Drug targets and mechanisms of resistance in the anaerobic protozoa

The anaerobic protozoa Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica infect up to a billion people each year. G. duodenalis and E. histolytica are primarily pathogens of the intestinal tract, although E. histolytica can form abscesses and invade other organs, where it can be fatal if left untreated. T. vaginalis infection is a sexually transmitted infection causing vaginitis and acute inflammatory disease of the genital mucosa. T. vaginalis has also been reported in the urinary tract fallopian tubes, and pelvis and can cause pneumonia, bronchitis, and oral lesions. Respiratory infections can be acquired perinatally. T. vaginalis infections have been associated with preterm delivery, low birth weight, and increased mortality as well as predisposing to human immunodeficiency virus infection, AIDS, and cervical cancer. All three organisms lack mitochondria and are susceptible to the nitroimidazole metronidazole because of similar low-redox-potential anaerobic metabolic pathways. Resistance to metronidazole and other drugs has been observed clinically and in the laboratory. Laboratory studies have identified the enzyme that activates metronidazole, pyruvate:ferredoxin oxidoreductase, to its nitroso form and distinct mechanisms of decreasing drug susceptibility that are induced in each organism. Although the nitroimidazoles have been the drug family of choice for treating the anaerobic protozoa, G. duodenalis is less susceptible to other antiparasitic drugs, such as furazolidone, albendazole, and quinacrine. Resistance has been demonstrated for each agent and the mechanism of resistance has been investigated. Metronidazole resistance in T. vaginalis is well documented, and the principal mechanisms have been defined Bypass metabolism, such as alternative oxidoreductases, have been discovered in both organisms. Aerobic versus anaerobic resistance in T. vaginalis is discussed. Mechanisms of metronidazole resistance in E. histolytica have recently been investigated ruing laboratory-induced resistant isolates. Instead of downregulation of the pyruvate:ferredoxin oxidoreductase and ferredoxin pathway as seen in G. duodenalis and T. vaginalis, E. histolytica induces oxidative stress mechanisms, including superoxide dismutase and peroxiredoxin. The review examines the value of investigating both clinical and laboratory-induced syngeneic drug-resistant isolates and dissection of the complementary data obtained. Comparison of resistance mechanisms in anaerobic bacteria and the parasitic protozoa is discussed as well as the value of studies of the epidemiology of resistance.

Codon modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity

Polynucleotide immunisation with the E7 gene of human papillomavirus (HPV) type 16 induces only moderate levels of immune response, which may in part be due to limitation in E7 gene expression influenced by biased HPV codon usage. Here we compare for expression and immunogenicity polynucleotide expression plasmids encoding wild-type (pWE7) or synthetic codon optimised (pHE7) HPV16 E7 DNA. Cos-1 cells transfected with pHE7 expressed higher levels of E7 protein than similar cells transfected with pW7. C57BL/6 mice and F1 (C57X FVB) E7 transgenic mice immunised intradermally with E7 plasmids produced high levels of anti-E7 antibody. pHE7 induced a significantly stronger E7-specific cytotoxic T-lymphocyte response than pWE7 and 100% tumour protection in C57BL/6 mice, but neither vaccine induced CTL in partially E7 tolerant K14E7 transgenic mice. The data indicate that immunogenicity of an E7 polynucleotide vaccine can be enhanced by codon modification. However, this may be insufficient for priming E7 responses in animals with split tolerance to E7 as a consequence of expression of E7 in somatic cells. (C) 2002 Elsevier Science (USA).

Limitations of HLA-transgenic mice in presentation of HLA-restricted cytotoxic T-cell epitopes from endogenously processed human papillomavirus type 16 E7 protein

