Photodynamic therapy for the treatment of Pseudomonas aeruginosa infections: A scoping review.

BACKGROUND: Pseudomonas aeruginosa is a Gram-negative bacillus that causes superficial and deep infections, which can be minor to life-threatening. Recently, P. aeruginosa has gained significant relevance due to the increased incidence of multidrug-resistant (MDR) strains that complicate antibiotic treatment. Due to MDR strains, alternative therapies, such as antimicrobial photodynamic therapy (PDT), are presented as a good option to treat nonsystemic infections. PDT combines a photosensitizer agent (PS), light, and oxygen to generate free radicals that destroy bacterial structures such as the envelope, matrix, and genetic material. This work aimed to identify the development stage of the PDT applied to P. aeruginosa to conclude which research stage should be emphasized more. METHODS: Systematic bibliographic search in various public databases was performed. Related articles were identified using keywords, and relevant ones were selected using inclusion and exclusion criteria according to the PRISMA protocol. RESULTS: We found 29 articles that meet the criteria, constituting a good body of evidence associated with using PDT against P. aeruginosa in vitro and less developed for in vivo research. CONCLUSIONS: We conclude that PDT could become an effective adjunct to antimicrobial therapy against P. aeruginosa. This effectiveness depends on the PS used and the location of the infection. Many PS already demonstrated efficacy in PDT, but the evidence is supported significantly by in vitro and very few in vivo studies. Therefore, we conclude that further research efforts should focus on demonstrating the safety and efficacy of these PSs in vivo in animal infection models.

Effect of photodynamic therapy on multidrug-resistant Acinetobacter baumannii: A scoping review.

BACKGROUND: Acinetobacter baumannii is a Gram-negative, non-fermenting coccobacillus of the Moraxellaceae family. It is an opportunistic pathogen responsible for several hospital-acquired infections (HAIs) associated with skin and tissue infections at surgical sites, catheter-associated urinary tract infections, and central line catheters. Multidrug-resistant (MDR) A. baumannii has caused hospital outbreaks that are difficult to eradicate and represent one of the leading producers of HAIs. MDR-A. baumannii presents a broad range of resistance to different antimicrobials, including carbapenems. Due to the low sensitivity to conventional antibiotic therapies, it is necessary to identify other therapeutic options. Antimicrobial photodynamic therapy (aPDT) is a promising alternative and complementary approach to address the shortage of antimicrobials in MDR-A. baumannii. APDT combines a photosensitizer agent, light, and oxygen to achieve a bactericidal/bacteriostatic effect. The effect is given by producing reactive oxygen species (ROS) that produce photooxidative stress over bacterial structures, such as the envelope and the DNA. METHODS: This study aims to systematically collect bibliographic information from databases such as PubMed, Scopus, and google scholar to analyze the relevant articles critically. RESULTS: An increasing body of evidence demonstrates the efficacy of photodynamic inactivation in eliminating A. baumannii strains, both in vitro and in vivo. CONCLUSIONS: The evidence supports that photodynamic inactivation is an alternative capable of eliminating strains of Acinetobacter baumannii and may considerably improve the treatment of MDR strains. Although they do exist, aPDT studies on MDR strains of A. baumannii are scarce and should increase since it is on these strains that photodynamic therapy becomes attractive.

Synergistic effect of Ru(II)-based type II photodynamic therapy with cefotaxime on clinical isolates of ESBL-producing Klebsiella pneumoniae.

