Mono- and poly-functional T cells in nontuberculous mycobacteria lung disease patients: Implications in analyzing risk of disease progression.

AT A GLANCE: The diagnosis and progression of nontuberculous mycobacteria lung disease (NTN-LD) are important for clinical judgement but cannot easily be predicted. The immunological response of mono- and poly-functional T cells, a representative of host reactivity to NTM, could be a surrogate biomarker for disease and progression prediction. BACKGROUND: Mycobacterium avium complex (MAC) and M. abscessus (MAB) induced lung disease (LD) have become a clinical concern. Predicting clinical disease relevance and progression is important, but suitable biomarkers are lacking. The host immune response of mono- and poly-functional T cells might aid in clinical judgement. METHODS: We enrolled 140 participants, including 42 MAC-LD, 25 MAB-LD, 31 MAC airway colonization (MAC-Co), 15 MAB-Co patients, and 27 healthy controls. Their blood mono- and poly-functional T cells were measured and analyzed after in-vitro stimulation. RESULTS: Patients with MAC-LD generally had lower total IFN-γ+, total TNF-α+ and triple-positive T cells but higher mono-IL-2+ expression than the controls and MAC-Co group. The MAB-LD group had lower total IL-2 and triple positive cells than the controls and colonization group. Multivariate analysis revealed that body mass index (BMI), mono-IL2+ CD4+ and triple positive-CD8+ cells (PMA stimulation) significantly predicted MAC-LD from the controls. By contrast, male gender and triple positive-CD4+ cells predicted MAC-LD from colonization. On the other hand, the triple positive-CD4+ cells (PMA stimulation) alone or together with the mock/MAB ratio of IL-2+/TNF-α+ CD4 cells could predict MAB-LD in the MAB-Co group or the controls. Among MAC/MAB-LD patients without anti-mycobacterial treatment, MAC-specific mono-IFN-γ+ CD4+ cells and PMA-induced triple positive-CD4+ cells were correlated with progression, with an area under the ROC curve of 0.875. CONCLUSIONS: The patients with MAC/MAB-LD had attenuated poly-functional T cells. The triple-positive CD4+ cells could be useful in diagnosing disease from colonization. MAC-specific mono-IFN-γ+ CD4+ cells and triple positive-CD4+ might predict radiographic progression, which could be useful in making treatment decisions.

Poly-functional T helper cells in human tonsillar mononuclear cells.

Tonsils are important lymphoid organs in which B cells and T cells complete their maturation and identify cells that are infected by pathogens. However, the functions of T cells in human tonsils remain unclear, especially the characteristics of polyfunctional CD4+ T helper cells. In this study, we used multi-color flow cytometry to analyze the expression or co-expression of effector cytokines in CD4+ T cells from tonsillar tissues. We have demonstrated that tonsillar CD4+ T cell can express various Th effector cytokines after short-term polyclonal stimulation, and that cytokine-producing CD4+ T cells were CD45RO+ T cells. In addition, we analyzed the co-expression of two or more kinds of cytokines at the level of a single cell. The results showed that tonsillar CD4+ T cells exhibited polyfunctionality by co-expressing two to five kinds of cytokines in the same time. These data furnished a basic theory for further understanding the differentiation of polyfunctional Th cells in human tonsils and their functions in resisting invasive microorganisms.

Intranasal Vaccination with Mannosylated Chitosan Formulated DNA Vaccine Enables Robust IgA and Cellular Response Induction in the Lungs of Mice and Improves Protection against Pulmonary Mycobacterial Challenge.

Induction of specific humoral and cellular immunity in the lung airways is proposed to be critical for vaccine protection against Mycobacterium tuberculosis (M. tb). To facilitate airway delivery and antigen targeting to the antigen presenting cells in the alveoli, we employed mannosylated chitosan (MCS) to formulate a multi-T-epitope DNA vaccine, pPES, as an intranasal TB vaccine. MCS-DNA nanoparticles appeared spherical with the average particle sizes as 400 nm. HSP65-specific bronchoalveolar lavage fluid SIgA level was significantly elevated by 4 doses of MCS-pPES intranasal immunization as compared to chitosan (CS)-DNA and BCG vaccine. I.n. immunization with MCS-DNA induced a modest peptide-specific Th1(IFN-γ, TNF-α, and IL-2) response in the spleen, while a potent poly-functional CD4+ T response that largely produced TNF-α and IFN-γ, as well as IL-2 in the lung, qualitatively better than that induced by CS-DNA and BCG vaccination. Such response by i.n. immunization with MCS-DNA provided improved protection in the lung against airway Mycobacterial bovis BCG challenge over i.n. CS-DNA and DNA, that is comparable to protection achieved by s.c. BCG vaccination. This enhanced protection was correlated with much greater accessibility of DNA particles to the alveolar macrophages in the lung mediated by man-chitosan. Thus, man-chitosan TB vaccine represents a promising vaccine platform capable of eliciting robust multi-functional T response in the lung mucus and achieving enhanced mucosal immune protection against pulmonary TB.

