RESUMO
Peptoanaerobacter stomatis is a newly appreciated taxon associated with periodontal diseases; however, little is known about the organism's pathogenic potential or its interaction with the host immune response. Neutrophils are the most abundant innate immune cell present in the gingival tissue and function to constrain the oral microbial challenge. However, some periodontal pathogens have developed strategies to evade phagocytosis and killing by neutrophils. Therefore, to begin to understand the role of P. stomatis in periodontitis, we studied its interactions with human neutrophils. Our data showed that after 30 min of incubation, neutrophils failed to engulf P. stomatis efficiently; however, when P. stomatis was internalized, it was promptly eradicated. P. stomatis challenge induced a robust intracellular respiratory burst; however, this response did not contribute to bacterial killing. Minimal superoxide release was observed by direct bacterial challenge; however, P. stomatis significantly increased N-formyl-methionyl-leucyl phenylalanine (fMLF)-stimulated superoxide release to an extent similar to that of cells primed with tumor necrosis factor alpha (TNF-α). When neutrophils were challenged with P. stomatis, 52% of the bacterium-containing phagosomes were enriched for the specific granule marker lactoferrin and 82% with the azurophil granule marker elastase. P. stomatis challenge stimulated exocytosis of the four neutrophil granule subtypes. Moreover, P. stomatis susceptibility to extracellular killing could be attributed to the exocytosis of antimicrobial components present in neutrophil granules. Priming neutrophils for an enhanced respiratory burst together with promoting granule content release could contribute to the chronic inflammation and tissue destruction that characterize periodontal diseases.
Assuntos
Clostridiales/imunologia , Grânulos Citoplasmáticos/metabolismo , Exocitose , Neutrófilos/imunologia , Humanos , Explosão Respiratória , Superóxidos/metabolismoRESUMO
Most of the microbial diversity on our planet cannot be cultivated, and remains inaccessible. To bring the missing species into culture, microbiologists have introduced over the past decade a number of innovations aiming to meet the demands of new microbes and better mimic their natural conditions. This resulted in a significant increase in microbial recovery yet the real reasons why so many microbes do not grow on artificial media remain largely unknown. The recently proposed scout model of microbial life cycle may provide a partial explanation for the phenomenon. It postulates that transition from dormancy to activity is a stochastic process originating in noise-driven bistability. The model helps explain several otherwise perplexing observations, and informs the future cultivation efforts.
Assuntos
Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Fenômenos Fisiológicos Bacterianos , Microbiologia Ambiental , Técnicas Microbiológicas/métodosRESUMO
In this study, we examine the temporal pattern of colony appearance during cultivation experiments, and whether this pattern could inform on optimizing the process of microbial discovery. In a series of long-term cultivation experiments, we observed an expected gradual increase over time of the total number of microbial isolates, culminating in a 700-fold colony count increase at 18 months. Conventional thought suggests that long-term incubations result in a culture collection enriched with species that are slow growing or rare, may be unavailable from short-term experiments, and likely are novel. However, after we examined the phylogenetic novelty of the isolates as a function of the time of their isolation, we found no correlation between the two. The probability of discovering either a new or rare species late in the incubation matched that of species isolated earlier. These outcomes are especially notable because of their generality: observations were essentially identical for marine and soil bacteria as well as for spore formers and non-spore formers. These findings are consistent with the idea of the stochastic awakening of dormant cells, thus lending support to the scout model. The process of microbial discovery is central to the study of environmental microorganisms and the human microbiome. While long-term incubation does not appear to increase the probability of discovering novel species, the technology enabling such incubations, i.e., single-cell cultivation, may still be the method of choice. While it does not necessarily allow more species to grow from a given inoculum, it minimizes the overall isolation effort and supplies needed.
