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Serum levels of reactive nitrogen intermediates (RNI; nitrate + nitrite), interferon gamma (IFN gamma) and tumour necrosis factor (TNF) were measured in 177 Papua New Guinean children with different clinical manifestations of malaria. The groups investigated were asymptomatic parasitaemic, mild malaria, cerebral malaria survivors and cerebral malaria non-survivors. The levels of TNF were highest among the cases of cerebral malaria who died and lowest among the asymptomatic parasitaemic children (mean log TNF levels 2.183 pg/mL vs. 1.455 pg/mL; P = 0.001). Similarly, the levels of IFN gamma were highest among the cerebral and lowest among the asymptomatic patients (mean log TNF levels 0.338 pg/mL vs 0.054 pg/mL; P < 0.0001). RNI levels were high among both the asymptomatic parasitaemic group and those who died due to cerebral malaria (mean log RNI levels 1.56 microM vs. 1.412 microM; P = 0.18). The ratio of RNI to TNF, however, was significantly higher among the asymptomatic parasitaemic children and lowest among those who died due to cerebral malaria (mean log (RNI:TNF) ratio 0.118 vs. -0.789; P < 0.001). We concluded that the ratio of serum RNI to serum TNF is a more useful indicator of outcome of falciparum malaria in this population than the absolute levels of either alone.

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In this review we summarise the arguments that inflammatory cytokines, triggered by material released from the parasite at schizogony (malarial toxin), might induce the illness and pathology seen in malaria. These pro-inflammatory cytokines can generate inducible nitric oxide synthase and cause nitric oxide to be released, as can low concentrations of malarial toxin itself provided interferon-gamma, which has only low activity in the absence of malarial toxin, is present. We suggest here that recently described hypermetabolic functions of these mediators provide a much more plausible explanation for malarial hyperlactataemia and hypoglycaemia, the chief prognostic indicators in falciparum malaria, than does hypoxia secondary to mechanical blockage of vessels by sequestering parasites, which is the dominant current theory. We also review the arguments that rationalise, through these mediators, the reversibility of the coma of cerebral malaria. Although not yet tested at a cellular level, the proposal that nitric oxide generated in cerebral vascular walls contributes to this coma continues to gather indirect support. In addition, new evidence incriminating nitric oxide in the mechanism of tolerance to endotoxin rationalises the raised nitric oxide generation seen in malarial tolerance.

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Autoimmune disease is generally rare in tropical rural populations. Plasma concentrations of nitrite plus nitrate (reactive nitrogen intermediates), reflecting high nitric-oxide production somewhere in the body, can be high in patients who have cerebral malaria, but even higher in symptom-free parasitised individuals, who are termed malaria-tolerant. We propose that the nitric oxide causing high serum levels of reactive nitrogen intermediates in malaria-tolerant individuals is generated in macrophages during the establishment and maintenance of malarial tolerance, and makes autoimmune disease rare in many tropical rural populations by minimising proliferation of autoreactive T cells. Conversely, innately low levels of nitric-oxide generation in these populations, selected by malarial disease in tropical areas, could rationalise their high frequency of autoimmune disease and hypertension when living in western societies.

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Serum levels of reactive nitrogen intermediates (RNI; nitrate plus nitrite) were measured in 92 patients with cerebral malaria in the Madang Province of Papua New Guinea. RNI levels were compared to disease severity and clinical outcome, and correlated with both the depth of coma on admission and its duration. Median levels were higher among children with deeper coma than among those with lighter coma (35.6 microM vs. 16.7 microM; P = 0.008) and also among children with longer duration of coma (72 h; 59.3 microM vs. 19.3 microM; P = 0.004). RNI levels also correlated with clinical outcome, fatal cases having significantly higher RNI levels than survivors (41.2 microM vs. 18.5 microM; P = 0.014). Thus, high RNI levels are associated with indices of disease severity and may predict outcome in children with cerebral malaria. These data are consistent with the hypothesis that nitric oxide is involved in the pathogenesis of coma in human cerebral malaria.