Multidrug-resistant bacteria, such as ESBL producing-Klebsiella pneumoniae, have increased substantially, encouraging the development of complementary therapies such as photodynamic inactivation (PDI). PDI uses photosensitizer (PS) compounds that kill bacteria using light to produce reactive oxygen species. We test Ru-based PS to inhibit K. pneumoniae and advance in the characterization of the mode of action. The PDI activity of PSRu-L2, and PSRu-L3, was determined by serial micro dilutions exposing K. pneumoniae to 0.612 J/cm 2 of light dose. PS interaction with cefotaxime was determined on a collection of 118 clinical isolates of K. pneumoniae. To characterize the mode of action of PDI, the bacterial response to oxidative stress was measured by RT-qPCR. Also, the cytotoxicity on mammalian cells was assessed by trypan blue exclusion. Over clinical isolates, the compounds are bactericidal, at doses of 8 µg/mL PSRu-L2 and 4 µg/mL PSRu-L3, inhibit bacterial growth by 3 log10 (>99.9%) with a lethality of 30 min. A remarkable synergistic effect of the PSRu-L2 and PSRu-L3 compounds with cefotaxime increased the bactericidal effect in a subpopulation of 66 ESBL-clinical isolates to > 6 log10 with an FIC-value of 0.16 and 0.17, respectively. The bacterial transcription response suggests that the mode of action occurs through Type II oxidative stress. The upregulation of the extracytoplasmic virulence factors mrkD, magA, and rmpA accompanied this response. Also, the compounds show little or no toxicity in vitro on HEp-2 and HEK293T cells. Through the type II effect, PSs compounds are bactericidal, synergistic on K. pneumoniae, and have low cytotoxicity in mammals.

Effective Treatment against ESBL-Producing Klebsiella pneumoniae through Synergism of the Photodynamic Activity of Re (I) Compounds with Beta-Lactams.

BACKGROUND: Extended-spectrum beta-lactamase (ESBL) and carbapenemase (KPC+) producing Klebsiella pneumoniae are multidrug-resistant bacteria (MDR) with the highest risk to human health. The significant reduction of new antibiotics development can be overcome by complementing with alternative therapies, such as antimicrobial photodynamic therapy (aPDI). Through photosensitizer (PS) compounds, aPDI produces local oxidative stress-activated by light (photooxidative stress), nonspecifically killing bacteria. METHODOLOGY: Bimetallic Re(I)-based compounds, PSRe-µL1 and PSRe-µL2, were tested in aPDI and compared with a Ru(II)-based PS positive control. The ability of PSRe-µL1 and PSRe-µL2 to inhibit K. pneumoniae was evaluated under a photon flux of 17 µW/cm2. In addition, an improved aPDI effect with imipenem on KPC+ bacteria and a synergistic effect with cefotaxime on ESBL producers of a collection of 118 clinical isolates of K. pneumoniae was determined. Furthermore, trypan blue exclusion assays determined the PS cytotoxicity on mammalian cells. RESULTS: At a minimum dose of 4 µg/mL, both the PSRe-µL1 and PSRe-µL2 significantly inhibited in 3log10 (>99.9%) the bacterial growth and showed a lethality of 60 and 30 min of light exposure, respectively. Furthermore, they were active on clinical isolates of K. pneumoniae at 3-6 log10. Additionally, a remarkably increased effectiveness of aPDI was observed over KPC+ bacteria when mixed with imipenem, and a synergistic effect from 3 to 6log10 over ESBL producers of K. pneumoniae clinic isolates when mixed with cefotaxime was determined for both PSs. Furthermore, the compounds show no dark toxicity and low light-dependent toxicity in vitro to mammalian HEp-2 and HEK293 cells. CONCLUSION: Compounds PSRe-µL1 and PSRe-µL2 produce an effective and synergistic aPDI effect on KPC+, ESBL, and clinical isolates of K. pneumoniae and have low cytotoxicity in mammalian cells.

Effective Photodynamic Therapy with Ir(III) for Virulent Clinical Isolates of Extended-Spectrum Beta-Lactamase Klebsiella pneumoniae.

