

The 5-nitroimidazoles, MK-436 and fexinidazole dissolved in dimethylsulphoxide can be converted by the addition of hydroxypropylcellulose into gels which facilitates the ease and accuracy of administration. When these gels are used in combination with melarsoprol gel they are capable of curing experimental murine CNS-trypanosomiasis with a one-day treatment. The use of melarsoprol/MK-436 was more efficient than melarsoprol/fexinidazole gels. Thus while a single treatment with 0.1 ml 3.6% melarsoprol gel with 0.1 ml (14.3 mumol) fexinidazole gel cured the infected mice, the same dose of melarsoprol gel with 0.1 ml (4.0 mumol) of MK-436 gel was equally effective. It was also possible to prepare a combined melarsoprol/MK-436 gel which cured experimental CNS-trypanosomiasis with a single treatment. Topical treatment with this melarsoprol/MK-436 gel mixture also resolved clinically the hind leg paralysis which is associated with post-treatment reactive encephalopathy caused by non-curative treatment of CNS-trypanosomiasis.

The importance of 2,3-dimercaptopropinol (British anti-lewisite, BAL) in the trypanocidal activity of topical melarsoprol.

Both melarsomine dichlorhydrate (mel Cy, Cymelarsan) and melarsen oxide can be dissolved in dimethylsulfoxide and converted into a gel by the addition of hydroxypropylcellulose. When Trypanosoma brucei brucei-infected mice are treated topically with these gels the circulating trypanosomes are rapidly cleared from the circulation but the infections relapse soon after the last application. However, when these two compounds are allowed to react with 2,3-dimercaptopropinol (British anti-lewisite, BAL) and form "melarsoprol" their efficacy, especially in the case of mel Cy, is restored to that of commercial melarsoprol (Arsobal) and trypanosomes in the central nervous system (CNS) can be eliminated. This would indicate that the dimercaptopropinol portion of the molecule does not act solely as an "antidote" to arsenic toxicity, but also plays an important role in the absorption of melarsoprol through the skin and/or blood-brain barrier into the CNS and/or into the trypanosome.

Topical chemotherapy for experimental African trypanosomiasis with cerebral involvement: the use of melarsoprol combined with the 5-nitroimidazole, megazol.

Megazol, one of a number of related 5-nitroimidazoles, can be dissolved in dimethylsulphoxide and the solution can be converted into a gel by the addition of hydroxypropylcellulose which facilitates the ease and accuracy of administration. This megazol gel, when used in combination with melarsoprol (3.6%) in propylene glycol gel, will cure experimental CNS-trypanosomiasis in mice. A single application of 0.1 ml of melarsoprol (3.6%) gel plus 0.1 ml of either 8 or 16 mg/ml megazol gel successfully treated experimental CNS-trypanosomiasis while two consecutive days' treatment with 0.05 ml melarsoprol and 0.1 ml of 16 or 32 mg/ml megazol gels also produced satisfactory cures.

The effect of spiroarsoranes on Trypanosoma brucei brucei and T. b. rhodesiense.

Topical application and intraperitoneal administration of spiroarsoranes were carried out to cure central nervous system (CNS) trypanosomiasis in the chronic Trypanosoma brucei GVR 35 mouse model. Topical application appeared more efficient than intraperitoneal injection. The periods of aparasitaemia after treatment were longer but none of the mice was permanently cured. Combination treatment with eflornithine (DFMO) and the spiroarsoranes failed to show any synergistic effect. In addition, spiroarsorane I was evaluated against the T. b. rhodesiense KETRI 2634 strain, whereby 60-mg/kg treatment produced a noticeable prolongation of the life span of trypanosome-positive animals. These in vivo results suggests that the spiroarsoranes have difficulty in crossing the blood-brain barrier (BBB) and clearing the parasites from the CNS or, alternatively, that these strains are less sensitive to pentavalent arsenicals than the T. b. brucei CMP fast strain, which in the present study was more sensitive to spiroarsoranes whose lipophilicity corresponded to a log-P value ranging from 2.5 to 3.7.

Successful treatment of experimental murine Trypanosoma brucei infection with topical melarsoprol gel.

Melarsoprol gel applied topically (0.1 mL for at least 2 d) can cure late-stage Trypanosoma brucei brucei and T. b. rhodesiense infections in mice. The best regimen was 3 applications at approximately 0, 6, and 24 h. The melarsoprol gel retained its activity at room temperature for at least 63 d. There was only minimal skin irritation and no sign of toxicity.