Assuntos
Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Biodiversidade , Bactérias/classificação , Humanos , Metagenoma , Dados de Sequência Molecular , Análise de Sequência de DNA , Microbiologia do Solo , Fatores de Tempo , Microbiologia da ÁguaRESUMO
We recently proposed a scout model of the microbial life cycle (S. S. Epstein, Nature 457:1083, 2009), the central element of which is the hypothesis that dormant microbial cells wake up into active (so-called scout) cells stochastically, independently of environmental cues. Here, we check the principal prediction of this hypothesis: under growth-permissive conditions, dormant cells initiate growth at random time intervals and exhibit no species-specific lag phase. We show that a range of microorganisms, including environmental species, Escherichia coli, and Mycobacterium smegmatis, indeed wake up in a seemingly stochastic manner and independently of environmental conditions, even in the longest incubations conducted (months to years long). As is implicit in the model, most of the cultures we obtained after long incubations were not inherently slow growers. Of the environmental isolates that required ≥7 months to form visible growth, only 5% needed an equally long incubation upon subculturing, with the majority exhibiting regrowth within 24 to 48 h. This apparent change was not a result of adaptive mutation; rather, most microbial species that appear to be slow growers were in fact fast growers with a delayed initiation of division. Genuine slow growth thus appears to be less significant than previously believed. Random, low-frequency exit from the nongrowing state may be a key element of a general microbial survival strategy, and the phylogenetic breadth of the organisms exhibiting such exit indicates that it represents a general phenomenon. The stochasticity of awakening can also provide a parsimonious explanation to several microbiological observations, including the apparent randomness of latent infections and the existence of viable-but-nonculturable cells (VBNC).
Assuntos
Adaptação Fisiológica , Escherichia coli/fisiologia , Mycobacterium smegmatis/fisiologia , Ciclo Celular , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Viabilidade Microbiana , Dados de Sequência Molecular , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/metabolismo , Análise de Sequência de DNA , Fatores de TempoRESUMO
A significant number of microorganisms from the human oral cavity remain uncultivated. This is a major impediment to the study of human health since some of the uncultivated species may be involved in a variety of systemic diseases. We used a range of innovations previously developed to cultivate microorganisms from the human oral cavity, focusing on anaerobic species. These innovations include (i) in vivo cultivation to specifically enrich for species actively growing in the oral cavity (the "minitrap" method), (ii) single-cell long-term cultivation to minimize the effect of fast-growing microorganisms, and (iii) modifications of conventional enrichment techniques, using media that did not contain sugar, including glucose. To enable cultivation of obligate anaerobes, we maintained strict anaerobic conditions in most of our cultivation experiments. We report that, on a per cell basis, the most successful recovery was achieved using minitrap enrichment (11%), followed by single-cell cultivation (3%) and conventional plating (1%). Taxonomically, the richest collection was obtained using the single-cell cultivation method, followed by minitrap and conventional enrichment, comprising representatives of 13, 9, and 4 genera, respectively. Interestingly, no single species was isolated by all three methods, indicating method complementarity. An important result is the isolation and maintenance in pure culture of 10 strains previously only known by their molecular signatures, as well as representatives of what are likely to be three new microbial genera. We conclude that the ensemble of new methods we introduced will likely help close the gap between cultivated and uncultivated species from the human oral cavity.
Assuntos
Bactérias Anaeróbias/isolamento & purificação , Técnicas de Cultura de Células/métodos , Boca/microbiologia , Bactérias Anaeróbias/genética , Técnicas Bacteriológicas , Sequência de Bases , Técnicas de Cultura de Células/instrumentação , Meios de Cultura , DNA Bacteriano/análise , DNA Bacteriano/genética , Humanos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , FilogeniaRESUMO
One of the oldest unresolved microbiological phenomena is why only a small fraction of the diverse microbiological population grows on artificial media. The "uncultivable" microbial majority arguably represents our planet's largest unexplored pool of biological and chemical novelty. Previously we showed that species from this pool could be grown inside diffusion chambers incubated in situ, likely because diffusion provides microorganisms with their naturally occurring growth factors. Here we utilize this approach and develop a novel high-throughput platform for parallel cultivation and isolation of previously uncultivated microbial species from a variety of environments. We have designed and tested an isolation chip (ichip) composed of several hundred miniature diffusion chambers, each inoculated with a single environmental cell. We show that microbial recovery in the ichip exceeds manyfold that afforded by standard cultivation, and the grown species are of significant phylogenetic novelty. The new method allows access to a large and diverse array of previously inaccessible microorganisms and is well suited for both fundamental and applied research.
