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Mammary tumors are the most frequent type of neoplasms in intact female dogs. New therapies that target neoplastic cells without affecting normal cells are highly sought. The Bacillus anthracis toxin has been reengineered to target tumor cells that express urokinase plasminogen activators and metalloproteinases. In previous studies carried out in our laboratory, the reengineered anthrax toxin had inhibitory effects on canine oral mucosal melanoma and canine osteosarcoma cells. In this study, five canine neoplastic epithelial cell lines (four adenocarcinomas and one adenoma) and one non-neoplastic canine mammary epithelial cell line were treated with different concentrations of reengineered anthrax toxin components. Cell viability was quantified using an MTT assay and half-maximal inhibitory concentration (IC 50 ) values. Cell lines were considered sensitive when the IC 50 was lower than 5000 ng/ml. One canine mammary adenocarcinoma cell line and one mammary adenoma cell line showed significantly decreased viability after treatment, whereas the non-neoplastic cell line was resistant. We conclude that the reengineered anthrax toxin may be considered a targeted therapy for canine mammary neoplasms while preserving normal canine mammary epithelial cells., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Ethnopharmacological Relevance: Helicobacter pylori (H. pylori) is a major pathogen colonized in the human stomach and is implicated in gastritis, peptic ulcer, and gastric carcinoma. Antibiotics are useful for eradicating H. pylori but failed for drug resistance, making it urgent to develop effective and safe drugs. Rhizoma Coptidis was reported as one of the most effective Chinese medicines to treat H. pylori-related gastrointestinal diseases, while the precise antimicrobial mechanism remains unclear. Thus, it is of great significance to study the antimicrobial ingredients and corresponding mechanisms of Rhizoma Coptidis., Aim of the Study: To search for the most effective alkaloid against H. pylori in Rhizoma Coptidis and illustrate the probable mechanisms., Materials and Methods: Five main alkaloids in Rhizoma Coptidis were isolated. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were tested to determine the most effective one. Bacterial growth experiments, Annexin V-FITC/PI staining, TUNEL staining, and transmission electron microscopy (TEM) were performed to further study the anti-H. pylori activity of coptisine (Cop). The in vivo effect of Cop on H. pylori eradication rate and H. pylori-induced inflammation was investigated in mice. Transcriptomics was used to understand the underlying mechanism of eradicating H. pylori and reducing host inflammation. Western blot, RT-PCR, and ELISA experiments were utilized and confirmed that cagA was one of the targets of Cop., Results: According to the MIC and MBC, Cop was the most effective alkaloid against H. pylori, especially with no drug resistance developed. In vitro experiments showed that Cop inhibited H. pylori by inducing DNA fragmentation, phosphatidylserine exposure, and membrane damage. Cop (150 mg/kg/day) effectively eradicated H. pylori in mice and reduced the levels of IL-2 and IL-6 to relieve gastric inflammation. Transcriptomic analysis revealed that virulence factor cagA was one of the hub genes associated with the inflammation-improving effect of Cop. That is, Cop could decrease the expression of CagA and subsequently reduce the translocation of CagA to gastric epithelial cells, thereby improving the morphology of hummingbird-like phenotype induced by CagA and alleviating inflammation., Conclusions: Cop is the most effective alkaloid in Rhizoma Coptidis and might act through multiple mechanisms for H. pylori eradication along with reducing the expression of CagA to alleviate inflammation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites. Preclinical studies demonstrate that toxin treatment induces tumor regression in several in vivo ovarian cancer models, including patient-derived xenografts, without adverse side effects, supportive of progression toward clinical evaluation. These data lay the groundwork for developing therapeutics for treating women with late-stage and recurrent ovarian cancers, utilizing a mechanism distinct from current anticancer therapies.

Background: The role of Helicobacter pylori (H. pylori) virulence factors of such as vacA s1m1 and cagA in designating clinical outcomes and eradication rate has been deeply challenged in the last decade. The goal of this analysis was to identify the potential relevance between cagA and vacA genotypes with reported antibiotic resistance observed in clinical H. pylori isolates., Methods: This literature search was conducted in databases such as Clarivate analytics, PubMed, Scopus, EMBASE, DOAJ, and Google Scholar by April 2022, regardless of language restrictions and publication date. Quality of the included studies was assessed by the Newcastle-Ottawa scale. Statistical analysis of retrieved studies was fulfilled using Comprehensive Meta-Analysis software version 2.2. Following quality appraisal of eligible studies, potential association between the status of cagA and vacA genes with resistance to clarithromycin, metronidazole, amoxicillin, tetracycline, and levofloxacin was measured using odds ratio with 95% confidence interval. We also used sensitivity analyses and meta-regression to eliminate the source of heterogeneity from the overall estimates. Publication bias was assessed using funnel plot, Egger's test, Begg's test with the trim and fill procedure to assess the presence and magnitude of publication bias in the included studies., Results: Our findings suggested that a significant relationship between cagA status ‎and increase resistance ‎to metronidazole (OR: 2.69; 95% CI: 1.24-5.83‎‏‎). In subgroup analysis, we ‎found that in the Western ‎population, infection with cagA-positive strains could be led to increase in ‎the resistance to ‎metronidazole (OR: 1.59; 95% CI: ‎0.78-3.21‎‏‎), ‎amoxicillin (OR: ‎19.68‎; 95% CI: 2.74-‎‎141.18), ‎and ‎levofloxacin (OR: ‎11.33; 95% CI: ‎1.39-‎‎91.85). After implementation of trim and fill method, the adjusted OR was not significantly differed from original estimates which in turn represented our subgroup analysis was statistically robust. On the other hand, vacA ‎genotypes usually ‎reduce the antibiotic resistance of this bacterium, so that vacA s1m1 significantly reduces the ‎resistance to ‎metronidazole (OR: 0.41; 95% CI: 0.20-0.86‎‏‎). Surprisingly, resistance of vacA s2m2 strains to antibiotics was low, the reason may be due ‎to the non-inflammatory properties of strains containing vacA s2m2. The meta-regression and sensitivity analyses successfully reduced the effect of heterogeneity from the overall estimates. In addition, although the pooled OR is reduced after trim and fill adjustment but results do not change the conclusion regarding vacA genotypes and antibiotic resistance., Conclusions: According to our findings, it was clearly demonstrated that cagA-positive strains are resistance to metronidazole, especially in Western countries. In Western countries, vacA s1m1 increases resistance to amoxicillin and levofloxacin. Based on the present findings, the vacA s1m1 genotype significantly increases resistance to metronidazole, while the vacA s1m2 decreases resistance to clarithromycin and metronidazole. Resistance to antibiotics in less virulent (vacA s2m2) strains is statistically significant lower than others., (© 2022. The Author(s).)

The human immune system detects potentially pathogenic microbes with receptors that respond to microbial metabolites. While the overall immune signaling pathway is known in considerable detail, the initial molecular signals, the microbially produced immunogens, for important diseases like Lyme disease (LD) are often not well-defined. The immunogens for LD are produced by the spirochete Borrelia burgdorferi , and a galactoglycerolipid ( 1 ) has been identified as a key trigger for the inflammatory immune response that characterizes LD. This report corrects the original structural assignment of 1 to 3 , a change of an α-galactopyranose to an α-galactofuranose headgroup. The seemingly small change has important implications for the diagnosis, prevention, and treatment of LD.

Objective: This study determined the effects of chemical adjuvants, incomplete Freund's adjuvant (IFA) and aluminum hydroxide (Alum), mycobacteria, and a DNA plasmid as delivery systems on the induction of protective Th1 (interferon-gamma (IFN-γ)) and nonprotective Th2 (IL-5) and Treg (IL-10) cytokine responses to Rv3619c and its peptides. Rv3619c is an immunodominant Mycobacterium tuberculosis-specific antigen and belongs to the early-secreted antigenic target of 6 kDa-family of proteins. Delivery systems are needed to deliver such antigens in animal models and induce protective immune responses., Methods: The rv3619c gene was amplified from the genomic DNA of M. tuberculosis and cloned into appropriate vectors for expression in Escherichia coli, Mycobacterium smegmatis, and eukaryotic cells. Spleen cells from mice immunized with rv3619c using different delivery systems were stimulated in vitro with synthetic peptides (P1 to P6) of Rv3619c, and secreted cytokines were estimated by ELISA., Results: The recombinant M. smegmatis and DNA plasmid induced the secretion of the protective cytokine IFN-γ in response to peptide-pool of Rv3619c and all the individual peptides, whereas rv3619c/IFA induced the secretion of IFN-γ in response to the peptide pool, and the peptides P5 and P6. However, the secretions of the nonprotective cytokines IL-5 and IL-10 were induced to none of the peptides with the delivery systems used., Conclusion: Rv3619c is a major antigen of M. tuberculosis with multiple immunogenic epitopes; however, immune responses to individual epitopes can vary based on delivery systems used., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

