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Pseudomonas aeruginosa (P.a.) infection accounts for nearly 20% of all cases of hospital acquired pneumonia with mortality rates >30%. P.a. infection induces a robust inflammatory response, which ideally enhances bacterial clearance. Unfortunately, excessive inflammation can also have negative effects, and often leads to cardiac dysfunction with associated morbidity and mortality. However, it remains unclear how P.a. lung infection causes cardiac dysfunction. Using a murine pneumonia model, we found that P.a. infection of the lungs led to severe cardiac left ventricular dysfunction and electrical abnormalities. More specifically, we found that neutrophil recruitment and release of S100A8/A9 in the lungs activates the TLR4/RAGE signaling pathways, which in turn enhance systemic inflammation and subsequent cardiac dysfunction. Paradoxically, global deletion of S100A8/A9 did not improve but aggravated cardiac dysfunction and mortality likely due to uncontrolled bacterial burden in the lungs and heart. Our results indicate that P.a. infection induced release of S100A8/9 is double-edged, providing increased risk for cardiac dysfunction yet limiting P.a. growth. Author summary: Over 5 million people develop pneumonia annually in the United States and is the 8th leading cause of death worldwide. Surprisingly, up to 30% of patients admitted to the hospital for community acquired pneumonia develop cardiovascular complications but the mechanisms are not clearly understood. Pseudomonas aeruginosa infection is very common in hospital settings and accounts for up to 20% of all cases of hospital-acquired pneumonia with a mortality rate of ~30%. P. aeruginosa infection and severe lung and systemic inflammation increases levels of S100A8/9 (calprotectin) in the blood stream. Systemic inflammation or dissemination of pathogens into heart tissue can induce cardiac inflammation, cardiomyocyte death and cardiac dysfunction. We found that P. aeruginosa infection increases infiltration of inflammatory immune cells in the heart, leading to arrhythmia and left ventricular dysfunction. Our study provides evidence that S100A8/9 provides protection against bacterial infection yet when its production is uncontrolled it activates host immune responses leading to severe cardiovascular complications. [ABSTRACT FROM AUTHOR]

Leishmaniasis is considered as one of the 20 neglected tropical diseases. Current methods of leishmanial diagnosis depend on conventional laboratory‐based techniques, which are time‐consuming, costly and require special equipment and trained personnel. In this context, we aimed to provide an immuno field effect transistors (ImmunoFET) biosensor that matches the conventional standards for point‐of‐care (POC) monitoring and detection of Leishmania (L.) donovani/Leishmania major. Crude antigens prepared by repeated freeze thawing of L. donovani/L. major stationary phase promastigotes were used for ELISA and ImmunoFETs. Lesishmania‐specific antigens were serially diluted in 1× PBS from a concentration of 106–102 parasites/mL. A specific polyclonal antibody‐based sandwich ELISA was established for the detection of Leishmania antigens. An immunoFET technology‐based POC novel assay was constructed for the detection of Leishmania antigens. Interactions between antigen–antibody at the gate surface generate an electrical signal that can be measured by semiconductor field‐effect principles. Sensitivity was considered and measured as the change in current divided by the initial current. The final L. donovani/L. major crude antigen protein concentrations were measured as 1.50 mg/mL. Sandwich ELISA against the Leishmania 40S ribosomal protein detected Leishmania antigens could detect as few as 100 L. donovani/L. major parasites. An immunoFET biosensor was constructed based on the optimization of aluminium gallium nitride/gallium nitride (AlGaN/GaN) surface oxidation methods. The device surface was composed by an AlGaN/GaN wafer with a 23 nm AlGaN barrier layer, a 2 μm GaN layer on the silicon carbide (SiC) substrate for Leishmania binding, and coated with a specific antibody against the Leishmania 40S ribosomal protein, which was successfully detected at concentrations from 106 to 102 parasites/mL in 1× PBS. At the concentration of 104 parasites, the immunoFETs device sensitivities were 13% and 0.052% in the sub‐threshold regime and the saturation regime, respectively. Leishmania parasites were successfully detected by the ImmunoFET biosensor at a diluted concentration as low as 150 ng/mL. In this study, the developed ImmunoFET biosensor performed well. ImmunoFET biosensors can be used as an alternative diagnostic method to ELISA. Increasing the sensitivity and optimization of immuno‐FET biosensors might allow earlier and faster detection of leishmaniasis. [ABSTRACT FROM AUTHOR]

