Dihydromyricetin treats pulmonary hypertension by modulating CKLF1/CCR5 axis-induced pulmonary vascular cell pyroptosis.

Pulmonary hypertension (PH) is a progressive cardiopulmonary disease characterized by elevated pulmonary artery pressure and vascular remodeling, resulting in poor prognosis and increased mortality rates. Chemokine-like factor 1 (CKLF1) plays a significant role in inducing inflammation and cell proliferation, both of which are critical processes in the pathogenesis of various diseases. Dihydromyricetin (DMY) has garnered attention for its potent anti-inflammatory properties. This study evaluated the protective effects of DMY against PH, demonstrating that DMY treatment can mitigate pyroptosis in pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells (PASMCs) in vivo via the CKLF1/CCR5 axis. Results indicated significant improvements in hemodynamics, inflammatory responses, fibrosis, vascular remodeling, and right ventricular hypertrophy in PH rats following DMY treatment. Furthermore, the interaction between CKLF1 and CCR5 was investigated in CKLF1-/- rats after PH induction. DMY was found to downregulate CKLF1 expression and the inflammatory response in the lungs, with its therapeutic efficacy diminished following CKLF1 knockdown. This study underscores the therapeutic potential of DMY in the management of PH and lays a foundation for future research and clinical applications.

Farnesol ameliorates DSS-induced IBD by regulating inflammatory cytokines, repairing the intestinal barrier, reversing the gut microbiota imbalance, and influencing fecal metabolome in C57BL/6 mice.

The incidence of inflammatory bowel disease (IBD) is rising globally, increasing interest in food ingredients for its prevention and control. This study evaluated the effect of farnesol (FAR), a key component of pomelo flower volatile oil, on dextran sodium sulfate (DSS)-induced colitis in C57BL/6 mice. FAR significantly alleviated DSS-induced colitis and secondary liver injury, as shown by improved body weight, DAI, colon length, and pathology, as well as liver function and blood lipid indices. The mechanism involves FAR-mediated regulation of inflammatory cytokines, increased expression of tight junction protein genes, and decreased expression of lipid metabolism-related proteins. FAR also enhanced gut microbiota diversity, balancing harmful and probiotic bacteria. Fecal metabolome analysis indicated FAR's role in reversing metabolic disturbances related to inflammation and liver lipid metabolism. These findings support developing functional foods for IBD treatment using pomelo flower volatile oil.

Target modulation of glycolytic pathways as a new strategy for the treatment of neuroinflammatory diseases.

Neuroinflammation is an innate and adaptive immune response initiated by the release of inflammatory mediators from various immune cells in response to harmful stimuli. While initially beneficial and protective, prolonged or excessive neuroinflammation has been identified in clinical and experimental studies as a key pathological driver of numerous neurological diseases and an accelerant of the aging process. Glycolysis, the metabolic process that converts glucose to pyruvate or lactate to produce adenosine 5'-triphosphate (ATP), is often dysregulated in many neuroinflammatory disorders and in the affected nerve cells. Enhancing glucose availability and uptake, as well as increasing glycolytic flux through pharmacological or genetic manipulation of glycolytic enzymes, has shown potential protective effects in several animal models of neuroinflammatory diseases. Modulating the glycolytic pathway to improve glucose metabolism and ATP production may help alleviate energy deficiencies associated with these conditions. In this review, we examine six neuroinflammatory diseases-stroke, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and depression-and provide evidence supporting the role of glycolysis in their treatment. We also explore the potential link between inflammation-induced aging and glycolysis. Additionally, we briefly discuss the critical role of glycolysis in three types of neuronal cells-neurons, microglia, and astrocytes-within physiological processes. This review highlights the significance of glycolysis in the pathology of neuroinflammatory diseases and its relevance to the aging process.

Progress of Exosomal MicroRNAs and Traditional Chinese Medicine Monomers in Neurodegenerative Diseases.