We investigated the use of mice transgenic for human leucocyte antigen (HLA) A*0201 antigen-binding domains to test vaccines composed of defined HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitopes of human papillomavirus (HPV) type 16 E7 oncoprotein. HPV is detected in >90% of cervical carcinomas. HPV16 E7 oncoprotein transforms cells of the uterine cervix and functions as a tumour-associated antigen to which immunotherapeutic strategies may be directed. We report that although the HLA A*0201 E7 epitope peptides function both to prime for E7 CTL responses, and to sensitize target cells for E7-directed CTL killing in situations where antigen processing is not required, the epitopes are not processed out of either endogenously expressed or immunization-introduced E7, by the mouse antigen-processing and presentation machinery. Thus (1) CTL induced by HLA A*0201 peptide immunization killed E7 peptide-pulsed target cells, but did not kill target cells expressing whole E7; (2) immunization with whole E7 protein did not elicit CTL directed to HLA A*0201-restricted E7 CTL epitopes; (3) HLA A*0201-restricted CTL epitopes expressed in the context of a DNA polytope vaccine did not activate E7-specific T cells either in 'conventional' HLA A*0201 transgenic (A2.1K(b) ) mice, or in HHD transgenic mice in which expression of endogenous H-2 class 1 is precluded; and (4) HLA A*0201 E7 peptide epitope immunization was incapable of preventing the growth of an HLA A*0201- and E7-expressing tumour. There are generic implications for the universal applicability of HLA-class 1 transgenic mice for studies of human CTL epitope presentation in murine models of human infectious disease where recognition of endogenously processed antigen is necessary. There are also specific implications for the use of HLA A2 transgenic mice for the development of E7-based therapeutic vaccines for cervical cancer.

Drug resistance in the sexually transmitted protozoan Trichomonas vaginalis

Trichomoniasis is the most common, sexually transmitted infection. It is caused by the flagellated protozoan parasite Trichomonas vaginalis. Symptoms include vaginitis and infections have been associated with preterm delivery, low birth weight and increased infant mortality, as well as predisposing to HIV/AIDS and cervical cancer. Trichomoniasis has the highest prevalence and incidence of any sexually transmitted infection. The 5-nitroimidazole drugs, of which metronidazole is the most prescribed, are the only approved, effective drugs to treat trichomoniasis. Resistance against metronidazole is frequently reported and cross-resistance among the family of 5-nitroimidazole drugs is common, leaving no alternative for treatment, with some cases remaining unresolved. The mechanism of metronidazole resistance in T. vaginalis from treatment failures is not well understood, unlike resistance which is developed in the laboratory under increasing metronidazole pressure. In the latter situation, hydrogenosomal function which is involved in activation of the prodrug, metronidazole, is down-regulated. Reversion to sensitivity is incomplete after removal of drug pressure in the highly resistant parasites while clinically resistant strains, so far analysed, maintain their resistance levels in the absence of drug pressure. Although anaerobic resistance has been regarded as a laboratory induced phenomenon, it clearly has been demonstrated in clinical isolates. Pursuit of both approaches will allow dissection of the underlying mechanisms. Many alternative drugs and treatments have been tested in vivo in cases of refractory trichomoniasis, as well as in vitro with some successes including the broad spectrum anti-parasitic drug nitazoxanide. Drug resistance incidence in T. vaginalis appears to be on the increase and improved surveillance of treatment failures is urged.

Provision of 4-1BB ligand enhances effector and memory CTL responses generated by immunization with dendritic cells expressing a human tumor-associated antigen

Up-regulation of receptor-ligand pairs during interaction of an MHC-presented epitope on dendritic cells (DCs) with cognate TCR may amplify, sustain, and drive diversity in the ensuing T cell immune response. Members of the TNF ligand superfamily and the TNFR superfamily contribute to this costimulatory molecule signaling. In this study, we used replication deficient adenoviruses to introduce a model tumor-associated Ag (the E7 oncoprotein of human papillomavirus 16) and the T cell costimulatory molecule 4-IBBL into murine DCs, and monitored the ability of these recombinant DO to elicit E7-directed T cell responses following immunization. Splenocytes from mice immunized with DCs expressing E7 alone elicited E7-directed effector and memory CTL responses. Coexpression of 4-1BBL in these E7-expressing DO increased effector and memory CTL responses when they were used for immunization. 4-1BBL expression up-regulated CD80 and CD86 second signaling molecules in DO. We also report an additive effect of 4-IBBL and receptor activator of NF-kappaB/receptor activator of NF-kappaB ligand coexpression in E7-transduced DC inummogens on E7-directed effector and memory CTL responses and on MHC class II and CD80/86 expression in DCs. Additionally, expression of 4-1BBL in E7-transduced DCs reduced nonspecific T cell activation characteristic of adenovirus vector-associated immunization. The results have generic implications for improved or tumor Ag-expressing DC vaccines by incorporation of exogenous 4-1BBL. There are also specific implications for an improved DC-based vaccine for human papillomavirus 16-associated cervical carcinoma.