Mycobacterium indicus pranii as a booster vaccine enhances BCG induced immunity and confers higher protection in animal models of tuberculosis.

BCG, the only approved vaccine protects against severe form of childhood tuberculosis but its protective efficacy wanes in adolescence. BCG has reduced the incidence of infant TB considerably in endemic areas; therefore prime-boost strategy is the most realistic measure for control of tuberculosis in near future. Mycobacterium indicus pranii (MIP) shares significant antigenic repertoire with Mtb and BCG and has been shown to impart significant protection in animal models of tuberculosis. In this study, MIP was given as a booster to BCG vaccine which enhanced the BCG mediated immune response, resulting in higher protection. MIP booster via aerosol route was found to be more effective in protection than subcutaneous route of booster immunization. Pro-inflammatory cytokines like IFN-γ, IL-12 and IL-17 were induced at higher level in infected lungs of 'BCG-MIP' group both at mRNA expression level and in secretory form when compared with 'only BCG' group. BCG-MIP groups had increased frequency of multifunctional T cells with high MFI for IFN-γ and TNF-α in Mtb infected mice. Our data demonstrate for the first time, potential application of MIP as a booster to BCG vaccine for efficient protection against tuberculosis. This could be very cost effective strategy for efficient control of tuberculosis.

Effector and Central Memory Poly-Functional CD4(+) and CD8(+) T Cells are Boosted upon ZOSTAVAX(®) Vaccination.

ZOSTAVAX(®) is a live attenuated varicella-zoster virus (VZV) vaccine that is licensed for the protection of individuals ≥50 years against shingles and its most common complication, postherpetic neuralgia. While IFNγ responses increase upon vaccination, the quality of the T cell response has not been elucidated. By using polychromatic flow cytometry, we characterized the breadth, magnitude, and quality of ex vivo CD4(+) and CD8(+) T cell responses induced 3-4 weeks after ZOSTAVAX vaccination of healthy adults. We show, for the first time that the highest frequencies of VZV-specific CD4(+) T cells were poly-functional CD154(+)IFNγ(+)IL-2(+)TNFα(+) cells, which were boosted upon vaccination. The CD4(+) T cells were broadly reactive to several VZV proteins, with immediate early (IE) 63 ranking the highest among them in the fold rise of poly-functional cells, followed by IE62, gB, open reading frame (ORF) 9, and gE. We identified a novel poly-functional ORF9-specific CD8(+) T cell population in 62% of the subjects, and these were boosted upon vaccination. Poly-functional CD4(+) and CD8(+) T cells produced significantly higher levels of IFNγ, IL-2, and TNFα compared to mono-functional cells. After vaccination, a boost in the expression of IFNγ by poly-functional IE63- and ORF9-specific CD4(+) T cells and IFNγ, IL-2, and TNFα by ORF9-specific poly-functional CD8(+) T cells was observed. Responding poly-functional T cells exhibited both effector (CCR7(-)CD45RA(-)CD45RO(+)), and central (CCR7(+)CD45RA(-)CD45RO(+)) memory phenotypes, which expressed comparable levels of cytokines. Altogether, our studies demonstrate that a boost in memory poly-functional CD4(+) T cells and ORF9-specific CD8(+) T cells may contribute toward ZOSTAVAX efficacy.

IL-12 induced the generation of IL-21- and IFN-γ-co-expressing poly-functional CD4+ T cells from human naive CD4+ T cells.