Assuntos
Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Água do Mar/microbiologia , Microbiologia do Solo , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Microorganisms comprise the bulk of biodiversity, but only a small fraction of this diversity grows on artificial media. This phenomenon was noticed almost a century ago, repeatedly confirmed, and termed the "great plate count anomaly." Advances in microbial cultivation improved microbial recovery but failed to explain why most microbial species do not grow in vitro. Here we show that at least some of such species can form domesticated variants capable of growth on artificial media. We also present evidence that small signaling molecules, such as short peptides, may be essential factors in initiating growth of nongrowing cells. We identified one 5-amino-acid peptide, LQPEV, that at 3.5 nM induces the otherwise "uncultivable" strain Psychrobacter sp. strain MSC33 to grow on standard media. This demonstrates that the restriction preventing microbial in vitro growth may be different from those offered to date to explain the "great plate count anomaly," such as deficiencies in nutrient composition and concentrations in standard media, medium toxicity, and inappropriate incubation time. Growth induction of MSC33 illustrates that some microorganisms do not grow in vitro because they are removed from their native communities and the signals produced therein. "Uncultivable" species represent the largest source of unexplored biodiversity, and provide remarkable opportunities for both basic and applied research. Access to cultures of some of these species should be possible through identification of the signaling compounds necessary for growth, their addition to standard medium formulations, and eventual domestication.
Assuntos
Substâncias de Crescimento/farmacologia , Oligopeptídeos/farmacologia , Pantoea/crescimento & desenvolvimento , Pseudoalteromonas/crescimento & desenvolvimento , Psychrobacter/crescimento & desenvolvimento , Difusão , Pantoea/efeitos dos fármacos , Pantoea/isolamento & purificação , Pseudoalteromonas/efeitos dos fármacos , Pseudoalteromonas/isolamento & purificação , Psychrobacter/efeitos dos fármacos , Psychrobacter/isolamento & purificação , Água do Mar/microbiologiaRESUMO
Molecular surveys suggest that communities of microbial eukaryotes are remarkably rich, because even large clone libraries seem to capture only a minority of species. This provides a qualitative picture of protistan richness but does not measure its real extent either locally or globally. Statistical analysis can estimate a community's richness, but the specific methods used to date are not always well grounded in statistical theory. Here we study a large protistan molecular survey from an anoxic water column in the Cariaco Basin (Caribbean Sea). We group individual 18S rRNA gene sequences into operational taxonomic units (OTUs) using different cutoff values for sequence similarity (99 to 50%) and systematically apply parametric models and nonparametric estimators to the OTU frequency data to estimate the total protistan diversity. The parametric models provided statistically sound estimates of protistan richness, with biologically meaningful standard errors, maximal data usage, and extensive model diagnostics and were preferable to the available nonparametric tools. Our clone library exceeded 700 clones but still covered only a minority of species and less than half of the larger protistan clades. Our estimates of total protistan richness portray the target community as very rich at all OTU levels, with hundreds of different populations apparently co-occurring in the small (3-liter) volume of our sample, as well as dozens of clades of the highest taxonomic order. These estimates are among the first for microbial eukaryotes that are obtained using state-of-the-art statistical methods and can serve as benchmark numbers for the local diversity of protists.
Assuntos
Anaerobiose , Células Eucarióticas/fisiologia , Oxigênio/metabolismo , Água do Mar/parasitologia , Ecossistema , Células Eucarióticas/classificação , RNA Ribossômico 18S/análise , RNA Ribossômico 18S/genética , Água do Mar/químicaRESUMO
The majority (>99%) of microorganisms from the environment resist cultivation in the laboratory. Ribosomal RNA analysis suggests that uncultivated organisms are found in nearly every prokaryotic group, and several divisions have no known cultivable representatives. We designed a diffusion chamber that allowed the growth of previously uncultivated microorganisms in a simulated natural environment. Colonies of representative marine organisms were isolated in pure culture. These isolates did not grow on artificial media alone but formed colonies in the presence of other microorganisms. This observation may help explain the nature of microbial uncultivability.
