BCG is widely used as a vaccine against tuberculosis due to
(Mtb), which kills millions of people each year. BCG variably protects children, but not adults against tuberculosis. BCG evades phagosome ...maturation, autophagy, and reduces MHC-II expression of antigen-presenting cells (APCs) affecting T-cell activation. To bypass these defects, an autophagy-inducing, TLR-2 activating C5 peptide from Mtb-derived CFP-10 protein was overexpressed in BCG in combination with Ag85B. Recombinant BCG
induced a robust MHC-II-dependent antigen presentation to CD4 T cells in vitro, and elicited stronger T
1 cytokines (IL-12, IL-1β, and TNFα) from APCs of C57Bl/6 mice increasing phosphorylation of p38MAPK and ERK. BCG
also enhanced MHC-II surface expression of MΦs by inhibiting MARCH1 ubiquitin ligase that degrades MHC-II. BCG
infected APCs from MyD88 or TLR-2 knockout mice showed decreased antigen presentation. Furthermore, BCG
induced LC3-dependent autophagy in macrophages increasing antigen presentation. Consistent with in vitro effects, BCG
markedly expanded both effector and central memory T cells in C57Bl/6 mice protecting them against both primary aerosol infection with Mtb and reinfection, but was less effective among TLR-2 knockout mice. Thus, BCG
induces stronger and longer lasting immunity, and is better than BCG against tuberculosis of mice.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death due to bacterial infections in mankind, and BCG, an attenuated strain of Mycobacterium bovis, is an ...approved vaccine. BCG sequesters in immature phagosomes of antigen presenting cells (APCs), which do not fuse with lysosomes, leading to decreased antigen processing and reduced Th1 responses. However, an Mtb derived ΔfbpA attenuated mutant underwent limited phagosome maturation, enhanced immunogenicity and was as effective as BCG in protecting mice against TB. To facilitate phagosome maturation of ΔfbpA, we disrupted an additional gene sapM, which encodes for an acid phosphatase. Compared to the wild type Mtb, the ΔfbpAΔsapM (double knock out; DKO) strain was attenuated for growth in mouse macrophages and PMA activated human THP1 macrophages. Attenuation correlated with increased oxidants in macrophages in response to DKO infection and enhanced labeling of lysosomal markers (CD63 and rab7) on DKO phagosomes. An in vitro Antigen 85B peptide presentation assay was used to determine antigen presentation to T cells by APCs infected with DKO or other mycobacterial strains. This revealed that DKO infected APCs showed the strongest ability to present Ag85B to T cells (>2500 pgs/mL in 4 hrs) as compared to APCs infected with wild type Mtb or ΔfbpA or ΔsapM strain (<1000 pgs/mL in 4 hrs), indicating that DKO strain has enhanced immunogenicity than other strains. The ability of DKO to undergo lysosomal fusion and vacuolar acidification correlated with antigen presentation since bafilomycin, that inhibits acidification in APCs, reduced antigen presentation. Finally, the DKO vaccine elicited a better Th1 response in mice after subcutaneous vaccination than either ΔfbpA or ΔsapM. Since ΔfbpA has been used in mice as a candidate vaccine and the DKO (ΔfbpAΔsapM) mutant is more immunogenic than ΔfbpA, we propose the DKO is a potential anti-tuberculosis vaccine.
Acute and chronic stresses are implicated in cardiovascular diseases including coronary artery disease. The present study was designed to examine the direct effects of the stress hormone cortisol on ...nitric oxide (NO) release and endothelial NO synthase (eNOS) expression in cultured bovine coronary artery endothelial cells (BCAEC). Nitrate, nitrite, and NO (NO(x)) were measured by the chemiluminescence method. At 24 h after treatment, cortisol (1 nM-10 microM) produced a dose-dependent decrease in NO(x) release, which was attenuated in the presence of the 11beta-hydroxysteroid dehydrogenase inhibitor carbenoxolone (3 microM). In accordance, eNOS protein levels were significantly decreased by cortisol in a dose-dependent manner. Cortisol pretreatment significantly increased the rate of eNOS protein degradation in the presence of cycloheximide. In addition, cortisol pretreatment decreased ATP-induced intracellular Ca(2+) elevation and NO(x) release in BCAEC. The presence of glucocorticoid receptors in BCAEC was demonstrated by Western blot. The results suggest that cortisol, through activation of glucocorticoid receptors, suppresses NO(x) release in BCAEC by downregulating eNOS proteins and inhibiting intracellular Ca(2+) mobilization. Decreased NO(x) is likely to result in an increase in contraction of coronary arteries, leading to a decrease in coronary blood flow.
