Overcoming ethical barriers to childhood tuberculosis research in developing countries.

In 2002, an international Workshop on Tuberculosis in Children was held to determine priority areas for basic, clinical and programmatic research to improve paediatric TB practice. During the workshop, issues related to the ethical treatment of children as research subjects, particularly in resource-poor settings, were identified as major constraints to the goal of expanding paediatric TB research. Based on participation in the workshop discussions, this article proposes concrete activities that can begin to address the ethical barriers. The time and costs associated with creating collaborative scientific and ethical research projects between high and low-income countries should be supported in grants for paediatric TB research studies in developing countries. Paediatric TB researchers in developing countries should be given opportunities to participate in international programmes that develop bioethics expertise. Relevant case studies of paediatric research in developing countries can assist ethical review committees in industrialised countries in understanding the special issues related to paediatric research in resource-limited settings. Paediatricians in developing countries need to be included in childhood TB research studies and ethical review committees.

The neglected burden of Plasmodium vivax malaria.

We estimate that the global burden of malaria due to Plasmodium vivax is approximately 70-80 million cases annually. Probably approximately 10-20% of the world's cases of P. vivax infection occur in Africa, south of the Sahara. In eastern and southern Africa, P. vivax represents around 10% of malaria cases but < 1% of cases in western and central Africa. Outside of African, P. vivax accounts for > 50% of all malaria cases. About 80-90% of P. vivax outside of Africa occurs in the Middle East, Asia, and the Western Pacific, mainly in the most tropical regions, and 10-15% in Central and South America. Because malaria transmission rates are low in most regions where P. vivax is prevalent, the human populations affected achieve little immunity to this parasite; as a result, in these regions, P. vivax infections affect people of all ages. Although the effects of repeated attacks of P. vivax through childhood and adult life are only rarely directly lethal, they can have major deleterious effects on personal well-being, growth, and development, and on the economic performance at the individual, family, community, and national levels. Features of the transmission biology of P. vivax give this species greater resilience than the less robust Plasmodiumfalciparum in the face of conditions adverse to the transmission of the parasites. Therefore, as control measures become more effective, the residual malaria burden is likely increasingly to become that of P. vivax.

Effects of initial dose on eastern equine encephalomyelitis virus dependent mortality in intrathoracically inoculated Culiseta melanura (Diptera: Culicidae).

Viral growth characteristics that favor rapid and prodigious virion production may increase virus transmission but be detrimental to infected hosts. Several arboviruses, including eastern equine encephalomyelitis (EEE) virus, negatively affect the survival of their infected mosquito vectors. To test the hypothesis that the mosquito virulent properties of EEE virus are caused by the presence of intrinsic viral growth properties, we investigated the effects of infecting dose on the survival of intrathoracically inoculated Culiseta melanura (Coquillett). Daily survival of age-matched females inoculated with either a low initial dose of 10(1.5) plaque-forming units (PFUs) per mosquito or a high initial dose of 10(5.5) PFUs per mosquito was monitored for 8 wk. Compared with diluent inoculated controls, mosquitoes from both dosage groups displayed highly significant decreases in survival. No significant differences in daily survival were detected between the two infected groups. Virus production within inoculated mosquitoes was assessed by sampling mosquitoes every 12 h for 96 h after inoculation. Rapid virus amplification occurred in both dosage groups, and by 24 h after exposure the mean viral loads in mosquitoes inoculated with the low dose were comparable to those inoculated with the high dose. Likewise, although detectable virions appeared sooner in the saliva of high dosage mosquitoes, by 72 h after inoculation no significant differences in virus transmission were detected between the two exposure groups. These results indicate that the virulence of EEE virus for its enzootic North American mosquito vector is not dosage dependent and likely reflects the inherent growth properties of this virus within infected mosquitoes.

Plasmodium vivax: a monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.

The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells. Plasmodium vivax CS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with other Plasmodium species, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with all P. vivax sporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.

Characterization of a sporozoite antigen common to Plasmodium falciparum and Plasmodium berghei.

Previous studies demonstrated that immunization with Plasmodium falciparum sporozoites protected mice against Plasmodium berghei sporozoite infection and that this cross-protection was mediated, at least in part, by anti-sporozoite antibody. The experiments presented in this report show that serum and monoclonal antibodies derived from these protected mice identify a novel 42/54-kDa antigen (designated Circumsporozoite Protein 2 or CSP-2) in both P. falciparum and P. berghei sporozoites. Anti-CSP-2 monoclonal antibody blocks invasion of P. falciparum and P. berghei sporozoites into hepatoma cells in vitro and binds the cell surface of sporozoites. Passive transfer of anti-CSP-2 monoclonal antibody protected mice from P. berghei sporozoite infection. Therefore, CSP-2 appears to play a role in the cross-protective immune response observed.

Plasmodium falciparum sporozoite immunization protects against Plasmodium berghei sporozoite infection.

