Though biofilms were first described by Antonie van Leeuwenhoek, the theory describing the biofilm process was not developed until 1978. We now understand that biofilms are universal, occurring in aquatic and industrial water systems as well as a large number of environments and medical devices relevant for public health. Using tools such as the scanning electron microscope and, more recently, the confocal laser scanning microscope, biofilm researchers now understand that biofilms are not unstructured, homogeneous deposits of cells and accumulated slime, but complex communities of surface-associated cells enclosed in a polymer matrix containing open water channels. Further studies have shown that the biofilm phenotype can be described in terms of the genes expressed by biofilm-associated cells. Microorganisms growing in a biofilm are highly resistant to antimicrobial agents by one or more mechanisms. Biofilm-associated microorganisms have been shown to be associated with several human diseases, such as native valve endocarditis and cystic fibrosis, and to colonize a wide variety of medical devices. Though epidemiologic evidence points to biofilms as a source of several infectious diseases, the exact mechanisms by which biofilm-associated microorganisms elicit disease are poorly understood. Detachment of cells or cell aggregates, production of endotoxin, increased resistance to the host immune system, and provision of a niche for the generation of resistant organisms are all biofilm processes which could initiate the disease process. Effective strategies to prevent or control biofilms on medical devices must take into consideration the unique and tenacious nature of biofilms. Current intervention strategies are designed to prevent initial device colonization, minimize microbial cell attachment to the device, penetrate the biofilm matrix and kill the associated cells, or remove the device from the patient. In the future, treatments may be based on inhibition of genes involved in cell attachment and biofilm formation.

Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms

The past several years have witnessed the evolution of gene medicine from an experimental technology into a viable strategy for developing therapeutics for a wide range of human disorders. Numerous prototype DNA-based biopharmaceuticals can now control disease progression by induction and/or inhibition of genes. These potent therapeutics include plasmids containing transgenes, oligonucleotides, aptamers, ribozymes, DNAzymes, and small interfering RNAs. Although only 2 DNA-based pharmaceuticals (an antisense oligonucleotide formulation, Vitravene, (USA, 1998), and an adenoviral gene therapy treatment, Gendicine (China, 2003), have received approval from regulatory agencies; numerous candidates are in advanced stages of human clinical trials. Selection of drugs on the basis of DNA sequence and structure has a reduced potential for toxicity, should result in fewer side effects, and therefore should eventually yield safer drugs than those currently available. These predictions are based on the high selectivity and specificity of such molecules for recognition of their molecular targets. However, poor cellular uptake and rapid in vivo degradation of DNA-based therapeutics necessitate the use of delivery systems to facilitate cellular internalization and preserve their activity. This review discusses the basis of structural design, mode of action, and applications of DNA-based therapeutics. The mechanisms of cellular uptake and intracellular trafficking of DNA-based therapeutics are examined, and the constraints these transport processes impose on the choice of delivery systems are summarized. Finally, the development of some of the most promising currently available DNA delivery platforms is discussed, and the merits and drawbacks of each approach are evaluated.

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DNA-based therapeutics and DNA delivery systems: a comprehensive review.

The only natural reservoir of Neisseria meningitidis is the human nasopharyngeal mucosa. Depending on age, climate, country, socioeconomic status, and other factors, approximately 10% of the human population harbors meningococci in the nose. However, invasive disease is relatively rare, as it occurs only when the following conditions are fulfilled: (i) contact with a virulent strain, (ii) colonization by that strain, (iii) penetration of the bacterium through the mucosa, and (iv) survival and eventually outgrowth of the meningococcus in the bloodstream. When the meningococcus has reached the bloodstream and specific antibodies are absent, as is the case for young children or after introduction of a new strain in a population, the ultimate outgrowth depends on the efficacy of the innate immune response. Massive outgrowth leads within 12 h to fulminant meningococcal sepsis (FMS), characterized by high intravascular concentrations of endotoxin that set free high concentrations of proinflammatory mediators. These mediators belonging to the complement system, the contact system, the fibrinolytic system, and the cytokine system induce shock and diffuse intravascular coagulation. FMS can be fatal within 24 h, often before signs of meningitis have developed. In spite of the increasing possibilities for treatment in intensive care units, the mortality rate of FMS is still 30%. When the outgrowth of meningococci in the bloodstream is impeded, seeding of bacteria in the subarachnoidal compartment may lead to overt meningitis within 24 to 36 h. With appropriate antibiotics and good clinical surveillance, the mortality rate of this form of invasive disease is 1 to 2%. The overall mortality rate of meningococcal disease can only be reduced when patients without meningitis, i.e., those who may develop FMS, are recognized early. This means that the fundamental nature of the disease as a meningococcus septicemia deserves more attention.

