Beyond the microcirculation: sequestration of infected red blood cells and reduced flow in large draining veins in experimental cerebral malaria.

Sequestration of infected red blood cells (iRBCs) in the microcirculation is a hallmark of cerebral malaria (CM) in post-mortem human brains. It remains controversial how this might be linked to the different disease manifestations, in particular brain swelling leading to brain herniation and death. The main hypotheses focus on iRBC-triggered inflammation and mechanical obstruction of blood flow. Here, we test these hypotheses using murine models of experimental CM (ECM), SPECT-imaging of radiolabeled iRBCs and cerebral perfusion, MR-angiography, q-PCR, and immunohistochemistry. We show that iRBC accumulation and reduced flow precede inflammation. Unexpectedly, we find that iRBCs accumulate not only in the microcirculation but also in large draining veins and sinuses, particularly at the rostral confluence. We identify two parallel venous streams from the superior sagittal sinus that open into the rostral rhinal veins and are partially connected to infected skull bone marrow. The flow in these vessels is reduced early, and the spatial patterns of pathology correspond to venous drainage territories. Our data suggest that venous efflux reductions downstream of the microcirculation are causally linked to ECM pathology, and that the different spatiotemporal patterns of edema development in mice and humans could be related to anatomical differences in venous anatomy.

Ca2+ sensitization in idiopathic dilated human myocardium. Differential in vitro effects of (+)-(5-methyl-6-phenyl)-1,3,5,6-tetrahydro-3,6-methano-1,5-benzodiazoci ne-2,4-dione, a novel purely Ca2+sensitizing agent, and (+)-5-(1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydroquinolin-6-yl)-6-meth yl-3, 6-dihydro-2H-1,3,4-thiadiazin-2-one on skinned fibres and isolated ventricular strips.

(+)-(5-Methyl-6-phenyl)-1,3,5,6-tetrahydro-3,6-methano-1, 5-benzodiazocine-2,4-dione (CAS 165755-40-8, CGP 48506) is a novel Ca2+ sensitizing agent devoid of any other positive inotropic mechanism, particularly phosphodiesterase (PDE) III inhibition. 5-(1-(3,4-Dimethoxybenzoyl)-1,2,3,4-tetrahydroquinolin-6-yl)-6-met hyl-3, 6-dihydro-2H-1,3,4-thiadiazin-2-one (CAS 120223-04-3, EMD 53998) is a PDE III inhibitor with a Ca2+ sensitizing activity residing in its (+)-enantiomer, EMD 57033 (CAS 147527-31-9). In skinned fibres and electrically stimulated left ventricular strips from idiopathic dilated human hearts, New York Heart Association (NYHA) class IV, the Ca2+ sensitizing and inotropic effects of the benzodiazocine CGP 48506 and the thiadiazinones EMD 53998 or EMD 57033 were compared. Both CGP 48506 and EMD 53998 induce a left shift of the Ca2+ activation curve of force towards lower Ca2+ concentrations in skinned fibres, which indicates Ca2+ sensitization. Only EMD 53998, but not CGP 48506, increases skinned fibre force at both minimum (resting) and maximally activating Ca2+ concentrations. This is taken as an argument for a principal difference in the mechanisms of the Ca2+ sensitizing actions of the two compounds. CGP 48506 is shown not to influence the amplitude of the Ca2+ transient in rat cardiomyocytes. On the other hand, both CGP 48506 and EMD 57033 show comparable, though quantitatively different, positive inotropic effects in electrically stimulated left ventricular strip preparations. It is unclear whether the PDE III inhibitory component of the profile of actions of EMD 57033 may play a role in preventing the increase in diastolic tension as expected from the skinned fibre experiments. It is noteworthy that both Ca2+ sensitizing agents act as positive inotropic compounds in the end-stage failing human heart where other inotropic agents like beta 1-adrenergic agonists or PDE inhibitors have been described to fail.

5-Methyl-6-phenyl-1,3,5,6-tetrahydro-3,6-methano-1,5-benzodiazocine-2,4-dione (BA 41899): representative of a novel class of purely calcium-sensitizing agents.