BACKGROUND: The extended-spectrum beta-lactamase (ESBL) Klebsiella pneumoniae is one of the leading causes of health-associated infections (HAIs), whose antibiotic treatments have been severely reduced. Moreover, HAI bacteria may harbor pathogenic factors such as siderophores, enzymes, or capsules, which increase the virulence of these strains. Thus, new therapies, such as antimicrobial photodynamic inactivation (aPDI), are needed. METHOD: A collection of 118 clinical isolates of K. pneumoniae was characterized by susceptibility and virulence through the determination of the minimum inhibitory concentration (MIC) of amikacin (Amk), cefotaxime (Cfx), ceftazidime (Cfz), imipenem (Imp), meropenem (Mer), and piperacillin-tazobactam (Pip-Taz); and, by PCR, the frequency of the virulence genes K2, magA, rmpA, entB, ybtS, and allS. Susceptibility to innate immunity, such as human serum, macrophages, and polymorphonuclear cells, was tested. All the strains were tested for sensitivity to the photosensitizer PSIR-3 (4 µg/mL) in a 17 µW/cm2 for 30 min aPDI. RESULTS: A significantly higher frequency of virulence genes in ESBL than non-ESBL bacteria was observed. The isolates of the genotype K2+, ybtS+, and allS+ display enhanced virulence, since they showed higher resistance to human serum, as well as to phagocytosis. All strains are susceptible to the aPDI with PSIR-3 decreasing viability in 3log10. The combined treatment with Cfx improved the aPDI to 6log10 for the ESBL strains. The combined treatment is synergistic, as it showed a fractional inhibitory concentration (FIC) index value of 0.15. CONCLUSIONS: The aPDI effectively inhibits clinical isolates of K. pneumoniae, including the riskier strains of ESBL-producing bacteria and the K2+, ybtS+, and allS+ genotype. The aPDI with PSIR-3 is synergistic with Cfx.

Photodynamic therapy for treatment of Staphylococcus aureus infections.

BACKGROUND: Staphylococcus aureus is a Gram-positive spherical bacterium that commonly causes various infections which can range from superficial to life-threatening. Hospital strains of S. aureus are often resistant to antibiotics, which has made their treatment difficult in recent decades. Other therapeutic alternatives have been postulated to overcome the drawbacks of antibiotic multi-resistance. Of these, photodynamic therapy (PDT) is a promising approach to address the notable shortage of new active antibiotics against multidrug-resistant S. aureus. PDT combines the use of a photosensitizer agent, light, and oxygen to eradicate pathogenic microorganisms. Through a systematic analysis of published results, this work aims to verify the usefulness of applying PDT in treating multidrug-resistant S.aureus infections. METHODS: This review was based on a bibliographic search in various databases and the analysis of relevant publications. RESULTS: There is currently a large body of evidence demonstrating the efficacy of photodynamic therapy in eliminating S.aureus strains. Both biofilm-producing strains, as well as multidrug-resistant strains. CONCLUSION: We conclude that there is sufficient scientific evidence that PDT is a useful adjunct to traditional antibiotic therapy for treating S. aureus infections. Clinical application through appropriate trials should be introduced to further define optimal treatment protocols, safety and efficay.

The mode of action of the PSIR-3 photosensitizer in the photodynamic inactivation of Klebsiella pneumoniae is by the production of type II ROS which activate RpoE-regulated extracytoplasmic factors.

BACKGROUND: Due to increased bacterial multi-drug resistance (MDR), there is an antibiotic depletion to treat infectious diseases. Consequently, other promising options have emerged, such as the antimicrobial photodynamic inactivation therapy (aPDI) based on photosensitizer (PS) compounds to produce light-activated local oxidative stress (photooxidative stress). However, there are scarce studies regarding the mode of action of PS compounds to induce photooxidative stress on pathogenic γ-proteobacteria such as MDR-Klebsiella pneumoniae. METHODOLOGY: The mode of action exerted by the cationic Ir(III)-based PS (PSIR-3) to inhibit the growth of K. pneumoniae was analyzed. RT-qPCR determined the transcriptional response induced by PSIR-3 on bacteria treated with aPDI. The expression levels of genes associated with a bacterial oxidative response, such as oxyR and sodA, and the extracytoplasmic, regulators rpoE and hfq were determined. Also, were determined the transcriptional response of the extracytoplasmic factors mrkD, acrB, magA, and rmpA. RESULTS: At 17 µW/cm2 photon flux and 4 µg/mL of the PSIR-3 compound, the K. pneumoniae growth was inhibited in 3 log10. Compared with untreated bacteria, the transcriptional response induced by PSIR-3 occurs via the extracytoplasmic sigma factor rpoE and hfq. In contrast, no participation in the oxyR pathway or induction of the sodA gene was observed. This response was accompanied by the upregulation of the extracytoplasmic virulence factors mrkD, magA, and rmpA. CONCLUSIONS: PDI aPDI produced by PSIR-3 kills K. pneumoniae and may induce damage to the bacterial envelope. The bacterium tries to avoid this injury by activation of extracytoplasmic factors mediated through the rpoE regulon.

