Microstructure, mechanical and corrosion properties of Mg-Dy-Gd-Zr alloys for medical applications.

In previous investigations, a Mg-10Dy (wt.%) alloy with a good combination of corrosion resistance and cytocompatibility showed great potential for use as a biodegradable implant material. However, the mechanical properties of Mg-10Dy alloy are not satisfactory. In order to allow the tailoring of mechanical properties required for various medical applications, four Mg-10(Dy+Gd)-0.2Zr (wt.%) alloys were investigated with respect to microstructure, mechanical and corrosion properties. With the increase in Gd content, the number of second-phase particles increased in the as-cast alloys, and the age-hardening response increased at 200°C. The yield strength increased, while the ductility reduced, especially for peak-aged alloys with the addition of Gd. Additionally, with increasing Gd content, the corrosion rate increased in the as-cast condition owing to the galvanic effect, but all the alloys had a similar corrosion rate (~0.5 mm year(-1)) in solution-treated and aged condition.

Effect of laser power and specimen temperature on atom probe analyses of magnesium alloys.

The influence of laser power, wave length, and specimen temperature on laser assisted atom probe analyses for Mg alloys was investigated. Higher laser power and lower specimen temperature led to improved mass and spatial resolutions. Background noise and mass resolutions were degraded with lower laser power and higher specimen temperature. By adjusting the conditions for laser assisted atom probe analyses, atom probe results with atomic layer resolutions were obtained from all the Mg alloys so far investigated. Laser assisted atom probe investigations revealed detailed chemical information on Guinier-Preston zones in Mg alloys.

Transcriptional response signature of human lymphoid cells to staphylococcal enterotoxin B.

Two shock-inducing toxins that result in similar eventual outcome of disease were studied to determine host gene expression responses, for correlation of both similar and unique gene patterns. We initially used differential display (DD)-PCR and identified 859 cDNA fragments that were differentially expressed after 16 h of in vitro exposure of human peripheral blood mononuclear cells (PBMC) to staphylococcal enterotoxin B (SEB). Upon further examination using custom cDNA microarrays and RT-PCR analysis, we found unique set of genes to each toxin (SEB or lipopolysaccharide (LPS)), especially at early time periods. By 16 h, there was a convergence of some gene expression responses and many of those genes code for proteins such as proteinases, transcription factors, vascular tone regulators, and respiratory distress. In an attempt to replicate the findings in vivo, monkeys were challenged with SEB and the resultant gene expression responses indicated a pattern typical of SEB exposure when compared to LPS, with a similar outcome. We provide evidence that vastly diverse global gene analysis techniques used in unison can not only effectively identify pathogen-specific genomic markers and provide a solid foundation to mechanistic insights but also explain some of the toxin-related symptoms through gene functions.

Anopheles arabiensis and An. funestus are equally important vectors of malaria in Matola coastal suburb of Maputo, southern Mozambique.

Transmission characteristics of malaria were studied in Matola, a coastal suburb of Maputo, the capital City, in southern Mozambique, from November 1994 to April 1996. The local climate alternates between cool dry season (May-October) and hot rainy season (November-April) with mean annual rainfall 650-850 mm. Saltmarsh and freshwater pools provide mosquito breeding sites in Matola. Malaria prevalence reached approximately 60% among people living nearest to the main breeding sites of the vectors. Plasmodium falciparum caused 97% of malaria cases, others being P. malariae and P. ovale. Potential malaria vector mosquitoes (Diptera: Culicidae) collected at Matola during daytime indoor-resting (n = 1021) and on human bait at night (n = 5893) comprised 12% Anopheles coustani Laveran (93% biting outdoors), 46% An. funestus Giles (68% biting indoors) and 42% An. gambiae Giles sensu lato (60% biting outdoors). All 215 specimens of An. gambiae s.l. identified genetically were An. arabiensis Patton. Anopheles funestus populations remained stable throughout the year, whereas densities of the An. gambiae complex fluctuated considerably, with An. arabiensis peaking during the rainy season. No concomitant rise in malaria incidence was observed. Human landing indices of An. funestus and An. arabiensis averaged 1.8 and 3.8 per man-night, respectively. Overall Plasmodium sporozoite rates were 2.42+/-1.24% in 2181 An. funestus and 1.11+/-1.25% in 1689 An. arabiensis dissected and examined microscopically. Mean daily survival rates were 0.79 for both vector species. Estimated infective bites/person/year were 15 An. funestus and 12 An. arabiensis. Biting rates were greatest at 2100-24.00 hours for An. funestus (68% endophagic) and 21.00-03.00 hours for An. arabiensis (40% endophagic). The entomological inoculation rate (EIR) declined sharply over very short distances (50% per 90m) away from breeding-sites of the vectors. Consequently, P. falciparum prevalence among Matola residents was halved 350 m within the town. Implications for the protective effectiveness of a 'cordon sanitaire' by residual house-spraying and/or the use of insecticide-treated bednets are discussed.