Combination chemotherapy of CNS trypanosomiasis.

The progress which has been made in the treatment of experimental CNS trypanosomiasis with combination chemotherapy is reviewed. The most significant has been the use of four specific 5-nitroimidazoles in combination with either suramin or the arsenicals. The latter combination of MK 436 and Mel Cy, producing a rapid cure of CNS trypanosomiasis with only a two dose regimen and thus, would make an ideal universal treatment for both early- and late-stage trypanosomiasis. However, the 5-nitroimidazoles, because they are Ames' positive, are unlikely to be developed for use in humans. The combination chemotherapeutic regimen of eflornithine and arsenicals would allow the use of reduced quantities of melarsoprol to be used with similar or increased efficacy. As these drugs are already approved for use in humans, they could be applied immediately to the human disease; however, the quantities of eflornithine required for cures and the basic cost of this compound may limit its use in human medicine. Investigations of the post-treatment reactive encephalopathies (PTRE) which occur after non-curative treatment of trypanosome infections have shown that they are essentially caused by the presence of a residual focus of living trypanosomes in the CNS. If all trypanosomes are eliminated from the CNS (curative treatment) then there are no PTRE and when non-curative treatment is used the reaction can be reduced or ameliorated by supportive treatment with anti-inflammatory drugs such as prednisolone, dexamethasone or azathioprine.

Chemotherapy of Trypanosoma brucei infection of the central nervous system: the use of a rapid chemotherapeutic regimen and the development of post-treatment encephalopathies.

The use of a rapid curative chemotherapeutic regimen for experimental infection of the central nervous system (CNS) of mice by Trypanosoma brucei has indicated that this 'aggressive' treatment does not precipitate the development of post-treatment meningo-encephalopathy. If meningoencephalitis is already established at the time of the treatment there is no exacerbation of the reaction and the CNS pathology rapidly returns to normal. Paralysis is not precipitated by the rapid curative treatment of either primary or relapse infections, in contrast to non-curative treatments. Mice showing this overt clinical paralysis in addition to histological meningoencephalitis soon regain mobility and the CNS pathology is rapidly resolved after curative chemotherapy. These experiments provide no support for the concept that the rapid release of trypanosome antigens in situ in the brain exacerbates the post-treatment encephalopathy. They do support the concept that it is viable trypanosomes remaining in the brain which are responsible.

Correlation of autoantibody titres with central nervous system pathology in experimental African trypanosomiasis.

CD-1 mice infected with the protozoan parasite Trypanosoma brucei brucei developed few signs of central nervous system pathology associated with the invasion of the central nervous system by these parasites and did not survive beyond 5-6 weeks with deaths common before this time point. However, use of the trypanocidal drug diminazene aceturate (40 mg/kg), which fails to cross the blood-brain barrier, on day 21 post-infection led to the development of central nervous system pathology similar to that seen in the fatal post-treatment reactive encephalopathies that can occur in human African trypanosomiasis. Enzyme-linked immunosorbent assays were used to measure autoantibody titres to double-stranded DNA, myelin basic protein and to the myelin-specific galactocerebrosides and gangliosides in groups of infected mice, with or without the post-treatment reaction, on day 30 post-infection and compared with uninfected controls. Infection with T. brucei brucei raised the titres of all of these autoantibodies. Treatment of infected mice with diminazene aceturate resulted in elevated levels of all of these autoantibodies compared to the untreated animals. There was a strong positive correlation between the central nervous system pathology and the levels of autoantibodies to myelin basic protein, galactocerebrosides and gangliosides, but not to double-stranded DNA. The elevated titres observed may be a consequence of the polyclonal B cell activation that is believed to occur in African trypanosomiasis, parasite epitopes that are cross-reactive with these central nervous system (CNS)-specific antigens or result from the CNS damage associated with sub-curative chemotherapy.

The use of azathioprine to ameliorate post-treatment encephalopathy associated with African trypanosomiasis.

The treatment of human African sleeping sickness is complicated by a post-treatment meningoencephalitis that may be fatal. Using a mouse model this study assesses the use of the non-steroidal anti-inflammatory drug, azathioprine, in the management of this post-treatment reaction. Female NIH mice treated with the trypanocidal compound diminazene aceturate (40 mg/kg), 28 days after infection, developed a similar post-treatment reaction to that seen in humans. Administration of azathioprine (100 mg/kg) for 5 days before and 5 days after trypanocidal chemotherapy abrogated the pathology in the central nervous system although this returned approximately 15 days after cessation of azathioprine. Activated astrocytes associated with the later stages of the infection did not appear to be affected by the use of azathioprine.