Assuntos
Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Técnicas Bacteriológicas , Sedimentos Geológicos/microbiologia , Água do Mar , Bactérias/classificação , Bactérias/citologia , Contagem de Colônia Microbiana , Meios de Cultura , DNA Bacteriano/análise , DNA Bacteriano/genética , DNA Ribossômico/análise , DNA Ribossômico/genética , Cultura em Câmaras de Difusão , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , Dióxido de SilícioRESUMO
Mammography screening is a profit-driven technology posing risks compounded by unreliability. In striking contrast, annual clinical breast examination (CBE) by a trained health professional, together with monthly breast self-examination (BSE), is safe, at least as effective, and low in cost. International programs for training nurses how to perform CBE and teach BSE are critical and overdue.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Autoexame de Mama , Mamografia/efeitos adversos , Programas de Rastreamento/métodos , Adulto , Idoso , American Cancer Society , Autoexame de Mama/normas , Conflito de Interesses , Reações Falso-Negativas , Reações Falso-Positivas , Feminino , Humanos , Mamografia/economia , Mamografia/normas , Programas de Rastreamento/efeitos adversos , Programas de Rastreamento/economia , Pessoa de Meia-Idade , National Institutes of Health (U.S.) , Palpação , Reprodutibilidade dos Testes , Fatores de Risco , Segurança , Estados UnidosRESUMO
Bacterial food poisoning can be readily prevented by long overdue basic sanitary measures rather than by ultrahazardous irradiation technologies.
Assuntos
Contaminação de Alimentos/legislação & jurisprudência , Contaminação de Alimentos/prevenção & controle , Indústria Alimentícia/economia , Irradiação de Alimentos/efeitos adversos , Irradiação de Alimentos/legislação & jurisprudência , Exposição Ambiental/efeitos adversos , Indústria Alimentícia/legislação & jurisprudência , Indústria Alimentícia/organização & administração , Abastecimento de Alimentos/normas , Órgãos Governamentais , Humanos , Manobras Políticas , Mutagênese/efeitos da radiação , Política , Privatização , Saneamento , Estados UnidosAssuntos
Hormônio do Crescimento/efeitos adversos , Leite , Neoplasias/induzido quimicamente , Neoplasias/prevenção & controle , Somatomedinas/efeitos adversos , Animais , Bovinos , Progressão da Doença , Hormônio do Crescimento/metabolismo , Humanos , Absorção Intestinal , Neoplasias/sangue , Neoplasias/dietoterapia , Proteínas Recombinantes/efeitos adversosRESUMO
An interlocking legislative complex is proposed for the control of carcinogenic and other adverse impacts of established run-away petrochemical and radionuclear technologies, with particular reference to winning the losing war against cancer. These proposals are also applicable to the poorly recognized, potentially adverse public health and environmental hazards of emerging technologies, particularly genetically engineered food production. The proposals embody fundamental democratic rights--the right to know and balanced and transparent decision making--the "Precautionary Principle," reduction in the use of toxics, incentives for the development of safe industrial technologies, and criminal sanctions for suppression or manipulation of information.
Assuntos
Carcinógenos/efeitos adversos , Qualidade de Produtos para o Consumidor/legislação & jurisprudência , Saúde Ambiental/legislação & jurisprudência , Indústrias/legislação & jurisprudência , Neoplasias/prevenção & controle , Adulto , Criança , Feminino , Humanos , Masculino , Neoplasias/epidemiologia , Neoplasias/etiologia , Formulação de Políticas , Tecnologia , Estados Unidos/epidemiologiaRESUMO
The American Cancer Society is fixated on damage control--diagnosis and treatment--and basic molecular biology, with indifference or even hostility to cancer prevention. This myopic mindset is compounded by interlocking conflicts of interest with the cancer drug, mammography, and other industries. The "nonprofit" status of the Society is in sharp conflict with its high overhead and expenses, excessive reserves of assets and contributions to political parties. All attempts to reform the Society over the past two decades have failed; a national economic boycott of the Society is long overdue.