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A cDNA expression library was constructed in lambda gt11 using poly A mRNA from the metacestode stage of Taenia crassiceps. The library was screened with rabbit antiserum to a previously defined protein fraction from Taenia hydatigena immunodiagnostic for bovine cysticercosis and with sera from cattle with experimentally induced cysticercosis. One clone (lambda TcA2) containing a 279-bp cDNA insert, reacted strongly with both antisera. A second clone (lambda TcA5.5) revealed the full-length cDNA sequence to be 361 bp. Data from Southern blots and enzymatically amplified genomic DNA segments were consistent with multiple copies or a gene family within the genome. The lambda TcA2 cDNA insert was subcloned into the plasmid pPR987 which generated a 47-kDa maltose-binding fusion protein (TcA2-MBP). Affinity-purified TcA2-MBP antigen reacted positively by ELISA with sera from cattle with experimentally induced T. saginata infections but not with sera from cattle with Fasciola hepatica or common gastrointestinal parasite infections. Rabbit polyclonal, monospecific antisera to TcA2-MBP recognized a 10-kDa protein in the cyst fluid, body wall and excretory/secretory products of the metacestode stage of T. crassiceps and immonolocalized this protein to organelles within the matrix of the cyst wall.

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Protoscolices of Echinococcus granulosus isolated from hydatid cysts of sheep and donkeys in Jordan were cultured in vitro using a modified diphasic culture system. Protoscolices from these two sources manifested differences in the mode of development, evagination and growth rates. Protoscolices isolated from sheep cysts grew in vitro in the polyzoic direction up to the three- to four-segmented mature worms, reaching a length of 2.9 mm. In contrast, donkey protoscolices failed to develop beyond the early stages, even after 67 days of culturing. On prolonged culturing, few worms of donkey origin reached the banding and segmentation stages, attaining a maximal length of 1.6 mm at periods ranging between 81 and 114 days of culturing. None of these segmented worms showed genital differentiation or analgen. The evagination and growth rates of protoscolices isolated from donkey liver cysts were compared with those obtained from sheep liver or lung cysts. The most significant difference in these rates occurred at the commencement of the segmentation stage. Differences in the development, growth and evagination rates observed between the donkey and sheep forms may reflect the strain variation of E. granulosus in this country.

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The 70% ammonium sulfate-soluble fraction of the cyst fluid of Taenia hydatigena (designated ThFAS) was previously shown to have potential as an immunodiagnostic reagent for bovine cysticercosis. Western blot analysis indicated that the specific reactivity with antibodies in sera of T. saginata-infected cattle was associated with a 10 kDa component. Rabbit antiserum to ThFAS identified a homologous antigenic protein from the cestode Taenia crassiceps. Consequently, a cDNA expression library was constructed in lambda gt11 using poly A mRNA purified from T. crassiceps metacestodes and screened with rabbit antiserum to ThFAS. One strongly reactive clone (designated lambda TCA-2) produced a 123 kDa beta-galactosidase fusion protein which reacted in Western blot with sera from calves experimentally-infected with T. saginata and did not react with sera from uninfected calves or from cattle infected with Fasciola hepatica or with common gastrointestinal cattle parasites.

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The continuous culturing of Trypanosoma acomys in the presence of a murine areolar-adipose cell line (A9) was possible for the 1st time. The trypanosomes were cultured at 37 degrees C with A9 in DMEM supplemented with 20% heat inactivated fetal bovine serum, using an initial inoculum from primary cultures of lung or blood clots from infected spiny mice. The cultures were maintained for 115 days and underwent 15 passages before termination and cryopreservation. Using this culture system T. acomys subcultures were initiated from 3 different initial inocula (3 x 10(4), 1.5 x 10(5) and 7.4 x 10(5) parasites/ml) and growth curves revealed that the lowest inoculum gave the best growth pattern. This inoculum yielded a population doubling time of less than 12 h for 4 days, a high peak density of 7 x 10(6) parasites/ml and the most gradual decline compared to the other 2 inocula. Rosetting epimastigotes and nests of amastigotes were observed in close association with the feeder layer cells. Epimastigotes were the most predominant form in culture supernatants but other morphological forms observed included trypomastigotes and sphaeromastigotes.