The differing impact of chloroquine and pyrimethamine/sulfadoxine upon the infectivity of malaria species to the mosquito vector.

Using serum or infected blood from Danish volunteers and Plasmodium falciparum-infected Mozambican patients, respectively, the impact of curative doses of chloroquine and pyrimethamine/sulfadoxine upon infectivity of P. falciparum to Anopheles arabiensis and An. gambiae or of P. berghei to An. stephensi was studied. Both treatments cleared circulating P. falciparum gametocytes within 28 days. Before this clearance, chloroquine enhanced infectivity to An. arabiensis, whereas pyrimethamine/sulfadoxine decreased infectivity. Patients harboring chloroquine-resistant parasites as opposed to -sensitive ones were 4.4 times more likely to have gametocytes following treatment. In contrast, pyrimethamine/sulfadoxine-resistant parasites were 1.9 times less likely to produce gametocytes. In laboratory infections using replicated P. berghei or P. falciparum preparations, serum from chloroquine-treated, uninfected, nonimmune volunteers enhanced gametocyte infectivity with increasing efficiency for 21 days following treatment, whereas pyrimethamine/sulfadoxine significantly suppressed infectivity. The observed enhancement in infectivity induced by the use of chloroquine combined with increased gametocytemias in chloroquine-resistant strains may in part explain the rapid spread of chloroquine resistance in endemic populations.

The Matola malaria project: a temporal and spatial study of malaria transmission and disease in a suburban area of Maputo, Mozambique.

A temporal and spatial study of malaria transmission in a suburban area of Maputo, Mozambique with a mean population density of 2,737/km2 was made from December 1992 to June 1995. A steep but continuous gradient was observed in the Plasmodium falciparum prevalence from 59.0% adjacent to the breeding sites to 5.4% only a few hundred meters distant. The entomologic inoculation rate ranged from a number too low to be determined in some districts to 20 infectious bites per person per year in the others. The risk of malaria was 6.2 times higher for individuals living less than 200 meters from the breeding sites than for individuals living 500 meters or more away from the breeding sites. In areas of high human density, mosquito and parasite dispersion is very limited, and therefore malaria control strategies could be more specifically targeted.

Plasmodium yoelii sporozoite infectivity varies as a function of sporozoite loads in Anopheles stephensi mosquitoes.