Bacterial infections cause huge losses in aquaculture and a wide range of health issues in humans. A vaccine is the most economical, efficient, and environment-friendly agent for protecting hosts against bacterial infections. This study aimed to identify broad, cross-protective antigens from the extracellular secretory proteome of the marine bacterium Vibrio alginolyticus . Of the 69 predicted extracellular secretory proteins in its genome, 16 were randomly selected for gene cloning to construct DNA vaccines, which were used to immunize zebrafish (Danio rerio). The innate immune response genes were also investigated. Among the 16 DNA vaccines, 3 (AT730_21605, AT730_22220, and AT730_22910) were protective against V. alginolyticus infection with 47-66.7% increased survival compared to the control, while other vaccines had lower or no protective effects. Furthermore, AT730_22220, AT730_22910, and AT730_21605 also exhibited cross-immune protective effects against Pseudomonas fluorescens and/or Aeromonas hydrophila infection. Mechanisms for cross-protective ability was explored based on conserved epitopes, innate immune responses, and antibody neutralizing ability. These results indicate that AT730_21605, AT730_22220, and AT730_22910 are potential polyvalent vaccine candidates against bacterial infections. Additionally, our results suggest that the extracellular secretory proteome is an antigen pool that can be used for the identification of cross-protective immunogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Peng, Tao, Kuang, Jiang, Peng and Li.)

Purpose: Tuberculosis, a cost and life threatening disease, was being subjected for improving vaccine strategies beyond BCG. Thus, a novel particulate delivery system using alginate-coated chitosan nanoparticles including PPE17 protein and CpG were administered through intranasal (IN) and subcutaneous (SC) routes., Methods: The encapsulated nanoparticles were first characterized for size, surface charge, encapsulation efficiency and in vitro release of PPE17 antigen. The nanoparticles were then administered intranasal and subcutaneously to evaluate the induction of systemic and/or mucosal immune responses in mice., Results: According to our result, the mean size of nanoparticles was measured about 427 nm, and exhibited a negative zeta potential of -37 mV. Following subcutaneous and intranasal administration, the results from cytokines assay showed that an increasing in the level of IFN-γ, and adversely a decrease in the level of IL-4 (presumptive Th1 biased immune response) was happened and also a notable elicitation in IL-17 cytokine was observed., Conclusion: In conclusion, our study demonstrated that alginate-coated chitosan nanoparticles showed to be an effective way to improve BCG efficiency as booster strategy for subcutaneous vaccine, and also can induce strong immune responses as prime strategy through intranasal vaccination., (Copyright © 2021. Published by Elsevier Inc.)

We studied the effects of M. tuberculosis secretory proteins ESAT-6 and CFP-10 on the properties of vaccinal mycobacteria BCG not producing these proteins. Phagocytosis of M. bovis by macrophages, proliferation of mycobacteria in macrophages, apoptosis and necrosis of macrophages, and the production of reactive oxygen and nitrogen species were studied. It was shown that both ESAT-6 and CFP-10 significantly increased the number of phagocytized mycobacteria by increasing the number of phagocytic-active macrophages and augment the intracellular proliferation of the pathogen. At the same time, macrophages preincubated with ESAT-6 and CFP-10 reduce the production of reactive oxygen and nitrogen species and are more susceptible to apoptosis and necrosis in the presence of mycobacteria. In summary, these proteins suppress macrophage-mediated mechanisms of anti-tuberculosis resistance and impart pronounced pathogenic properties to non-pathogenic mycobacteria that do not secrete ESAT-6 and CFP-10., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Microvascular changes and retinal degeneration precede diabetic retinopathy. Oxidative stress alters several intracellular signaling pathways, which form the basis of diabetic retinopathy. Many antioxidants have been investigated as possible preventive and therapeutic remedies for diabetic retinopathy. The current study investigated the modulatory effects of trans-resveratrol on streptozotocin-induced type 1 diabetes mediated changes in the transcription and levels of apoptosis-related proteins and mitogen-activated protein kinases (MAPKs) in the retinal pigment epithelium (RPE) of adult male dark Agouti rats. In control rats, 5 mg/kg/d trans-resveratrol administration for 30 days increased gene expressions of tumor suppressor protein 53, Bcl2-associated X protein, B-cell lymphoma-2 (Bcl2), Caspase-3 (CASP3), CASP8 and CASP9, p38αMAPK, c-Jun N-terminal kinase-1 (JNK1), and extracellular signal-regulated kinase-1 (ERK1). On the other hand, diabetes decreased gene expressions of CASP3, CASP8, p38αMAPK, JNK, and ERK1. Trans-resveratrol reversed the inhibited gene expressions of CASP8, p38αMAPK, JNK, and ERK1 to normal control levels in diabetic rats. Trans-resveratrol normalized diabetes-induced upregulation of CASP3 and -9, cytochrome-c, Bcl-2, and ERK1 proteins. In conclusion, Trans-resveratrol-induced alterations in gene expressions do not seem to affect RPE functions as they do not reflect as altered protein functions. Trans-resveratrol imparts its protective effects by normalizing apoptosis-related proteins and ERK1 but does not affect JNK proteins. Trans-resveratrol causes cytostasis in RPE of normal rats by upregulating Bcl2 protein and apoptotic proteins., (Copyright © 2021 Elsevier B.V. All rights reserved.)

BACKGROUND Previously, we demonstrated that the chlamydial protein pGP3 forms a stable complex with LL-37 to neutralize its proinflammatory activity during the pathogenesis of psoriasis. The middle domain of pGP3 (pGP3M) is critical for the binding and neutralization of LL-37. Here, we further examined the mechanism underlying pGP3-mediated inhibition of psoriasis progression and evaluated the inhibitory effect of pGP3M on the development of psoriasis-like skin lesions in mice. MATERIAL AND METHODS Stock solutions of pGP3M and pGP3 (100 μg/mL) were prepared using sterile ultrapure water and intramuscularly injected into the left leg of the imiquimod (IMQ)-induced psoriasis mouse model. The severity of skin lesions was evaluated based on the psoriasis area and severity index score and ear skin thickness. The skin biopsy and blood samples were collected on the 8th day for histological analysis and inflammatory cytokines detection. RESULTS Erythema, scaling, and thickening were observed on the dorsal skin and the right ear skin of IMQ-treated mice. Treatment with pGP3 and pGP3M alleviated the IMQ-induced erythema, inflammatory cell infiltration, and scaly plaques. Compared with IMQ-treated and PBS-treated mice, pGP3- and PGP3M-treated mice had less inflammatory cell infiltration in skin tissues and had significantly reduced IL-17A, IFN-γ, and IL-22 levels in serum. CONCLUSIONS The anti-psoriatic efficacy of exogenous pGP3M was similar to that of pGP3. This indicated that pGP3M attenuated the IMQ-induced inflammatory and psoriatic symptoms in mice by binding and inhibiting LL-37. Further research is needed to examine the toxicity of pGP3 and pGP3M before clinical trial evaluation.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human cancers, including Kaposi's sarcoma (KS) and primary effusion lymphoma, which are mostly seen in immunocompromised patients, such as human immunodefeciency virus (HIV)+ individuals. Tuberculosis (TB), caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb), remains one of the deadliest infectious diseases in the world. The risk of developing TB is dramatically higher in people living with HIV than among those without HIV infection. Case reports link cutaneous or pulmonary KS in HIV+ patients with mycobacterial co-infections, however, impacts of Mtb infection or its products on KSHV-infected cells are not known. We report here that ESAT-6, a secreted Mtb virulence factor, induces viral reactivation from KSHV-infected cells. KSHV-infected pulmonary endothelial cells were resistant to ESAT-6 induced inhibition of cell growth. Our data demonstrate that Mtb virulence factors influence the biology of KSHV-infected cells, highlighting the need to study the interactions between these two pathogens commonly found in people living with HIV., (© 2020 Wiley Periodicals LLC.)

Vaccines based on live attenuated Chlamydia elementary bodies (EBs) can cause disease in vaccinated animals and the comparably safer inactivated whole EBs are only marginally protective. Recent studies show that a vaccine formulation comprising UV-inactivated EBs (EB) and appropriate mucosal delivery systems and/or adjuvants induced significant protective immunity. We tested the hypothesis that intranasal delivery of UV-inactivated C. psittaci EB formulated in Vibrio cholerae ghosts (VCG)-chitosan nanoparticles will induce protective immunity against intranasal challenge in SPF chickens. We first compared the impact of VCG and CpG adjuvants on protective immunity following IN mucosal and IM systemic delivery of EB formulated in chitosan hydrogel/microspheres. Immunologic analysis revealed that IN immunization in the presence of VCG induced higher levels of IFN-γ response than IM delivery or the CpG adjuvanted groups. Also, vaccine efficacy evaluation showed enhanced pharyngeal bacterial clearance and protection against lung lesions with the VCG adjuvanted vaccine formulation, thereby establishing the superior adjuvanticity of VCG over CpG. We next evaluated the impact of different concentrations of VCG on protective immunity following IN mucosal immunization. Interestingly, the adjuvanticity of VCG was concentration-dependent, since protective immunity induced following IN mucosal immunization showed dose-dependent immune responses and protection. These studies reveal that formulation of inactivated chlamydial antigens with adjuvants, such as VCG and chitosan increases their ability to induce protective immune responses against challenge.

