Expression, domain structure, and enzymatic properties of an active recombinant human DNA topoisomerase II beta.

Human cells express two genetically distinct isoforms of DNA topoisomerase II, alpha and beta, which catalyze ATP-dependent DNA strand passage and are an important antitumor drug target. Here we report for the first time the successful overexpression of human topoisomerase II beta in yeast by cloning a topoisomerase II beta cDNA in a yeast shuttle vector under the control of a galactose-inducible promoter. Recombinant human topoisomerase II beta (residues 46-1621 fused to the first 5 residues of yeast topoisomerase II) was purified to homogeneity, yielding an enzymatically active polypeptide in sufficient quantity to allow analysis of its domain structure and comparison with that of recombinant human topoisomerase II alpha. Partial digestion of beta with either trypsin or protease SV8 generated fragments of approximately 130, 90, 62, and 45-50 kDa, arising from cleavage at three limited and discrete regions of the protein (A, B, and C) indicating the presence of at least four structural domains. Recombinant human topoisomerase II alpha and beta induced DNA breakage which was promoted by a variety of agents. Isoform differences in drug-induced DNA breakage were observed. These studies of human topoisomerase II beta in concert with alpha should aid the determination of their individual roles in cancer chemotherapy and should facilitate the design, targeting, and testing of cytotoxic antitumor agents.

Trypanosoma brucei rhodesiense: use of an antigen detection enzyme immunoassay for evaluation of response to chemotherapy in infected vervet monkeys (Cercopithecus aethiops).

Thirty eight Trypanosoma brucei rhodesiense-infected vervet monkeys (Cercopithecus aethiops) in the late (meningoencephalitic) stage of disease, treated with various trypanocidal drugs, were monitored for a period of more than 600 days to assess the rate of clearance of trypanosome antigens from serum and cerebrospinal fluid (CSF). There was a complete but gradual reduction in antigen titres, as assessed by ELISA, in animals treated intravenously with melarsoprol, the standard drug for the late stage disease. In 8 of the 9 monkeys treated with melarsoprol, the antigen titres, as assessed by optical density values, dropped by 50% within 252 days (mean value 68 days for antigens in CSF and 116 for serum) following treatment. The remaining animal in this group, that displayed persistent antigenaemia, had been treated with a sub-curative drug dosage level. Thus, if time to 50% reduction in antigen levels were to be taken as an index to predict cure, the follow-up period after melarsoprol treatment could have been reduced from 600 to 252 days for 8 of the 9 animals, leaving only one animal for further follow up. The animals treated with experimental drug combinations displayed a variable picture. Five monkeys showed a persistence of antigens in both serum and CSF throughout the observation period, suggesting failure of the drugs to cure the infection. Parasitologically confirmed relapse of the infection was indeed observed in all the five monkeys. In some monkeys, the parasite antigens eventually cleared from serum and CSF completely, but this took a longer time duration than in the melarsoprol treated animals; others showed persistence of parasite antigens in serum, but the parasites were not detected in blood or CSF throughout the entire follow-up period. These results suggest that the experimental drug combinations used were not effective in clearing the parasites from cryptic foci and hence the persistence of antigens in serum and/or CSF.(ABSTRACT TRUNCATED AT 250 WORDS)

Synergistic activity of 5-substituted 2-nitroimidazoles (Ro 15-0216 and benznidazole) and DL-alpha-difluoromethylornithine on Trypanosoma brucei brucei.

The antitrypanosomal activity of two 5-substituted 2-nitro-imidazoles (Ro 15-0216 and benznidazole) and alpha-DL-difluoro-methylornithine (DFMO) was tested in four stocks of Trypanosoma brucei brucei in vitro. The IC50 (drug concentration which inhibits growth of trypanosome populations by 50%) values ranged from 0.27-1.0 for Ro 15-0216, 84-265 for benznidazole, and 147-691 microM for DFMO. Potentiation of antitrypanosomal activity of the combination of Ro 15-0216 and DFMO was demonstrated in a 24 h growth inhibition test. A synergistic effect was also demonstrated when benznidazole and DFMO were combined in a long term viability assay in vitro. Although 40 microM DFMO and 20 microM benznidazole were ineffective when used individually, trypanosomes of all stocks were killed when both drugs were present simultaneously at these concentrations. The combination of 40 microM DFMO and 4 microM benznidazole led to growth suppression. At an early stage of infection, a single injection of 100 mg/kg Ro 15-0216 at the end of a 3-day treatment period with DFMO (2% in drinking water) resulted in a 100% cure of T. b. brucei-infected mice, whereas monotherapy with either drug at the same dose levels was completely ineffective. Nitroimidazoles and DFMO given simultaneously might improve the therapy of human sleeping sickness.