Mechanisms by which Plasmodium sporozoites survive and maintain their infectivity within the salivary glands of mosquitoes are unknown. In this study we establish a relationship between the number of sporozoites present in the salivary glands of individual mosquitoes (sporozoite load) and sporozoite infectiousness (or "quality") as measured by infections in BALB/c or ICR mice. When Plasmodium yoelii-infected Anopheles stephensi mosquitoes were each allowed to feed on a single mouse, we noted that sporozoites from mosquitoes with higher sporozoite loads were more infectious in 13 of 30 (43%) mice. In a second experiment, we inoculated mice with known numbers of sporozoites from individual mosquitoes. Eleven of 18 (61%) and 16 of 18 (89%) mice that received 25 and 100 sporozoites, respectively, became infected. For inoculations using 100 sporozoites, again we noted that sporozoites from mosquitoes with higher sporozoite loads were more infectious to mice. In a third and final experiment, the overall infectiousness of sporozoites from individual mosquitoes was evaluated first by allowing individual mosquitoes to feed on individual mice and then by intravenous inoculations of 100 sporozoites in a second mouse. There was a significant difference in host infections as a function of sporozoite loads in 14 of 19 (74%) mice. Analysis of the feeding times for infected versus noninfected mosquitoes did not show a significant difference between the 2 groups. The mean total feeding times for 50 infected and 45 noninfected An. stephensi mosquitoes were 306 (standard deviation [SD] = +/-230) and 441 (SD = +/-273) sec, respectively. Further, among infected An. stephensi mosquitoes there was no difference in probing times between cohorts that transmitted infectious sporozoites to mice and cohorts that failed to transmit infectious sporozoites. Our findings that sporozoite load influences sporozoite infectiousness or quality suggest that this may be an important factor in malaria parasite transmission.

Exflagellation responses of cultured Plasmodium falciparum (Haemosporida: Plasmodiidae) gametocytes to human sera and midguts of anopheline mosquitoes (Diptera: Culicidae).

The process of exflagellation was quantified for cultured Plasmodium falciparum gametocytes exposed to human sera and midgut homogenates from six vector species of Anopheles mosquitoes. Neither serum factors related to malaria exposure nor factors in the midguts of taxonomically diverse anophelines had significant inhibitory effects on the exflagellation of P. falciparum microgametocytes. Therefore, differences in vector competence among anopheline species most likely are caused by vector-parasite interactions occurring after microgametogenesis.

Anti-malarial drug development using models of enzyme structure.

BACKGROUND: The trophozoite stage of the malaria parasite infects red blood cells. During this phase of their life-cycle, the parasites use hemoglobin as their principal source of amino acids, using a cysteine protease to degrade it. We have previously reported a three-dimensional model of this cysteine protease, based on the structures of homologous proteases, and the use of the program DOCK to identify a ligand for the malaria protease. RESULTS: Here we describe the design of improved ligands starting from this lead. Ligand design was based on the predicted configuration of the lead compound docked to the model three-dimensional structure of the protease. The lead compound has an IC50 of 6 microM, and our design/synthesis strategy has resulted in increasingly potent derivatives that block the ability of the parasites to infect and/or mature in red blood cells. The two best derivatives to date have IC50(s) of 450 nM and 150 nM. CONCLUSIONS: A new class of anti-malarial chemotherapeutics has resulted from a computational search that was based on a model of the target protease. Despite the lack of a detailed experimental structure of the target enzyme or the enzyme-inhibitor complex, we have been able to identify compounds with increased potency. These compounds approach the activity of chloroquine (IC50 = 20 nM), but have a distinct mechanism of action. This series of compounds could thus lead to new therapies for chloroquine-resistant malaria.

Diel oviposition periodicity of anopheline mosquitoes (Diptera: Culicidae) from the Americas: Anopheles albimanus Wieldemann and Anopheles freeborni Aitken.

The diel oviposition patterns of Anopheles albimanus and An. freeborni derived from Panama and California, U.S.A., respectively, were studied in the laboratory by recording the egg-laying of individuals and colonies at 2-h intervals. Anopheles freeborni oviposition patterns were almost exclusively nocturnal, with 80% of eggs being laid during the scotophase. A large, well-defined peak of oviposition, comprising about 70% of eggs laid, occurred 4-6 h before sunrise. Anopheles albimanus oviposition patterns were bimodal with crepuscular and nocturnal components, 68% of eggs being laid shortly before lights went 'off' or 'on' and 32% during the scotophase. A small, well-defined peak of oviposition, comprising about 42% of eggs laid, occurred immediately following lights off (i.e. 18.00-20.00 hours). Nulliparous females allowed to engorge on human blood matured, on average, 104 follicles (range 80-142) for An. albimanus and 114 follicles (range 31-201) for An. freeborni.