NK cells play an important role in the control of tuberculosis infection: they are not only able to kill the infected cells, but also control the activity of macrophages and development of the adaptive immune response. Still, there is little information on the role of specific NK cell subsets in this network. In this study, we focused on the mycobacteria-driven responses of the NK cells expressing HLA-DR - a type of MHC class II. We have revealed that this subset is increased in the peripheral blood of patients with primary diagnosed tuberculosis, and expands in response to in vitro stimulation with ultrasonically destroyed Mycobacterium tuberculosis cells (sonicate). The expanded HLA-DR + NK cells had less differentiated phenotype, higher proliferative activity and increased expression of NKp30 and NKp46 receptors. HLA-DR + CD56 dim NK cells showed higher IFNγ production and degranulation level than the respective HLA-DR - NK cells in response to both 24 h and 7 day stimulation with sonicate, while HLA-DR + CD56 bright NK cells mostly demonstarted similar high responsiveness to the same stimulating conditions as their HLA-DR - CD56 bright counterparts. After preliminary incubation with destroyed mycobacteria, cytokine-activated HLA-DR-expressing NK cells were able to mediate mycobacteria-induced and HLA-DR-dependent cytokine production in autologous CD4 + T cells. Thus, functionally active HLA-DR + cells seem to be one of the NK cell subsets providing an important link to the adaptive immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kust, Streltsova, Panteleev, Karpina, Lyadova, Sapozhnikov and Kovalenko.)

Q fever is caused by the obligate intracellular bacterium, Coxiella burnetii , a designated potential agent of bioterrorism because of its route of transmission, resistance to disinfectants, and low infectious dose. The only vaccine licensed for human use is Q-VAX ® (Seqirus, licensed in Australia), a formalin-inactivated whole-cell vaccine, which produces severe local and systemic reactogenic responses in previously sensitized individuals. Accordingly, the U.S. Food and Drug Administration and other regulatory bodies around the world, have been reluctant to approve Q-VAX for widespread use. To obviate these adverse reactions, we prepared recombinant protein subunit vaccine candidates containing purified CBU1910, CBU0307, CBU0545, CBU0612, CBU0891, and CBU1398 proteins and TLR triagonist adjuvants. TLR triagonist adjuvants combine different TLR agonists to enhance immune responses to vaccine antigens. We tested both the protective efficacy and reactogenicity of our vaccine candidates in Hartley guinea pigs using intratracheal infection with live C. burnetii . While all of our candidates showed varying degrees of protection during challenge, local reactogenic responses were significantly reduced for one of our vaccine candidates when compared with a formalin-inactivated whole-cell vaccine. Our findings show that subunit vaccines combined with novel TLR triagonist adjuvants can generate protective immunity to C. burnetii infection while reducing reactogenic responses., Competing Interests: PF, DD, JF, LL, SJ, RN, and AJ own shares in Nanommune Inc. Nanommune does not sell the arrays described in this paper, nor funded any part of the work described herein. Neither Nanommune or its shareholders are likely to benefit from the results described in this publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fratzke, Jan, Felgner, Liang, Nakajima, Jasinskas, Manna, Nihesh, Maiti, Albin, Esser-Kahn, Davies, Samuel, Felgner and Gregory.)

Regarding the role of micro RNAs (miRNA) in the proliferation and differentiation of T cells as well as the controversy around the role of bacteria in the pathogenesis of abdominal aortic aneurysm (AAA), the effects of Helicobacter pylori (Hp) and Lactobacillus acidophilus (La) were investigated in the induction of miRNAs and apoptosis in CD4+ memory T (Tem) cells of AAA patients and controls. Signature atherosclerosis miRNAs 21, 92a, 146a, 155, 326 and 663 were measured in the sera and tissues of AAA patients and control. PBMCs separately and in co-culture with HUVEC were treated with Hp-water-extract (HpWE) and La-conditioned-medium (LaCM). Apoptosis and miRNA levels were assessed in the isolated Tem by flowcytometry and real-time-PCR. In single-culture, HpWE increased apoptosis and miR-155 and LaCM decreased apoptosis and increased miR-21. In co-culture, apoptosis decreased in both groups in response to CagA+HpWE. Also, all miRNAs increased in patients Tem but in controls, only miR- 146a and 21 showed changes. Although, apoptosis was similar in Tem of patients and controls, the effects of Hp and La were different on the induction of apoptosis and miRNAs and also these bacteria showed different impacts in single and co-culture conditions. Beyond the direct effects of these bacteria on the pathogenesis of diseases, their effects on miRNAs expression may shed light on their roles in the development and the prevention of AAA., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cancer is considered as a disease with high rates of mortality and morbidity. The limitations and side effects of common treatments have prompted the need for innovative cancer therapies. Furthermore, selectivity and targeting of cancer cells are crucial factors to successful treatment of cancer. One of these methods is the use of bacterial toxins including Bacillus anthracis toxin to aid cancer therapy. This toxin is composed of three polypeptides: protective factor (PA), lethal factor (LF), and edema factor (EF). PA can bind to various surface receptors of all types of human cells and it internalizes the lethal factor and edema factor subunits of the toxin in the cytosol. In the present study, we cloned and expressed the lef gene of B. anthracis as the lethal part of the toxin in Bacillus subtilis WB600 by a shuttle expression vector PHT4. The rLF made in B. subtilis is efficiently secreted by the host into the culture medium which facilitates downstream processing. The rLF can be used to study cancer treatment. Abbreviations: EF: edema factor; LF: lethal factor; PA: protective factor; rLF: recombinant lethal factor; rPAm: recombinant protective factor mutants; uPA: urokinase-type plasminogen activator; uPAR: urokinase-type plasminogen activator receptor.

This work demonstrates the presence of immune regulatory cells in the cervical lymph nodes draining Bacillus Calmette-Guérin (BCG) vaccinated site on the dorsum of the ear in guinea pigs. It is shown that whole cervical lymph node cells did not proliferate in vitro in the presence of soluble mycobacterial antigens (PPD or leprosin) despite being responsive to whole mycobacteria. Besides, T cells from these lymph nodes separated as a non-adherent fraction on a nylon wool column, proliferated to PPD in the presence of autologous antigen presenting cells. Interestingly, addition of as low as 20% nylon wool adherent cells to these, sharply decreased the proliferation by 83%. Looking into what cells in the adherent fraction suppressed the proliferation, it was found that neither the T cell nor the macrophage enriched cell fractions of this population individually showed suppressive effect, indicating that their co-presence was necessary for the suppression. Since BCG induced granulomas resolve much faster than granulomas induced by other mycobacteria such as Mycobacterium leprae the present experimental findings add to the existing evidence that intradermal BCG vaccination influences subsequent immune responses in the host and may further stress upon its beneficial role seen in Covid-19 patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