Differential susceptibility to DL-alpha-difluoromethylornithine in clinical isolates of Trypanosoma brucei rhodesiense.

DL-alpha-Difluoromethylornithine is an enzyme-activated inhibitor of ornithine decarboxylase and an antagonist of polyamine metabolism that has been successful in clinical trials against West African sleeping sickness caused by Trypanosoma brucei gambiense. Its potential for use against the more virulent East African form of the disease, caused by T. brucei rhodesiense, is not certain. We examined 14 East African clinical isolates from the Kenya Trypanosomiasis Research Institute strain bank plus 2 established isolates for susceptibility to DL-alpha-difluoromethylornithine and to standard trypanocides. Seven of 16 strains were partially or totally refractory to DL-alpha-difluoromethylornithine in our test system. Four strains were also refractory to arsenical drugs, and five were refractory to diamidines. The results indicate that other novel agents or combinations of established agents may be needed for chemotherapy of East African disease.

Homidium bromide as a chemoprophylactic for cattle trypanosomiasis in Kenya.

Homidium bromide was used in a strategic chemoprophylactic regime to control trypanosomiasis in Boran cattle in Kenya. Trypanosome infection rates in cattle receiving homidium bromide prophylaxis were compared with those in control cattle which received no prophylaxis but were treated with diminazene aceturate when infected. Homidium bromide was administered twice during the year after which no infections were detected for periods of nineteen weeks and seventeen weeks respectively. The drug sensitivity of the infecting trypanosomes is believed to be a major factor in determining the duration of prophylaxis.

Serodiagnosis of African sleeping sickness in vervet monkeys by detection of parasite antigens.

Sera of vervet monkeys experimentally infected with T. b. rhodesiense were examined using a double antibody sandwich ELISA and Procyclic Agglutination Trypanosomiasis Test (PATT) for the presence of circulating trypanosomal antigens and anti-procyclic surface antibodies, respectively. Trypanosomal antigens were detected at 7 days post infection and remained at a detectable level thereafter during the infection. Antigens were not detected in sera prior to experimental infection or at 26 days after trypanocidal drug treatment. Although both the PATT and the sandwich ELISA results correlated with the infection status of the animals, the sandwich ELISA gave a better indication of the disease progression than the PATT, especially during trypanocidal drug therapy. The results illustrate the potential utility of the double antibody sandwich ELISA for diagnosis of African sleeping sickness.

Evidence of blood cerebrospinal fluid barrier permeability impairment in Trypanosoma rhodesiense infection in vervet monkeys.

Blood-cerebrospinal fluid (CSF) barrier damage in 11 vervet monkeys was estimated before infection and during the early and late phases of Trypanosoma rhodesiense disease, using the method given by Tibbling, Link and Ohman (1977). Of the 11, only one monkey showed signs of barrier impairment that ranged from a slight (12.6) to total barrier impairment (285); the latter occurring just before the height of clinical encephalitis. The barrier reverted to normal after melarsoprol treatment at 1.8 mg/kg X 4. Between the two extremes of barrier impairment, there were periods of remission during which time the barrier reverted to normal. This monkey however continued to suffer epileptic fits for a period of three months, a condition that started soon after the treatment.

Improved clinical approach to the diagnosis of canine ehrlichiosis.

Canine ehrlichiosis is being recognised with increasing frequency in many parts of the world. Based upon a detailed clinical and laboratory examination including a simple in vitro blood culture diagnostic test 373 cases have been classified into seven broad groups. These groups include acute, haemorrhagic, chronic, uraemic, subclinical, carrier state and those with babesiosis. While many of these groups overlap they will assist the clinician in diagnosis and allow the application of specific therapy before the disease progresses to a chronic irreversible stage.

Use of procyclic trypanosomes for detection of antibodies in sera from vervet monkeys infected with Trypanosoma rhodesiense: an immunodiagnostic test for African sleeping sickness.

Uncoated procyclic culture forms of African trypanosomes were used in immunofluorescence and simple agglutination assays to detect antibodies in the sera of vervet monkeys infected with T. b. rhodesiense. Antibodies to procyclic surface antigens were found in sera from animals with active, untreated infections or sera taken soon after treatment with trypanocidal drugs. The antibodies were detectable within 7 days of infection. No specific antibodies were detected in sera prior to infection or long after drug cure. The results indicate that antigens expressed on the surface of procyclic culture forms of T. brucei spp. are useful for the detection of antibodies produced in response to infection with T. b. rhodesiense and may allow the development of a simple immunodiagnostic test for African sleeping sickness. In addition, the use of a form of the trypanosome of a different differentiation state from the infecting organism illustrates the utility of this approach for detection of antibodies to common antigens.