Age is a major risk factor for chronic infections, including tuberculosis (TB). Elderly TB patients also suffer from elevated levels of psychological stress. It is not clear how psychological stress impacts immune response to Mycobacterium tuberculosis (M.tb). In this study, we used social disruption stress (SDR) to investigate effects of psychological stress in young and old mice. Unexpectedly, we found that SDR suppresses lung inflammation in old mice as evidenced by lower pro-inflammatory cytokine levels in bronchial lavage fluid and decreased cytokine mRNA expression by alveolar macrophages. To investigate effects of stress on M.tb infection, mice were subjected to SDR and then infected with M.tb. As previously reported, old mice were better at controlling infection at 30 days than young mice. This control was transient as CFUs at 60 days were higher in old control mice compared to young mice. Consistently, SDR significantly increased M. tb growth at 60 days in old mice compared to young mice. In addition, SDR in old mice resulted in accumulation of IL-10 mRNA and decreased IFN-g mRNA at 60 days. Also, confocal microscopy of lung sections from old SDR mice showed increased number of CD4 T cells which express LAG3 and CD49b, markers of IL-10 secreting regulatory T cells. Further, we also demonstrated that CD4 T cells from old SDR mice express IL-10. Thus, we conclude that psychological stress in old mice prior to infection, increases differentiation of IL-10 secreting T cells, which over time results in loss of control of the infection. [ABSTRACT FROM AUTHOR]

Antitumor activity of immune cells such as T cells and NK cells has made them auspicious therapeutic regimens for adaptive cancer immunotherapy. Enhancing their cytotoxic effects against malignancies and overcoming their suppression in tumor microenvironment (TME) may improve their efficacy to treat cancers. Clustered, regularly interspaced short palindromic repeats (CRISPR) genome editing has become one of the most popular tools to enhance immune cell antitumor activity. In this review we highlight applications and practicability of CRISPR/Cas9 gene editing and engineering strategies for cancer immunotherapy. In addition, we have reviewed several approaches to study CRISPR off-target effects. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in occasional skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen−/−). Notably, LmCen−/− is a genetically engineered centrin gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with LmCen−/− have no visible lesions following challenge with L. major-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden. LmCen−/− immunization results in protection and an immune response comparable to leishmanization. LmCen−/− is safe since it is unable to cause disease in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials. Here, the authors engineer an attenuated knock-out Leishmania (LmCen−/−) vaccine that is safe in immunocompromised mice and induces an immune response and protection similar to leishmanization with wild-type Leishmania. Since LmCen−/− is antibiotic resistant marker free, it is a candidate for clinical development. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis is the most severe form of leishmaniasis caused by Leishmania (L.) donovani complex. Drug-resistant strains have been developed as a consequence of the current chemotherapeutic interventions, which has increased the need for advanced preventive and therapeutic strategies. A2-CPA-CPB-CTE-recombinant strain of L. tarentolae, which is non-pathogenic to humans, was shown protective in live vaccine as well as its DNA vaccine counter-part in both murine and canine models. Methods: We evaluated the effectiveness of these DNA and live vaccination harboring A2-CPA-CPB-CTEin protecting hamsters against L. infantum infection using prime-boost regimens, namely DNA/DNA and Live/Live (n=9 hamsters per group). Cationic solid lipid nanoparticles (cSLN) were utilized as an adjuvant for DNA priming and electroporation for boosting DNA. At different time points post-challenge, parasite burden and body weight as well as humoral immune responses were measured. Results: Both immunization strategies partially protect hamsters against L. infantum challenge. This protective immunity is associated with remarkable decrease in parasite load in liver and spleen of vaccinated hamsters eight weeks after challenge compared to control group. Conclusion: Both test groups (DNA/DNA and Live/Live) elicited high levels of IgG2 and total IgG as humoral immune responses and lower level of parasite propagation in both liver and spleen. [ABSTRACT FROM AUTHOR]

