A mechanism for improved talc pleurodesis via foam delivery.

Talcum powder is recognized as the leading drug for pleurodesis, a treatment of choice for malignant pleural effusions. Recently, it was shown that hydrogel foam delivery systems significantly enhanced the number of adhesions between the chest wall and the lung in a New Zealand rabbit model due to the sol-gel transition. However, many questions still remain regarding the cause of improved efficacy, such as: (1) Would only hydrogel foams improve the efficacy of talc pleurodesis? (2) Is it possible to achieve the same efficacy of hydrogels using non-hydrogel foams? 3) What are the physicochemical properties that can be correlated to the efficacy of talc pleurodesis? In this study, we use non-hydrogel foam formulations to determine the efficacy of pleurodesis. Foam stability and rheology of the formulations were correlated to adhesion formation. The results clearly suggest a correlation of pleurodesis efficacy to the viscosity and modulus of the foam delivery system.

Translational Activities to Enable NTD Vaccines.

There is an urgent need to develop new vaccines for tuberculosis, HIV/AIDS, and malaria, as well as for chronic and debilitating infections known as neglected tropical diseases (NTDs). The term "NTD" emerged at the beginning of the new millennium to describe a set of diseases that are characterized as (1) poverty related, (2) endemic to the tropics and subtropics, (3) lacking public health attention and inadequate industrial investment, (4) having poor research funding and a weak research and development (R&D) pipeline, (5) usually associated with high morbidity but low mortality, and (6) often having no safe and long-lasting treatment available. Many additional challenges to the current control and elimination programs for NTDs exist. These include inconsistent performance of diagnostic tests, regional differences in access to treatment and in treatment outcome, lack of integrated surveillance and vector/intermediate host control, and impact of ecological climatic changes particularly in regions where new cases are increasing in previously nonendemic areas. Moreover, the development of NTD vaccines, including those for schistosomiasis, leishmaniasis, leprosy, hookworm, and Chagas disease are being led by nonprofit product development partnerships (PDPs) working in partnership with academic and industrial partners, contract research organizations, and in some instances vaccine manufacturers in developing countries. In this review, we emphasize global efforts to fuel the development of NTD vaccines, the translational activities needed to effectively move promising vaccine candidates to Phase-I clinical trials and some of the hurdles to ensuring their availability to people in the poorest countries of Africa, Asia, Latin America, and the Caribbean.

Leishmania vaccine development: exploiting the host-vector-parasite interface.

Visceral leishmaniasis (VL) is a disease transmitted by phlebotomine sand flies, fatal if untreated, and with no available human vaccine. In rodents, cellular immunity to Leishmania parasite proteins as well as salivary proteins of the sand fly is associated with protection, making them worthy targets for further exploration as vaccines. This review discusses the notion that a combination vaccine including Leishmania and vector salivary antigens may improve vaccine efficacy by targeting the parasite at its most vulnerable stage just after transmission. Furthermore, we put forward the notion that better modeling of natural transmission is needed to test efficacy of vaccines. For example, the fact that individuals living in endemic areas are exposed to sand fly bites and will mount an immune response to salivary proteins should be considered in pre-clinical and clinical evaluation of leishmaniasis vaccines. Nevertheless, despite remaining obstacles there is good reason to be optimistic that safe and effective vaccines against leishmaniasis can be developed.

Biomarkers for intracellular pathogens: establishing tools as vaccine and therapeutic endpoints for visceral leishmaniasis.

Visceral leishmaniasis in South Asia is a serious disease affecting children and adults. Acute visceral leishmaniasis develops in only a fraction of those infected individuals, the majority being asymptomatic with the potential to transmit infection and develop disease. We followed 56 individuals characterized as being asymptomatic by seropositivity with rk39 rapid diagnostic test in a hyperendemic district of Bangladesh to define the utility of Leishmania-specific antibodies and DNA in identifying infection. At baseline, 54 of the individuals were seropositive with one or more quantitative antibody assays and antibody levels persisted at follow up. Most seropositive individuals (47/54) tested positive by quantitative PCR at baseline, but only 16 tested positive at follow up. The discrepancies among the different tests may shed light on the dynamics of asymptomatic infections of Leishmania donovani, as well as underscore the need for standard diagnostic tools for active surveillance as well as assessing the effectiveness of prophylactic and therapeutic interventions.

Adjuvants for malaria vaccines.