Abstract
BCG is the only human-approved vaccine used to protect against childhood tuberculosis although it fails to protect against adult tuberculosis. BCG vaccine localizes to an immature phagosomal ...compartment and avoids lysosomal fusion. Furthermore, it reduces the expression of MHC-II in macrophages in vitro and in phagocytic cells of mice after infection. We hypothesized that immunogenicity of BCG is reduced because of reduced MHC-II expression by APCs and the addition of Toll-like receptor (TLR) - ligands will strengthen and prolong immunity conferred by BCG vaccine and perhaps protect against adult disease. Since TLR ligands activate macrophages, we tested their effect on MHC-II expression. BCG infection down regulated the expression of MHC-II while a combination of BCG vaccine with ligands for TLR-7, and 9 markedly induced an up-regulation that correlated with the ability of macrophages to present antigens to T cells. Furthermore, we identified that TLR-7, and 9 but not TLR-1/2, TLR-3 or TLR-5 induced a down regulation of the ubiquitin ligase MARCH1 that in turn correlated with a decreased ubiquitination and enhanced clearance of MHC-II from macrophages. These data suggest that BCG down regulates immune responses while TLR-ligands can be used to up regulate MHC-II contributing to a novel mode of adjuvant action.
Mycobacterium tuberculosis
(Mtb) replicates within the human macrophages and we investigated the activating effects of retinoic acid (RA) and vitamin D
3
(VD) on macrophages in relation to the ...viability of intracellular Mtb. A combination of these vitamins (RAVD) enhanced the levels of DC-SIGN and mannose receptors on THP-1 macrophages that increased mycobacterial uptake but inhibited the subsequent intracellular growth of Mtb by inducing reactive oxygen species and autophagy. RAVD also enhanced antigen presenting and chemotactic receptors on THPs suggesting an activated phenotype for RAVD activated THPs. RAVD mediated activation was also associated with a marked phenotypic change in Mtb infected THPs that fused with adjacent THPs to form multinucleated giant cells (MNGCs). Typically, MNGCs occurred over 30 days of
in vitro
culture and contained non-replicating persisting Mtb for more than 60 days in culture. Latent tuberculosis occurs in over a third of mankind and we propose that RAVD mediated induction of persistent Mtb within human macrophages provides a novel model to develop therapeutic approaches and investigate pathogenesis of latency.
Abstract 3729
The serine/threonine kinase Akt plays a critical signaling role downstream of phosphatidylinositol-3-kinase (PI3K) and is important in promoting cell survival and inhibiting apoptosis. ...Indeed, Akt activation and overexpression is often associated with resistance to chemotherapy or radiotherapy. Previous studies demonstrated the potential therapeutic value of targeting the PI3K pathway in lymphoma, as both the selective PI3Kδ inhibitor CAL-101, and everolimus and temsirolimus, which target PI3K and mTOR, produce clinical responses in a variety of lymphomas. We evaluated the effect of the novel allosteric Akt inhibitor, MK-2206, in a panel of lymphoma cell lines and primary lymphoma cells. We found that Akt, and activated pAkt, are highly expressed in lymphoma cells. After 72 hours of incubation, the Akt inhibitor MK-2206 demonstrated antiproliferative activity in a variety of lymphoma cell lines, with an IC50 ranging between 0.1 and 5μM. There was no correlation between pre-treatment levels of pAKT, PI3K isoforms, or PTEN protein expression and sensitivity to MK-2206. Within the diffuse large B cell lymphoma cell lines, those of GCB cell of origin were more sensitive to MK-2206, compared with the ABC-derived cell lines. Resistant cell lines tended to had weak or absent expression of p-GSK3 and p-4EBPI. Mechanistically, MK-2206 treatment decreased the level of p-Akt (Ser473), and p-Akt (Thr308), irrespective of drug sensitivity. Furthermore, MK-2206 decreased the phosphorylation level of Akt downstream targets, including p-GSK3 beta and p-PRAS40, upregulated p27. and dephosphorylated p70S6K. Moreover, MK-2206 treatment decreased HIF-1 alpha and VEGF expression. Depending on the cell of origin, the antiproliferative effect resulted from cycle arrest at the G0/G1 phase, autophagy, orapoptosis. MK-2206 showed synergistic effect in combination with the HDAC inhibitor, Vorinostat. Using pathway-specific protein arrays focusing on apoptosis, kinases, and transcription factors, the combination of MK-2206 and Vorinostat effectively altered p53 and p27 levels, which were associated with increased PARP cleavage and induction of apoptosis. Our data demonstrate that AKT is a promising target for the treatment of lymphoma, and provide a rationale for an ongoing trial, evaluating MK-2206 for the treatment of patients with relapsed lymphoma.