Exosomes, extracellular vesicles secreted by various cells, actively participate in intercellular communication by facilitating the exchange of crucial molecular information such as DNA, RNA, and lipids. Within this intricate network, microRNAs, endogenous non-coding small RNAs, emerge as pivotal regulators of post-transcriptional gene expression, significantly influencing the development of neurodegenerative diseases. The historical prominence of traditional Chinese medicine (TCM) in clinical practice in China underscores its enduring significance. Notably, TCM monomers, serving as active constituents within herbal medicine, assume a critical role in the treatment of neurodegenerative diseases, particularly in mitigating oxidative stress, inhibiting apoptosis, and reducing inflammation. This comprehensive review aims to delineate the specific involvement of exosomal microRNAs in various neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke, and amyotrophic lateral sclerosis. Furthermore, the exploration extends to the application of TCM monomers, elucidating their efficacy as therapeutic agents in these conditions. Additionally, the review examines the utilization of exosomes as drug delivery carriers in the context of neurodegenerative diseases, providing a nuanced understanding of the potential synergies between TCM and modern therapeutic approaches. This synthesis of knowledge aims to contribute to the advancement of our comprehension of the intricate molecular mechanisms underlying neurodegeneration and the potential therapeutic avenues offered by TCcom interventions.

Asymmetric Cycloaddition of N-2,2,2-Trifluoroethylisatin Ketimines and Unsymmetrical Dicarbonyl-Activated Alkenes: Construction of 5'-Trifluoromethylated 3,2'-Pyrrolidinyl Spirooxindoles with Three Carbonyl Groups.

The asymmetric cycloaddition between N-2,2,2-trifluoroethylisatin ketimines and unsymmetrical dicarbonyl-activated alkenes catalyzed by a bifunctional squaramide has been discovered. The present study demonstrates an efficient approach for the regio-, diastereo-, and enantioselective synthesis of densely functionalized 5'-trifluoromethylated 3,2'-pyrrolidinyl spirooxindoles featuring three different types of carbonyl groups.

CCR5 and inflammatory storm.

Chemokines and their corresponding receptors play crucial roles in orchestrating inflammatory and immune responses, particularly in the context of pathological conditions disrupting the internal environment. Among these receptors, CCR5 has garnered considerable attention due to its significant involvement in the inflammatory cascade, serving as a pivotal mediator of neuroinflammation and other inflammatory pathways associated with various diseases. However, a notable gap persists in comprehending the intricate mechanisms governing the interplay between CCR5 and its ligands across diverse and intricate inflammatory pathologies. Further exploration is warranted, especially concerning the inflammatory cascade instigated by immune cell infiltration and the precise binding sites within signaling pathways. This study aims to illuminate the regulatory axes modulating signaling pathways in inflammatory cells by providing a comprehensive overview of the pathogenic processes associated with CCR5 and its ligands across various disorders. The primary focus lies on investigating the pathomechanisms associated with CCR5 in disorders related to neuroinflammation, alongside the potential impact of aging on these processes and therapeutic interventions. The discourse culminates in addressing current challenges and envisaging potential future applications, advocating for innovative research endeavors to advance our comprehension of this realm.

[Exosome and prevention of Alzheimer's disease: based on theory of kidney storing essence].

The theory of kidney storing essence storage, an important part of the basic theory of traditional Chinese medicine(TCM), comes from the Chapter 9 Discussion on Six-Plus-Six System and the Manifestations of the Viscera in the Plain Questions, which says that "the kidney manages closure and is the root of storage and the house of Jing(Essence)". According to this theory, essence is the fundamental substance of human life activities and it is closely related to the growth and development of the human body. Alzheimer's disease(AD) is one of the common neurodegenerative diseases, with the main pathological features of Aß deposition and Tau phosphorylation, which activate neurotoxic reactions and eventually lead to neuronal dysfunction and cell death, severely impairing the patient's cognitive and memory functions. Although research results have been achieved in the TCM treatment of AD, the complex pathogenesis of AD makes it difficult to develop the drugs capable of curing AD. The stem cell therapy is an important method to promote self-repair and regeneration, and bone marrow mesenchymal stem cells(BMSCs) as adult stem cells have the ability of multi-directional differentiation. By reviewing the relevant literature, this paper discusses the association between BMSCs and the TCM theory of kidney storing essence, and expounds the material basis of this theory from the perspective of molecular biology. Studies have shown that TCM with the effect of tonifying the kidney in the treatment of AD are associated with BMSCs. Exosomes produced by such cells are one of the main substances affecting AD. Exosomes containing nucleic acids, proteins, and lipids can participate in intercellular communication, regulate cell function, and affect AD by reducing Aß deposition, inhibiting Tau protein phosphorylation and neuroinflammation, and promoting neuronal regeneration. Therefore, discussing the prevention and treatment of exosomes and AD based on the theory of kidney storing essence will provide a new research idea for the TCM treatment of AD.