Synergistic effect of combined imipenem and photodynamic treatment with the cationic Ir(III) complexes to polypyridine ligand on carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: Carbapenemase-producing strains of Klebsiella pneumoniae (KPC+) are one of the multi-drug resistant bacteria with the highest risk for human health. The colistin is the only antibiotic option against KPC+; however, due to its emerging resistance, therapies such as antimicrobial photodynamic inactivation (aPDI), are needed. APDI uses photosensitizer compounds (PS) to produce light-activated local oxidative stress (photooxidative stress). Within the PSs variety, cationic PSs are thought to interact closely with the bacterial envelope producing an increased cytotoxic effect. METHODOLOGY: The Ir(III)-based cationic compounds, PSIR-3, and PSIR-4 were tested on aPDI and compared to a positive control of Ru(II)-based PS. The PSIR-3 and PSIR-4 abilities to inhibit the growth of KPC+ and KPC- bacteria were evaluated, under 17 µW/cm2 photon flux. Also, the cytotoxicity of the PSs in eukaryotic cells was determined by MTS and trypan blue exclusion assays. RESULTS: After light-activation, only the PSIR-3 compound inhibited 3 log10 (> 99.9 %) bacterial growth in a minimum dose of 4 µg/mL with the lethality of 30 min of light exposure. Outstandingly, the compound PSIR-3 showed a synergistic effect with imipenem, significantly increasing the bacterial inhibition of KPC+ to 6 log10, which was not observed in the control compound. In normal immortalized gastric cell line GES-1, the compound PSIR-3 showed no significant cytotoxicity, although increased cytotoxicity under light-activation was observed on gastric cancer-derived cells AGS. CONCLUSION: The PSIR-3 compound produces an efficient aPDI, killing K. pneumoniae KPC+- strains, and increasing its susceptibility in conjunction with imipenem, exhibiting low cytotoxicity to normal eukaryotic cells.

The 5' untranslated region of the anti-apoptotic protein Survivin contains an inhibitory upstream AUG codon.

Survivin (BIRC5) is an anti-apoptotic protein that is important in cancer. Mechanisms responsible for controlling Survivin levels in cells include transcriptional regulation and modulation of protein stability via post-translational modifications; however to date, translational control has been poorly studied. Here, we focused particularly on the primary control elements present in the Survivin 5' untranslated region (5'UTR). Bioinformatic analysis of ribosome occupancy on the Survivin 5'UTR revealed the presence of elongating ribosomes upstream of the canonical initiator AUG, suggesting an alternative upstream initiator AUG (uAUG) might exist. This uAUG was found out-of-frame at position -71 and appeared as a conserved element in mammals. RACE analysis revealed different transcriptional start sites for BIRC5, which indicated that translational control by this uAUG is restricted to longer 5'UTR variants. We studied the activity of the uAUG in different cell types by cloning the Survivin 5'UTR DNA sequence (wild-type and mutated variants) upstream of renilla luciferase (RLuc) into a pcDNA3 plasmid. Changes in RLuc activity were determined by luminescence assays and Western blotting. Results showed that when this uAUG was mutated to AUU or AGG in the cloned Survivin 5'UTR, RLuc activity was significantly increased. Similar results were obtained when uAUG was positioned inframe with the RLuc initiator AUG. Immunodetection of Renilla (35 kDa) by Western blotting revealed the presence of a second band (37 kDa approximately) in cells transfected with the Inframe reporter constructs, indicating that the uAUG was functional in our experimental conditions. In conclusion, our experimental data demonstrate the presence of an alternative and inhibitory initiator uAUG in the Survivin 5' UTR. This inhibitory uAUG may help understanding how Survivin expression is downregulated under physiological or pathological conditions.