Infectivity of Plasmodium vivax and P. falciparum to Anopheles tessellatus; relationship between oocyst and sporozoite development.

The assessment of malarial infectivity, for example in the evaluation of transmission blocking immunity, is generally based on counting oocysts in mosquitoes fed on infected blood. Ultimate transmission of the disease may, however, depend on the sporozoite load in the mosquito and its relationship to the size of the inoculum introduced to man. We conducted a laboratory study on Anopheles tessellatus infected with 108 different natural isolates of Plasmodium vivax from patients and 24 of P. falciparum to determine the relationship between oocyst numbers, sporozoite loads, and the effect of these on mosquito mortality. It was found that the P. vivax parasite density was positively correlated with the proportion of mosquitoes infected by a given feed at both the midgut and gland stages of parasite development (correlation coefficient [r] = 0.77, P < 0.001 and r = 0.6, P < 0.05 respectively). A significant positive linear correlation was observed between the number of oocysts and sporozoites in P. vivax (r = 0.5; P < 0.05); the proportions of mosquitoes infected with oocysts and sporozoites were also similarly related, although in general about 15% of mosquitoes infected with oocysts failed to develop salivary gland infections with sporozoites. The number of mosquitoes infected with P. falciparum parasites was too low for statistical analysis. Infection with either species of parasite did not appear to affect mosquito survival, nor was parasite density in the mosquito correlated with mosquito mortality.

Method to estimate relative transmission efficiencies of Anopheles species (Diptera: Culicidae) in human malaria transmission.

A mathematical expression was derived to estimate the relative malaria transmission efficiency of an anopheline species with respect to a standard well-characterized species for which all vector parameters can be sufficiently determined. The method is particularly useful in situations where multiple anopheline species contribute to human malaria transmission and requires the estimation of the man-biting rate, the sporozoite rate, and the human malaria incidence. Under stable conditions of vector abundance, the average sporozoite rate in a species during a transmission season would by itself reflect its relative transmission efficiency. This "efficiency" then was used to calculate the "effective human-biting rate"; i.e., the human-biting rate of that species if it were to have ecological properties identical to those of the standard species. The standard well-characterized species then could be used with the effective human-biting rate of all species to quantify transmission, thus overcoming the need to measure vector parameters for all anopheline species contributing to transmission. An expression also was derived to calculate the relative contribution made by each species to malaria transmission. The usefulness of this method was illustrated using entomological and epidemiological data from Kataragama, Sri Lanka.

Anti-circumsporozoite protein antibodies measure age related exposure to malaria in Kataragama, Sri Lanka.

Antibodies to two peptides DDAAD and (NANP)40 representing the repetitive sequence of circumsporozoite antigens (CS protein) of P. vivax and P. falciparum respectively were measured in a cohort of 149 and 107 individuals respectively at four, 6 monthly blood surveys performed on residents of Kataragama, a P. vivax malaria endemic region in southern Sri Lanka. The prevalence of antibodies to the CS protein of both species was relatively low being less than 20% to either peptide in the population as a whole, this being consistent with the low entomological inoculation rates in the area. A marked age related prevalence pattern was evident, with the prevalence of antibodies increasing with age to reach between 25 to 30% in the 25-50 year age group in both P. vivax and P. falciparum. The population had had a life long exposure to P. vivax malaria but not to P. falciparum, an epidemic of which occurred in this region a few months prior to the beginning of this study. Nevertheless, the age-related prevalence of these antibodies was identical with respect to the two species. This suggests that the age-related prevalence pattern reflected differences in inoculation rates between the age groups due to differences in exposure to inoculation rather than an age acquired response resulting from a cumulative experience over several years. An analysis of antibody prevalence in individuals showed first, that sporozoite inoculations must have been clustered rather than homogeneously distributed in the population and secondly, that sero-conversion did not correlate with malaria infections in these individuals.