Irradiated sporozoites are generally thought to elicit protective immune responses that are parasite stage and species specific. But immunization with Plasmodium falciparum sporozoites delivered by the bite of infected mosquitoes protects an average of 60% mice from Plasmodium berghei sporozoite infection. Protection appears to be specific as P. falciparum sporozoite-immunized mice protected against P. berghei remain susceptible to Plasmodium yoelii sporozoite infection. Passively transferred immunoglobulin from P. falciparum sporozoite-immunized mouse serum protected naive mice against challenge with P. berghei sporozoites, indicating that cross-protection is mediated, at least in part, by anti-sporozoite antibody. Antibody-mediated protection may not be due to cross-reaction between P. falciparum and P. berghei CS proteins because mice immunized with the recombinant P. falciparum CS protein repeat vaccine candidate, R32tet32, remain susceptible to P. berghei sporozoite infection.

Expression of members of the heat-shock protein 70 family in the exoerythrocytic stages of Plasmodium berghei and Plasmodium falciparum.

Exoerythrocytic stages of Plasmodium berghei cultured in HepG2-A16 hepatoma cells and those of P. falciparum in human hepatocytes transplanted under the kidney capsule of CB-17/ICr scid/scid mice were used to evaluate expression of heat-shock-related stress proteins. Although undetectable in the sporozoites, the expression of proteins similar in sequence of a heat-shock protein of 70 kDa and a glucose-regulated protein of 78 kDa was markedly induced in the hepatic stages of malaria parasites. Expression of these proteins in the exoerythrocytic stages of the malaria parasite warrants a systematic evaluation of their potential role in eliciting cellular immune responses directed against infected hepatocytes.

A protective monoclonal antibody with dual specificity for Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins.

An IgM monoclonal antibody (Mab 36) which reacts with the circumsporozoite (CS) proteins of both P. falciparum and P. berghei was isolated from Plasmodium falciparum sporozoite-immunized mice. In assays of biological activity, Mab 36 induces the CS precipitation reaction with live sporozoites and blocks the invasion of hepatoma cells by sporozoites in vitro at concentrations much lower than those observed for previously reported CS protein-specific monoclonal antibodies. Mab 36 also provided complete protection against P. berghei sporozoite challenge in mice at low doses. Linear epitope mapping revealed that the epitope specificities recognized by Mab 36 are completely encompassed by other monoclonals previously shown to be associated in vivo with protection against P. falciparum or P. berghei sporozoite infection. These results suggest that the ability to make high-affinity IgM antibody to specific CS protein repeat epitopes may be important for eliciting protection against malarial infection.

Nuclear transcription in preblastoderm Drosophila embryos.

Drosophila preblastoderm (0 - 2.5 hr post-oviposition) embryos incorporate [32P] phosphate into newly synthesized RNA. A fraction of this RNA can be ascribed to nuclear transcription by virtue of its hybridization to nuclear DNA. This confirms the electron microscopic observation of McKnight and Miller (1) that nuclear transcription takes place at a low level in preblastoderm embryos. These nuclear transcripts are relatively small (7 - 12S), poly A(+) and appear on polysomes. Translation of newly synthesized nuclear transcripts during preblastoderm indicates that a zygotic genome contribution to embryonic phenotype may occur earlier in development than previously thought.

Genomic clones coding for some of the initial genes expressed during Drosophila development.

Preblastoderm Drosophila embryos were made permeable and labeled in vivo with [32P]phosphate-containing medium. Cytoplasmic polyadenylylated RNA was extracted from these embryos and used to screen a library of Drosophila genomic DNA sequences cloned in phage lambda. Ten cloned sequences were selected for further study. These sequences were not complementary to mitochondrial DNA, nor did they contain the repeated nuclear genes coding for rRNA or histones. The cloned sequences each encode one or more unique genes expressed in preblastoderm embryos. RNA blot analysis indicated that some of these genes are also expressed at other times during embryogenesis. These results show that, in spite of the rapid nuclear divisions taking place during the preblastoderm stage, Drosophila nuclear genes are transcribed and that a subset of these genes show variable, stage-specific levels of expression during early embryogenesis.

Correlation between phosphorylated H1 histones and satellite DNAs in Drosophila virilis.

Drosophila virilis DNA contains satellites I, II, and III. D. novamexicana DNA contains satellite I. D. virilis H1 histone contains subfractions a, b, c, d, and e; D. novamexicana H1 contains subfractions a, b, and c. Therefore, satellites II and III might be correlated with H1d and H1e. To test the validity of this correlation, the H1 histones of polytene nuclei, which contain less than 1% satellite DNA, were analyzed. Polytene nuclei of D. virilis contain substantially decreased levels of H1c and H1e and marginally decreased levels of H1d. Polytene nuclei of D. novamexicana contain decreased levels of H1c.H1c is correlated with satellite I (common to D. virilis and D. novamexicana); H1e is correlated with satellites II and III; H1d is not correlated with any satellite DNA, because its level is virtually unchanged in polytene cells lacking detectable amounts of satellite DNA. Alkaline phosphatase digestion of the H1 histones reveals that H1c is the phosphorylated form of H1b and H1e is the phosphorylated form of H1d. Therefore, the under-replication of satellite DNAs is correlated with the decreased phosphorylation of H1 histones. In vitro, D. virilis H1 histones preferentially bind D. virilis DNAs in the progression III greater than II greater than I greater than main band, whereas D. virilis core histones do not preferentially bind any D. virilis DNA. As an extension of these results, we suggest that phosphorylated H1 histones bind D. virilis satellite DNAs in vivo and are involved in the compaction of heterochromatin.