Chapter 16: Prophylactic Human Papillomavirus Vaccines

Candidate prophylactic vaccines based on papillomavirus L1 virus-like particles (VLPs) are currently in human clinical trials. The main long-term goal of the vaccine is to reduce the incidence of cervical cancer and its precursors. In animal papillomavirus models, systemic immunization with L1 VLPs can induce high titers of neutralizing antibodies that confer protection against high-dose experimental papillomavirus challenge. In humans, systemic vaccination with L1 VLPs has been well tolerated and induced high serum antibody titers (at least 40 times higher than titers seen following natural infection). A recent proof of principle HPV16 L1 VLP efficacy trial has shown excellent protection against persistent HPV16 infection and associated cytological abnormalities. Large scale efficacy trials of L1 VLPs from HPV16 and 18 (the HPV types found most frequently in cervical cancer), with or without HPV6 and 11 (the HPV types responsible for most genital warts), are planned. If the results of these large trials support the encouraging results of the early trials, they should lead to a commercial prophylactic HPV vaccine. Implementation issues may include how to make the vaccine available in the developing world, where the majority of cervical cancer cases occur, the appropriate age of vaccination, and the role of male vaccination. Because a VLP vaccine is likely to provide type-specific protection, increasing the number of cancer-associated HPV types in the vaccine is a likely approach to broadening the protection to additional types. There will probably also be efforts to develop alternative vaccine formulations better suited to implementation in developing countries as well as attempts to develop vaccines with a therapeutic activity against established HPV infection because a combined prophylactic/therapeutic vaccine may be expected to have an even greater impact than a purely prophylactic vaccine on HPV induced disease.

Enhanced effector and memory CTL responses generated by incorporation of receptor activator of NF-kappa B(RANK)/RANK ligand costimulatory molecules into dendritic cell immunogens expressing a human tumor-specific antigen

The outcome of dendritic cell (DC) presentation of Ag to T cells via the TCR/MHC synapse is determined by second signaling through CD80/86 and, importantly, by ligation of costimulatory ligands and receptors located at the DC and T cell surfaces. Downstream signaling triggered by costimulatory molecule ligation results in reciprocal DC and T cell activation and survival, which predisposes to enhanced T cell-mediated immune responses. In this study, we used adenoviral vectors to express a model tumor Ag (the E7 oncoprotein of human papillomavirus 16) with or without coexpression of receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL) or CD40/CD40L costimulatory molecules, and used these transgenic DCs to immunize mice for the generation of E7-directed CD8(+) T cell responses. We show that coexpression of RANK/RANKL, but not CD40/CD40L, in E7-expressing DCs augmented E7-specific IFN-gamma-secreting effector and memory T cells and E7-specific CTLs. These responses were also augmented by coexpression of T cell costimulatory molecules (RANKL and CD40L) or DC costimulatory molecules (RANK and CD40) in the E7-expressing DC immunogens. Augmentation of CTL responses correlated with up-regulation of CD80 and CD86 expression in DCs transduced with costimulatory molecules, suggesting a mechanism for enhanced T cell activation/survival. These results have generic implications for improved tumor Ag-expressing DC vaccines, and specific implications for a DC-based vaccine approach for human papillomavirus 16-associated cervical carcinoma.