Ginsenoside Rg1 treats ischemic stroke by regulating CKLF1/CCR5 axis-induced neuronal cell pyroptosis.

BACKGROUND: Ischemic stroke, a severe and life-threatening neurodegenerative condition, currently relies on thrombolytic therapy with limited therapeutic window and potential risks of hemorrhagic transformation. Thus, there is a crucial need to explore novel therapeutic agents for ischemic stroke. Ginsenoside Rg1 (Rg1), a potential neuroprotective agent, exhibits anti-ischemic effects attributed to its anti-inflammatory, anti-oxidant, and anti-apoptotic properties. Nevertheless, the precise underlying mechanism of action remains to be fully elucidated. PURPOSE: This study aimed to explore whether Rg1 exerts anti-ischemic stroke effects by inhibiting pyroptotic neuronal cell death through modulation of the chemokine like factor 1 (CKLF1)/ C-C chemokine receptor type 5 (CCR5) axis. METHODS: In this study, the MCAO model was used as an ischemic stroke model, and experimental tests were performed after 6 hours of ischemia. The anti-ischemic effect of Rg1 was examined by TTC staining, nissl-staining and neurobehavioral tests. In the in vitro experiments, PC12 cells were subjected to stimulation with CKLF1's mimetic peptide C27 to assess the potential of CKLF1 to induce focal neuronal cell death. Additionally, the impact of CKLF1 mimetic peptide C27, antagonistic peptide C19, and CCR5 inhibitor MVC on PC12 cells subjected to oxygen-glucose deprivation (OGD) and subsequently treated with Rg1 was investigated. In vivo, Rg1 treatment was examined by quantitative real-time PCR (qPCR), ELISA, immunohistochemistry (IHC), immunofluorescence (IF), western blot (WB), and co-immunoprecipitate (Co-IP) assays to perspective whether Rg1 treatment reduces CKLF1/CCR5 axis-induced pyroptotic neuronal cell death. In addition, to further explore the biological significance of CKLF1 in ischemic stroke, CKLF1-/- rats were used as the observation subjects in this study. RESULTS: The in vitro results suggested that CKLF1 was able to induce neuronal cells to undergo pyroptosis. In vivo pharmacodynamic results showed that Rg1 treatment was able to significantly improve symptoms in ischemic stroke rats. In addition, Rg1 treatment was able to inhibit the interaction between CKLF1 and CCR5 after ischemic stroke and inhibited CKLF1/CCR5 axis-induced pyroptosis. The results of related experiments in CKLF1-/- rats showed that Rg1 lost its therapeutic effect after CKLF1 knockdown. CONCLUSION: Our findings indicate that the activation of the NLRP3 inflammasome is initiated by the CKLF1/CCR5 axis, facilitated through the activation of the NF-κB pathway, ultimately resulting in the pyroptosis of neuronal cells. Conversely, Rg1 demonstrates the capability to mitigate neuronal cell damage following CKLF1-induced effects by suppressing the expression of CKLF1. Thus, CKLF1 represents a crucial target for Rg1 in the context of cerebral ischemia treatment, and it also holds promise as a potential target for drug screening in the management of ischemic stroke.

CC chemokines Modulate Immune responses in Pulmonary Hypertension.