Photodynamic treatment with cationic Ir(III) complexes induces a synergistic antimicrobial effect with imipenem over carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: Bacteria prevalent in the hospital environment have developed multi-drug resistance (MDR), such as the carbapenemase-producing Klebsiella pneumoniae (KPC+). Photodynamic therapy (PDT), which uses light-activated photosensitizer compounds (PSs), has emerged as an alternative to antibiotics. Cationic-PSs have a better bactericidal effect by interacting more closely with the bacterial envelope. METHODS: Two PSs based on cationic Ir (III) compounds (PSIR-1 and PSIR-2) were studied in photodynamic therapy against KPC+ and KPC- bacteria, and their PDT activities were compared with a cationic Ru(II) control compound (PS -Ru). RESULTS: Similar to the behavior of PS-Ru control, the cytotoxicity of PSIR-1 and 2, showing a bacterial inhibition growth of more than 3log10 (>99.9 % inactivation), at light fluency of 17 µW/cm2. The minimal dose to accomplish the inhibition in 3log10 was determined for PSIR-1 and PSIR-2 at 4 and 2 µg/mL, respectively and the lethality was 30 min of light exposure for both compounds. Notably, the PSIR-1 and 2 compounds showed a synergistic effect with imipenem by significantly increasing (up to 6 log10) the photodynamic bactericidal effect for KPC+ strains. This synergy is specific for PSIR-1 and 2 compounds, since it was not observed with the PS-Ru control. On normal gastric cells GES-1, both PSIR-1 and 2 showed significant cytotoxicity; however, the highest cytotoxicity was found in gastric tumor cells (AGS). CONCLUSION: The compounds PSIR-1 and 2 are bactericidal photosensitizers and represent a promising alternative for complementing the treatment of infections by MDR bacteria since they should not be toxic in the dark.

Photodynamic treatment for multidrug-resistant Gram-negative bacteria: Perspectives for the treatment of Klebsiella pneumoniae infections.

The emergence of multi-drug resistance for pathogenic bacteria is one of the most pressing global threats to human health in the 21st century. Hence, the availability of new treatment becomes indispensable to prevent morbidity and mortality caused by infectious agents. This article reviews the antimicrobial properties of photodynamic therapy (PDT), which is based on the use of photosensitizers compounds (PSs). The PSs are non-toxic small molecules, which induce oxidative stress only under excitation with light. Then, the PDT has the advantage to be locally activated using phototherapy devices. We focus on PDT for the Klebsiella pneumoniae, as an example of Gram-negative bacteria, due to its relevance as an agent of health-associated infections (HAI) and a multi-drug resistant bacteria. K. pneumoniae is a fermentative bacillus, member of the Enterobacteriaceae family, which is most commonly associated with producing infection of the urinary tract (UTI) and pneumonia. K. pneumoniae infections may occur in deep organs such as bladder or lungs tissues; therefore, activating light must get access or penetrate tissues with sufficient power to produce effective PDT. Consequently, the rationale for selecting the most appropriate PSs, as well as photodynamic devices and photon fluence doses, were reviewed. Also, the mechanisms by which PDT activates the immune system and its importance to eradicate the infection successfully, are discussed.

Recombinant BCG Vaccines Reduce Pneumovirus-Caused Airway Pathology by Inducing Protective Humoral Immunity.

The Human Respiratory Syncytial Virus (hRSV) and the Human Metapneumovirus (hMPV) are two pneumoviruses that are leading agents causing acute lower respiratory tract infections (ALRTIs) affecting young infants, the elderly, and immunocompromised patients worldwide. Since these pathogens were first discovered, many approaches for the licensing of safe and effective vaccines have been explored being unsuccessful to date. We have previously described that immunization with recombinant strains of Mycobacterium bovis Bacillus Calmette-Guérin (rBCG) expressing the hRSV nucleoprotein (rBCG-N) or the hMPV phosphoprotein (rBCG-P) induced immune protection against each respective virus. These vaccines efficiently promoted viral clearance without significant lung damage, mainly through the induction of a T helper 1 cellular immunity. Here we show that upon viral challenge, rBCG-immunized mice developed a protective humoral immunity, characterized by production of antibodies specific for most hRSV and hMPV proteins. Further, isotype switching from IgG1 to IgG2a was observed in mice immunized with rBCG vaccines and correlated with an increased viral clearance, as compared to unimmunized animals. Finally, sera obtained from animals immunized with rBCG vaccines and infected with their respective viruses exhibited virus neutralizing capacity and protected naïve mice from viral replication and pulmonary disease. These results support the notion that the use of rBCG strains could be considered as an effective vaccination approach against other respiratory viruses with similar biology as hRSV and hMPV.