There is a renewed enthusiasm about subunit vaccines for malaria coincident with the formation of new alliances and partnerships raising international public awareness, attracting increased resources and the re-focusing of research programs on adjuvant development for infectious disease vaccines. It is generally accepted that subunit vaccines for malaria will require adjuvants to induce protective immune responses, and availability of suitable adjuvants has in the past been a barrier to the development of malaria vaccines. Several novel adjuvants are now in licensed products or in late stage clinical development, while several others are in the earlier development pipeline. Successful vaccine development requires knowing which adjuvants to use and knowing how to formulate adjuvants and antigens to achieve stable, safe, and immunogenic vaccines. For the majority of vaccine researchers this information is not readily available, nor is access to well-characterized adjuvants. In this minireview, we outline the current state of adjuvant research and development as it pertains to effective malaria vaccines.

Applying weight gain in Pomacea lineata (Spix 1824) (Mollusca: Prosobranchia) as a measure of herbicide toxicity

Pomacea lineata, um molusco amplamente distribuído e considerado como peste em plantações de arroz no Oriente, pode ser considerado como um valioso recurso para monitorar a qualidade da água no Nordeste do Brasil. Neste trabalho, apresentamos dados que demonstram que o incremento de peso em moluscos neonatos é uma medida consistente que responde eficientemente ao estresse imposto por concentrações tóxicas subletais dos herbicidas Paraquat e Round-up. Os resultados de crescimento para avaliar a toxicidade crônica foram obtidos em experimentos de quatro e quatro, oito, doze e dezesseis dias para Paraquat e Round-up, respectivamente. A maior concentração de efeito não observado (NOEC) e a menor concentração de efeito observado (LOEC) para Paraquat, após 96 horas, foram respectivamente de 0,12 e 0,25 mg/L. Para o Round-up, os valores de NOEC e LOEC estimados foram respectivamente de 0,25 e 0,5 mg/L. Todas as concentrações de Round-up testadas após 192 horas de exposição provocaram diminuições nas taxas de crescimento, sendo significativamente diferentes do controle. Conseqüentemente não pode ser estimado o valor de NOEC. O valor de LOEC foi menor do que 0,12 mg/L. Além disso, não houve nenhuma mortalidade durante o teste. Por conseguinte, nenhum NOEC pôde ser derivado e o LOEC era < 0,12 mg/L. Para as mais baixas concentrações de Paraquat testadas (0,005 mg/L), houve um aumento do crescimento que foi significativamente maior que o controle, sugerindo a ocorrência de um efeito hormético.

Applying weight gain in Pomacea lineata (SPIX 1824) (Mollusca: Prosobranchia) as a measure of herbicide toxicity.

Pomacea lineata, an extremely ubiquitous snail and pest to rice farmers throughout Asia, holds promise as a valuable resource for monitoring water quality in northeast Brazil. In this paper, we present data demonstrating the rate of weight gain in P. lineata neonates as a consistent measure of the stress imposed by sublethal concentrations of the herbicides Paraquat and Round-up. Our secondary agenda is to demonstrate the feasibility of incorporating bioassay into the standard municipal and state procedure of monitoring water quality. Growth data to assess chronic toxicity were generated in experiments of four and four, eight, twelve and sixteen days for Paraquat and Round-up, respectively. We estimated a 96 h no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for Paraquat of 0.12 and 0.25 mg/L. The 96 h Round-up data yielded NOEC and LOEC values, respectively, of 0.25 and 0.5 mg/L. All concentrations of Round-up tested for the 192 h exposure yielded significantly lower growth than the control. Consequently, no NOEC could be derived. The LOEC was < 0.12 mg/L. Furthermore, there was no mortality during the test. At the lowest concentrations of Paraquat tested (0.005 mg/L) there was a significant increase in growth compared with the controls, suggesting a hormetic effect.

Vaccination with plasmid DNA encoding TSA/LmSTI1 leishmanial fusion proteins confers protection against Leishmania major infection in susceptible BALB/c mice.

