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In the current study, an Immersed Boundary Method for simulating cavitating flows with complex or moving boundaries is presented, which follows the discrete direct forcing approach. Although the Immersed Boundary Methods are widely used in various applications of single phase, multiphase and particulate flows, either incompressible or compressible, and numerous alternative formulations exist, to the best of the authors' knowledge, a handful of computational works employ such methodologies on cavitating flows. The herein proposed method, following the works of the author's group1,2,3, tries to fill this gap and to solidify the development of a computational tool of a simple formulation capable to tackle complex numerical problems of cavitation modelling. The method aims to be used in a wide range of applications of industrial interest and treat flows of engineering scales. Therefore, a validation of the method is performed by numerous benchmark test-cases, of progressively increasing complexity, from incompressible low Reynolds number to compressible and highly turbulent cavitating flows.

Although mouse infection models have been extensively used to study the host response to Mycobacterium tuberculosis, their validity in revealing determinants of human tuberculosis (TB) resistance and disease progression has been heavily debated. Here, we show that the modular transcriptional signature in the blood of susceptible mice infected with a clinical isolate of M. tuberculosis resembles that of active human TB disease, with dominance of a type I interferon response and neutrophil activation and recruitment, together with a loss in B lymphocyte, natural killer and T cell effector responses. In addition, resistant but not susceptible strains of mice show increased lung B cell, natural killer and T cell effector responses in the lung upon infection. Notably, the blood signature of active disease shared by mice and humans is also evident in latent TB progressors before diagnosis, suggesting that these responses both predict and contribute to the pathogenesis of progressive M. tuberculosis infection.

Cavitation induction is of high interest for a wide range of applications, from hydraulic machines to bioengineering applications. Numerous experimental and numerical studies have aimed to unveil the dynamics of cavitation to enhance the performance and lower the impact of erosion on machinery but also to employ its mechanics in advanced noninvasive medical procedures. The current work provides a comprehensive review of the methodologies that have been developed in the framework of computational fluid dynamics in order to study cavitating flows, highlighting the link of the application with the utilized approach. The methods are presented and assessed according to the class of physical problems addressed, which, in turn, are classified into problems of single-bubble dynamics, bubble cluster dynamics, and cavitating flows at engineering scales.

Accurate wall-shear stress (WSS) in-vitro measurements within complex geometries such as the human aortic arch under pulsatile flow are still difficult to achieve, meanwhile such data are important for classifying impacts of prosthetic valves on aortic walls. Micro-cantilever beams can serve to sense the WSS in such flows for applications in in-vitro flow tester. However, within pulsatile flows and complex 3D curved geometries such as the aortic arch, the flexible sensor structures are subject to oscillating boundary layer thickness and profile shape, which may not have been taken into account in the calibration procedure. The fluid–structure interaction is sensitive to these changes, thus reflecting also the flow-induced deflection of the sensor tip which is actually the sensing signal. We develop herein a methodology for in-situ calibration of the response of the sensors directly in the complex geometry of the aortic arch, assisted by reference data from numerical simulations of the flow under the same boundary conditions. For this procedure, a quick exchange of the heart valve in the tester with a tubular insert is done to provide a smooth contour in the curved aorta model. Arrays of 500 μ m long micro-pillar WSS sensors in the aorta model are calibrated under physiological pulsatile flow and used then for mapping the WSS evolution in the arch induced by two different heart valve, showing their difference of impact. The developed methodology completes the in-house built in-vitro flow tester with a reliable WSS measurement technique and provides a unique hydrodynamic testing facility for heart valve prostheses and their impact on the WSS distal along the aortic walls.

Tuberculosis (TB) is a leading cause of mortality due to infectious disease, but the factors determining disease progression are unclear. Transcriptional signatures associated with type I IFN signalling and neutrophilic inflammation were shown to correlate with disease severity in mouse models of TB. Here we show that similar transcriptional signatures correlate with increased bacterial loads and exacerbate pathology during Mycobacterium tuberculosis infection upon GM-CSF blockade. Loss of GM-CSF signalling or genetic susceptibility to TB (C3HeB/FeJ mice) result in type I IFN-induced neutrophil extracellular trap (NET) formation that promotes bacterial growth and promotes disease severity. Consistently, NETs are present in necrotic lung lesions of TB patients responding poorly to antibiotic therapy, supporting the role of NETs in a late stage of TB pathogenesis. Our findings reveal an important cytokine-based innate immune effector network with a central role in determining the outcome of M. tuberculosis infection., GM-CSF is involved in control over M. tuberculosis infection. Here the authors show that GM-CSF reduces type 1 interferon driven neutrophil recruitment, NETosis and bacterial growth in the lungs of infected mice, and provide evidence that this NETosis occurs in infected humans who are not responsive to antibiotic therapy.