BA 41899 (5-methyl-6-phenyl-1,3,5,6-tetrahydro-3,6-methano-1,5- benzodiazocine-2,4-dione, 6) is a structurally novel 1,5-benzodiazocine derivative and represents the prototype of a hitherto unknown class of positive inotropic Ca(2+)-sensitizing agents. It is completely devoid of phosphodiesterase (PDE) III inhibitory activity or any other known inotropic mechanism. BA 41899 (6) exhibits a pharmacological in vitro profile comprising Ca(2+)-sensitizing, positive inotropic, and negative chronotropic effects. CGP 48506 ((+)-6), the (+)-enantiomer of BA 41899 (6), enantiospecifically carries Ca2+ sensitization by up to a full pCa unit and a corresponding positive inotropic effect. Conversely, the negative chronotropic action resides in the corresponding (-)-enantiomer, CGP 48508 ((-)-6). All the effects are exerted in the low micromolar range. The positive inotropic action of CGP 48506 ((+)-6) is associated with a decelerating effect on contraction and, more prominently, relaxation dynamics in isolated guinea pig atria. In contrast to Ca(2+)-sensitizing PDE inhibitors, CGP 48506 ((+)-6) does not increase maximum Ca(2+)-activated force in myocardial skinned fibers.

Turnover of microfilariae in small mammals 2. Disintegration of microfilariae (Acanthocheilonema viteae) (Filarioidea: Nematoda) after intravenous injection into the jird, Meriones unguiculatus.

After i.v. injection of 305 x 10(3) microfilariae (mf) per animal (50 g) into naive jirds, 50.8% of them could be recovered at autopsy 15 min later. Of these, 65.8% were calculated to be in the peripheral circulating blood (PCB) and were completely intact; 18.6% were recovered by perfusion of the lungs and 13.6% from the liver. In both organs about half the mf were associated with adherent lymphocytes and neutrophils but a few were partly disintegrated. Only 2.6% were recovered from the kidneys and the spleen. In long-term injection experiments using the same inoculum size the autopsy was done 15 min and 1, 3 and 6 weeks post-injection (p.i.) of mf into naive jirds. Throughout the experimental period the density of mf remained more or less constant in the PCB, but 3 weeks p.i. the density in the lungs increased up to 14 times to that in the PCB, whereas in the liver it decreased at the same time to a density similar to that in the PCB. In patent animals with adult worms delivering mf these were distributed as follows: 34.7% were calculated to be in the PCB; 24.4% were obtained by perfusion from the lungs and 22.0% from the liver; the rest were found in the kidneys (16.6%) and spleen (2.3%). In the lungs and the liver about 5/6 were associated with adherent cells, partly disintegrated or as fragments. In view of the fact that very few mf become disintegrated immediately after i.v. injection and also from their extremely long sojourn in the PCB, a low turnover rate of mf is presumed.(ABSTRACT TRUNCATED AT 250 WORDS)

Turnover of microfilariae in small mammals. 1. Disintegration of microfilariae (Litomosoides sigmodontis) (Filarioidea: Nematoda) after intravenous injection into Sigmodon hispidus, the cotton rat.

After i.v. injection of 833 x 10(3) microfilariae (mf) per animal (150 g) into naive recipient cotton rats, at autopsy 15 min thereafter 30.4% of them could be recovered as a total: 19.1% were proved in the peripheral circulating blood (PCB) completely intact. 6.5% were recovered by perfusion of the lungs, of which 3/5 were associated with adherent macrophages and neutrophils or partly disintegrated. By perfusion of the liver only 3.8% were obtained, in spite of the four times greater volume of blood, of which 2/3 had adherent cells or were partly disintegrated. 0.7% and 0.3% were recovered from kidneys and spleen, respectively. In patent animals with adult worms the permanently delivered mf were distributed as follows: 41.6% were proved in the PCB; by perfusion 19.1% were obtained from the lungs and 32.9% from the liver; the rest of 6.4% were found in kidneys and spleen. In the capillary systems of lungs as well as the liver the proportion of normal mf (1/3), with adherent cells (1/3), partly disintegrated ones (1/5) and fragments (1/20) were quite similar. In long term mf injection experiments using the same dosage the autopsy was done 30 min and 1, 2, 3 and 28 days p. inj. of mf into naive animals. 30 min p.inj. 56% of the mf injected could be recovered as a total: 28.6% were obtained from the PCB, 16.0% from the lungs and 7.1% from the liver as normal mf (no perfusion), the rest of 4.1% from heart muscle, kidneys and spleen.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-dependent recovery of adult Acanthocheilonema viteae (Nematoda: Filarioidea) after single and trickle inoculations in jirds.