Caracterização molecular de papilomavírus humano (HPV) e vírus adeno-associado (AAV) em lesões intraepiteliais de colo uterino: um estudo de seguimento

O câncer de colo uterino (CCU), cujo agente etiológico é o papilomavírus humano (HPV), é um dos tipos de câncer mais frequentes em mulheres em todo o mundo, não só em incidência como também em mortalidade. Alguns genótipos de HPV, denominados de alto risco (HR-HPV), e suas variantes gênicas, estão mais associados à indução de lesões malignas, sendo HPV16 e 18 os mais frequentes. Algumas infecções do trato genital podem atuar como cofatores da progressão carcinogênica do CCU, porém a infecção por vírus adeno-associado (AAV) parece estar inversamente relacionada, o que pode refletir em um papel protetor no desenvolvimento do CCU induzido pelo HPV. Portanto, este estudo objetivou investigar o papel da infecção mista AAV-HPV e das variantes oncogênicas de HPV na progressão das lesões intraepiteliais de colo de útero e acompanhar a eliminação / persistência viral em relação à progressão / regressão das lesões cervicouterinas. Exames citológicos foram realizados em amostras de espécime cervical, coletadas em dois momentos, de mulheres atendidas no Hospital Universitário Cassiano Antonio Moraes – HUCAM e seguiram para tratamento conforme preconizado. DNA foi extraído pelo kit comercial QIAamp® DNA Mini Kit, seguindo instruções do fabricante. DNA de AAV foi investigado por PCR e nPCR e, de HPV, por PCR e Captura Híbrida® (CH). Genotipagem de AAV e HPV foram realizadas por RFLP e RLB, respectivamente. Dos casos encaminhados ao ambulatório de colposcopia, 57,3% tiveram citologia normal, 23,1% lesões de baixo grau e 19,6% lesões de alto grau. Dos casos com citologia normal, 78% permaneceram normais, enquanto 22% progrediram à lesão; dos casos com lesão de baixo grau, 74% regrediram para citologia normal, enquanto 78,6% dos casos com lesão de alto grau apresentaram lesão de baixo grau ou citologia normal na segunda coleta. Foram positivas para HPV, 56% e 36,5% das amostras da primeira e segunda coletas, respectivamente. Foi observada boa correlação (kappa= 0,66) entre os testes de PCR e CH para detecção de HPV. Os HR-HPV foram detectados em mais de 90% das amostras de ambas as coletas, sendo os mais frequentes os HPV16, 58, 51, 52 e 53. Variante não-europeia esteve associada ao desenvolvimento de lesão cervical de alto grau, enquanto a presença de AAV foi inversamente relacionada à progressão da lesão cervical induzida por HPV.

Mammography in asymptomatic women aged 40-49 years

OBJECTIVE To assess findings of mammography of and interventions resulting from breast cancer screening in women aged 40-49 years with no increased risk (typical risk) of breast cancer. METHODS This cross-sectional study evaluated women aged 40-49 years who underwent mammography screening in a mastology reference center in Recife, PE, Northeastern Brazil, between January 2010 and October 2011. Women with breast-related complaints, positive findings in the physical examination, or high risk of breast cancer were excluded. RESULTS The 1,000 mammograms performed were classified into the following Breast Imaging-Reporting and Data System (BI-RADS) categories BI-RADS 0, 232; BI-RADS 1, 294; BI-RADS 2, 294; BI-RADS 3, 16; BI-RADS 4A, 2; BI-RADS 5, 1. There was one case of grade II invasive ductal carcinoma and various interventions, including 469 ultrasound scans, 53 referrals to mastologists, 11 cytological examinations, and 8 biopsies. CONCLUSIONS Mammography screening in women aged 40-49 years with typical risk of breast cancer led to the performance of other interventions. However, it also resulted in increased costs without demonstrable efficacy in decreasing mortality.