BACKGROUND: Pulmonary hypertension (PH) represents a progressive condition characterized by the remodeling of pulmonary arteries, ultimately culminating in right heart failure and increased mortality rates. Substantial evidence has elucidated the pivotal role of perivascular inflammatory factors and immune dysregulation in the pathogenesis of PH. Chemokines, a class of small secreted proteins, exert precise control over immune cell recruitment and functionality, particularly with respect to their migration to sites of inflammation. Consequently, chemokines emerge as critical drivers facilitating immune cell infiltration into the pulmonary tissue during inflammatory responses. This review comprehensively examines the significant contributions of CC chemokines in the maintenance of immune cell homeostasis and their pivotal role in regulating inflammatory responses. The central focus of this discussion is directed towards elucidating the precise immunoregulatory actions of CC chemokines concerning various immune cell types, including neutrophils, monocytes, macrophages, lymphocytes, dendritic cells, mast cells, eosinophils, and basophils, particularly in the context of pH processes. Furthermore, this paper delves into an exploration of the underlying pathogenic mechanisms that underpin the development of PH. Specifically, it investigates processes such as cellular pyroptosis, examines the intricate crosstalk between bone morphogenetic protein receptor type 2 (BMPR2) mutations and the immune response, and sheds light on key signaling pathways involved in the inflammatory response. These aspects are deemed critical in enhancing our understanding of the complex pathophysiology of PH. Moreover, this review provides a comprehensive synthesis of findings from experimental investigations targeting immune cells and CC chemokines. AIM OF REVIEW: In summary, the inquiry into the inflammatory responses mediated by CC chemokines and their corresponding receptors, and their potential in modulating immune reactions, holds promise as a prospective avenue for addressing PH. The potential inhibition of CC chemokines and their receptors stands as a viable strategy to attenuate the inflammatory cascade and ameliorate the pathological manifestations of PH. Nonetheless, it is essential to acknowledge the current state of clinical trials and the ensuing progress, which regrettably appears to be less than encouraging. Substantial hurdles exist in the successful translation of research findings into clinical applications. The intention is that such emphasis could potentially foster the advancement of potent therapeutic agents presently in the process of clinical evaluation. This, in turn, may further bolster the potential for effective management of PH.

Sophoridine inhibits endotoxin-induced acute lung injury by enhancing autophagy of macrophage and reducing inflammation.

Acute lung injury (ALI) is characterized by uncontrolled inflammation, which can lead to respiratory distress syndrome and cause patient death. In this study, we sought to determine the role of sophoridine, a compound purified from sophora, in ALI. A mouse model of ALI was established by treating mice with LPS through nonexposed tracheal instillation. After LPS-induced mice were treated with sophoridine, LPS-induced alveolar wall thickening, alveolar interstitial inflammatory exudation and thickening, and the degree of pulmonary edema were found to be inhibited. Macrophages play an important role in inflammation, and in vitro experiments have demonstrated that sophoridine reduces the LPS-induced expression of inflammatory factors by macrophages, suggesting that sophoridine may inhibit lung inflammation in LPS-treated mice through reduces the secretion of inflammatory factors. Further, treatment with sophoridine up-regulated autophagy in macrophage cells in vitro and mouse lung tissues in vivo. LPS can bind to TLRs and activate the MyD88/NF-κB pathways, leading to increased inflammation in the pathogenesis of ALI. Our findings revealed that sophoridine down-regulated the expression of TLR4/MyD88/NF-κB and mTOR mRNA and protein in mouse pulmonary tissue. Collectively, these findings indicate that sophoridine may inhibit LPS-induced ALI by enhancing autophagy of macrophages and reducing inflammation.

Growth Hormone Receptor Controls Adipogenic Differentiation of Chicken Bone Marrow Mesenchymal Stem Cells by Affecting Mitochondrial Biogenesis and Mitochondrial Function.

Growth hormone receptor (GHR) can activate several signaling pathways after binding to growth hormone (GH) to regulate cell growth and development. Sex-linked dwarf (SLD) chickens, normal protein functions are prevented because of exon mutations in the GHR gene, have more severe fat deposition. However, the specific molecular mechanisms responsible for this phenotype remains unclear. We therefore investigated the effect of the GHR gene on adipogenic differentiation of chicken bone marrow mesenchymal stem cells (BMSCs). We found that bone marrow fat deposition was more severe in SLD chickens compared to normal chickens, and the expression of genes related to adipogenic differentiation was enhanced in SLD chicken BMSCs. We also detected enhanced mitochondrial function of BMSCs in SLD chickens. In vitro, overexpression of GHR in chicken BMSCs increased mitochondrial membrane potential but decreased reactive oxygen and ATP contents, oxidative phosphorylation complex enzyme activity, and mitochondrial number. Expression of genes associated with mitochondrial biogenesis and function was repressed during adipogenic differentiation in chicken BMSCs, the adipogenic differentiation capacity of chicken BMSCs was also repressed. With knockdown of GHR, opposite results were observed. We concluded that GHR inhibited adipogenic differentiation of chicken BMSCs by suppressing mitochondrial biogenesis and mitochondrial function.