Increasing single doses of 5, 15, 30, 60 and 90 third-stage Acanthocheilonema viteae larvae per animal were inoculated into jirds. The adult worm load increased steadily, whereas the recovery rate decreased significantly, i.e. the correlation between dose and recovery was negative (rs, -0.90; n = 5; alpha, 0.05). The same inoculation doses were given as trickle inoculations of 5 L3 each (3 x 5, 6 x 5, 12 x 5, 18 x 5) at intervals of 2-6 days throughout the prepatency period. Irrespective of the number of repeated inoculations, a rather constant but low load of 7-10 worms/animal was reached. The recovery rate decreased drastically (rs, -1.0; n = 5). When trickle inoculations were carried out in animals exhibiting patient infections, the superinoculated larvae seemed to be destroyed almost completely; thus, a parasite-host equilibrium was guaranteed in all cases. The immunological background is discussed.

Pharmacological properties of five diclofenac metabolites identified in human plasma.

Five metabolites of diclofenac sodium (Voltarol) have been identified in human plasma. All five metabolites were more than 50 times less potent than diclofenac in inhibiting PGE2 production in zymosan-stimulated mouse macrophages and LTC4 synthesis was not inhibited in these cells. Anti-inflammatory activity (adjuvant arthritis and carragheenan-induced paw oedema in rats) and analgesic activity (phenyl-p-benzoquinone writhing, mouse) of the metabolites were at least 10 times lower when compared to diclofenac. There was a good correlation between in vitro PGE2 inhibition and in vivo activities for diclofenac and its metabolites indicating that inhibition of prostaglandin synthesis is a major mechanism responsible for their pharmacological actions.

Recovery of adult stages and microfilaraemia after low dose inoculation of third stage larvae of Litomosoides carinii in Sigmodon hispidus.

Because of the negative binomial distribution of filarial third stage larvae (L3) in their vectors, under natural conditions only a few are usually transferred per bite. After an inoculation of 5 L3 per animal into eight host animals at least one developed a long lasting patency based on one reproductive female only. After an inoculation of 15 L3, three of eight animals developed long lasting patency, harbouring between two and five fertile females. The rates of adult stages recovered were 0.43 and 0.30 respectively. The parasitaemias of the six patent animals in both experimental groups increased with the number of reproductive females present (r = 0.89, p = less than 0.005). All non-patent animals which were mf-negative in the pleural fluid and lung blood as well had a single sex or no worm load. In only one animal was there an apparently normal but non-reproductive pair of worms without any pathological alterations of the host animal. Encapsulated adult worms were found rarely, but independent of the final worm load or inoculation dose and always beside normal adults. In three of the 16 animals inoculated with 5 or 15 L3 patency passed after 10-12 weeks p.i., in two others it seemed to pass soon. After inoculation of 30, 40 or 60 L3 per animal patency passed early in about one half of 105 animals, when they were observed up to 24-36 weeks p.i. In conclusion all types of host defensive reactions are already visible after inoculation with such small doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental onchocerciasis in chimpanzees. Cell-mediated immune responses, and production and effects of IL-1 and IL-2 with Onchocerca volvulus infection.