Sistemas de prevenção de cuidados de saúde

A tomada de decisão na saúde pode-se tornar um processo complexo e moroso. A complexidade associada ao processo de decisão na saúde advém da diversidade de opções clinicamente razoáveis, ou seja, nenhuma opção se sobrepõem _a outra, visto que cada uma possui os seus riscos e benefícios, que são normalmente interpretados de modo diferente entre os indivíduos. Desta forma, cabe ao paciente e _a sua equipa médica optarem pela opção que melhor se enquadra na situação clínica do paciente Para tornar este processo menos complexo, cada vez mais se utiliza as chamadas "ferramentas de decisão", que se caraterizam por fornecer informação sobre as diferentes opções clínicas, traduzindo-se numa diminuição da dificuldade da tomada de decisão. De uma forma geral, as ferramentas de decisão são desenvolvidas com o intuito de facilitar a tomada de decisão, através do aumento do conhecimento científico sobre um determinado problema (tomada de decisão informada) e uma mudança de atitude do paciente face aos seus cuidados de saúde. Na realização da presente dissertação foi desenvolvido um sistema de informação na web, que engloba informação relativa ao rastreio do cancro da próstata. Este sistema também surge acoplado a um conjunto de componentes de decisão, que têm como objetivo auxiliar os indivíduos no processo de decisão para a realização do rastreio do cancro da próstata, assim como a prevenção de doenças relacionadas com a próstata. A implementação desta aplicação web teve como base as necessidades do indivíduo, ou seja informações clínicas sobre possíveis riscos e benefícios associados ao rastreio, assim como fornecer uma maior interatividade com o utilizador. A primeira versão da aplicação já foi testada e avaliada através da participação de um conjunto de indivíduos que compõem o público-alvo para este tipo de aplicações. Os resultados obtidos permitiram concluir que os requisitos definidos para esta aplicação, permitem o aumento do conhecimento do indivíduo e o auxílio na tomada de decisão para a realização do rastreio do cancro da próstata.

Development and Assessment of an E-Learning Course on Breast Imaging for Radiographers: A Stratified Randomized Controlled Trial

Background: Mammography is considered the best imaging technique for breast cancer screening, and the radiographer plays an important role in its performance. Therefore, continuing education is critical to improving the performance of these professionals and thus providing better health care services. Objective: Our goal was to develop an e-learning course on breast imaging for radiographers, assessing its efficacy , effectiveness, and user satisfaction. Methods: A stratified randomized controlled trial was performed with radiographers and radiology students who already had mammography training, using pre- and post-knowledge tests, and satisfaction questionnaires. The primary outcome was the improvement in test results (percentage of correct answers), using intention-to-treat and per-protocol analysis. Results: A total of 54 participants were assigned to the intervention (20 students plus 34 radiographers) with 53 controls (19+34). The intervention was completed by 40 participants (11+29), with 4 (2+2) discontinued interventions, and 10 (7+3) lost to follow-up. Differences in the primary outcome were found between intervention and control: 21 versus 4 percentage points (pp), P<.001. Stratified analysis showed effect in radiographers (23 pp vs 4 pp; P=.004) but was unclear in students (18 pp vs 5 pp; P=.098). Nonetheless, differences in students’ posttest results were found (88% vs 63%; P=.003), which were absent in pretest (63% vs 63%; P=.106). The per-protocol analysis showed a higher effect (26 pp vs 2 pp; P<.001), both in students (25 pp vs 3 pp; P=.004) and radiographers (27 pp vs 2 pp; P<.001). Overall, 85% were satisfied with the course, and 88% considered it successful. Conclusions: This e-learning course is effective, especially for radiographers, which highlights the need for continuing education.

Sistema de apoio à decisão do rastreio do cancro da mama

O estudo do rastreio do cancro da mama funciona não só como um benefício, mas também como um método que pode causar controvérsias nas mulheres consideradas saudáveis antes de se submeterem ao rastreio. Assim, a necessidade do utente adquirir conhecimento sobre as vantagens e desvantagens do rastreio do cancro da mama é fundamental para decidir se, se submete ou não, a este rastreio. A decisão do paciente é crucial na decisão final e as consequências já devem ser do seu conhecimento. Com este estudo, pretendeu-se o desenvolvimento de um Sistema de Informação (SI) na Web, que funcione como auxiliar no processo de tomada de decisão para a realização do rastreio do cancro da mama. A decisão do componente do SI é fundamental, com o objetivo de auxiliar os utilizadores no processo de tomada de decisão para a realização do rastreio do cancro da mama. O desenvolvimento da plataforma “Decidir”, apresenta-se numa aplicação crucial no domínio médico da decisão do rastreio do cancro da mama, onde referências às suas controvérsias e benefícios são fundamentais para um melhor cuidado de saúde. Para esse efeito foram implementadas duas ferramentas para ajudar na decisão de realizar ou não o rastreio do cancro da mama. A primeira ferramenta resulta na decisão pessoal de cada indivíduo (paciente/profissionais de saúde), que pode ser variável, conforme um conjunto de proposições relevantes no aspeto pessoal e clínico para a tomada de decisão do rastreio nesta área.