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Two parasite antigens have been isolated from Echinococcus granulosus hydatid cyst fluid using hydrophobic interaction chromatography, anion exchange chromatography and gel filtration chromatography. Initial characterization of the antigens indicates that both are glycoproteins, of approximately 20 and 48 kDa (Eg20 and Eg48). When the two antigens were tested with a battery of antisera from patients with heterologous parasitic infections, only Eg20 was found to be specific for E. granulosus. The Eg48 antigen cross-reacted with the sera of 33% of E. multilocularis patients. In both antigens, some of the epitopes recognized by antibodies in the sera of hydatid patients were periodate-sensitive. This suggests the involvement of carbohydrates in at least some of the antigenic determinants. Due to the abundance of the Eg48 antigen in the hydatid cyst fluid, it would be the more practically useful antigen for disease diagnosis, especially in countries where only E. granulosus is endemic.

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A total of 380 ewes of different age groups were inspected for spleen hydatidosis during the summer of 1985. Of these, 7.6% had cysts in the spleen. There was an increase in the prevalence of the disease with increasing age of the animal, reaching 11.8% in greater than or equal to 8-year-old ewes. Out of all the animals with spleen hydatidosis, 61.5% were found to have fertile cysts and, when classified, 46.7% were found to be fertile with a mean total number of protoscolices of 13.4 x 10(3) per cyst. The majority of the protoscolices were found to be viable (74%). Spleen hydatidosis which is mainly solitary seems to be secondary to the involvement of the liver or the liver and lung simultaneously. The importance of these aspects of spleen hydatidosis are discussed.

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Trypanosoma acomys (Stercoraria, Trypanosomatidae) was studied in the laboratory-bred spiny mouse Acomys cahirinus. Only trypomastigotes were found in peripheral blood, whereas the thymus of experimentally infected A. cahirinus showed amastigote syncytial reproductive forms whose nuclei of nuclei ranged from 2 to 128. These reproductive forms were detected 9-10 days after the i.p. injection of spiny mice with trypanosomes obtained from primary lung cultures. Transitional trypomastigote-like forms were observed in the thymus; no reproductive or diving forms were encountered in any other tissue, including blood. The course of the parasitemia in spiny mice inoculated i.p. with 1.2 x 10(6) parasites from 26- to 32-day-old cultures initiated from infected lungs was studied. A similar, characteristic pattern was observed in both male and female hosts. Four phases were recognized: latent, logarithmic, stationary, and decline. These phases ended on days 2, 14, 98, and 154 postinoculation, respectively. The decline phase was gradual and the parasites could not be detected by day 154 postinoculation. In contrast, inoculation from older cultures (35-50 days old) showed longer latent and logarithmic phases, a shorter stationary phase, and a sharp drop in parasite density during the decline phase; the parasites were not detected in the peripheral blood by 120 days postinoculation.

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Epidermal keratinocytes grow clonally when provided with a 3T3 feeder layer and medium supplemented with 20% foetal bovine serum, hydrocortisone and cholera toxin. In this culture system the proliferative response of freshly isolated human and mouse keratinocytes to a short exposure (24 h) of the tumour promoter phorbol, 12-myristate, 13-acetate (PMA) was dependent on the donor age but was independent of the species or the biopsy site. Human keratinocytes from early (16-18 week) foetal donors displayed a strong proliferative response to PMA as determined by a 5- to 7-fold increase in colony number and an increase in the average colony size. In contrast, adult and juvenile keratinocytes of all ages from both mice and humans displayed an inhibition of colony forming efficiency and a reduction in colony size. When continuously passaged in culture human foetal keratinocytes gradually changed the pattern of their response to PMA so that they were inhibited from growing rather than being stimulated and after 21 days (three passages) their response was quantitatively similar to juvenile keratinocytes. Co-culture of juvenile keratinocytes with irradiated foetal keratinocytes did not alter their response to PMA so that the observed proliferative response of foetal keratinocytes to PMA is not readily explained by the autosecretion of mitogens or other regulatory molecules by these cells. Late foetal (17 days gestation), neonatal and post-neonatal (5-10 days old) mouse keratinocytes were also inhibited from growing by PMA but the magnitude of the effect was directly related to the age of the mouse and was in all cases less than that observed with adult mice. The relationship of these results to the mechanism of action of phorbol esters in epidermis is discussed.