Nine of eighteen chimpanzees inoculated with infective third-stage larvae of Onchocerca volvulus developed patent infection with microfilariae in skin biopsies. In all infected chimpanzees the in vitro cellular reactivity to O. volvulus adult worm-derived Ag (OvAg) increased significantly after exposure to third-stage larvae. However, during prepatency the in vitro cellular responses to OvAg decreased gradually in subsequently mf positive (patent) animals, and returned with patency to values not different to those before infection. In non-patent chimpanzees cellular responses remained significantly higher than before infection. Stimulation of PBMC in vitro with bacterial Ag and mitogen did not show any differences between the experimental groups through 20 months p.i. The addition of exogenous IL-2 did not restore the impaired responses of PBMC to OvAg in patent animals. Exogenous IL-2 elicited an additive increase of the cellular response to OvAg in nonpatent, and a mitogenic effect to OvAg in patent animals. Selective depletion of adherent, suppressor/cytotoxic (CD8+), NK cells (CD16+) and the use of autologous serum had no effect on antigenic and mitogenic cellular responsiveness. OvAg-induced IL-2 production decreased after patency, whereas, IL-1 production was significantly greater in both patent and nonpatent than in control chimpanzees. In summary, these data demonstrate that experimental O. volvulus infection in chimpanzees stimulated a substantial cell-mediated immune response. In patent chimpanzees an OvAg-specific cellular hyporesponsiveness occurred before onset of patency, possibly due to decreased IL-2 production and responsiveness.

The vector host link in filariasis.

Only a minority of microfilariae taken up by the appropriate vector develop to the infestive stage, and the distribution of the resulting third-stage larvae in the vector population is overdispersed. The larvae which reach the final host at first stimulate an immune defence reaction, but this is later suppressed. The importance of these factors in the life cycles of vector-borne parasites is considered.

Identification of a potential protective microfilarial antigen from Litomosoides carinii (Nematoda, Filarioidea).

The proteins of the filarial parasite Litomosoides carinii that are presented to the host in the course of a natural infection were assessed by surface and intrinsic labelling, microfilarial agglutination assays and ELISA. Surface labelling revealed several proteins present on all parasite stages which were recognised by infected animals. Only one major surface protein of 55 KD was found on microfilariae, while in all other stages (adult males, adult females and infective larvae isolated from pool animals) a range of proteins could be labelled. Immunisation with live microfilariae obtained from blood provoked sera which agglutinated microfilariae in vitro and co-precipitated the 55 KD surface protein. A proportion of animals quantitatively infected with 30 infective larvae (L3) developed antibodies during prepatency that were capable of agglutinating microfilariae, suggesting a common epitope between L3/L4 and microfilariae. Intrinsic labelling of microfilarial proteins was used to identify three additional proteins of 16, 29 and 43 KD which may also be important during infection. The anti-microfilarial antibody titre was quantified by ELISA using microfilarial antigen and sera from various time points during the course of a natural infection.

Homologous and crossreacting immune response of the jird and cotton rat against microfilariae of Dipetalonema viteae and Litomosoides carinii (Nematoda: Filarioidea).

Consecutive injections of microfilariae of L. carinii and D. viteae were carried out in the homologous or heterologous sequence into the corresponding natural or into the experimental hosts. Microfilariae of D. viteae are imunogenic in Sigmodon specifically and crossreacting as well. However, in the natural host, Meriones, they are doubtlessly tolerogenic. The sojourn and level of microfilaraemia are prolonged resp. higher when living D. viteae-Mf are injected s.c. followed by an i.v. injection. However, freeze-killed microfilariae of D. viteae are immunogenic in Meriones. Obviously the two filarial worms living in small mammals realize the balance with their natural hosts in quite a different manner. Which one of the two possibilities, or if one of them at all, reflects the relationship in human pathogenic filariae remains open.

Structure-activity profile of a series of novel triazoloquinazoline adenosine antagonists.