Growth hormone receptor gene influences mitochondrial function and chicken lipid metabolism by AMPK-PGC1α-PPAR signaling pathway.

BACKGROUND: Adipose tissue is an important endocrine and energy-storage organ in organisms, and it plays a crucial role in the energy-metabolism balance. Previous studies have found that sex-linked dwarf (SLD) chickens generally have excessively high abdominal fat deposition during the growing period, which increases feeding costs. However, the underlying mechanism of this fat deposition during the growth of SLD chickens remains unknown. RESULTS: The Oil Red O staining showed that the lipid-droplet area of SLD chickens was larger than that of normal chickens in E15 and 14d. Consistently, TG content in the livers of SLD chickens was higher than that of normal chickens in E15 and 14d. Further, lower ΔΨm and lower ATP levels and higher MDA levels were observed in SLD chickens than normal chickens in both E15 and 14d. We also found that overexpression of GHR reduced the expression of genes related to lipid metabolism (AMPK, PGC1α, PPARγ, FAS, C/EBP) and oxidative phosphorylation (CYTB, CYTC, COX1, ATP), as well as reducing ΔΨm and ATP levels and increasing MDA levels. In addition, overexpression of GHR inhibited fat deposition in CPPAs, as measured by Oil Red O staining. On the contrary, knockdown of GHR had the opposite effects in vitro. CONCLUSIONS: In summary, we demonstrate that GHR promotes mitochondrial function and inhibits lipid peroxidation as well as fat deposition in vivo and in vitro. Therefore, GHR is essential for maintaining the stability of lipid metabolism and regulating mitochondrial function in chicken.

[Th1 immune response induced by Mycobacterium tuberculosis H37Ra in NOD2 knockout mice].

Objective To investigate the roles of Th1 cytokines tumor necrosis factor α (TNF-α), interferon gamma (IFN-γ) and multifunctional T cells in nucleotides binding oligomer domain 2 knockout (NOD2-/-) mice infected with Mycobacterium tuberculosis (MTB) H37Ra. Methods Mouse models of pulmonary infection were established by tracheal instillation of MTB strain H37Ra into NOD2-/- mice and C57BL/6 mice (n=10 each group). Lung tissues were removed and stained by HE staining and pathological scores were evaluated 4 weeks after infection. The levels of TNF-α and IFN-γ in the lung homogenates were detected by ELISA, and the ratio of multifunctional CD4+ T and CD8+ T cells in the spleen were examined by flow cytometry. Results MTB infection promoted lung inflammation of NOD2-/- mice. The levels of TNF-α and IFN-γ in the lung tissues of NOD2-/- mice increased. Compared with normal saline group, TNF-α+, IFN-γ+ cells and TNF-α+IFN-γ+ cells in CD4+/CD8+T cells significantly increased in NOD2-/- mice and C57BL/6 mice after the infection. TNF-α+CD4+T cells, IFN-γ+CD4+T cells and IFN-γ+CD8+T cells in MTB-infected NOD2-/- mice were significantly higher than those in MTB-infected C57BL/6 mice. Conclusion H37Ra can induce Th1 immune response in NOD2-/- mice.

Tuning the Protein Phosphorylation by Receptor Type Protein Tyrosine Phosphatase Epsilon (PTPRE) in Normal and Cancer Cells.

Tyrosine phosphorylation is an important post-translation modification of proteins that is controlled by tyrosine kinases and phosphatases. Disruption of the balance between the activity of tyrosine kinases and phosphatases may result in diseases. Receptor type protein tyrosine phosphatase epsilon (PTPRE) is closely related with receptor type protein tyrosine phosphatase alpha (PTPRA). PTPRE has been studied in osteoclast cells, nerve cells, hematopoietic cells, cancer cells and others, and it has different functions among various tissues. In this review, we summarized the current knowledge about the regulation of PTPRE on cellular signal transduction and its function under normal and pathological conditions.