Astrocyte activation correlates with cytokine production in central nervous system of Trypanosoma brucei brucei-infected mice.

BACKGROUND: During the late-stage disease associated with human African trypanosomiasis, caused by infection with either Trypanosoma gambiense or T. rhodesiense, parasites invade the central nervous system (CNS), eventually leading to development of CNS pathology. This can be exacerbated by subcurative chemotherapy. The mechanisms through which the inflammatory processes within the CNS are controlled remain unclear. EXPERIMENTAL DESIGN: Mice infected with T. b. brucei were treated with a trypanocidal drug regimen on day 28 postinfection that cleared parasites from all sites except the brain. Brains of mice killed at different times during infection and after chemotherapy were analyzed, using immunocytochemistry for astrocyte activation and polymerase chain reaction assisted amplification of RNA to detect cytokine transcripts. RESULTS: Drug-treated animals developed a posttreatment meningoencephalitis similar to that which can occur in humans with late-stage African trypanosomiasis. Between days 14 and 21 postinfection, before chemotherapy and the subsequent development of inflammatory lesions in the brain, astrocytes became activated. The production of several cytokines correlated with this astrocyte activation. Low levels of interleukin-1 alpha transcripts were detected in uninfected controls, but levels increased with astrocyte activation in the infected animals. Transcripts for the macrophage inflammatory protein-1 and tumor necrosis factor-alpha were first detected on day 21 postinfection, with higher levels in mice after development of the posttreatment meningoencephalitis, whereas granulocyte macrophage-colony stimulating factor was detected only in animals that developed posttreatment reaction. Interleukin-6 and interferon-gamma were also first detected on day 21 postinfection, correlating with astrocyte activation but subsequently declined with time in both treated and untreated mice. CONCLUSIONS: These results indicate that cytokines are being produced within the CNS before any inflammation and that astrocytes may be the source of some of these cytokines. Thus astrocyte activation may be key in the control and development of the CNS inflammatory processes that occur in African sleeping sickness.

Chemotherapy of CNS-trypanosomiasis: the combined use of diminazene aceturate or pentamidine with DL-alpha-difluoromethylornithine (DFMO).

The chemotherapy of CNS-trypanosomiasis with DL-alpha-difluoromethylornithine (Eflornithine, DFMO) with the diamidine, pentamidine (Lomidine) or diminazene aceturate (Berenil) was examined using the mouse model for CNS-trypanosomiasis. Although combined DFMO/pentamidine therapy can give complete cures, one or more doses of 100 mg/kg pentamidine was required with continuous administration of DFMO for 14-16 days, and even this treatment failed to cure the mice in a repeat experiment. Diminazene aceturate was more efficient and provided a 40 mg-kg dose was utilised, it was possible to reduce the period of DFMO to approximately 12 days. Attempts to reduce the dose rate of diminazene to less than 40 mg-kg failed. Of the two compounds, diminazene aceturate was the more successful and could possibly be used in combination with DFMO in the treatment of CNS-trypanosomiasis.

Isolation of drug-resistant strains of Trypanosoma congolense from the lower Shabelle region of southern Somalia.

Drug resistance by pathogenic trypanosomes in Somali livestock has been suspected for some time but there have been few attempts to examine this problem in detail. Field isolations from two areas in the Lower Shabelle Region were obtained by injecting blood from trypanosome infected cattle into a recipient calf. Once the calf became parasitaemic it was treated with a standard dose of isometamidium chloride (Samorin, RMB) at 0.5 mg/kg. When a subsequent relapse infection developed, indicative of drug resistance, blood was taken and injected into groups of cattle and mice and these were treated with a range of doses of isometamidium chloride and diminazene aceturate (Berenil, Hoechst AC) to determine the degree of drug resistance. Both isolates showed remarkably high levels of drug resistance to both isometamidium chloride and diminazene aceturate, with minimum curative doses in cattle of > 2.0 mg/kg and 7.5 mg/kg for the two drugs respectively. Minimum curative doses in mice were approximately ten-fold those in cattle. Fortunately there have been a very few reports from Africa of such high levels of resistance of Trypanosoma congolense to this normal "sanative pair" of drugs. The results indicate that drug resistance could be an important constraint on the use of trypanocidal drugs to control trypanosomosis in Somalia.