Prevalence of human papillomavirus infection in rural villages of the Bolivian Amazon

Cervical cancer constitutes a major health problem in developing countries like Bolivia. The roles of certain genotypes of human papillomaviruses (HPVs) in the pathogenesis of cervical cancer is well established. The prevalence of HPV infection among sexually active women varies greatly. Information regarding HPV infection in Bolivia is very much scarce, specially in regions like the Amazonian lowland. We studied 135 healthy women living in four rural localities of the Bolivian Amazon. Presence of HPV in DNA extracted from cervical swabs was analyzed using a reverse line hybridization assay. The estimated overall HPV infection prevalence among the studied rural localities was 5.9% (ranging from 0-16.6%). These values were unexpectedly low considering Bolivia has a high incidence of cervical cancer. The fact that Amazonian people seem to be less exposed to HPV, makes it likely that some other risk factors including host lifestyle behaviors and genetic background may be involved in the development of cervical cancer in this population.

Level of behavior and knowledge concerning human papillomavirus among university students of a nursing college

INTRODUCTION: Human pappilomavirus is one of the most common sexually transmitted diseases, and persistent HPV infection is considered the most important cause of cervical cancer. It is detected in more than 98% of this type of cancer. This study aimed to determine the level of knowledge concerning human papillomavirus among nursing college students of a private educational institution located in the City of Bauru, SP, and correlate their knowledge according to the course year. METHODS: A descriptive study with a quantitative approach, performed with a questionnaire that permitted the quantification of data and opinions, thus guaranteeing the precision of the results without distortions in analysis or interpretation. The survey was applied to randomly selected 1st, 2nd, 3rd, and 4th-year nursing college students. Twenty students from each level were selected during August 2009, totaling 80 students of both genders. RESULTS: Observation revealed that 4th-year students had greater knowledge than 1st-year students, reflecting the greater period of study, the lack of knowledge of 1st-year students was due to the low level of information acquired before entering college. CONCLUSIONS: The need for complementary studies which determine the profile and knowledge of a larger number of teenagers in relation to HPV was established. The need for educational programs that can overcome this lack of information is undeniable, especially those aimed at making adolescents less susceptible to HPV and other STDs.

Efeito de um resultado falso positivo da leitura da mamografia na não participação no rastreio do cancro da mama : estudo de coorte histórico de um rastreio de base populacional da região de saúde do centro em Portugal

RESUMO - Introdução: O cancro da mama é uma das principais causas de mortalidade por doença oncológica. O rastreio contribui para o aumento da sobrevivência, mas apresenta riscos como a obtenção de um resultado falso positivo com efeitos controversos sobre a participação subsequente. Métodos: Realizou-se um estudo de coorte histórico (2006-2012) de 170.835 mulheres com 45-67 anos, elegíveis para o programa de rastreio do cancro da mama da ARSC,IP. Calcularam-se as medidas de efeito de um falso positivo da leitura na não participação na volta consecutiva de rastreio do cancro da mama, e a associação entre o evento em estudo e factores sociodemográficos, relacionados com o rastreio e com a anamnese, através de análise de regressão de Poisson. Resultados: A incidência de não participação foi 12,13%. A exposição a falso positivo da leitura aumentou 8,01% o risco absoluto de não participação. O falso positivo da leitura da mamografia revelou-se um factor de risco para a não participação (RRa=1,17; IC 1,10-1,25). O efeito protector da existência de participações anteriores foi superior ao efeito dos factores de risco identificados. Identificaram-se outros factores de risco e de protecção. Discussão: De acordo com os factores de risco e de protecção identificados recomendaram-se alterações à operacionalização do programa de rastreio, a manutenção das estatégias adequadas e a realização de estudos futuros para avaliar o efeito de outros factores não incluídos neste estudo. A comunicação do risco associado a um resultado anormal da mamografia é importante para diminuir a ansiedade consequente ao rastreio, devendo ser oferecidas intervenções que promovam a participação no rastreio.