Background: Ibrutinib is a Bruton's tyrosine kinase (BTK) and interleukin-2-inducible kinase (ITK) inhibitor used for treating chronic lymphocytic leukaemia (CLL) and other cancers. Although ibrutinib is known to inhibit the growth of breast cancer cell growth in vitro, its impact on the treatment and metastasis of breast cancer is unclear.Methods: Using an orthotopic mouse breast cancer model, we show that ibrutinib inhibits the progression and metastasis of breast cancer.Results: Ibrutinib inhibited proliferation of cancer cells in vitro, and Ibrutinib-treated mice displayed significantly lower tumour burdens and metastasis compared to controls. Furthermore, the spleens and tumours from Ibrutinib-treated mice contained more mature DCs and lower numbers of myeloid-derived suppressor cells (MDSCs), which promote disease progression and are linked to poor prognosis. We also confirmed that ex vivo treatment of MDSCs with ibrutinib switched their phenotype to mature DCs and significantly enhanced MHCII expression. Further, ibrutinib treatment promoted T cell proliferation and effector functions leading to the induction of antitumour TH1 and CTL immune responses.Conclusions: Ibrutinib inhibits tumour development and metastasis in breast cancer by promoting the development of mature DCs from MDSCs and hence could be a novel therapeutic agent for the treatment of breast cancer. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis is caused by Mycobacterium tuberculosis. Recent emergence of multidrug-resistant (MDR) tuberculosis strains seriously threatens tuberculosis control and prevention. However, the role of macrophage multidrug resistance gene MDR1 on intracellular M. tuberculosis survival during antituberculosis drug treatment is not known.Methods: We used the human monocyte-derived macrophages to study the role of M. tuberculosis in regulation of MDR1 and drug resistance.Results: We discovered that M. tuberculosis infection increases the expression of macrophage MDR1 to extrude various chemical substances, including tuberculosis drugs, resulting in enhanced survival of intracellular M. tuberculosis. The pathway of regulation involves M. tuberculosis infection of macrophages and suppression of heat shock factor 1, a transcriptional regulator of MDR1 through the up-regulation of miR-431. Notably, nonpathogenic Mycobacterium smegmatis did not increase MDR1 expression, indicating active secretion of virulence factors in pathogenic M. tuberculosis contributing to this phenotype. Finally, inhibition of MDR1 improves antibiotic-mediated killing of M. tuberculosis.Conclusion: We report a novel finding that M. tuberculosis up-regulates MDR1 during infection, which limits the exposure of M. tuberculosis to sublethal concentrations of antimicrobials. This condition promotes M. tuberculosis survival and potentially enhances the emergence of resistant variants. [ABSTRACT FROM AUTHOR]

Background New drugs are needed for leishmaniasis because current treatments such as pentavalent antimonials are toxic and require prolonged administration, leading to poor patient compliance. Ibrutinib is an anticancer drug known to modulate T-helper type 1 (Th1)/Th2 responses and has the potential to regulate immunity against infectious disease. Methods In this study, we evaluated the efficacy of oral ibrutinib as a host-targeted treatment for visceral leishmaniasis (VL) caused by Leishmania donovani using an experimental mouse model. Results We found that oral ibrutinib was significantly more effective than the pentavalent antimonial sodium stibogluconate (70 mg/kg) for the treatment of VL caused by L. donovani. Ibrutinib treatment increased the number of interleukin 4– and interferon γ–producing natural killer T cells in the liver and spleen and enhanced granuloma formation in the liver. Further, ibrutinib treatment reduced the influx of Ly6Chi inflammatory monocytes, which mediate susceptibility to L. donovani. Finally, ibrutinib treatment was associated with the increased production of the cytokines interferon γ, tumor necrosis factor α, interleukin 4, and interleukin 13 in the liver and spleen, which are associated with protection against L. donovani. Conclusions Our findings show that oral ibrutinib is highly effective for the treatment of VL caused by L. donovani and mediates its antileishmanial activity by promoting host immunity. Therefore, ibrutinib could be a novel host-targeted drug for the treatment of VL. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate mannose-anchored thiolated chitosan (MTC) based nanocarriers (NCs) for enhanced permeability, improved oral bioavailability and anti-parasitic potential of amphotericin B (AmB). Transgenic Leishmania donovani parasites expressing red fluorescent protein DsRed2 and imaging-flow cytometry was used to investigate parasitic burdens inside bone marrow-derived macrophages ex vivo. Cytokine estimation revealed that MTC nanocarriers activated the macrophages to impart an explicit immune response by higher production of TNF-α and IL-12 as compared to control. Cells treated with MTC NCs showed a significantly higher magnitude of nitrite and propidium iodide (PI) fluorescence intensity in contrast to cells treated with AmB. Concerning to apparent permeability coefficient (Papp) results, the MTC NCs formulation displayed more specific permeation across the Caco-2 cell monolayer as compared to AmB. The half-life of MTC NCs was about 3.3-fold persistent than oral AmB used as positive control. Also, t oral bioavailability of AmB was increased to 6.4-fold for MTC NCs compared to AmB for positive control. Acute oral evaluation indicated that MTC NCs were significantly less toxic compared to the AmB. Based on these findings, MTC NCs seems to be promising for significant oral absorption and improved oral bioavailability of AmB in leishmaniasis chemotherapy. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate mannose-anchored thiolated chitosan (MTC) based nanocarriers (NCs) for enhanced permeability, improved oral bioavailability and anti-parasitic potential of amphotericin B (AmB). Transgenic Leishmania donovani parasites expressing red fluorescent protein DsRed2 and imaging-flow cytometry was used to investigate parasitic burdens inside bone marrow-derived macrophages ex vivo. Cytokine estimation revealed that MTC nanocarriers activated the macrophages to impart an explicit immune response by higher production of TNF-α and IL-12 as compared to control. Cells treated with MTC NCs showed a significantly higher magnitude of nitrite and propidium iodide (PI) fluorescence intensity in contrast to cells treated with AmB. Concerning to apparent permeability coefficient (Papp) results, the MTC NCs formulation displayed more specific permeation across the Caco-2 cell monolayer as compared to AmB. The half-life of MTC NCs was about 3.3-fold persistent than oral AmB used as positive control. Also, t oral bioavailability of AmB was increased to 6.4-fold for MTC NCs compared to AmB for positive control. Acute oral evaluation indicated that MTC NCs were significantly less toxic compared to the AmB. Based on these findings, MTC NCs seems to be promising for significant oral absorption and improved oral bioavailability of AmB in leishmaniasis chemotherapy. [ABSTRACT FROM AUTHOR]

Vaccination with durable immunity is the main goal and fundamental to control leishmaniasis. To stimulate the immune response, small numbers of parasites are necessary to be presented in the mammalian host. Similar to natural course of infection, strategy using live vaccine is more attractive when compared to other approaches. Live vaccines present the whole spectrum of antigens to the host immune system in the absence of any adjuvant. Leishmanization was the first effort for live vaccination and currently used in a few countries against cutaneous leishmaniasis, in spite of their obstacle and safety. Then, live attenuated vaccines developed with similar promotion of creating long-term immunity in the host with lower side effect. Different examples of attenuated strains are generated through long-term in vitro culturing, culturing under drug pressure, temperature sensitivity, and chemical mutagenesis, but none is safe enough and their revision to virulent form is possible. Attenuation through genetic manipulation and disruption of virulence factors or essential enzymes for intracellular survival are among other approaches that are intensively under study. Other designs to develop live vaccines for visceral form of leishmaniasis are utilization of live avirulent microorganisms such as Lactococcus lactis, Salmonella enterica, and Leishmania tarentolae called as vectored vaccine. Apparently, these vaccines are intrinsically safer and can harbor the candidate antigens in their genome through different genetic manipulation and create more potential to control Leishmania parasite as an intracellular pathogen. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans Leishmania (L.) tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB without its unusual C-terminal extension (CPB-CTE)) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB-CTE-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB-CTE-recombinant L. tarentolae as a safe live vaccine candidate against VL. Author Summary: Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and has emerged as an opportunistic infection in HIV-1 infected patients in many parts of the world. Drug-resistant forms have developed so emergence and increased the need for advanced preventive strategies. Using live avirulent organisms as a vaccine has been proven to be more effective than other regimens. The lizard protozoan parasite Leishmania tarentolae is considered as nonpathogenic to humans. In our previous work, a recombinant L. tarentolae strain expressing the amastigote-specific L. donovani A2 antigen as a vaccine candidate elicited protection against L. infantum challenge in mice. Furthermore, combinations of CPA/CPB cysteine proteinases were more protective against visceral and cutaneous Leishmania infections than the individual forms. Herein, we used DNA/Live and Live/Live prime-boost vaccination strategies against visceral leishmaniasis in BALB/c mice consisting of the A2-CPA-CPB-CTE tri-fusion genes formulated with cationic solid lipid nanoparticles and a recombinant L. tarentolae expressing the tri-fusion. Assessments of cytokine production, humoral responses, parasite burden and histopathological studies support that the recombinant L. tarentolae A2-CPA-CPB-CTE candidate vaccine elicits a protective response against visceral leishmaniasis in mice and represents an important step forward in the development of new vaccine combinations against Leishmania infections. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans Leishmania (L.) tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB without its unusual Cterminal extension (CPB-CTE)) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB-CTE-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB-CTE-recombinant L. tarentolae as a safe live vaccine candidate against VL. [ABSTRACT FROM AUTHOR]

Background: New drugs are needed for leishmaniasis because current treatments such as pentavalent antimonials are toxic and require prolonged administration, leading to poor patient compliance. Ibrutinib is an anticancer drug known to modulate T-helper type 1 (Th1)/Th2 responses and has the potential to regulate immunity against infectious disease.Methods: In this study, we evaluated the efficacy of oral ibrutinib as a host-targeted treatment for visceral leishmaniasis (VL) caused by Leishmania donovani using an experimental mouse model.Results: We found that oral ibrutinib was significantly more effective than the pentavalent antimonial sodium stibogluconate (70 mg/kg) for the treatment of VL caused by L. donovani. Ibrutinib treatment increased the number of interleukin 4- and interferon γ-producing natural killer T cells in the liver and spleen and enhanced granuloma formation in the liver. Further, ibrutinib treatment reduced the influx of Ly6Chi inflammatory monocytes, which mediate susceptibility to L. donovani. Finally, ibrutinib treatment was associated with the increased production of the cytokines interferon γ, tumor necrosis factor α, interleukin 4, and interleukin 13 in the liver and spleen, which are associated with protection against L. donovani.Conclusions: Our findings show that oral ibrutinib is highly effective for the treatment of VL caused by L. donovani and mediates its antileishmanial activity by promoting host immunity. Therefore, ibrutinib could be a novel host-targeted drug for the treatment of VL. [ABSTRACT FROM AUTHOR]