A species-specific activation of Toll-like receptor signaling in bovine and sheep bronchial epithelial cells triggered by Mycobacterial infections.

Pulmonary tuberculosis caused by a Mycobacterium infection remains a major public health problem in most part of the world, in part owing to the transmission of its pathogens between hosts including human, domestic and wild animals. To date, molecular mechanisms of the pathogenesis of TB are still incompletely understood. In addition to alveolar macrophages, airway epithelial cells have also been recently recognized as main targets for Mycobacteria infections. In an effort to understand the pathogen-host interaction between Mycobacteria and airway epithelial cells in domestic animals, in present study, we investigated the Toll-like receptor (TLR) signaling in bovine and sheep airway epithelial cells in response to an infection of Mycobacterium tuberculosis avirulent H37Ra stain or Mycobacterium bovis BCG vaccine strain, using primary air-liquid interface (ALI) bronchial epithelial culture models. Our results revealed a host and pathogen species-specific TLR-mediated recognition of pathogen-associated molecular patterns (PAMPs), induction and activation of TLR signaling pathways, and substantial induction of inflammatory response in bronchial epithelial cells in response to Mycobacteria infections between these two species. Interestingly, the activation TLR signaling in bovine bronchial epithelial cells induced by Mycobacteria infection was mainly through a myeloid differentiation factor 88 (MyD88)-independent TLR signaling pathway, while both MyD88-dependent and independent TLR signaling cascades could be induced in sheep epithelial cells. Equally noteworthy, a BCG infection was able to induce both MyD88-dependent and independent signaling in sheep and bovine airway epithelial cells, but more robust inflammatory responses were induced in sheep epithelial cells relative to the bovines; whereas an H37Ra infection displayed an ability to mainly trigger a MyD88-independent TLR signaling cascade in these two host species, and induce a more extent expression of inflammatory cytokines in bovine cells in comparison with that in sheep. These data thus provide an evidence for a host and pathogen species-specific response in bovine and sheep airway epithelial cells in response to Mycobacteria infections, which also suggest there is a need to consider in the interpretation of data generated using a species other than the primary host for analysis of a function role or mechanism of ligands or pathogens.

Enhanced and durable protective immune responses induced by a cocktail of recombinant BCG strains expressing antigens of multistage of Mycobacterium tuberculosis.

Although Bacillus Calmette-Guérin (BCG) vaccine confers protection from Mycobacterium tuberculosis infection in children, its immune protection gradually wanes over time, and consequently leads to an inability to prevent the reactivation of latent infection of M. tuberculosis. Therefore, improving BCG for better control of tuberculosis (TB) is urgently needed. We thus hypothesized that recombinant BCG overexpressing immunodominant antigens expressed at different growth stages of M. tuberculosis could provide a more comprehensive protection against primary and latent M. tuberculosis infection. Here, a novel cocktail of recombinant BCG (rBCG) strains, namely ABX, was produced by combining rBCG::85A, rBCG::85B, and rBCG::X, which overexpressed respective multistage antigens Ag85A, Ag85B, and HspX of M. tuberculosis. Our results showed that ABX was able to induce a stronger immune protection than individual rBCGs or BCG against primary TB infection in C57BL/6 mice. Mechanistically, the immune protection was attributed to stronger antigen-specific CD4(+) Th1 responses, higher numbers of IFN-γ(+) CD4(+) TEM and IL-2(+) CD8(+) TCM cells elicited by ABX. These findings thus provide a novel strategy for the improvement of BCG efficacy and potentially a promising prophylactic TB vaccine candidate, warranting further investigation.

High level of IFN-γ released from whole blood of human tuberculosis infections following stimulation with Rv2073c of Mycobacterium tuberculosis.