During a search for benzodiazepine receptor modulators, a highly potent adenosine antagonist (CGS 15943) was discovered. The compound was defined as a resonance-stabilized hybrid of the canonical structures 9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (2a) and 9-chloro-2-(2-furyl)-5,6-dihydro[1,2,4]triazolo[1,5-c]-quinazolin- 5-imine (2b). Spectroscopic evidence and chemical reactivity in polar media favor the amine form 2a as the major contributor of the two canonical structures. The synthesis of 2 and some of its analogues and the structure-activity relationships in four biological test systems are described. Replacement of the 9-chloro group by hydrogen, hydroxyl, or methoxyl gave compounds with comparable binding potency at the A1 and A2 receptors but much less activity as antagonists of 2-chloroadenosine in guinea pig tracheal strips. Alkylation of the 5-amino group caused, in general, a loss of binding activity, particularly at the A2 receptor, as well as complete loss of activity in the tracheal model. Modification of the 2-furyl group caused a pronounced loss of activity in all of the test systems.

Studies on the dynamics of transmission of onchocerciasis in a Sudan-savanna area of North Cameroon I. Prevailing Simulium vectors, their biting rates and age-composition at different distances from their breeding sites.

Along the main water courses in the sparsely populated areas of the Sous-Préfecture of Tcholliré, the vectors of onchocerciasis were mainly Simulium damnosum s. str. and S. sirbanum, together with a small proportion of S. squamosum. Over a period of one to three years, vector biting rates were measured at 23 fly-catching sites in the vicinities of nine villages with different endemicities of onchocerciasis. Annual Biting Rates (ABR) on man were estimated as 26,100-83,800 fly-bites per man per year along the rivers Mayo Rey and Vina du Nord, and 11,000-37,400 at rainy season tributaries. Biting rates decreased rapidly at increasing distances from the river, and were between 10,700 and 2400 at 2-10 km cross-country from the breeding site. Lowest biting rates (50-6000) were measured at the village centres. The ABR varied from year to year in relation to the water-discharge of the main rivers, the coefficient of variation of the mean being 34-49%. The parous rates were 64-73% at the perennial breeding sites and only 17-44% away from the breeding sites, indicating dispersal mainly of young nulliparous flies and a reduced flight-range after oviposition.

Studies on the dynamics of transmission of onchocerciasis in a Sudan-savanna area of North Cameroon V. What is a tolerable level of Annual Transmission Potential?

The prevalence and intensity of infection with Onchocerca volvulus were assessed in population surveys in nine villages, situated at different distances from Simulium damnosum s.l. breeding sites. The prevalence varied from 48 to 89%, the arithmetic mean densities of microfilariae per skin snip were between 16 and 109, and severe ocular lesions were found in from 1 to 22% of patients. Annual Transmission Potentials (ATP) were measured for up to three years in the near vicinity of nine villages at several fly-catching sites. Weighted means of the ATP over the three years, and of the sojourn times of the human population, were calculated at three of the villages, where the prevalence of onchocerciasis was 51, 61 and 89%. An average ATP of 100 larvae or less in the head, thorax and abdomen of the flies was associated with an onchocerciasis prevalence of 50 to 60%, a mean microfilarial density below 40 microfilariae per skin-snip, less than 5% of ocular lesions, and no onchocercal blindness. This value might therefore be considered to be an indication of the level to which the transmission must be reduced in the savanna in order to prevent the occurrence of severe ocular lesions or blindness. It is lower than the present level accepted by the Onchocerciasis Control Programme in the Volta River Basin.

T-cell dependent and T-cell independent immune response in normal and in with Litomosoides carinii infected cotton rats.

The immune responses to SRBC and LPS were examined in immunized and non-immunized inbred cotton rats at different times after an infection with Litomosoides carinii. During the infection, the T-cell dependent immune response to SRBC was significantly suppressed in non-immunized animals as compared to non infected controls. In contrast, after immunization with SRBC the antibody levels of infected animals were significantly higher in three of five events. At the onset of patency the response to SRBC was significantly depressed transiently in immunized animals. A corresponding depression could be provoked by injections of isolated microfilariae as well as by FCS in immunized animals, infected and non-infected. The level of the antibodies was dependent on the density of microfilariae in the peripheral blood. The T-cell independent response to LPS was equal in non-immunized infected animals as in controls. After immunization with LPS during prepatency and at onset of patency a distinct but commensurate reaction was observed. Only after immunization at the peak of patency, the immune response of infected animals was significantly suppressed. In general, the antibody levels to LPS were not influenced by the microfilaremia.