Evaluation of monoclonal antibodies that detect conserved proteins from Respiratory Syncytial Virus, Metapneumovirus and Adenovirus in human samples.

Human Respiratory Syncytial Virus (hRSV), human Metapneumovirus (hMPV) and Adenovirus (ADV), are three of the most prevalent viruses responsible for pneumonia and bronchiolitis in children and elderly worldwide, accounting for a high number of hospitalizations annually. Diagnosis of these viruses is required to take clinical actions that allow an appropriate patient management. Thereby, new strategies to design fast diagnostic methods are highly required. In the present work, six monoclonal antibodies (mAbs, two for each virus) specific for conserved proteins from hRSV, hMPV and ADV were generated and evaluated through different immunological techniques, based on detection of purified protein, viral particles and human samples. In vitro evaluation of these antibodies showed higher specificity and sensitivity than commercial antibodies tested in this study. These antibodies were used to design a sandwich ELISA tests that allowed the detection of hRSV, hMPV, and ADV in human nasopharyngeal swabs. We observed that hRSV and ADV were detected with sensitivity and specificity equivalent to a current Direct Fluorescence Assay (DFA) methodology. However, hMPV was detected with more sensitivity than DFA. Our data suggest that these new mAbs can efficiently identify infected samples and discriminate from patients infected with other respiratory pathogens.

Modulation of Host Immunity by the Human Metapneumovirus.

Globally, as a leading agent of acute respiratory tract infections in children <5 years of age and the elderly, the human metapneumovirus (HMPV) has gained considerable attention. As inferred from studies comparing vaccinated and experimentally infected mice, the acquired immune response elicited by this pathogen fails to efficiently clear the virus from the airways, which leads to an exaggerated inflammatory response and lung damage. Furthermore, after disease resolution, there is a poor development of T and B cell immunological memory, which is believed to promote reinfections and viral spread in the community. In this article, we discuss the molecular mechanisms that shape the interactions of HMPV with host tissues that lead to pulmonary pathology and to the development of adaptive immunity that fails to protect against natural infections by this virus.

Understanding Lung Immunopathology Caused by the Human Metapneumovirus: Implications for Rational Vaccine Design.

Acute respiratory tract infections (ARTIs) are the major cause of child mortality worldwide. The human metapneumovirus (hMPV) is one of the leading causes of child hospitalizations due to pneumonia. The adaptive immune response generated by the host against hMPV is usually inefficient at protecting from reinfections, which is repeat throughout life, from childhood to old age. Despite considerable research efforts, to date there are no licensed vaccines to prevent respiratory disease caused by hMPV infection. In this article we review current vaccine strategies tested in animal models and the implication of such studies in understanding the different immune cell populations that contribute to hMPV clearance and the prevention and resolution of lung inflammation upon exposure to the virus.

Elevated IL-3 and IL-12p40 levels in the lower airway of infants with RSV-induced bronchiolitis correlate with recurrent wheezing.

Respiratory Syncytial Virus (RSV) is the first cause of hospitalization due to bronchiolitis in infants. RSV bronchiolitis has been linked to asthma and recurrent wheezing, however the mechanisms behind this association have not been elucidated. Here, we evaluated the cytokine and chemokine profiles in the airways in infants with RSV bronchiolitis. Nasopharyngeal Aspirates (NPA) and Bronchoalveolar Lavage Fluids (BALF) from infants hospitalized due to RSV bronchiolitis and healthy controls were analyzed for cytokine and chemokine production. We observed elevated levels of Th2 cytokines (IL-3, IL-4, IL-10 and IL-13), pro-inflammatory cytokines and chemokines (IL-1ß, IL-6, TNF-ß, MCP-1/CCL2, MIP-1α/CCL3 and IL-8/CXCL8) in BALF from infants with RSV bronchiolitis, as compared to controls. We found a direct correlation of IL-3 and IL-12p40 levels with the development of recurrent wheezing later in life. These results suggest that IL-3 and IL-12p40 could be considered as molecular predictors for recurrent wheezing due to RSV infection.