Tuberculosis (TB), caused by Mycobacterium tuberculosis infection, is a leading cause of mortality and morbidity, causing ∼1.5 million deaths annually. CD4+ T cells and several cytokines, such as the Th1 cytokine IFN-γ, are critical in the control of this infection. Conversely, the immunosuppressive cytokine IL-10 has been shown to dampen Th1 cell responses to M. tuberculosis infection impairing bacterial clearance. However, the critical cellular source of IL-10 during M. tuberculosis infection is still unknown. Using IL-10 reporter mice, we show in this article that during the first 14 d of M. tuberculosis infection, the predominant cells expressing IL-10 in the lung were Ly6C+ monocytes. However, after day 21 postinfection, IL-10–expressing T cells were also highly represented. Notably, mice deficient in T cell–derived IL-10, but not mice deficient in monocyte-derived IL-10, showed a significant reduction in lung bacterial loads during chronic M. tuberculosis infection compared with fully IL-10–competent mice, indicating a major role for T cell–derived IL-10 in TB susceptibility. IL-10–expressing cells were detected among both CD4+ and CD8+ T cells, expressed high levels of CD44 and Tbet, and were able to coproduce IFN-γ and IL-10 upon ex vivo stimulation. Furthermore, during M. tuberculosis infection, Il10 expression in CD4+ T cells was partially regulated by both IL-27 and type I IFN signaling. Together, our data reveal that, despite the multiple immune sources of IL-10 during M. tuberculosis infection, activated effector T cells are the major source accounting for IL-10–induced TB susceptibility.

Understanding how immune challenges elicit different responses is critical for diagnosing and deciphering immune regulation. Using a modular strategy to interpret the complex transcriptional host response in mouse models of infection and inflammation, we show a breadth of immune responses in the lung. Lung immune signatures are dominated by either IFN-γ and IFN-inducible, IL-17-induced neutrophil- or allergy-associated gene expression. Type I IFN and IFN-γ-inducible, but not IL-17- or allergy-associated signatures, are preserved in the blood. While IL-17-associated genes identified in lung are detected in blood, the allergy signature is only detectable in blood CD4+ effector cells. Type I IFN-inducible genes are abrogated in the absence of IFN-γ signaling and decrease in the absence of IFNAR signaling, both independently contributing to the regulation of granulocyte responses and pathology during Toxoplasma gondii infection. Our framework provides an ideal tool for comparative analyses of transcriptional signatures contributing to protection or pathogenesis in disease., The authors present an extensive profile of host transcriptional respones to a diverse group of pathogens and allergens. In doing so, they identify TH1, type I IFN, TH17, and TH2 responses, that underlie each immune response in both the blood and lung, which represents a global profile of host-pathogen immune responses.

Analysis of the mouse transcriptional response to Listeria monocytogenes infection reveals that a large set of genes are perturbed in both blood and tissue and that these transcriptional responses are enriched for pathways of the immune response. Further we identified enrichment for both type I and type II interferon (IFN) signaling molecules in the blood and tissues upon infection. Since type I IFN signaling has been reported widely to impair bacterial clearance we examined gene expression from blood and tissues of wild type (WT) and type I IFNαβ receptor-deficient (Ifnar1-/-) mice at the basal level and upon infection with L. monocytogenes. Measurement of the fold change response upon infection in the absence of type I IFN signaling demonstrated an upregulation of specific genes at day 1 post infection. A less marked reduction of the global gene expression signature in blood or tissues from infected Ifnar1-/- as compared to WT mice was observed at days 2 and 3 after infection, with marked reduction in key genes such as Oasg1 and Stat2. Moreover, on in depth analysis, changes in gene expression in uninfected mice of key IFN regulatory genes including Irf9, Irf7, Stat1 and others were identified, and although induced by an equivalent degree upon infection this resulted in significantly lower final gene expression levels upon infection of Ifnar1-/- mice. These data highlight how dysregulation of this network in the steady state and temporally upon infection may determine the outcome of this bacterial infection and how basal levels of type I IFN-inducible genes may perturb an optimal host immune response to control intracellular bacterial infections such as L. monocytogenes.

Vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) remains the only prophylactic vaccine against tuberculosis, caused by Mycobacterium tuberculosis, but gives variable protection against pulmonary disease. The generation of host Th1 responses following BCG vaccination is accepted as the major mechanism of protection against M. tuberculosis infection. Early production of IL-17 in the lungs following M. tuberculosis challenge of mice previously vaccinated with M. tuberculosis peptides in adjuvant has been shown to be required for efficient Th1 cell recruitment. IL-10 regulates various processes involved in generation of Th1 and Th17 responses. Previous studies have shown IL-10 as a negative regulator of the immune response to primary M. tuberculosis infection, with Il10−/− mice having reduced lung bacterial loads. In this study we show that inhibition of IL-10 signaling during BCG vaccination enhances host-generated Ag-specific IFN-γ and IL-17A responses, and that this regimen gives significantly greater protection against aerogenic M. tuberculosis challenge in both susceptible and relatively resistant strains of mice. In M. tuberculosis-susceptible CBA/J mice, Ab blockade of IL-10R specifically during BCG vaccination resulted in additional protection against M. tuberculosis challenge of >1-log10 compared with equivalent isotype-treated controls. The protection observed following BCG vaccination concurrent with anti–IL-10R mAb treatment was sustained through chronic M. tuberculosis infection and correlated with enhanced lung Th1 and Th17 responses and increased IFN-γ and IL-17A production by γδ T cells and an innate-like Thy1.2+CD3− lymphoid population. We show that IL-10 inhibits optimal BCG-elicited protection, therefore suggesting that antagonists of IL-10 may be of great benefit as adjuvants in preventive vaccination against tuberculosis.

A series of large-ringed calix[6,7,8]arene analogues have been synthesised and their affect against Mycobacterium tuberculosis in vivo established. In general, when p-phenylcalixarenes and tert-butylcalixarenes were not functionalised at the lower rim, low biological activities were observed. However on going from partially to fully lower rim pegylated calixarenes the anti-mycobacterial properties improved. The addition of cyanopropoxy groups at the lower rim gave rise to low activities, whereas the addition of acetate moieties interestingly had pro-TB effects. Two upper rim sulfonated calixarenes showed promising properties. In the course of this work, a high yielding procedure to synthesise p-phenylcalix[7]arene was also established.

IL-10 regulates the balance of an immune response between pathogen clearance and immunopathology. We show here that Mycobacterium tuberculosis (Mtb) infection in the absence of IL-10 (IL-10(-/-) mice) results in reduced bacterial loads in the lung. This reduction was preceded by an accelerated and enhanced IFN-gamma response in the lung, an increased influx of CD4(+) T cells into the lung, and enhanced production of chemokines and cytokines, including CXCL10 and IL-17, in both the lung and the serum. Neutralization of IL-17 affected neither the enhanced production of CXCL10 nor the accumulation of IFN-gamma-producing T cells in the lungs, but led to reduced numbers of granulocytes in the lung and reduced bacterial loads in the spleens of Mtb-infected mice. This suggests that IL-17 may contribute to dissemination of Mtb.