S. aureus is associated with atopic dermatitis (AD). Several staphylococcal products including cell wall components, protease, and exotoxins, are thought to be involved in allergic inflammation of AD via activating immune cells such as T cells and mast cells. None of the staphylococcal exotoxins has been reported to activate a primary IL-4 inducer, basophils, that are known to produce large amounts of IL-4 in response to allergens as well as IgE-independent stimuli such as mites and helminth proteases. In this study, we investigated the ability of staphylococcal superantigen-like (SSL) family to activate basophils. SSL12, reported its activity to activate mast cells, induced the production of IL-4 in bone marrow derived basophils. SSL12 also evoked the release of IL-4 in freshly isolated murine basophils in bone marrow cells, as the depletion of basophils by basophils-specific antibodies against high-affinity IgE receptor and CD49b diminished the responsiveness of bone marrow cells for SSL12. These results propose the novel immune regulatory activity of SSL12 by inducing IL-4 in basophils, that contributes to the development of allergic inflammation disorders and the immune evasion of the cocci., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Mycobacterium tuberculosis (Mtb)-specific perforin were significantly increased in patients with tuberculosis. This study aims to evaluate the diagnosis value of Mtb-specific perforin in pediatric patients with tuberculosis., Methods: Diagnostic performance of perforin levels induced by 6-kDa early secreted antigen target (ESAT6) or culture filtered protein 10 (CFP10) were evaluated in eighty-six samples from children participants by receiver operating characteristic curve analysis. Flow cytometry was used to detect the expression of perforin and INF-γ of CD4 + , CD8 + T cells in response to CFP10 stimulation., Results: After ex vivo stimulation, levels of ESAT6/CFP10-specific perforin in LTBI patients were significantly higher than active TB (ATB) patients, non-tuberculosis infection (non-TB), and health control (HC) individuals. The diagnostic efficacy of CFP10-specific perforin for TB diagnosis was significantly higher than ESAT6-specific perforin and T-SPOT assay, and when 0.74 ng/mL was taken as the cutoff value, the sensitivity, specificity, and accuracy were 97.83%, 87.5%, and 93.02%. CFP10-specific perforin in both CD4 + and CD8 + T cells were significantly higher in ATB patients compared to HCs and further increased in LTBI patients. However, INF-γ was mainly secreted by CD4 + T cells and showed no significant difference between LTBI and ATB patients. In addition, CFP10-specific perforin can effectively distinguish between ATB and LTBI with the cutoff value of 1.80 ng/mL. Sensitivity and specificity were 88.46% and 85.62%, respectively., Conclusions: CFP10-specific perforin may be used as a novel cellular immunity-based diagnostic marker of pediatric patients with tuberculosis, and with the potential for discriminating ATB from LTBI., (© 2020 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Inhalational anthrax, a disease caused by inhaling Bacillus anthracis spores, leads to respiratory distress, vascular leakage, high-level bacteremia, and often death within days. Anthrax lethal toxin and edema toxin, which are composed of protective antigen (PA) plus either lethal factor (LF) or edema factor (EF), respectively, play an important yet incompletely defined role in the pulmonary pathophysiology. To better understand their contribution, we examined the structural integrity of the alveolar-capillary barrier in archival formalin-fixed lungs of cynomolgus monkeys challenged with the fully virulent B. anthracis Ames wild-type strain or the isogenic toxin-deficient mutants ΔEF, ΔLF, and ΔPA. Pulmonary spore challenge with the wild-type strain caused high mortality, intra-alveolar hemorrhages, extensive alveolar septal sequestration of bacteria and neutrophils, diffuse destabilization of epithelial and endothelial junctions, increased markers of coagulation and complement activation (including tissue factor and C5a), and multifocal intra-alveolar fibrin deposition. ΔEF challenge was lethal and showed similar alveolar-capillary alterations; however, intra-alveolar hemorrhages, bacterial deposition, and markers of coagulation or complement were absent or markedly lower. In contrast, ΔLF or ΔPA challenges were nonlethal and showed no signs of alveolar bacterial deposition or alveolar-capillary changes. These findings provide evidence that lethal toxin plays a determinative role in bacterial dissemination and alveolar-capillary barrier dysfunction, and edema toxin may significantly exacerbate pulmonary pathologies in a systemic infection., (Published by Elsevier Inc.)

Selective IgM deficiency (SIgMD) is a rare immunodeficiency characterized by serum IgM below two standard of mean, and normal IgG and IgA levels. Both in human and mice with selective IgM deficiency, germinal centers cells are decreased. The development of germinal center and humoral immunity are regulated in part by follicular helper T (T FH ) and follicular regulatory T (T FR ) cells. However, the analysis of circulating T FH (cT FH ) and T FR (cT FR ) cells in the pathogenesis of SIgMD has not been explored. We observed lower percentage of cT FR cells in SIgMD patients than in control group. However, we did not observe any significant difference in the percentage of cT FH cells and their subsets between both experimental groups. When data were analyzed according to specific antibody response to pneumococcal polysaccharide, we observed a higher percentage of cT FH cells in SIgMD patients with specific antibody deficiency than in SIgMD patients with normal specific antibody response. Our results suggest that cT FH cells and their subsets are preserved in SIgMD patients. However, the role of lower percentage of cT FR cells in the pathogenesis of this immunodeficiency is not clear., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Canine and human osteosarcomas (OSA) share similarities. Novel therapies are necessary for these tumours. The Bacillus anthracis toxin was reengineered to target and kill cells with high expressions of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA). Since canine OSA express MMPs and uPA, we assessed whether the reengineered toxin could show efficacy against these tumours. Two OSA cell lines (canine D17 and human MG63) and a non-neoplastic canine osteoblastic cell line (COBS) were used. Cells were treated with different concentrations of the reengineered anthrax toxin and cell viability was quantified using MTT assay. The cell cycle, apoptosis, and necrosis were analysed by flow cytometry. The wound-healing assay was performed to quantify the migration capacity of treated cells. D17 and MG63 cells had significantly decreased viability after 24 h of treatment. Cell cycle analysis revealed that OSA cells underwent apoptosis when treated with the toxin, whereas COBS cells arrested in the G1 phase. The wound-healing assay showed that D17 and MG63 cells had a significantly reduced migration capacity after treatment. These results point for the first time towards the in vitro inhibitory effects of the reengineered anthrax toxin on OSA cells; this reengineered toxin could be further tested as a new therapy for OSA.

The study focuses on whether antigenic proteins encapsulated in biopolymeric nanoparticles can augment protective efficacy. Chitosan nanoparticles (ChN) were prepared by ionic gelation method and Culture Filtrate Proteins (CFP) - CFP-10 and CFP-21 of Mycobacterium tuberculosis (Mtb) were encapsulated in ChN. The binding efficiency of nanoparticles with CFP-10 and CFP-21 proteins was confirmed by UV-Spectrophotometer. The efficacy of nanoparticles-encapsulated antigenic proteins administered intraperitoneal against Mtb aerosol infection was evaluated in Balb/c mice. Protection study was done by bacterial counts [CFU]. CFP-10 and CFP-21 proteins primed cells demonstrated a Th1 bias T cell response in an ex vivo assay. ChN-CFP10 and ChN-CFP21 nanoparticles have both protective and therapeutic potential against Mtb. In the group of mice immunized with CHN-CFP-10 the number of colonies reduced significantly from day 15 to day 60. ChN-CFP-21 showed maximum protection in ChN-CFP-21 immunized mice. ChN-CFP-10 and ChN-CFP-21 clearly showed enhanced protection against Mtb., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Anthrax lethal toxin (LT) is a protease that activates the NLRP1b inflammasome sensor in certain rodent strains. Unlike better-studied sensors, relatively little is known about the priming requirements for NLRP1b. In this study, we investigate the rapid and striking priming-independent LT-induced release of IL-1β in mice within hours of toxin challenge. We find IL-1β release to be a NLRP1b- and caspase-1-dependent, NLRP3 and caspase-11-independent event that requires both neutrophils and peptidyl arginine deiminiase-4 (PAD4) activity. The simultaneous LT-induced IL-18 response is neutrophil-independent. Bone marrow reconstitution experiments in mice show toxin-induced IL-1β originates from hematopoietic cells. LT treatment of neutrophils in vitro did not induce IL-1β, neutrophil extracellular traps (NETs), or pyroptosis. Although platelets interact closely with neutrophils and are also a potential source of IL-1β, they were unable to bind or endocytose LT and did not secrete IL-1β in response to the toxin. LT-treated mice had higher levels of cell-free DNA and HMGB1 in circulation than PBS-treated controls, and treatment of mice with recombinant DNase reduced the neutrophil- and NLRP1-dependent IL-1β release. DNA sensor AIM2 deficiency, however, did not impact IL-1β release. These data, in combination with the findings on PAD4, suggest a possible role for in vivo NETs or cell-free DNA in cytokine induction in response to LT challenge. Our findings suggest a complex interaction of events and/or mediators in LT-treated mice with the neutrophil as a central player in induction of a profound and rapid inflammatory response to toxin., (©2020 Society for Leukocyte Biology.)

Tuberculosis (TB) claims 1.5 million lives per year. This situation is largely due to the low efficacy of the only licensed TB vaccine, Bacillus Calmette-Guérin (BCG) against pulmonary TB. The metabolic disease type 2 diabetes (T2D) is a risk factor for TB and the mechanisms underlying increased TB susceptibility in T2D are not well understood. Furthermore, it is unknown if new TB vaccines will provide protection in the context of T2D. Here we used a diet-induced murine model of T2D to investigate the underlying mechanisms of TB/T2D comorbidity and to evaluate the protective capacity of two experimental TB vaccines in comparison to conventional BCG. Our data reveal a distinct immune dysfunction that is associated with diminished recognition of mycobacterial antigens in T2D. More importantly, we provide compelling evidence that mucosal delivery of recombinant BCG strains expressing the Mycobacterium tuberculosis ( Mtb ) ESX-1 secretion system (BCG::RD1 and BCG::RD1 ESAT-6 ∆92-95) are safe and confer superior immunity against aerosol Mtb infection in the context of T2D. Our findings suggest that the remarkable anti-TB immunity by these recombinant BCG strains is achieved via augmenting the numbers and functional capacity of antigen presenting cells in the lungs of diabetic mice., Competing Interests: The authors declare no competing interest.