Human metapneumovirus infection activates the TSLP pathway that drives excessive pulmonary inflammation and viral replication in mice.

Human metapneumovirus (hMPV) is a leading cause of acute respiratory tract infections in children and the elderly. The mechanism by which this virus triggers an inflammatory response still remains unknown. Here, we evaluated whether the thymic stromal lymphopoietin (TSLP) pathway contributes to lung inflammation upon hMPV infection. We found that hMPV infection promotes TSLP expression both in human airway epithelial cells and in the mouse lung. hMPV infection induced lung infiltration of OX40L(+) CD11b(+) DCs. Mice lacking the TSLP receptor deficient mice (tslpr(-/-) ) showed reduced lung inflammation and hMPV replication. These mice displayed a decreased number of neutrophils as well a reduction in levels of thymus and activation-regulated chemokine/CCL17, IL-5, IL-13, and TNF-α in the airways upon hMPV infection. Furthermore, a higher frequency of CD4(+) and CD8(+) T cells was found in tslpr(-/-) mice compared to WT mice, which could contribute to controlling viral spread. Depletion of neutrophils in WT and tslpr(-/-) mice decreased inflammation and hMPV replication. Remarkably, blockage of TSLP or OX40L with specific Abs reduced lung inflammation and viral replication following hMPV challenge in mice. Altogether, these results suggest that activation of the TSLP pathway is pivotal in the development of pulmonary pathology and pulmonary hMPV replication.

Surface expression of the hRSV nucleoprotein impairs immunological synapse formation with T cells.

Human respiratory syncytial virus (hRSV) is the leading cause of bronchiolitis and pneumonia in young children worldwide. The recurrent hRSV outbreaks and reinfections are the cause of a significant public health burden and associate with an inefficient antiviral immunity, even after disease resolution. Although several mouse- and human cell-based studies have shown that hRSV infection prevents naïve T-cell activation by antigen-presenting cells, the mechanism underlying such inhibition remains unknown. Here, we show that the hRSV nucleoprotein (N) could be at least partially responsible for inhibiting T-cell activation during infection by this virus. Early after infection, the N protein was expressed on the surface of epithelial and dendritic cells, after interacting with trans-Golgi and lysosomal compartments. Further, experiments on supported lipid bilayers loaded with peptide-MHC (pMHC) complexes showed that surface-anchored N protein prevented immunological synapse assembly by naive CD4(+) T cells and, to a lesser extent, by antigen-experienced T-cell blasts. Synapse assembly inhibition was in part due to reduced T-cell receptor (TCR) signaling and pMHC clustering at the T-cell-bilayer interface, suggesting that N protein interferes with pMHC-TCR interactions. Moreover, N protein colocalized with the TCR independently of pMHC, consistent with a possible interaction with TCR complex components. Based on these data, we conclude that hRSV N protein expression at the surface of infected cells inhibits T-cell activation. Our study defines this protein as a major virulence factor that contributes to impairing acquired immunity and enhances susceptibility to reinfection by hRSV.

Immunization with a recombinant bacillus Calmette-Guerin strain confers protective Th1 immunity against the human metapneumovirus.