Assuntos
American Cancer Society/economia , Organizações sem Fins Lucrativos/economia , Política , Conflito de Interesses , Humanos , Neoplasias/prevenção & controle , Política Organizacional , Serviços Preventivos de Saúde/economia , Estados UnidosRESUMO
OBJECTIVE: The increasing involvement of insurers and hospitals in monitoring patient care is encroaching on the psychiatrist's autonomy in making clinical decisions. This study examined the prevalence of constraints on psychiatric inpatient practices, as well as how characteristics of psychiatrists affect the type and the degree of these external pressures. METHOD: About 2,500 psychiatrists with active hospital affiliations were surveyed by mail, as a subset of APA's 1988 national survey of psychiatrists. They were questioned about whether the hospital or insurers had pressured them to change their inpatient practices or had attempted to discourage admission of certain types of patients. Characteristics of the psychiatrists' background, available from the main survey, were used as independent variables in a set of regression models, with frequency of different constraints as the dependent variables. RESULTS: More than three quarters of those surveyed reported pressure from insurers for early discharge; nearly two-thirds said hospitals limited length of stay; and about half had been discouraged from admitting severely ill patients, the uninsured, or Medicaid recipients. Characteristics of psychiatrists, such as length of time in practice, income, sex, and medical school education outside the United States, were associated with the prevalence of external pressures. CONCLUSIONS: Constraints on psychiatrists' practices are widespread. Their ability to resist pressures depends on their bargaining power, which seems to be lowest for those who have relatively little experience, who are female, or who have gone to medical school outside the United States. Psychiatrists appear to be willing to trade off more constraints for higher incomes. Severely ill patients and those with little or no insurance are more likely than others to be affected by these limits on psychiatrists' autonomy.
Assuntos
Hospitalização , Programas de Assistência Gerenciada , Transtornos Mentais/terapia , Psiquiatria/organização & administração , Feminino , Médicos Graduados Estrangeiros , Relações Hospital-Médico , Humanos , Renda , Seguradoras , Seguro de Hospitalização , Seguro Psiquiátrico , Tempo de Internação , Masculino , Medicaid , Privilégios do Corpo Clínico , Transtornos Mentais/classificação , Padrões de Prática Médica , Índice de Gravidade de Doença , Fatores Sexuais , Estados UnidosRESUMO
Levels of insulin-like growth factor-1 (IGF-1) are substantially elevated and more bioactive in the milk of cows hyperstimulated with the biosynthetic bovine growth hormones rBGH, and are further increased by pasteurization. IGF-1 is absorbed from the gastrointestinal tract, as evidenced by marked growth-promoting effects even in short-term tests in mature rats, and absorption is likely to be still higher in infants. Converging lines of evidence incriminate IGF-1 in rBGH milk as a potential risk factor for both breast and gastrointestinal cancers.
Assuntos
Rotulagem de Alimentos , Hormônio do Crescimento/efeitos adversos , Fator de Crescimento Insulin-Like I/efeitos adversos , Fator de Crescimento Insulin-Like I/análise , Leite/química , Animais , Neoplasias da Mama/induzido quimicamente , Bovinos , Feminino , Rotulagem de Alimentos/legislação & jurisprudência , Neoplasias Gastrointestinais/induzido quimicamente , Humanos , Lactente , Recém-Nascido , Absorção Intestinal , Ratos , Fatores de Risco , Estados Unidos , United States Food and Drug AdministrationRESUMO
There were clear differences in our study between the management strategies employed by public agencies and those favored by private agencies. These differences, however, appeared to reflect the realities of financing rather than any fundamental differences in their orientation toward public service. There was no clear evidence that particular management practices affected an agency's performance on measures of financial access or acceptance of referrals from public hospitals. Government regulation and pressure from advocacy groups probably helped to maintain private agencies' focus on these and other public goals. From a public policy perspective, choosing a provider solely on the basis of ownership status is, at best, a naive approach to providing public mental health treatment. Not only is there great variation in process and practices within both private and public groups, but external factors such as competition from private practitioners may also exert a stronger influence on agency behavior than does ownership status. Because most current proposals for health care reform rely heavily on increased competition among providers to achieve their goals, the importance of ownership status as a predictor of conduct or performance may be further diminished. The emphasis on competition could increase differences between urban agencies and those in rural areas where there is less competition and, therefore, require different contracting approaches. As we move toward a health care system based on competition, administrators and policy makers will be forced to abandon their reliance on stereotypical public/private agency behavior as guides for policy decisions. Instead, they will have to consider more carefully the effects of political and market influences as well as agency characteristics when choosing community mental health providers.