Animal infection models to study Mycobacterium avium subsp. paratuberculosis (MAP) infection are useful for evaluating the efficacy of vaccines and other therapeutics for the prevention or treatment of infection. The goal of the present study was to compare smaller ruminants, sheep and goats, with calves as infection models. Neonatal sheep, goats, and calves (n = 4) received 10 9 cfu of a cattle isolate of MAP in milk replacer on days 0, 3 and 6 in a 12-month study and sampled monthly thereafter. Results demonstrated a robust antigen-specific IFN-γ response at 90 days post-inoculation for sheep and goats, with lower responses noted for calves. By 360 days, IFN-γ responses were 50 and 82% higher for calves than for goats and sheep, respectively. Although MAP-specific antibody responses were first observed in sheep at 90 days, calves had higher antibody responses throughout the remainder of the study. Following pass-through shedding on day 7, fecal shedding was fairly negligible across treatments but remained higher for calves throughout the study. Colonization of tissues was variable within treatment group and was higher for calves and sheep for the majority of tissues. Upon antigen stimulation of PBMCs, higher populations of CD4 + T cells cells and lower populations of γδ TCR + and NK cells were observed for goats and calves compared to sheep. Relative gene expression of IL-4, IL-12, and IL-17 in PBMCs was higher in goats, corresponding to lower tissue colonization with MAP. These data suggest that ruminant species are fairly comparable as infection models for MAP, but discrete differences in host responses to MAP infection exist between species., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest with respect to the research, authorship, and/or publication of this article. Mention of tradenames or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture., (Published by Elsevier B.V.)

Abdominal aortic aneurysm (AAA) is a degenerative inflammatory disease with unknown etiology. AAA is characterized by abdominal aortic dilatation more than 3 cm and is often asymptomatic, but the rupture of aneurysm can lead to death. Age, smoking and male sex are major predisposing factors of AAA. This study compares the effect of Helicobacter (H.) pylori and Lactobacillus (L.) acidophilus on the cytokine profile of PBMCs of 5 men with abdominal aortic aneurysm (AAA) and 5 men with normal/insignificant angiography, CT-Scan and ultrasonography results in the single-culture and in the co-culture with HUVECs. IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17 F, IL-21, IL-22, IFN-γ and TNF-α were measured in culture supernatants using a commercial fluorescent-labeled-bead assay. In general, CagA+ H. pylori-extract induced higher production of IFN-γ, IL-13 and IL-21 by PBMCs. Treatment of patients' PBMCs with CagA + H. pylori-extract induced Th2 cytokines while treatment of controls' PBMCs with CagA + H. pylori-extract increased Th1 cytokines. In the co-culture, however, patients' PBMCs produced Th1 cytokines irrespective of extract treatment, while controls' PBMCs produced Th2 cytokines and decreased IL-10. CagA+ H. pylori- as well as L. acidophilus-extract induced higher levels of IL-9 by controls' PBMCs in co-culture with HUVECs than patients (P = 0.05 and P = 0.01). The cytokine pattern of PBMCs induced by CagA+ H. pylori- and L. acidophilus-extracts in the co-culture with HUVECs shows differences in AAA patients and in comparison to controls. Decreased secretion of IL-9, IL-21 and IL-22 by PBMCs of patients treated with CagA+ H. pylori extract in co-culture, as opposed to non-AAA controls may indicate the active role ECs play in AAA. Simultaneous production of IL-10 and Th1 cytokines in patients and pronounced Th2 cytokines in controls in response to both bacteria may point to the inherent differences between patients and controls, which need further investigation., Competing Interests: Declaration of Competing Interest The authors declare no competing interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The persistence of new leprosy cases in endemic areas such as India, Brazil, Bangladesh, and the Philippines has encouraged studies of chemoprophylaxis among contacts of patients. Epidemiological screening tools to enable early detection of infected individuals in endemic populations would be critical to target individuals most in need of intervention. Despite decades of attempts, however, there still are no tests available for the early detection of low-level infection with Mycobacterium leprae. In this report, we describe the development of a leprosy skin test using M. leprae-specific antigens. We selected the chimeric LID-1 fusion protein, formulated to achieve maximum performance at a minimal dose, as a skin test candidate based on its ability to elicit delayed-type hypersensitivity (DTH) reactions in M. leprae immune guinea pigs in a sensitive and specific manner, i.e., with no cross-reactivity observed with other mycobacterial species. Importantly, evaluations in armadillos indicated that intradermal inoculation of formulated LID-1 could distinguish uninfected from M. leprae-infected animals manifesting with symptoms distinctly similar to the PB presentation of patients. Together, our data provide strong proof-of-concept for developing an antigen-specific skin test to detect low-level M. leprae infection. Such a test could, when applied with appropriate use of chemo- and/or immunoprophylaxis, be instrumental in altering the evolution of clinical disease and M. leprae transmission, thus furthering the objective of zero leprosy.

Canine oral mucosal melanomas (OMM) are the most common oral malignancy in dogs and few treatments are available. Thus, new treatment modalities are needed for this disease. Bacillus anthracis (anthrax) toxin has been reengineered to target tumor cells that express urokinase plasminogen activator (uPA) and metalloproteinases (MMP-2), and has shown antineoplastic effects both, in vitro and in vivo. This study aimed to evaluate the effects of a reengineered anthrax toxin on canine OMM. Five dogs bearing OMM without lung metastasis were included in the clinical study. Tumor tissue was analyzed by immunohistochemistry for expression of uPA, uPA receptor, MMP-2, MT1-MMP and TIMP-2. Animals received either three or six intratumoral injections of the reengineered anthrax toxin prior to surgical tumor excision. OMM samples from the five dogs were positive for all antibodies. After intratumoral treatment, all dogs showed stable disease according to the canine Response Evaluation Criteria in Solid Tumors (cRECIST), and tumors had decreased bleeding. Histopathology has shown necrosis of tumor cells and blood vessel walls after treatment. No significant systemic side effects were noted. In conclusion, the reengineered anthrax toxin exerted inhibitory effects when administered intratumorally, and systemic administration of this toxin is a promising therapy for canine OMM.

Background: Pseudomonas aeruginosa has an important role in nosocomial infections., Objective: To evaluate biological activity of the detoxified LPS (D-LPS) entrapped into Poly lactic-co-glycolic acid (PLGA) nanoparticles., Materials: LPS was extracted and detoxified from the P. aeruginosa strain PAO1. The D-LPS, conjugated to the PLGA nanoparticles with 1-ethyl-3-dimethyl aminopropyl carbodiimide (EDAC) and N-hydroxy-succinimide (NHS). The connection was evaluated by FTIR (Fourier transform infrared), Zetasizer, and Atomic Force Microscope (AFM). The BALB/c mice injected intramuscularly with the D-LPS-PLGA with two-week intervals and then challenged two weeks after the last immunization. The bioactivity of the induced specific antisera and cytokines responses against D-LPS-PLGA antigen was assessed by ELISA., Results: D-LPS-PLGA conjugation was confirmed by FTIR, Zetasizer, and AFM. The ELISA results showed that D-LPS was successful in the stimulation of the humoral immune response. The immune responses raised against the D-LPS-PLGA, significantly decreased bacterial titer in the spleen of the immunized mice after challenge with PAO1 strain in comparison with the control groups., Conclusion: The conjugation of the bacterial LPS to the PLGA nanoparticle increased their functional activity by decrease in bacterial dissemination and increase the killing of opsonized bacteria.

Anthrax lethal toxin (LT) is a protease virulence factor produced by Bacillus anthracis that is required for its pathogenicity. LT treatment causes a rapid degradation of c-Jun protein that follows inactivation of the MEK1/2-Erk1/2 signaling pathway. Here we identify COP1 as the ubiquitin E3 ligase that is essential for LT-induced c-Jun degradation. COP1 knockdown using siRNA prevents degradation of c-Jun, ETV4, and ETV5 in cells treated with either LT or the MEK1/2 inhibitor, U0126. Immunofluorescence staining reveals that COP1 preferentially localizes to the nuclear envelope, but it is released from the nuclear envelope into the nucleoplasm following Erk1/2 inactivation. At baseline, COP1 attaches to the nuclear envelope via interaction with translocated promoter region (TPR), a component of the nuclear pore complex. Disruption of this COP1-TPR interaction, through Erk1/2 inactivation or TPR knockdown, leads to rapid COP1 release from the nuclear envelope into the nucleoplasm where it degrades COP1 substrates. COP1-mediated degradation of c-Jun protein, combined with LT-mediated blockade of the JNK1/2 signaling pathway, inhibits cellular proliferation. This effect on proliferation is reversed by COP1 knockdown and ectopic expression of an LT-resistant MKK7-4 fusion protein. Taken together, this study reveals that the nuclear envelope acts as a reservoir, maintaining COP1 poised for action. Upon Erk1/2 inactivation, COP1 is rapidly released from the nuclear envelope, promoting the degradation of its nuclear substrates, including c-Jun, a critical transcription factor that promotes cellular proliferation. This regulation allows mammalian cells to respond rapidly to changes in extracellular cues and mediates pathogenic mechanisms in disease states., Competing Interests: The authors declare no competing interest.