Update on Meningococcal Disease with Emphasis on Pathogenesis and Clinical Management

Brass AL, Dykxhoorn DM, Benita Y, et al. Science . 2008;319(5865):921–926 PURPOSE OF THE STUDY. The HIV genome encodes only 15 proteins and, therefore, must use multiple host-cell collaborators for successful replication and transmission. Required host-derived proteins include CD4 as the primary virus receptor and chemokine receptors as coreceptors. This study identified multiple other host proteins required for HIV activity. METHODS. Human cells known to be susceptible to HIV were exposed in vitro to HIV. Using small interfering RNAs able to inhibit each known gene in the human genome 1 at a time, the investigators tested whether HIV could establish an infection and copy itself. HIV dependence on >21 000 human genes was examined. RESULTS. More than 250 human genes were identified to be required for efficient HIV replication. Termed “HIV-dependency factors,” the products of these genes are known to participate in a broad array of cellular functions and implicate unsuspected pathways in the virus life cycle. CONCLUSIONS. The extraordinary dependence of HIV on human host proteins for efficient transmission and replication provides many new potential targets for antiretroviral therapy. REVIEWER COMMENTS. An example of targeting host proteins is the use of chemokine receptor 5 (CCR5) inhibitors. Many people with CCR5 deficiency are very resistant to HIV infection yet have limited if any clinical consequences. Maraviroc CCR5 inhibitor is approved for treatment for HIV infection. This study identified many more such potential targets.

Identification of Host Proteins Required for HIV Infection Through a Functional Genomic Screen

Stochastic gene expression has been implicated in a variety of cellular processes, including cell differentiation and disease. In this issue of Cell, Weinberger et al. (2005) take an integrated computational-experimental approach to study the Tat transactivation feedback loop in HIV-1 and show that fluctuations in a key regulator, Tat, can result in a phenotypic bifurcation. This phenomenon is observed in an isogenic population where individual cells display two distinct expression states corresponding to latent and productive infection by HIV-1. These findings demonstrate the importance of stochastic gene expression in molecular "decision-making."

Stochastic Gene Expression in a Lentiviral Positive Feedback Loop: HIV-1 Tat Fluctuations Drive Phenotypic Diversity

The late Nobel Laureate Sir Peter Medawar once memorably described viruses as ‘bad news wrapped in protein’. Virus assembly in HIV is a remarkably well coordinated process in which the virus achieves extracellular budding using primarily intracellular budding machinery and also the unusual phenomenon of export from the cell of an RNA. Recruitment of the ESCRT system by HIV is one of the best documented examples of the comprehensive way in which a virus hijacks a normal cellular process. This review is a summary of our current understanding of the budding process of HIV, from genomic RNA capture through budding and on to viral maturation, but centering on the proteins of the ESCRT pathway and highlighting some recent advances in our understanding of the cellular components involved and the complex interplay between the Gag protein and the genomic RNA.

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Wrapping up the bad news – HIV assembly and release

One of the cellular defenses against virus infection is the silencing of viral gene expression. There is evidence that at least two gene-silencing mechanisms are used against the human immuno-deficiency virus (HIV). Paradoxically, this cellular defense mechanism contributes to viral latency and persistence, and we review here the relationship of viral latency to gene-silencing mechanisms.

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Andrew Ml Lever

Papers

Wrapping up the bad news – HIV assembly and release

Chromatin, gene silencing and HIV latency

Fatal melioidosis in a tourist returning from Thailand

Treatment of native valve candida endocarditis with fluconazole

Reasons for delay in administration of antibiotics to patients with meningitis and meningococcaemia