No relevant conflicts of interest to declare.
Mycobacterium tuberculosis (Mtb) replicates within the human macrophages and we investigated the activating effects of retinoic acid (RA) and vitamin D(3) (VD) on macrophages in relation to the ...viability of intracellular Mtb. A combination of these vitamins (RAVD) enhanced the levels of DC-SIGN and mannose receptors on THP-1 macrophages that increased mycobacterial uptake but inhibited the subsequent intracellular growth of Mtb by inducing reactive oxygen species and autophagy. RAVD also enhanced antigen presenting and chemotactic receptors on THPs suggesting an activated phenotype for RAVD activated THPs. RAVD mediated activation was also associated with a marked phenotypic change in Mtb infected THPs that fused with adjacent THPs to form multinucleated giant cells (MNGCs). Typically, MNGCs occurred over 30 days of in vitro culture and contained non-replicating persisting Mtb for more than 60 days in culture. Latent tuberculosis occurs in over a third of mankind and we propose that RAVD mediated induction of persistent Mtb within human macrophages provides a novel model to develop therapeutic approaches and investigate pathogenesis of latency.
Epidemiologic studies showed an association between adverse intrauterine environment and ischemic heart disease in the adult. We tested the hypothesis that prenatal hypoxia increased the ...susceptibility of adult heart to ischemia-reperfusion (I-R) injury.
Time-dated pregnant rats were divided between normoxic and hypoxic (10.5% oxygen from day 15 to 21) groups. Hearts of 6-month-old male progeny were studied using Langendorff preparation and were subjected to two protocols of I-R: 10 minutes of ischemia and 3 hours of reperfusion (I-R(10)) or 25 minutes of ischemia and 3 hours of reperfusion (I-R(25)).
Prenatal hypoxia did not change basal left ventricular (LV) function. I-R(10) produced myocardial stunning and a transient decrease in LV function in control hearts but caused myocardial infarction and a persistent decrease in postischemic recovery of LV function in hypoxic hearts. I-R(25) caused myocardial infarction in both control and hypoxic hearts, which was significantly higher in hypoxic hearts. The postischemic recovery of LV function was significantly reduced in hypoxic hearts. I-R(25)-induced activation of caspase-3 and apoptosis in the left ventricle were significantly higher in hypoxic than control hearts. There was a significant decrease in LV heat shock protein 70 and endothelial nitric oxide synthase levels in hypoxic hearts. Prenatal hypoxia did not change beta(1)-adrenoreceptor levels but significantly increased beta(2)-adrenoreceptor in the left ventricle. In addition, it increased G(s)alpha but decreased G(i)alpha.
Prenatal chronic hypoxia increases the susceptibility of adult heart to I-R injury. Several possible mechanisms may be involved, including an increase in beta(2)-adrenoreceptor and the G(s)alpha/G(i)alpha ratio, and a decrease in heat shock protein 70 and endothelial nitric oxide synthase in the left ventricle.
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