Brucellosis is a worldwide bacterial zoonosis disease. Live attenuated Brucella vaccines have several drawbacks. Thus development of a safe and effective vaccine for brucellosis is a concern of many scientists. FliC protein contributes in virulence of Brucella; hence, it is a promising target for brucellosis vaccine. In this study, Mannosylated Chitosan Nanoparticles (MCN) loaded with FliC protein were synthesized as a targeted vaccine delivery system. The immunogenicity and protective efficacy of FliC and FliC-MCN against Brucella infection were evaluated in BALB/c mice. After cloning, expression and purification, FliC protein was loaded on MCN. The particle size, loading efficiency and in vitro release of the NPs were determined. Our investigation revealed that FliC and FliC-MCN could significantly increase specific IgG response (higher IgG2a titers). Besides, spleen cells from immunized mice produced high level of IFN-γ and IL-2 and low level IL-10 cytokines. Immunization with FliC and FliC-MCN conferred significant degree of protection against B. melitensis 16 M and B. abortus 544 infections. Overall these results indicate that FliC protein would be a novel potential antigen candidate for the development of a subunit vaccine against B. melitensis and B. abortus. Moreover, MCN could be used as an adjuvant and targeted vaccine delivery system., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

-20pt?>Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that causes contagious tuberculosis (TB). Recently, Mtb-secreted proteins have been considered virulence factors and candidates for drugs and vaccines. Among these proteins, 6-kDa early secreted antigenic target (ESAT-6) is known to be able to induce component of matrix metalloproteinase-9 (MMP-9) in epithelial cells, leading to recruitment of macrophages. However, detailed function of ESAT-6 during macrophage recruitment to inflammatory sites remains unknown. Thus, the objective of the present study was to elucidate such function of EAST-6 and mechanism(s) involved. In the present study, we have found that recombinant ESAT-6 purified in the form of ESAT-6 double-connected structure (2E6D) could inhibit lipopolysaccharide (LPS)-induced potential of cell migration and inflammation in murine macrophage cells. Interestingly, 2E6D suppressed LPS-induced MMP-9 expression at both protein and mRNA levels as well as its enzyme activity. Levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes as known upregulators of MMP-9 were significantly decreased when 2E6D has been treated. In addition, nitric oxide (NO) as a second messenger was also significantly decreased by treatment with the purified 2E6D. Furthermore, 2E6D inhibited LPS-induced phosphorylation of IκB and translocation of NF-κB. Moreover, 2E6D suppressed phosphorylation of MAPK signaling proteins. Taken together, these results suggest that ESAT-6 can suppress LPS-induced MMP-9 and inflammation by downregulating COX-2, iNOS, and NO through NF-κB and MAPK signaling.

Pneumococcal Surface Protein A (PspA) has been successfully tested as vaccine candidate against Streptococcus pneumoniae infections. Vaccines able to induce PspA-specific antibodies and Th1 cytokines usually provide protection in mice. We have shown that the whole cell pertussis vaccine (wP) or components from acellular pertussis vaccines, such as Pertussis Toxin or Filamentous Hemagglutinin (FHA), are good adjuvants to PspA, suggesting that combined pertussis-PspA vaccines would be interesting strategies against the two infections. Here, we evaluated the potential of wP as a delivery vector to PspA. Bordetella pertussis strains producing a PspA from clade 4 (PspA4Pro) fused to the N-terminal region of FHA (Fha44) were constructed and inactivated with formaldehyde for the production of wPPspA4Pro. Subcutaneous immunization of mice with wPPspA4Pro induced low levels of anti-PspA4 IgG, even after 3 doses, and did not protect against a lethal pneumococcal challenge. Prime-boost strategies using wPPspA4Pro and PspA4Pro showed that there was no advantage in using the wPPspA4Pro vaccine. Immunization of mice with purified PspA4Pro induced higher levels of antibodies and protection against pneumococcal infection than the prime-boost strategies. Finally, purified Fha44:PspA4Pro induced high levels of anti-PspA4Pro IgG, but no protection, suggesting that the antibodies induced by the fusion protein were not directed to protective epitopes., Competing Interests: ML Oliveira, EN Miyaji and PL Ho have a patent deposit for the use of the whole cell pertussis vaccine as adjuvant to PspA in Brazil (INPI PI 1003753-5 A2). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Leprosy, once considered as poor man's disease may cause severe neurological complications and physical disabilities. Classification of leprosy depends upon the cell mediated and humoral immune responses of the host, from tuberculoid to lepromatous stage. Current therapy to prevent the disease is not only very lengthy but also consists of expensive multiple antibiotics in combination. Treatment and the duration depend on the bacillary loads, from six months in paucibacillary to a year in multibacillary leprosy. Although as per WHO recommendations, these antibiotics are freely available but still out of reach to patients of many rural areas of the world. In this review, we have focused on the nutritional aspect during the multi-drug therapy of leprosy along with the role of nutrition, particularly malnutrition, on susceptibility of Mycobacterium leprae and development of clinical symptoms. We further discussed the diet plan for the patients and how diet plans can affect the immune responses during the disease., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Intracellular pathogenic structures or activities stimulate the formation of inflammasomes, which recruit and activate caspase-1 and trigger an inflammatory form of cell death called pyroptosis. The well-characterized mammalian inflammasome sensor proteins all detect one specific type of signal, for example double-stranded DNA or bacterial flagellin. Remarkably, NLRP1 was the first protein discovered to form an inflammasome, but the pathogenic signal that NLRP1 detects has not yet been identified. NLRP1 is highly polymorphic, even among inbred rodent strains, and it has been suggested that these diverse NLRP1 alleles may have evolved to detect entirely different stimuli. Intriguingly, inhibitors of the serine proteases DPP8 and DPP9 (DPP8/9) were recently shown to activate human NLRP1, its homolog CARD8, and several mouse NLRP1 alleles. Here, we show now that DPP8/9 inhibitors activate all functional rodent NLRP1 alleles, indicating that DPP8/9 inhibition induces a signal detected by all NLRP1 proteins. Moreover, we discovered that the NLRP1 allele sensitivities to DPP8/9 inhibitor-induced and Toxoplasma gondii-induced pyroptosis are strikingly similar, suggesting that DPP8/9 inhibition phenocopies a key activity of T. gondii. Overall, this work indicates that the highly polymorphic NLRP1 inflammasome indeed senses a specific signal like the other mammalian inflammasomes.

We established a murine model of multiwall carbon nanotube (MWCNT)-elicited chronic granulomatous disease that bears similarities to human sarcoidosis pathology, including alveolar macrophage deficiency of peroxisome proliferator-activated receptor γ (PPARγ). Because lymphocyte reactivity to mycobacterial antigens has been reported in sarcoidosis, we hypothesized that addition of mycobacterial ESAT-6 (early secreted antigenic target protein 6) to MWCNT might exacerbate pulmonary granulomatous pathology. MWCNTs with or without ESAT-6 peptide 14 were instilled by the oropharyngeal route into macrophage-specific PPARγ-knockout (KO) or wild-type mice. Control animals received PBS or ESAT-6. Lung tissues, BAL cells, and BAL fluid were evaluated 60 days after instillation. PPARγ-KO mice receiving MWCNT + ESAT-6 had increased granulomas and significantly elevated fibrosis (trichrome staining) compared with wild-type mice or PPARγ-KO mice that received only MWCNT. Immunostaining of lung tissues revealed elevated fibronectin and Siglec F expression on CD11c + infiltrating alveolar macrophages in the presence of MWCNT + ESAT-6 compared with MWCNT alone. Analyses of BAL fluid proteins indicated increased levels of transforming growth factor (TGF)-β and the TGF-β pathway mediator IL-13 in PPARγ-KO mice that received MWCNT + ESAT-6 compared with wild-type or PPARγ-KO mice that received MWCNT. Similarly, mRNA levels of matrix metalloproteinase 9, another requisite factor for TGF-β production, was elevated in PPARγ-KO mice by MWCNT + ESAT-6. Analysis of ESAT-6 in lung tissues by mass spectrometry revealed ESAT-6 retention in lung tissues of PPARγ-KO but not wild-type mice. These data indicate that PPARγ deficiency promotes pulmonary ESAT-6 retention, exacerbates macrophage responses to MWCNT + ESAT-6, and intensifies pulmonary fibrosis. The present findings suggest that the model may facilitate understanding of the effects of environmental factors on sarcoidosis-associated pulmonary fibrosis.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that triggers several survival mechanisms against the host immune system. Many studies show that the diverse components of Mtb can modulate apoptosis in various types of cells differently. So far, apoptosis induced by ESAT-6, an early secreted antigenic target of 6-kDa of Mtb, has been studied but the details of molecular mechanism and signaling pathway remain incompletely defined. This study investigated the role of recombinant ESAT-6 in inducing apoptosis in primary bone marrow-derived macrophages (BMDMs) of mice using Annexin V/PI assay with FACS analysis and Western blotting technique. It has been found that ESAT-6-induced apoptosis in BMDMs in a dose- and time-dependent pattern. Apoptosis induced by ESAT-6 was mainly via the intrinsic pathway with elevated protein levels of cleaved caspase-9 and -3. Furthermore, ESAT-6 also induced Bim activation during this process. Interestingly, this event was TLR2-dependent since the effect of ESAT-6 on apoptosis vanished in BMDM from mice with TLR2 deficiency. Furthermore, ROS generation and MAPKs phosphorylation induced by ESAT-6 were also involved in caspase-9 and caspase-3 activation. Taken together, these data suggest that ESAT-6-mediated apoptosis is involved in ROS-MAPKs signaling and further activating the intrinsic pathway, which provides new insights into the basic physiology of macrophage death in tuberculosis.