We have recently shown that a cocktail containing two leishmanial recombinant antigens (LmSTI1 and TSA) and interleukin-12 (IL-12) as an adjuvant induces solid protection in both a murine and a nonhuman primate model of cutaneous leishmaniasis. However, because IL-12 is difficult to prepare, is expensive, and does not have the stability required for a vaccine product, we have investigated the possibility of using DNA as an alternative means of inducing protective immunity. Here, we present evidence that the antigens TSA and LmSTI1 delivered in a plasmid DNA format either as single genes or in a tandem digene construct induce equally solid protection against Leishmania major infection in susceptible BALB/c mice. Immunization of mice with either TSA DNA or LmSTI1 DNA induced specific CD4(+)-T-cell responses of the Th1 phenotype without a requirement for specific adjuvant. CD8 responses, as measured by cytotoxic-T-lymphocyte activity, were generated after immunization with TSA DNA but not LmSTI1 DNA. Interestingly, vaccination of mice with TSA DNA consistently induced protection to a much greater extent than LmSTI1 DNA, thus supporting the notion that CD8 responses might be an important accessory arm of the immune response for acquired resistance against leishmaniasis. Moreover, the protection induced by DNA immunization was specific for infection with Leishmania, i.e., the immunization had no effect on the course of infection of the mice challenged with an unrelated intracellular pathogen such as Mycobacterium tuberculosis. Conversely, immunization of BALB/c mice with a plasmid DNA that is protective against challenge with M. tuberculosis had no effect on the course of infection of these mice with L. major. Together, these results indicate that the protection observed with the leishmanial DNA is mediated by acquired specific immune response rather than by the activation of nonspecific innate immune mechanisms. In addition, a plasmid DNA containing a fusion construct of the two genes was also tested. Similarly to the plasmids encoding individual proteins, the fusion construct induced both specific immune responses to the individual antigens and protection against challenge with L. major. These results confirm previous observations about the possibility of DNA immunization against leishmaniasis and lend support to the idea of using a single polygenic plasmid DNA construct to achieve polyspecific immune responses to several distinct parasite antigens.

Successful use of a defined antigen/GM-CSF adjuvant vaccine to treat mucosal Leishmaniasis refractory to antimony: A case report.

Immunotherapy has been proposed as a method to treat mucosal leishmaniasis for many years, but the approach has been hampered by poor definition and variability of antigens used, and results have been inconclusive. We report here a case of antimonial-refractory mucosal leishmaniasis in a 45 year old male who was treated with three single injections (one per month) with a cocktail of four Leishmania recombinant antigens selected after documented hypo-responsiveness of the patient to these antigens, plus 50 microg of GM-CSF as vaccine adjuvant. Three months after treatment, all lesions had resolved completely and the patient remains without relapse after two years. Side effects of the treatment included only moderate erythema and induration at the injection site after the second and third injections. We conclude that carefully selected microbial antigens and cytokine adjuvant can be successful as immunotherapy for patients with antimonial-refractory mucosal leishmaniasis.

Vaccination with the T cell antigen Mtb 8.4 protects against challenge with Mycobacterium tuberculosis.

The development of an effective vaccine against Mycobacterium tuberculosis is a research area of intense interest. Mounting evidence suggests that protective immunity to M. tuberculosis relies on both MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells. By purifying polypeptides present in the culture filtrate of M. tuberculosis and evaluating these molecules for their ability to stimulate PBMC from purified protein derivative-positive healthy individuals, we previously identified a low-m.w. immunoreactive T cell Ag, Mtb 8.4, which elicited strong Th1 T cell responses in healthy purified protein derivative-positive human PBMC and in mice immunized with recombinant Mtb 8.4. Herein we report that Mtb 8.4-specific T cells can be detected in mice immunized with the current live attenuated vaccine, Mycobacterium bovis-bacillus Calmette-Guérin as well as in mice infected i.v. with M. tuberculosis. More importantly, immunization of mice with either plasmid DNA encoding Mtb 8.4 or Mtb 8.4 recombinant protein formulated with IFA elicited strong CD4(+) T cell and CD8(+) CTL responses and induced protection on challenge with virulent M. tuberculosis. Thus, these results suggest that Mtb 8.4 is a potential candidate for inclusion in a subunit vaccine against TB.

Protection against cutaneous leishmaniasis induced by recombinant antigens in murine and nonhuman primate models of the human disease.

Leishmaniasis affects approximately 2 million people each year throughout the world. This high incidence is due in part to the lack of an efficacious vaccine. We present evidence that the recombinant leishmanial antigens LmSTI1 and TSA, which we identified and characterized previously, induce excellent protection in both murine and nonhuman primate (rhesus monkey) models of human cutaneous leishmaniasis. The remarkable protection induced by LmSTI1 and TSA in an animal model that is evolutionarily close to humans qualifies this antigen combination as a promising candidate subunit vaccine against human leishmaniasis.

Evaluation of DPPD, a single recombinant Mycobacterium tuberculosis protein as an alternative antigen for the Mantoux test.