Clustering of malaria infections within an endemic population: risk of malaria associated with the type of housing construction.

The occurrence of malaria infections due to Plasmodium vivax and P. falciparum was monitored in a population of 3,023 people living in six contiguous villages in Kataragama, an area of endemic malaria in southern Sri Lanka, over a period of 17 months. The annual incidence of malaria in this population during the study period was 25.8%. Malaria attacks were clustered, occurring more frequently than expected in certain individuals and housing groups and less frequently than expected in others. In one of these villages, the distribution of cases was examined in relation to locality and to the type of house construction. There was a strong association between the malaria incidence and house construction, independent of location. The risk of getting malaria was greater for inhabitants of the poorest type of house construction (incomplete, mud, or cadjan (palm) walls, and cadjan thatched roofs) compared to houses with complete brick and plaster walls and tiled roofs. Houses that were better constructed had a significantly lower malaria incidence rate (10.5%) than those that were poorly constructed (21.2%; P less than 0.01, by Student's t-test). There was also a significantly higher number of indoor resting mosquitoes collected from the poorly constructed houses than from those better constructed; the average (geometric mean) of mosquito densities found in houses of better versus poor construction were 0.97 and 1.89 per collection in the dry season, and 1.95 and 3.42 per collection in the wet season, respectively (P less than 0.05 in both seasons). This indicated that the higher malaria risk associated with poorly constructed houses was at least partly due to higher human-mosquito contact among their inhabitants.

A mathematical model for Plasmodium vivax malaria transmission: estimation of the impact of transmission-blocking immunity in an endemic area.

We have developed a multi-state mathematical model to describe the transmission of Plasmodium vivax malaria; the model accommodates variable transmission-blocking/enhancing immunity during the course of a blood infection, a short memory for boosting immunity, and relapses. Using the model, we simulated the incidence of human malaria, sporozoite rates in the vector population, and the level of transmission-blocking immunity for the infected population over a period of time. Field data from an epidemiological study conducted in Kataragama in the south of Sri Lanka were used to test the results obtained. The incidence of malaria during the study period was simulated satisfactorily. The impact of naturally-acquired transmission-blocking immunity on malaria transmission under different vectorial capacities was also simulated. The results show that at low vectorial capacities, e.g., just above the threshold for transmission, the effect of transmission-blocking immunity is very significant; however, the effect is lower at higher vectorial capacities.

Characteristics of malaria transmission in Kataragama, Sri Lanka: a focus for immuno-epidemiological studies.

Parasitological and entomological parameters of malaria transmission were monitored for 17 months in 3,625 residents in a Plasmodium vivax malaria endemic region in southern Sri Lanka; the study area consisted of 7 contiguous villages where routine national malaria control operations were being conducted. Malaria was monitored in every resident; fever patients were screened and 4 periodical mass blood surveys were conducted. An annual malaria incidence rate of 23.1% was reported during the period: 9.3% was due to P. vivax and 13.8% was due to P. falciparum; there had been a recent epidemic of the latter in this region, whereas the P. falciparum incidence rate in the previous 10 years had been negligible. There was a wide seasonal fluctuation in the malaria incidence, with the peak incidence closely following the monsoon rains. The prevalence of malaria due to both species detected at the 4 mass blood surveys ranged from 0.98% (at low transmission) to 2.35% (at peak transmission periods). Adults and children developed acute clinical manifestations of malaria. Entomological measurements confirmed a low degree of endemicity with estimated inoculation rates of 0.0029 and 0.0109 (infectious bites/man/night) for P. vivax and P. falciparum, respectively. Several anopheline species contributed to the transmission, and the overall man biting rates (MBR) showed a marked seasonal variation. Malaria at Kataragama, typical of endemic areas of Sri Lanka, thus presents characteristics of "unstable" transmission. Malaria was clustered in the population. There was a low clinical tolerance to P. falciparum malaria, to which most had only been at risk, compared to P. vivax, to which most had had a life-long exposure.