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Examination of 471 sheep, 118 goats, 157 cattle and 56 camels slaughtered in abattoirs in North Jordan was carried out during March-May 1984. Drought conditions that prevailed during the preceding winter led to slaughtering old female sheep (greater than or equal to 4 years) due to scarcity of food, which allowed us to analyse the prevalence of hydatidosis in various age groups of sheep. An overall infection rate of 27.8, 1.7, 5.8 and 10.7 percent was found in sheep, goats, cattle and camels, respectively. The infection rate was as low as 1.5 percent in male and 1.9 percent in female sheep under 2 years of age. However, the rate of hydatid infection increased with age and reached as high as 63.7 percent in ewes 4 years of age and older. The percentage of animals with fertile cysts was also highest in sheep (68.7 percent of infected animals) and increased with age reaching 100 percent in ewes which were 10 years of age or older. Analysis of all cysts recovered from the livers and lungs of infected ewes from various age groups revealed a sharp increase in the mean total number of cysts in age groups over 8 years of age. The fertility rate of the cysts in the liver was significantly greater in ewes 6 years old or more (64.8--78.6 percent) than in younger age groups (8.7-46.2 percent). In the lung, the fertility rate increased progressively with age reaching as high as 97.9 percent in ewes 10 years old or more. These findings of high infection and fertility rates of hydatid disease in sheep, particularly of older age groups, prompt plans for further epidemiological studies and control programmes.

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The prevalence of hydatid disease was investigated in 704 sheep, 391 goats, 280 cattle and 68 camels slaughtered at two abattoirs in North Jordan. The infection rates for these animals were 4.0, 3.6, 11.4 and 8.8%, respectively. In general, older animals had higher infection rates than younger ones. The percentage of infected animals that had fertile cysts was 66.7% in camels, 34.3% in cattle, 28.6% in goats and 7.1% in sheep. Out of 143 single cysts recovered from cattle, 52.4% were fertile; 29.2% of cysts in goats, 14.7% of cysts in camels and 8.0% of those in sheep were fertile. However, infected sheep had more fertile multiple cysts than other animals. The lung was the predominant location for hydatid cysts in camels. In cattle and goats more hydatid cysts were found in the lung than in the liver, while in sheep the reverse was true.

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Anti-tumor immune reactivity of lymphocytes derived from lymph nodes regional to and distant from tumor growth, as well as that of peripheral blood leucocytes, against autochthonous tumor cells, was investigated. Experiments were carried out in vitro using a 51Cr cytotoxic assay and in vivo by cannulating the afferent and efferent lymphatics of regional and distant lymph nodes and challenging via the afferent lymphatics with 10(7) cultivated autochthonous tumor cells. No anti-tumor cytotoxic reactivity was detected in vitro using lymphocytes derived from any of the sources studied. In vivo, while challenge with autochthonous tumor cells produced no response in the regional lymph node, significant blast cell response was obtained in the distant node. The response at the distant node was associated with the production of antibodies that could bind to tumor cells without causing their demise. The anergy observed at the regional lymph node, and the possibility of a relation between the events occurring at that node and those observed at the distant node, are discussed.

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Seven epithelial tumour cell lines derived from naturally occurring ovine squamous cell carcinomata (OSCC) were examined for their ability to stimulate allogeneic lymphocytes in vitro. All cell lines failed to stimulate any proliferative response; a failure which was not due to the absence of alloantigens on the tumour cells. It was found, however, that the cell lines tested caused a marked suppression of a third party mixed lymphocyte reaction. This suppression was mediated only by live tumour cells since the addition of heat-killed tumour cells had no effect on the response.

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The ability of three cultivated epithelial tumor cell lines, derived from naturally occurring ovine squamous cell carcinomata, to stimulate a response in normal allogeneic sheep, was investigated. All three chosen cell lines failed to stimulate a response in allogeneic lymphocytes in vitro. In two of the tumors, it was shown that the lack of stimulation could be due to a suppressive effect by the tumor cells on lymphocytes. In vivo, two of the cell lines stimulated a marked cellular response which was associated with the production of high levels of antibody and which induced cytotoxic cell formation. The remaining cell line stimulated only a marginal response. No suppressive activity of the tumor cells was detected in allogeneic sheep. The significance of the response obtained to challenge with tumor cells, as compared to challenge with allogeneic lymphocytes from the tumor cell donor, is also discussed.

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