More efficacious and specific biomarkers are urgently needed for better control of tuberculosis (TB), the second leading infectious cause of mortality worldwide. The region of difference 9 (RD9) presents the genome of the causative pathogen Mycobacterium tuberculosis rather than other species of the genus Mycobacterium, which might be promising targets for specific diagnosis, vaccine development and pathogenesis. In this study, two proteins Rv2073c and Rv2074, encoded by the RD9 were expressed and purified from Escherichia coli system. Following stimulation with both proteins, the levels of IFN-γ secreted by T cells from a total of 49 whole blood samples obtained from clinically diagnosed active TB patients, patients with latent TB infections (LTBIs), and healthy donors, were compared with those of the incubation with recombinant fusion protein of CFP21 and MPT64 (rCM). Our results demonstrated that only Rv2073c could induce a higher level of IFN-γ in TB infections than healthy controls and there was a positive correlation between Rv2073c- and rCM-specific IFN-γ levels in TB infections and healthy donors, respectively. These findings indicate that Rv2073c might be a promising antigen for specific diagnostic reagents and vaccine candidates of TB.

Protection against Mycobacterium tuberculosis infection offered by a new multistage subunit vaccine correlates with increased number of IFN-γ+ IL-2+ CD4+ and IFN-γ+ CD8+ T cells.

Protein subunit vaccines present a compelling new area of research for control of tuberculosis (TB). Based on the interaction between Mycobacterium tuberculosis and its host, five stage-specific antigens of M. tuberculosis that participate in TB pathogenesis--Rv1813, Rv2660c, Ag85B, Rv2623, and HspX--were selected. These antigens were verified to be recognized by T cells from a total of 42 whole blood samples obtained from active TB patients, patients with latent TB infections (LTBIs), and healthy control donors. The multistage polyprotein A1D4 was developed using the selected five antigens as a potentially more effective novel subunit vaccine. The immunogenicity and protective efficacy of A1D4 emulsified in the adjuvant MTO [monophosphoryl lipid A (MPL), trehalose-6,6'-dibehenate (TDB), components of MF59] was compared with Bacillus Calmette-Guerin (BCG) in C57BL/6 mice. Our results demonstrated that A1D4/MTO could provide more significant protection against M. tuberculosis infection than the PBS control or MTO adjuvant alone judging from the A1D4-specific Th1-type immune response; however, its efficacy was inferior to BCG as demonstrated by the bacterial load in the lung and spleen, and by the pathological changes in the lung. Antigen-specific single IL-2-secreting cells and different combinations with IL-2-secreting CD4+ T cells were beneficial and correlated with BCG vaccine-induced protection against TB. Antigen-specific IFN-γ+ IL-2+ CD4+ T cells were the only effective biomarker significantly induced by A1D4/MTO. Among all groups, A1D4/MTO immunization also conferred the highest number of antigen-specific single IFN-γ+ and IFN-γ+ TNF-α+ CD4+ T cells, which might be related to the antigen load in vivo, and single IFN-γ+ CD8+ T cells by mimicking the immune patterns of LTBIs or curable TB patients. Our strategy seems promising for the development of a TB vaccine based on multistage antigens, and subunit antigen A1D4 suspended in MTO adjuvant warrants preclinical evaluation in animal models of latent infection and may boost BCG vaccination.

Comparison of BCG prime-DNA booster and rBCG regimens for protection against tuberculosis.

Developing an effective adult prophylaxis vaccine is a high priority in the global control of tuberculosis (TB), because TB remains an important public health problem and the current widely used BCG vaccine provides effective protection only for children but variable protection against adult TB. BCG priming-heterologous vaccines booster and recombinant BCG technologies have been thought as two important regimens for inducing effective protection against adult TB. Obviously, defining the protective efficacy of the two regimens would benefit more rational design of the future adult TB vaccines. In this study, a recombinant BCG strain (rBCG::685A) expressing the fusion protein of ESAT-6 and Ag85A (r685A) of Mycobacterium tuberculosis was constructed successfully and the secretion of r685A protein from rBCG strain was confirmed by western blotting with anti-ESAT-6 and anti-Ag85A polyclonal antibodies, respectively. The immune responses and protective effects in rBCG::685A vaccinated C57BL/6 mice were compared with that of our previous reported BCG prime-pcD685A booster regimen. Boosting BCG with pcD685A DNA elicited higher level of r685A protein specific IFN-γ secreted by splenocytes and a more significant increase of both TNF-α and iNOS responses in the lung, thus providing better control of bacterial growth in both lung and spleen of immunized mice challenged with virulent M. tuberculosis, compared with mice vaccinated with rBCG::685A or BCG alone. Our results have implications for development of more effective adult TB vaccines for improved control of TB.