Mycobacterium tuberculosis remains one of the most successful pathogens of mankind, infecting one-third of the world's population and causing over 2 million deaths annually (35). The incidence of tuberculosis (TB) has increased in recent years and this is attributed to a number of factors, including coinfection with human immunodeficiency virus and the emergence of multidrug-resistant strains of the M. tuberculosis bacterium (24). The chemotherapeutic regimens available for treatment of TB are far from ideal, requiring the ingestion of multiple anti-TB drugs in combination over prolonged periods. The side effects of current drug regimens, combined with the protracted duration of treatment, frequently result in poor patient compliance, treatment failures, and associated emergence of drug resistance with major financial implications (26). The close supervision of treatment that is needed to raise efficacy to acceptable levels, such as the World Health Organization's DOTS program, pushes the cost beyond the reach of many of the world's populations most in need. The development of novel, shorter treatments for TB is now an urgent requirement. The possibilities of immunotherapy deserve more attention than they have received in the past, not least because immunotherapy could circumvent the problems of drug resistance. However, this must be approached with caution, because the disease is itself a consequence of the immune response and one must stimulate protective and not harmful aspects of the response. M. tuberculosis is a facultative intracellular pathogen, and it is cell-mediated Th1 type immunity, comprising cytokine-mediated monocyte activation and T-cell cytotoxicity toward infected macrophages, that is the major component of the protective immune response. The role of antibodies in protection is less clear but is being reevaluated in light of a number of recent publications (7, 13, 34). Indeed, it was the concern of one of us (S.J.) that antibodies that were being administered to patients in the form of high-dose intravenous immunoglobulin (hdIVIg) might theoretically exacerbate latent or undiagnosed tuberculosis that led to the present study. IVIg is a human blood product prepared from the plasma of from 1,000 to 15,000 donors per batch. It has been widely used in the treatment of primary and secondary antibody deficiencies and in these circumstances it is administered at replacement dose (200 to 400 mg/kg of body weight at 2- to 4-week intervals) (28). In contrast, hdIVIg, given at 2 g/kg/month and used as an immunomodulatory agent, was first described for immune-mediated thrombocytopenia (18) but is now widely used in treating a range of neurological, hematological, immunological, dermatological, and rheumatological immune and inflammatory disorders (29). Recently, the use of IVIg as an anti-infectious agent in viral and bacterial infections has been reviewed (4), and it has been demonstrated that IVIg given in combination with ampicillin is protective against pneumococcal pneumonia (10). In this study, we investigated the capacity of hdIVIg to influence the course of infection in a murine model of TB.

Mycobacterium tuberculosis infects one-third of the world's population, and it accounts for more deaths each year than any other infectious bacterium (13). The problem, associated with multiple-drug resistance (12), has prompted a great interest in understanding new alternatives in host-mediated mechanisms of disease intervention. A new therapeutic agent, with activity mediated through a host-derived effector mechanism, would be particularly attractive, since it could be less susceptible to selection for drug resistance; if the balance between the pathogenic mycobacteria and the macrophage can be manipulated in favor of the host macrophage, it may be possible to develop novel adjunctive therapies for tuberculosis control. Calixarenes have been used as building blocks for host molecules with numerous applications in supramolecular chemistry (5); some were identified as having antimycobacterial activity (3, 7). Most experimental work has been carried out with the compound Macrocyclon, also known as HOC 12.5EO, which was prepared by reacting the macrocycle HOC under basic conditions with ethylene oxide to give a heterogeneous compound with an average polyethylene glycol (PEG) chain of 12.5 U (3). The compound HOC was prepared from t-octylphenol and formaldehyde by a modified Zinke-Ziegler procedure; for many years, it was believed to be a cyclic tetrameric compound (3). Although the antibacterial mechanism of action of HOC compounds is not known, we have excluded extracellular inhibition of mycobacterial growth by Macrocyclon treatment (3, 7, 8). Therefore, it is believed that they work through a host-mediated mechanism (7), a view supported by reports showing activity in a wide range of in vivo models of infection in addition to tuberculosis (10). In this study, we have extended observations on the parent preparation, Macrocyclon, to show that it significantly affects mycobacterial growth in murine macrophages by a mechanism requiring inducible nitric oxide synthase (iNOS) activity. In addition, we show that Macrocyclon is effective in athymic and major histocompatibility complex class II−/− (MHC-II−/−) mice, and we have synthesized new structurally related calixarene compounds which show significant antimycobacterial activity.

Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites which are generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxifies reactive oxygen species, and DNA repair systems, which repair damage resulting from oxidative stress. To (i) determine the relative importance of the DNA repair system when oxidative stress is encountered by the Mycobacterium tuberculosis complex during infection of the host and to (ii) provide improved mycobacterial hosts as live carriers to express foreign antigens, the recA locus was inactivated by allelic exchange in Mycobacterium bovis BCG. The recA mutants are sensitive to DNA-damaging agents and show increased susceptibility to metronidazole, the first lead compound active against the dormant M. tuberculosis complex. Surprisingly, the recA genotype does not affect the in vitro dormancy response, nor does the defect in the DNA repair system lead to attenuation as determined in a mouse infection model. The recA mutants will be a valuable tool for further development of BCG as an antigen delivery system to express foreign antigens and as a source of a genetically stable vaccine against tuberculosis.

Summary Activated dendritic cells are critically important in the priming of T-cell responses. In this report we show that the infection of a conditionally immortalized dendritic cell line (tsDC) with Mycobacterium tuberculosis resulted in the up-regulation of B7-1 and B7-2 co-stimulatory molecules and the induction of several inflammatory cytokines, including tumour necrosis factor-α and interleukin-6, -1β and -12. In addition, we show that these activated dendritic cells were capable of eliciting antigen-specific T-cell responses and potent anti-mycobacterial protective immunity in a murine model of experimental tuberculosis infection.

Influenza followed by severe acute bacterial pneumonia is a major cause of mortality worldwide. Several mechanisms account for this enhanced susceptibility, including increased production of type I interferon (IFN). In individuals infected with Mycobacterium tuberculosis, the influence of acute viral infections on tuberculosis progression is unclear. We show that prior exposure of mice to influenza A virus, followed by M. tuberculosis infection, leads to enhanced mycobacterial growth and decreased survival. Following M. tuberculosis/influenza virus coinfection, mycobacterial growth is enhanced by a type I IFN signaling pathway. Our findings highlight the detrimental influence influenza virus infection can have before or during M. tuberculosis infection.

There are 3 million deaths per annum worldwide due to tuberculosis, and AIDS is compounding the problem. A better vaccine than the live mycobacterium currently in use, bacillus Calmette-Guérin (BCG), is needed. When mice were injected with plasmid DNA encoding a single mycobacterial antigen (65-kDa heat shock protein, hsp65) they made specific cellular and humoral responses to the protein and became immune to subsequent challenge with Mycobacterium tuberculosis. Protection was equivalent to that obtained by vaccinating with live BCG, whereas immunizing with the protein was ineffective. Protection was also obtained with DNA encoding another mycobacterial antigen (36-kDa proline-rich antigen). These results suggest that DNA vaccination might yield improved vaccines to replace BCG.

Mycobacterium tuberculosis continues to kill about 3 million people every year, more than any other single infectious agent. This is attributed primarily to an inadequate immune response towards infecting bacteria, which suffer growth inhibition rather than death and subsequently multiply catastrophically. Although the bacillus Calmette-Guerin (BCG) vaccine is widely used, it has major limitations as a preventative measure. In addition, effective treatment requires that patients take large doses of antibacterial drug combinations for at least 6 months after diagnosis, which is difficult to achieve in many parts of the world and is further restricted by the emergence of multidrug-resistant strains of M. tuberculosis. In these circumstances, immunotherapy to boost the efficiency of the immune system in infected patients could be a valuable adjunct to antibacterial chemotherapy. Here we show in mice that DNA vaccines, initially designed to prevent infection, can also have a pronounced therapeutic action. In heavily infected mice, DNA vaccinations can switch the immune response from one that is relatively inefficient and gives bacterial stasis to one that kills bacteria. Application of such immunotherapy in conjunction with conventional chemotherapeutic antibacterial drugs might result in faster or more certain cure of the disease in humans.

Tuberculosis is responsible for >2 million deaths a year, and the number of new cases is rising worldwide. DNA vaccination combined with Mycobacterium bovis bacillus Calmette Guérin (BCG) represents a potential strategy for prevention of this disease. Here, we used a heterologous prime-boost immunization approach using a combination of DNA plasmids and BCG in order to improve the efficacy of vaccination against Mycobacterium tuberculosis infection in mice. As model antigens, we selected the M. tuberculosis Apa (for alanine-proline-rich antigen) and the immunodominant Hsp65 and Hsp70 mycobacterial antigens combined with BCG. We demonstrated that animals injected with a combination of DNA vectors expressing these antigens, when boosted with BCG, showed increased specific antimycobacterial immune responses compared to animals vaccinated with BCG alone. More importantly, the protection achieved with this regimen was also significantly better than with BCG alone.