Although the tuberculin test has aided in the diagnosis of tuberculosis for more than 85 years, its interpretation is difficult particularly because sensitization with non-tuberculous mycobacteria leads to false positive tests. Using the guinea pig model of tuberculosis, we have recently described a recombinant antigen (DPPD) that could circumvent this problem. The DPPD gene is unique to the M. tuberculosis complex organisms and is absent in the organisms representative of all other members of the Mycobacterium genus. Moreover, DPPD induced strong DTH in 100% of the guinea pigs infected with M. tuberculosis and in none of the guinea pigs immunized with nine different species of Mycobacterium. Here we present results of a clinical investigation using DPPD. Mantoux test using both PPD and DPPD was initially performed in 26 patients with confirmed pulmonary tuberculosis and in 25 healthy PPD negative individuals. The results indicated that both PPD and DPPD elicited DTH in 24 out of the 26 patients. No DTH was observed in any of the PPD negative individuals. In addition, a small clinical trial was performed in a population of 270 clinically healthy and randomly selected individuals. DPPD produced a bimodal histogram of skin reaction size and PPD produced a skewed histogram. Because the DPPD gene is not present in non-tuberculous bacilli, these results suggest that this molecule can be an additional tool for a more specific diagnosis of tuberculosis.

Cloning of a Mycobacterium tuberculosis gene encoding a purifed protein derivative protein that elicits strong tuberculosis-specific delayed-type hypersensitivity.

The purified protein derivative (PPD) skin test has been used for the diagnosis of tuberculosis for more than 75 years. However, the test lacks specificity because all mycobacteria share antigens present in PPD. Therefore, sensitization with nontuberculous pathogenic or with environmental nonpathogenic mycobacteria can lead to positive skin tests. This communication describes a novel PPD protein present only in tuberculous complex mycobacteria. A recombinant protein was obtained and named DPPD on the basis of the first 4 amino acids of its N-terminus sequence. DPPD elicited delayed-type hypersensitivity (DTH) in 100% of Mycobacterium tuberculosis-infected guinea pigs but in no animals sensitized with several organisms representative of all members of the Mycobacterium genus. Preliminary results indicate that DPPD induces strong and specific DTH in humans. This work points to the definition of a single recombinant M. tuberculosis protein that may be an alternative to the PPD test.

Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(+) T cells.

Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) is likely to be dependent on the identification of T cell antigens that induce strong proliferation and interferon gamma production from healthy purified protein derivative (PPD)(+) donors. We have developed a sensitive and rapid technique for screening an Mtb genomic library expressed in Escherichia coli using Mtb-specific CD4(+) T cells. Using this technique, we identified a family of highly related Mtb antigens. The gene of one family member encodes a 9.9-kD antigen, termed Mtb9.9A. Recombinant Mtb9.9A protein, expressed and purified from E. coli, elicited strong T cell proliferation and IFN-gamma production by peripheral blood mononuclear cells from PPD(+) but not PPD(-) individuals. Southern blot analysis and examination of the Mtb genome sequence revealed a family of highly related genes. A T cell line from a PPD(+) donor that failed to react with recombinant Mtb9.9A recognized one of the other family members, Mtb9.9C. Synthetic peptides were used to map the T cell epitope recognized by this line, and revealed a single amino acid substitution in this region when compared with Mtb9.9A. The direct identification of antigens using T cells from immune donors will undoubtedly be critical for the development of vaccines to several intracellular pathogens.

Molecular characterization and human T-cell responses to a member of a novel Mycobacterium tuberculosis mtb39 gene family.

We have used expression screening of a genomic Mycobacterium tuberculosis library with tuberculosis (TB) patient sera to identify novel genes that may be used diagnostically or in the development of a TB vaccine. Using this strategy, we have cloned a novel gene, termed mtb39a, that encodes a 39-kDa protein. Molecular characterization revealed that mtb39a is a member of a family of three highly related genes that are conserved among strains of M. tuberculosis and Mycobacterium bovis BCG but not in other mycobacterial species tested. Immunoblot analysis demonstrated the presence of Mtb39A in M. tuberculosis lysate but not in culture filtrate proteins (CFP), indicating that it is not a secreted antigen. This conclusion is strengthened by the observation that a human T-cell clone specific for purified recombinant Mtb39A protein recognized autologous dendritic cells infected with TB or pulsed with purified protein derivative (PPD) but did not respond to M. tuberculosis CFP. Purified recombinant Mtb39A elicited strong T-cell proliferative and gamma interferon responses in peripheral blood mononuclear cells from 9 of 12 PPD-positive individuals tested, and overlapping peptides were used to identify a minimum of 10 distinct T-cell epitopes. Additionally, mice immunized with mtb39a DNA have shown increased protection from M. tuberculosis challenge, as indicated by a reduction of bacterial load. The human T-cell responses and initial animal studies provide support for further evaluation of this antigen as a possible component of a subunit vaccine for M.tuberculosis.