Along with the human respiratory syncytial virus (hRSV), the human metapneumovirus (hMPV) is one of the leading causes of childhood hospitalization and a major health burden worldwide. Unfortunately, owing to an inefficient immunological memory, hMPV infection provides limited immune protection against reinfection. Furthermore, hMPV can induce an inadequate Th2 type immune response that causes severe lung inflammation, leading to airway obstruction. Similar to hRSV, it is likely that an effective clearance of hMPV would require a balanced Th1 type immunity by the host, involving the activation of IFN-γ-secreting T cells. A recognized inducer of Th1 immunity is Mycobacterium bovis bacillus Calmette-Guérin (BCG), which has been used in newborns for many decades and in several countries as a tuberculosis vaccine. We have previously shown that immunization with BCG strains expressing hRSV Ags can induce an efficient immune response that protects against this virus. In this study, we show that immunization with rBCG strains expressing the phosphoprotein from hMPV also can induce protective Th1 immunity. Mice immunized with rBCG were protected against weight loss, airway inflammation, and viral replication in the lungs after hMPV infection. Our rBCG vaccine also induced the activation of hMPV-specific T cells producing IFN-γ and IL-2, which could protect from hMPV infection when transferred to recipient mice. These data strongly support the notion that rBCG induces protective Th1 immunity and could be considered as an efficient vaccine against hMPV.

Role of Salmonella enterica exposure in Chilean Crohn's disease patients.

AIM: To study the association between exposure to Salmonella enterica (SE) and Crohn's disease (CD) and its clinical implications in Chilean patients. METHODS: Ninety-four unrelated Chilean CD patients from CAREI (Active Cohort Registry of Inflammatory Bowel Disease) presenting to a single inflammatory bowel disease (IBD) unit of a University Hospital were prospectively included in this study. A complete clinical evaluation, including smoking history, was performed at the initial visit, and all the important data of clinical evolution of CD were obtained. Blood samples from these CD patients and 88 healthy sex- and age-matched control subjects were analyzed for exposure to SE and for their NOD2/CARD15 gene status using the presence of anti-Salmonella lipopolysaccharide antibodies [immunoglobulin-G type (IgG)] and polymerase chain reaction (PCR), respectively. We also evaluated exposure to SE in 90 sex- and age-matched patients without CD, but with known smoking status (30 smokers, 30 non-smokers, and 30 former smokers). RESULTS: CD patients comprised 54 females and 40 males, aged 35.5 ± 15.2 years at diagnosis with a mean follow-up of 9.0 ± 6.8 years. CD was inflammatory in 59 patients (62.7%), stricturing in 24 (25.5%) and penetrating in 15 (15.5%). Thirty cases (31.9%) had lesions in the ileum, 29 (30.8%) had ileocolonic lesions, 32 (34.0%) had colonic lesions and 23 (24.4%) had perianal disease. Sixteen CD patients (17%) were exposed to SE compared to 15 (17%) of 88 healthy control subjects (P = 0.8). Thirty-one CD patients (32.9%) were smokers, and 7 (7.4%) were former smokers at diagnosis. In the group exposed to SE, 10 of 16 patients (62.5%) were active smokers compared to 21 of 78 patients (26.9%) in the unexposed group (P = 0.01). On the other hand, 10 of 31 smoking patients (32%) were exposed to SE compared to 5 of 56 nonsmoking patients (9%), and one of the seven former smokers (14%) (P = 0.01). In the group of 90 patients without CD, but whose smoking status was known, there was no difference in exposure to SE [3 of 30 smokers (10%), 5 of 30 non-smokers (16%), and 5 of 30 former smokers (16%); P = 0.6]. There were no differences in disease severity between CD patients with and those without anti-SE IgG antibodies, estimated as the appearance of stricturing [2 (12.5%) vs 22 (28.2%); P = 0.2] or penetrating lesions [2 (12.5%) vs 13 (16.6%); P = 1.0]; or the need for immunosuppressants [11 (68.7%) vs 55 (70.5%); P = 1.0], anti-tumor necrosis factor therapy [1 (6.2%) vs 7 (8.9%); P = 1.0], hospitalization [13 (81.2%) vs 58 (74.3%); P = 0.7], or surgery [3 (18.7%) vs 12 (15.3%); P = 0.3), respectively]. No other factors were associated with SE, including NOD2/CARD15 gene status. Seventeen CD patients (18%) had at least one mutation of the NOD2/CARD15 gene. CONCLUSION: Our study found no association between exposure to SE and CD. We observed a positive correlation between SE exposure and cigarette smoking in Chilean patients with CD, but not with disease severity.