We have previously demonstrated that vaccination of mice with plasmid DNA vectors expressing immunodominant mycobacterial genes induced cellular immune responses and significant protection against challenge withMycobacterium tuberculosis. We demonstrate here, using in vitro-synthesized RNA, that vaccination with DNA or RNA constructs expressing theM. tuberculosisMPT83 antigen are capable of inducing specific humoral and T-cell immune responses and confer modest but significant protection againstM. tuberculosischallenge in mice. This is the first report of protective immunity conferred against intracellular bacteria by an RNA vaccine. This novel approach avoids some of the drawbacks of DNA vaccines and illustrates the potential for developing new antimycobacterial immunization strategies.

The widely used vaccine against tuberculosis, BCG, shows evidence of genetic instability. It has undergone major genetic rearrangements resulting in deletion and duplication of segments of its chromosome. In order to produce a BCG strain with more favourable genetic properties, we inactivated the recA gene. Targeted deletion of the recA gene of BCG resulted in a complete loss of recombination between homologous, chromosomally-located sequences, as well as between plasmid- and chromosomally-located sequences. The deltarecA mutant BCG was as effective as the wild-type in conferring protection in mice against an intravenous challenge with virulent Mycobacterium tuberculosis, indicating that the loss of an SOS response-mediated DNA repair mechanism did not compromise the immunological properties of BCG. The availability of a genetically stable, fully immunogenic BCG is important for the future development of BCG as a live vaccine.

Mycobacterium tuberculosis H37Ra is an attenuated tubercle bacillus closely related to the virulent type strain M. tuberculosis H37Rv. Despite extensive study, the reason for the decreased virulence of M. tuberculosis H37Ra has not been determined. A genomic approach was therefore initiated to identify genetic differences between M. tuberculosis H37Rv and M. tuberculosis H37Ra as a means of pinpointing the attenuating mutation(s). Digestion with the rare-cutting restriction endonuclease Dra I revealed two polymorphisms between the strains: a 480-kb fragment in M. tuberculosis H37Rv was replaced by two fragments of 220 and 260 kb in M. tuberculosis H37Ra, while there was a ∼7.9-kb Dra I fragment in M. tuberculosis H37Ra that had no counterpart in M. tuberculosis H37Rv. As the M. tuberculosis insertion sequence IS 6110 contains a single Dra I restriction site, it was considered possible that these polymorphisms were the result of IS 6110 transposition events in M. tuberculosis H37Ra, events that may have inactivated virulence genes. The 7.9-kb polymorphism was found to be due to the presence of the previously described H37Rv RvD2 deletion in M. tuberculosis H37Ra, with sequence analysis suggesting an IS 6110 -mediated deletion mechanism for loss of RvD2. Three other IS 6110 -catalyzed deletions from the M. tuberculosis H37Rv chromosome (RvD3 to RvD5) were also identified, suggesting that this mechanism plays an important role in genome plasticity in the tubercle bacilli. Comparative mapping and sequencing revealed that the 480-kb polymorphism was due to an IS 6110 insertion in M. tuberculosis H37Ra near oriC . Complementation of M. tuberculosis H37Ra with a 2.9-kb restriction fragment from M. tuberculosis H37Rv that encompassed the IS 6110 insertion did not increase the survival of recombinant M. tuberculosis H37Ra in mice. In conclusion, this study describes the presence and mechanisms of genomic variation between M. tuberculosis H37Ra and M. tuberculosis H37Rv, although the role that they play in the attenuation of M. tuberculosis H37Ra is unclear.

The availability of cloned mycobacterial genes and suitable vectors for expression in mammalian cells has now opened a new avenue in which individual mycobacterial protein antigens can be tested for their ability to confer protective immunity. Expression of protection against mycobacteria is largely a function of antigen specific CD4 and cytotoxic CD8 T cells. We have found that intramuscular injection of plasmid DNA expressing several mycobacterial immunodominant genes can induce both T cell populations and confer protection equivalent to BCG, suggesting that this approach may lead to a new vaccine.