Trypanotolerance in East African Orma Boran cattle.

Comparative studies on two types of large East African zebu (Bos indicus) Boran cattle, on a beef ranch in Kenya, have indicated that a Boran type bred by the Orma tribe has a superior response to tsetse fly challenge. The Orma Boran when compared with an improved Boran was found to have lower trypanosome infection rates and, when untreated, better control of anaemia and decreased mortality.

Cryosurgery of bovine squamous cell carcinoma of the vulva.

Sixty-two cattle with vulval carcinoma of various sizes were treated by cryosurgery using double freeze-thaw cycles. A cure rate of 88.7 per cent was achieved. It was confirmed that the smaller the lesions the better the response and that in larger lesions failure occurred because of the difficulty in freezing the mass of neoplastic tissue rapidly to the lethal temperature of -25 degrees C using the cryosurgical unit available. It was concluded that, if instituted early in the clinical course of the disease, cryotherapy of bovine vulval carcinoma is superior to surgical excision but the cost of the equipment may be a limiting factor in its application in the field.

Trypanosomal cardiac valvulitis in vervet monkeys.

Quarantined vervet monkeys (Cercopithecus aethiops) were infected with Trypanosoma brucei (10(4) parasites/animal) in a tsetse free area. Thirteen monkeys (11 infected with T.b. rhodesiense and 2 with T.b. brucei) were studied. Animals became parasitaemic within one week after infection. The infection time lasted between 21 and 129 days; in 8 monkeys it was between 50 and 70 days. Macroscopically massive pericarditis was observed in one, pericardial effusion in one, small apical aneurysms in three. Microscopically all had pancarditis of moderate to marked degree including all 4 chambers, the apices and the valvular appendages. Trypanosomes and inflammatory cells were found in the interstitium of the 3 cardiac layers. Lymphatic drainage of trypanosomes and inflammatory cells was seen in all hearts, the lymphatic vessels showing signs of stasis. By light microscopy trypanosomes and inflammatory cells were also shown to occur in all four types of heart valves implicating diffuse valvulitis. In two instances electron microscopy confirmed the intact presence of the trypanosomes clustering in great numbers in the pars elastica of the valves. In the collagenous part, some intact single trypanosomes were seen between collagen fibres. No amastigotes were observed. These results point to select sites in organ topography which in turn may be relevant to active disease, chemotherapy and healing. The possibility of hiding places within the heart - protecting trypanosomes from the host defense and/or chemotherapeutic agents - ought to be considered.

Tick survey on goats and sheep in Kenya.

A tick survey was done on sheep and goats in Siaya and Kakamega Districts, Kenya between October 1980 and October 1981. Most of the animals were found to carry one to 10 ticks with no significant difference between sheep and goats. The most abundant species was R. appendiculatus followed by R. evertsi. There were more male than female ticks found on the animals. Thirty per cent of the farms visited practised the hand picking method of tick control while 14% regularly dipped their animals.

Immunopathological aspects of trypanosomal meningoencephalitis in vervet monkeys after relapse following Berenil treatment.

Four quarantined vervet monkeys were treated with intramuscular Berenil in patent CNS infection after experimental trypanosome inoculation with Trypanosoma brucei rhodesiense or T. brucei brucei. All four animals relapsed in the post-therapeutic survival time of 37 to 209 days when they had fully developed meningoencephalitis in histological sections with the presence of interstitial intracerebral trypanosomes, which were confirmed in two monkeys by electron microscopy. In both, sequential samples of the serum and cerebrospinal fluid were analysed for circulating immune complexes, immunoglobulins and albumin. From these results the intracerebral IgG synthesis and the impairment of the blood-brain-barrier were calculated, both being present in advanced infection. Circulating immune complexes were present in the serum, but could not be demonstrated in the cerebrospinal fluid. The monkey model therefore permits the study of various aspects of cerebral trypanosomiasis. Berenil treatment is inefficient in patent CNS infection and leads to a protracted, less virulent disease course with terminal meningoencephalitis and intracerebral "persister" trypanosomes. This drug-induced trypanosome shift with meningoencephalitis could be used for chemotherapeutic purposes to test new compounds in late stage disease.