Molecular cloning and immunologic reactivity of a novel low molecular mass antigen of Mycobacterium tuberculosis.

Polypeptide Ags present in the culture filtrate of Mycobacterium tuberculosis were purified and evaluated for their ability to stimulate PBMC from purified protein derivative (PPD)-positive healthy donors. One such Ag, which elicited strong proliferation and IFN-gamma production, was further characterized. The N-terminal amino acid sequence of this polypeptide was determined and used to design oligonucleotides for screening a recombinant M. tuberculosis genomic DNA library. The gene (Mtb 8.4) corresponding to the identified polypeptide was cloned, sequenced, and expressed in Escherichia coli. The predicted m.w. of the recombinant protein without its signal peptide was 8.4 kDa. By Southern analysis, the DNA encoding this mycobacterial protein was found in the M. tuberculosis substrains H37Rv, H37Ra, Erdman, and "C" strain, as well as in certain other mycobacterial species, including Mycobacterium avium and Mycobacterium bovis BCG (bacillus Calmette-Guerin, Pasteur). The Mtb 8.4 gene appears to be absent from the environmental mycobacterial species examined thus far, including Mycobacterium smegmatis, Mycobacterium gordonae, Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium scrofulaceum. Recombinant Mtb 8.4 Ag induced significant proliferation as well as production of IFN-gamma, IL-10, and TNF-alpha, but not IL-5, from human PBMC isolated from PPD-positive healthy donors. Mtb 8.4 did not stimulate PBMC from PPD-negative donors. Furthermore, immunogenicity studies in mice indicate that Mtb 8.4 elicits a Th1 cytokine profile, which is considered important for protective immunity to tuberculosis. Collectively, these results demonstrate that Mtb 8.4 is an immunodominant T cell Ag of M. tuberculosis.

Paraiotonchium muscadomesticae n. sp. (Tylenchida: lotonchiidae) a Parasite of the House Fly (Musca domestica) in Brazil and a Key to Species of the Genus Paraiotonchium.

Paraiotonchium muscadomesticae n. sp., a parasite of the house fly, Musca domestica L., is described and illustrated from material collected in Brazil. The life cycle of P. muscadomesticae is similar to that of P. autumnale (Nickle), consisting of alternating gamogenetic and parthenogenetic generations. Paraiotonchium muscadomesticae n. sp. can be distinguished from P. nicholasi Slobodyanyuk, P. autumnale (Nickle) Slobodyanyuk, and P. crassirostris (Yatham &Rao) Siddiqi by the shorter body length of young heterosexual females, 652 g.m (530-709) for P. muscadomesticae compared to 750 mum or more (801-1,050) for the others. Paraiotonchium muscadomesticae is close to P. nicholosi but differs from it by V ratio and spicule length (V = 80-84; spicule = 16-21 p.m in P. muscadomesticae compared to V = 73-78; spicule = 25-35 mum in P. nicholasi). Paraiotonchium muscadomesticae and P. nicholasi differ from all species of this genus by the absence of a bursa on males of these two species.

Characterization and description of a virus causing salivary gland hyperplasia in the housefly, Musca domestica.

A double-stranded DNA virus was isolated from hyperplasic salivary glands of male and female houseflies, Musca domestica L. (Diptera: Muscidae), collected from a dairy in Alachua County, Florida, U.S.A. Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) of this housefly salivary gland hyperplasia (SGH) virus revealed the presence of two major and eight minor structural polypeptides. Restriction endonuclease analysis indicated that the c. 137 kilobase pair DNA was double-stranded. Weekly, sweep-net sampling of the fly population throughout the season (May-October, 1991) showed that 1.5-18.5% of the dissected flies possessed hyperplasic salivary glands. The virus replicated within the nuclei of the salivary gland cells and was transmitted per os to newly-emerged healthy adult flies.

The impact of depressed pH and elevated aluminum concentrations on specific dynamic action in Somatochlora cingulata (de Selys).

Differences in oxygen consumption attributable to apparent specific dynamic action (SDA) were measured in relation to feeding level in the dragonfly naiad Somatochlora cingulata exposed to low pH and sublethal aluminum concentration plus low pH. The average increases in respiration among the treatments following feeding were 50-60% that of controls. The average treatment peak height ratios (post/prefeeding respiration rates) were proportionately reduced and not significantly different from the average controls indicating that the resting metabolism, rather than SDA, was reduced.