Background: Angiogenesis can occur under pathological conditions when stimuli such as inflammation, vascular obstruction or hypoxia exist. These stimuli are present in cardiopulmonary dirofilariosis (Dirofilaria immitis). The aim of this study was to analyze the capacity of D. immitis antigens to modify the expression of angiogenic factors and trigger the formation of pseudocapillaries (tube-like structures) in an in vitro model of endothelial cells., Methods: The expression of VEGF-A, sFlt, mEndoglin and sEndoglin in cultures of canine microvascular endothelial cells stimulated with extract of adult worms of D. immitis obtained from an untreated dog (DiSA) and from a dog treated for 15 days with doxycycline (tDiSA), was determined by using commercial kits. The capacity of pseudocapillary formation was evaluated analyzing cell connections and cell groups in Matrigel cell cultures stimulated with DiSA and tDiSA. In both cases non-stimulated cultures were used as controls., Results: First, we demonstrated that worms obtained from the dog treated with doxycycline showed a significantly lower amount of Wolbachia (less than 60%) than worms removed from the untreated dog. Only DiSA was able to significantly increase the expression of the proangiogenic factor VEGF-A in the endotelial cells cultures. None of the D. immitis extracts modified the expression of sFlt. tDiSA extract was able to modify the expression of the endoglins, significantly decreasing the expression of the pro-angiogenic mEndoglin and increasing the anti-angiogenic sEndoglin. The formation of pseudocapillaries was negatively influenced by tDiSA, which reduced the organization and number of cellular connections., Conclusions: The ability of antigens from adult D. immitis worms to modify the expression of pro and anti-angiogenic factors in endotelial cell cultures was demonstrated, as well as the trend to form pseudocapillaries in vitro. The capacity of stimulation may be linked to the amount of Wolbachia present in the antigenic extracts.

In recent years, anthrax toxin has been reengineered to act as a highly specific antiangiogenic cancer therapeutic, shown to kill tumors in animal models. This has been achieved by modifying protective antigen (PA) so that its activation and toxicity require the presence of two proteases, matrix metalloproteinase (MMP) and urokinase plasminogen activator (uPA), which are upregulated in tumor microenvironments. These therapeutics consist of intercomplementing PA variants, which are individually nontoxic, but form functional toxins upon complementary oligomerization. Here, we have created a dual-protease requiring PA targeting system which utilizes bismaleimide cross-linked PA (CLPA) rather than the intercomplementing PA variants. Three different CLPA agents were tested and, as expected, found to exclusively form octamers. Two of the CLPA agents have in vitro toxicities equal to those of previous intercomplementing agents, while the third CLPA agent had compromised in vitro cleavage and was significantly less cytotoxic. We hypothesize this difference was due to steric hindrance caused by cross-linking two PA monomers in close proximity to the PA cleavage site. Overall, this work advances the development and use of the PA and LF tumor-targeting system as a practical cancer therapeutic, as it provides a way to reduce the drug components of the anthrax toxin drug delivery system from three to two, which may lower the cost and simplify testing in clinical trials. HIGHLIGHT: Previously, anthrax toxin has been reengineered to act as a highly specific antiangiogenic cancer therapeutic. Here, we present a version, which utilizes bismaleimide cross-linked protective antigen (PA) rather than intercomplementing PA variants. This advances the development of anthrax toxin as a practical cancer therapeutic as it reduces the components of the drug delivery system to two, which may lower the cost and simplify testing in clinical trials., (© 2019 The Protein Society.)

Developing effective mucosal subunit vaccine for the Streptococcus pneumoniae has been unsuccessful mainly because of their poor immunogenicity with insufficient memory T and B cell responses. We thus address whether such limitation can be overcome by introducing effective adjuvants that can enhance immunity and show here that polysorbitol transporter (PST) serves as a mucosal adjuvant for a subunit vaccine against the Streptococcus pneumoniae. Pneumococcal surface protein A (PspA) with PST adjuvant induced protective immunity against S. pneumoniae challenge, especially long-term T and B cell immune responses. Moreover, we found that the PST preferentially induced T helper (Th) responses toward Th2 or T follicular helper (Tfh) cells and, importantly, that the responses were mediated through antigen-presenting cells via activating a peroxisome proliferator-activated receptor gamma (PPAR-γ) pathway. Thus, these data indicate that PST can be used as an effective and safe mucosal vaccine adjuvant against S. pneumoniae infection. STATE OF SIGNIFICANCE: In this study, we suggested the nanoparticle forming adjuvant, PST works as an effective adjuvant for the pneumococcal vaccine, PspA. The PspA subunit vaccine together with PST adjuvant efficiently induced protective immunity, even in the long-term memory responses, against Streptococcus pneumoniae lethal challenge. We found that PspA with PST adjuvant induced dendritic cell activation followed by follicular helper T cell responses through PPAR-γ pathway resulting long-term memory antibody-producing cells. Consequently, in this paper, we suggest the mechanism for safe nanoparticle forming subunit vaccine adjuvant against pneumococcal infection., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

A safe and effective adjuvant is necessary to induce reliable protective efficacy of the protein-based vaccines against tuberculosis (TB). Mycobacterial components, such as synthetic cord factor and arabinogalactan, have been used as one of the adjuvant components. Mycobacterium bovis bacillus Calmette- Guérin cell-wall skeleton (BCG-CWS) has been used as an effective immune-stimulator. However, it is not proven whether BCG-CWS can be an effective adjuvant for the subunit protein vaccine of TB. In this study, we demonstrated that the BCG-CWS effectively coupled with Ag85B and enhanced the conjugated Ag85B activity on the maturation of dendritic cells (DCs). Ag85B-BCG-CWS-matured DCs induced significant Th1 and Th17 responses when compared to BCG-CWS or Ag85B alone. In addition, significant Ag85B-specific Th1 and Th17 responses were induced in Ag85B-BCG-CWS-immunized mice before infection with M. tuberculosis and maintained after infection. Moreover, Ag85B-BCG-CWS showed significant protective effect comparable to live BCG at 6 weeks after infection and maintained its protective efficacy at 32 weeks post-challenge, whereas live BCG did not. These results suggest that the BCG-CWS may be an effective adjuvant candidate for a protein-based vaccine against TB., Competing Interests: The authors have declared that no competing interests exist.

The Bacillus anthracis Edema Toxin (ET), composed of a Protective Antigen (PA) and the Edema Factor (EF), is a cellular adenylate cyclase that alters host responses by elevating cyclic adenosine monophosphate (cAMP) to supraphysiologic levels. However, the role of ET in systemic anthrax is unclear. Efferocytosis is a cAMP-sensitive, anti-inflammatory process of apoptotic cell engulfment, the inhibition of which may promote sepsis in systemic anthrax. Here, we tested the hypothesis that ET inhibits efferocytosis by primary human macrophages and evaluated the mechanisms of altered efferocytic signaling. ET, but not PA or EF alone, inhibited the efferocytosis of early apoptotic neutrophils (PMN) by primary human M2 macrophages (polarized with IL-4, IL-10, and/or dexamethasone) at concentrations relevant to those encountered in systemic infection. ET inhibited Protein S- and MFGE8-dependent efferocytosis initiated by signaling through MerTK and αVβ5 receptors, respectively. ET inhibited Rac1 activation as well as the phosphorylation of Rac1 and key activating sites of calcium calmodulin-dependent kinases CamK1α, CamK4, and vasodilator-stimulated phosphoprotein, that were induced by the exposure of M2(Dex) macrophages to Protein S-opsonized apoptotic PMN. These results show that ET impairs macrophage efferocytosis and alters efferocytic receptor signaling.