Interleukine-12 is critical for the differentiation of Th1 cells and can improve the development of Th1 cells with Tfh cell features in mouse model. Human effector CD4(+) T cells also exhibit poly-functionality by co-expressing IL-21 and IFN-γ. However, the effects of IL-12 on regulating generation of human IL-21- and IFN-γ-expressing CD4(+) T cells are still incompletely understood. Our studies found that IL-12 but not IL-21 could induce the differentiation of human naive CD4(+) T cells into multi-cytokine expressing CD4(+) T cells in vitro, which co-expressed IL-21 and IFN-γ with or without IL-2 and TNF-α. At early stage of differentiation, addition of excess exogenous IFN-γ could increase the generation of IL-21- and IFN-γ-expressing CD4(+) T cells, furthermore, anti-IFN-γ depressed the percentage of poly-functional CD4(+) T cells. Phenotypically, IL-21(+)IFN-γ(+)CD4(+) T cells exhibited more characteristic features about both of Th1 and Tfh cells than IL-21 or IFN-γ single-expressing CD4(+) T cells. Mechamistically, IL-12 modulated the differentiation of IL-21(+)IFN-γ(+)CD4(+) T cells from naive CD4(+) T cells via the pathways of STAT-1/4, T-bet and BCL(-)6. Different from naive CD4(+) T cells, IL-12 increasing the generation of IL-21(+)IFN-γ(+)CD4(+) T cells from memory CD4(+) T cells was only involved in STAT-4 pathway but not STAT-1. Poly-functional CD4(+) T cells were contributed to generation and progress of varies diseases and our studies provide basic theoretics for the designs of vaccine and therapies of diseases.

Crystal structure of N-[4-amino-5-cyano-6-(methyl-sulfan-yl)pyridin-2-yl]acetamide hemihydrate.

The title compound, C9H10N4OS·0.5H2O, crystallizes with two independent mol-ecules (A and B) in the asymmetric unit, together with a water mol-ecule of crystallization. The acetamide moiety, which has an extended conformation, is inclined to the pyridine ring by 7.95 (16)° in mol-ecule A and by 1.77 (16)° in mol-ecule B. In the crystal, the A and B mol-ecules are linked by two N-H⋯Ocarbon-yl hydrogen bonds, forming a dimer. The dimers are linked via N-H⋯N hydrogen bonds, forming ribbons that are linked by N-H⋯Owater hydrogen bonds to form sheets parallel to (110). The sheets are linked by O-H⋯N hydrogen bonds, forming slabs, and between the slabs there are weak slipped parallel π-π inter-actions [inter-centroid distance = 3.734 (2) Å, inter-planar distance = 3.3505 (11) Šand slippage = 1.648 Å], forming a three-dimensional structure.

Flavones: an important scaffold for medicinal chemistry.

Flavones have antioxidant, anti-proliferative, anti-tumor, anti-microbial, estrogenic, acetyl cholinesterase, anti-inflammatory activities and are also used in cancer, cardiovascular disease, neurodegenerative disorders, etc. Also, flavonoids are found to have an effect on several mammalian enzymes like protein kinases that regulate multiple cell signaling pathways and alterations in multiple cellular signaling pathways are frequently found in many diseases. Flavones have been an indispensable anchor for the development of new therapeutic agents. The majority of metabolic diseases are speculated to originate from oxidative stress, and it is therefore significant that recent studies have shown the positive effect of flavones on diseases related to oxidative stress. Due to the wide range of biological activities of flavones, their structure-activity relationships have generated interest among medicinal chemists. The outstanding development of flavones derivatives in diverse diseases in very short span of time proves its magnitude for medicinal chemistry research. The present review gives detail about the structural requirement of flavone derivatives for various pharmacological activities. This information may provide an opportunity to scientists of medicinal chemistry discipline to design selective, optimize as well as poly-functional flavone derivatives for the treatment of multi-factorial diseases.

Arsonium-containing lipophosphoramides, poly-functional nano-carriers for simultaneous antibacterial action and eukaryotic cell transfection.

Gene therapy of diseases like cystic fibrosis (CF) would consist of delivering a gene medicine towards the lungs via the respiratory tract into the target epithelial cells. Accordingly, poly-functional nano-carriers are required in order to overcome the various successive barriers of such a complex environment, such as airway colonization with bacterial strains. In this work, the antibacterial effectiveness of a series of cationic lipids is investigated before evaluating its compatibility with gene transfer into human bronchial epithelial cells. Among the various compounds considered, some bearing a trimethyl-arsonium headgroup demonstrate very potent biocide effects towards clinically relevant bacterial strains. In contrast to cationic lipids exhibiting no or insufficient antibacterial potency, arsonium-containing lipophosphoramides can simultaneously inhibit bacteria while delivering DNA into eukaryotic cells, as efficiently and safely as in absence of bacteria. Moreover, such vectors can demonstrate antibacterial activity in vitro while retaining high gene transfection efficiency to the nasal epithelium as well as to the lungs in mice in vivo. Arsonium-containing amphiphiles are the first synthetic compounds shown to achieve efficient gene delivery in the presence of bacteria, a property particularly suitable for gene therapy strategies under infected conditions such as within the airways of CF patients.