Assuntos
Centros Comunitários de Saúde Mental/organização & administração , Competição Econômica , Privatização/economia , Administração em Saúde Pública/economia , Centros Comunitários de Saúde Mental/economia , Centros Comunitários de Saúde Mental/estatística & dados numéricos , Serviços Contratados , Política de Saúde , Humanos , Propriedade , Política , Valores Sociais , Estados UnidosRESUMO
For over three decades, evidence has accumulated relating avoidable exposures to environmental and occupational carcinogens to the escalating incidence of breast cancer in the United States and other major industrialized nations. This evidence has until very recently been totally ignored by the cancer establishment, the National Cancer Institute, and the American Cancer Society, despite expenditures of over $1 billion on breast cancer research. Recognition of these environmental and occupational risk factors should lead to the belated development of public health policies directed to the primary prevention of breast cancer. Their recognition should also lend urgency to the need for radical reforms in the priorities and leadership of the cancer establishment.
Assuntos
Neoplasias da Mama/induzido quimicamente , Carcinógenos Ambientais/efeitos adversos , Doenças Profissionais/induzido quimicamente , Exposição Ocupacional/efeitos adversos , Neoplasias da Mama/prevenção & controle , Feminino , Humanos , Doenças Profissionais/prevenção & controle , Fatores de RiscoRESUMO
A statement by some 68 prominent national experts in industrial medicine, carcinogenesis, epidemiology, and public health, released at a February 4, 1992 press conference in Washington, D.C., charged that the National Cancer Institute (NCI) has confused the public by repeated claims of winning the war against cancer. In fact, age standardized incidence rates have increased sharply over recent decades, while ability to treat and cure most cancers has not materially improved. Furthermore, the NCI has minimized evidence for increasing cancer rates which are largely attributed to smoking and to diet. In so doing, NCI trivializes the importance of occupational carcinogens as non-smoking-attributable causes of lung and other cancers, and ignores the tenuous and inconsistent evidence for the causal role of diet per se and also the important role of carcinogenic dietary contaminants. Reflecting this near exclusionary blame-the-victim theory of cancer causation, with support from the American Cancer Society and industry, the NCI discounts the role of avoidable involuntary exposures to industrial carcinogens in air, water, food, the home, and the workplace. The NCI has also failed to provide scientific guidance to Congress and regulatory agencies on fundamental principles of carcinogenesis and epidemiology, and on the critical need to reduce avoidable exposures to environmental and occupational carcinogens. Contrary to NCI, analysis of their $2 billion budget reveals very limited allocations for research on primary cancer prevention, and for occupational cancer which receives only $19 million annually, 1% of NCI's total budget. Problems of professional mindsets in NCI leadership--fixation on diagnosis, treatment, and basic research (much of questionable relevance) and the neglect of cancer prevention--are exemplified by the composition of the Executive President's Cancer Panel and the National Cancer Advisory Board. Contrary to the explicit mandate of the National Cancer Act, the Board is virtually devoid of recognized authorities in occupational and environmental carcinogenesis. These problems are further compounded by institutionalized conflicts of interest reflected in the composition of past Cancer Panels, and of the current Board of Overseers of the Memorial Sloan Kettering Cancer Center, NCI's prototype comprehensive cancer center, with their closely interlocking financial interests with the cancer drug and other industries. Comprehensive reforms of NCI policies and priorities are overdue. Implementation of such reforms is, however, unlikely in the absence of further support from industrial medicine professionals, which is here solicited, besides action by Congress and concerned citizen groups.