Background: Leprosy is an ideal human disease to study T cell regulation as patients show correlation between cytokine skewed Th1-Th2 responses and clinical forms of the disease. The Role of transcription factors on the modulation of Th1 and Th2 responses by M. leprae antigens has not been adequately studied. In the present study, we studied the effect of M. leprae antigens on transcription factors STAT-4, STAT-6 and CREB and their correlation with Th1/Th2 cell mediated immune responses in leprosy., Methods: Leprosy patients of both categories of tuberculoid leprosy (BT/TT) and lepromatous leprosy (BL/LL) were selected from the OPD of NJ1L & OMD, (ICMR), Agra and healthy individuals (H) were chosen from the staff and students working in the institute. Peripheral blood mononuclear cells (PBMCs) of the study subjects were stimulated with M. leprae antigens (WCL, MLSA, and PGL-1). Sandwich ELISA was done in the culture supernatants of healthy and leprosy patients to detect IL-4, IL-10 and IFN-γ. Further, expression of IFN-γ and IL-4 and activation of STAT4, STAT6 and CREB transcription factors in CD4 + T cell with or without stimulation of M. leprae antigens was investigated by flow cytometry., Results: Lepromatous leprosy patients showed significantly lower IFN-γ and higher IL-4 levels in culture supernatant and significantly low expression of IFN-γ and higher expression of IL-4 by CD4 + T cells than healthy individuals with or without antigenic stimulation. Antigenic stimulation significantly increased IL-10 in BL/LL patients but not in BT/TT patients or healthy individuals. PGL-1 stimulation led to significantly higher activation of STAT-6 in BT/TT and BL/LL patients in comparison to healthy individuals. All the three antigens led to activation of CREB in healthy and BT/TT patients but not in BL/LL patients., Conclusion: Our findings show that M. leprae antigens differentially modulate activation of T cell transcription factors STAT-4/STAT-6 and CREB. These transcription factors are well known to regulate Th1 and Th2 mediated immune response which in turn could play vital role in the clinical manifestations of leprosy. These observations may help to determine how these T cell transcription factors affect the development of immune dysfunction and whether these new pathways have a role in immunomodulation in intracellular diseases like leprosy and TB.

Urinary tract infections (UTIs) are among the most common bacterial infections. Its management has become increasingly challenging due to antimicrobial resistance. The four mainstays to tackle this crisis rely on the development of new antibiotic agents, the introduction of preventive and alternative antimicrobial strategies, the concept of antimicrobial stewardship, and effective hygiene measures. One of the most effective approaches to prevent UTIs is the design of a potent vaccine. OM-89 is a lyophilised preparation of membrane proteins from 18 different uropathogenic Escherichia coli strains. The safety and efficacy of this immunoactive agent is well documented; therefore, it is recommended for the prophylaxis of UTI according to the current European Association of Urology guidelines on urological infections. In terms of a true vaccine designed to target specifically pathogenic bacteria, no substance is currently available. ExPEC4V, a novel tetravalent bioconjugate vaccine against extraintestinal pathogenic E. coli, was evaluated for safety, immunogenicity, and clinical efficacy in a randomised, single-blinded, placebo-controlled phase 1b trial. The vaccine was well tolerated and elicited a robust antibody response in patients suffering from recurrent UTIs. Although the first clinical data suggested a reduced incidence of UTIs after vaccination, especially for higher bacterial loads, further randomised controlled trials are necessary to determine its true clinical benefit., (Copyright © 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Bacterial vaccines have been widely used to prevent infectious diseases, especially in veterinary medicine. Although there are many reports on bacterin adjuvants, only a few contain innovations in bacterin adjuvants. Taking this into consideration, in this study we designed and synthesized a new aluminum (oxy) hydroxide (AlOOH) nanorod (Al-NR) with a diameter of 200 ± 80 nm and a length of 1.1 ± 0.6 μm. Using whole- Pseudomonas aeruginosa PAO1 as antigens, we showed that the bacterial antigens of P. aeruginosa PAO1 adsorbed on the Al-NRs induced a quick and stronger antigen-specific antibody response than those of the other control groups, especially in the early stage of immunization. Furthermore, the level of antigen-specific IgG was approximately 4-fold higher than that of the no adjuvant group and 2.5-fold higher than those of other adjuvant groups in the first week after the initial immunization. The potent adjuvant activity of the Al-NRs was attributed to the rapid presentation of antigen adsorbed on them by APCs. Additionally, Al-NRs induced a milder local inflammation than the other adjuvants. In short, we confirmed that Al-NRs, enhancing both humoral and cellular immune responses, are a potentially promising vaccine adjuvant delivery system for inhibiting the whole- Pseudomonas aeruginosa infection.

Background: Porphyromonas gingivalis adherence to oral streptococci is a key point in the pathogenesis of periodontal diseases (Honda in Cell Host Microbe 10:423-425, 2011). Previous work in our groups has shown that a region of the streptococcal antigen denoted BAR (SspB Adherence Region) inhibits P. gingivalis/S. gordonii interaction and biofilm formation both in vitro and in a mouse model of periodontitis (Daep et al. in Infect Immun 74:5756-5762, 2006; Daep et al. in Infect immun 76:3273-3280, 2008; Daep et al. in Infect Immun 79:67-74, 2011). However, high localized concentration and prolonged exposure are needed for BAR to be an effective therapeutic in the oral cavity., Methods: To address these challenges, we fabricated poly(lactic-co-glycolic acid) (PLGA) and methoxy-polyethylene glycol PLGA (mPEG-PLGA) nanoparticles (NPs) that encapsulate BAR peptide, and assessed the potency of BAR-encapsulated NPs to inhibit and disrupt in vitro two-species biofilms. In addition, the kinetics of BAR-encapsulated NPs were compared after different durations of exposure in a two-species biofilm model, against previously evaluated BAR-modified NPs and free BAR., Results: BAR-encapsulated PLGA and mPEG-PLGA NPs potently inhibited biofilm formation (IC50 = 0.7 μM) and also disrupted established biofilms (IC50 = 1.3 μM) in a dose-dependent manner. In addition, BAR released during the first 2 h of administration potently inhibits biofilm formation, while a longer duration of 3 h is required to disrupt pre-existing biofilms., Conclusions: These results suggest that BAR-encapsulated NPs provide a potent platform to inhibit (prevent) and disrupt (treat) P. gingivalis/S. gordonii biofilms, relative to free BAR.

Although IL-10 is known to be an important cytokine in the immune response to TB, very little is known about the role of IL-20 subfamily of cytokines in the host response to TB. To identify the role of CD4 + T and CD8 + T cells producing IL-20 subfamily of cytokines in human TB, we enumerated the frequencies of IL-10, IL-19 and IL-24 expressing CD4 + and CD8 + T cells following Mtb-specific antigen stimulation of cells from individuals with pulmonary TB (PTB) and latent TB (LTB). We first demonstrated that Mtb-specific antigen induce an expansion of CD4 + and CD8 + T cells expressing IL-10, IL-19 and IL-24 in PTB and LTB individuals, with frequencies being significantly higher in PTB. Next, we demonstrated that IL-10, IL-19 and IL-24 play an important role in the regulation of CD4 + and CD8 + T cells expressing Th1/Tc1 and Th17/Tc17 cytokines in PTB but not LTB individuals. Thus, active PTB is characterized by an IL-10, IL-19 and IL-24 mediated down modulation of Th1/Tc1 and/or Th17/Tc17 cytokines in CD4 + and CD8 + T cell subsets. This suggests that the IL-20 subfamily of cytokines, similar to IL-10 might play a potentially crucial role in the modulation of T cell responses in active TB disease., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

BACKGROUND The anti-microbial protein cathelicidin LL-37 plays an important role in the pathogenesis of psoriasis by inducing inflammation. Our previous study showed that the chlamydial plasmid-encoded protein pGP3 forms a stable complex with LL-37 to neutralize its pro-inflammatory activity. Here, we explored whether pGP3 can inhibit the development of lesions in mice with imiquimod-induced psoriasis. MATERIAL AND METHODS The protein pGP3 was expressed in bacteria and purified using glutathione-conjugated agarose beads and a precision protease. The ability of the purified pGP3 to block chemotaxis mediated by LL-37 was tested in vitro using bone marrow-derived neutrophils. The ability of the protein to inhibit the development of psoriasis-like lesions was tested by topically or subcutaneously administering pGP3 in doses of 10 or 50 μg to mice previously treated with imiquimod. Mouse skin was evaluated using the psoriasis area and severity index (PASI) score and photography. Skin biopsies were taken on day 8 and analyzed histologically. RESULTS Purified pGP3 inhibited LL-37-mediated chemotaxis. Mice treated with 50 μg pGP3 showed clinical improvement with less severe erythema, infiltration, and scales; these mice also showed thinner dermis and less hyperkeratosis, parakeratosis, and inflammatory cell infiltration than mice treated with without 10 μg pGP3. CONCLUSIONS PGP3 can inhibit the development of psoriasis-like lesions in mice, possibly through its ability to bind LL-37. Future work should examine the mechanisms underlying this therapeutic effect.