Evaluation of two different adjuvants with immunogenic uroplakin 3A-derived peptide for their ability to evoke an immune response in mice

Rationale

Organ- or tissue-specific antigens produced by normal tissue or by cancer cells could be used in cancer immunotherapy, to target the tumor. In our previous study, we induced T-cell-mediated, bladderspecific autoimmunity by targeting the bladder-specific protein Uroplakin 3A (UPK3A). UPK3A is a well-chosen target for developing an autoimmune response against bladder cancer since the antigen is also expressed in bladder tumors. To use this peptide, which was derived from the UPK3A protein in a bladder cancer vaccine study, it is necessary to induce a strong immune response. In this study, we aimed to develop a robust immune response in BALB/c mice using the well-characterized keyhole limpet hemocyanin (KLH)-conjugated peptide antigen (UPK3A 65-84) conjugated with an immunogenic carrier protein. In combination with the peptide, we used either Freund’s complete adjuvant (CFA) or CpG (cytosine-phosphate-guanine oligonucleotides) as effective adjuvants in order to overcome tumor tolerance.

Objectives

The immune response evoked by UPK3A 65-84 peptide, using two different adjuvants, was compared by detection of changes in the proliferative response of immune cells, in the cytokine profile, and in the immune cell populations.

Findings

We demonstrated that CpG, combined with KLH-UPK3A 65-84, promoted a more robust immune response, via induction of higher IL-2, IFN-γ, TNF-α, IL-17 production and activation of more immune cells (CD4⁺ T cells, CD8⁺ T cells, NK cells CD11b, CD45), than CFA and the KLHUPK3A 65-84.

Conclusion

CpG as an adjuvant combined with KLH-UPK3A 65-84 could be used in preclinical models of bladder cancer for the development of cancer immunotherapy strategies.

     

The IKK‐related kinase TBK1 activates mTORC1 directly in response to growth factors and innate immune agonists

The innate immune kinase TBK1 initiates inflammatory responses to combat infectious pathogens by driving production of type I interferons. TBK1 also controls metabolic processes and promotes oncogene‐induced cell proliferation and survival. Here, we demonstrate that TBK1 activates mTOR complex 1 (mTORC1) directly. In cultured cells, TBK1 associates with and activates mTORC1 through site‐specific mTOR phosphorylation (on S2159) in response to certain growth factor receptors (i.e., EGF‐receptor but not insulin receptor) and pathogen recognition receptors (PRRs) (i.e., TLR3; TLR4), revealing a stimulus‐selective role for TBK1 in mTORC1 regulation. By studying cultured macrophages and those isolated from genome edited mTOR S2159A knock‐in mice, we show that mTOR S2159 phosphorylation promotes mTORC1 signaling, IRF3 nuclear translocation, and IFN‐β production. These data demonstrate a direct mechanistic link between TBK1 and mTORC1 function as well as physiologic significance of the TBK1‐mTORC1 axis in control of innate immune function. These data unveil TBK1 as a direct mTORC1 activator and suggest unanticipated roles for mTORC1 downstream of TBK1 in control of innate immunity, tumorigenesis, and disorders linked to chronic inflammation.     

Plasma and urinary endothelin-1 concentrations in asphyxiated newborns [corrected

The aim of this study was to determine if there is any correlation between the hypoxia induced deterioration of renal functions and urinary excretions of endothelin (ET). Therefore using a sensitive and specific radioimmunoassay, we have investigated plasma ET-1 concentrations and urine ET-1 excretions in healthy and asphyxiated newborns. Sixteen newborns (10 boys, 6 girls) with perinatal asphyxia or hypoxia of variable seriousness which were followed at Newborn Intensive Care Unit in Eskisehir Osmangazi University Faculty of Medicine were enrolled. Simultaneously, gestation and weight matched 10 newborns (6 boys, 4 girls) with no asphyxia (first minute Apgar score >7) were enrolled as controls. Plasma ET-1 concentrations of the asphyxiated infants (61.8+/-79.3 pg/ml, between 23.4-125.2 pg/ml) were higher than in the control group (29.3+/-22.1 pg/ml, between 12.3 and 50.8 pg/ml, p<0.05). However creatinine clearance values were not different between the two groups (p>0.05), mean fractional excretion of sodium levels (FeNa%) were higher in the study group than the controls (p<0.01). Urinary ET-1 concentrations in the asphyxiated infants were 144.6+/-63.4 pg/ml versus 70.1+/-27.7 pg/ml in the control group (p<0.001). The ET clearance were more elevated in the asphyxiated newborns than in the healthy infants (p<0.05). Urinary ET-1/Cr ratio in the hypoxic infants were significantly elevated in the first day of life when compared with those of healthy infants (p<0.05). Total ET excretion was negatively correlated with FeNa (%) (r=-0.603, p<0.05). Plasma ET-1 concentrations of the asphyxiated infants reduced at 48 hours of age (p<0.001). Fifth minute Apgar score was negatively correlated with urinary ET-1 levels (r=-0.615, p<0.01), urinary Na excretion (r=-0.583, p<0.01), FeNa (%) (r=-0.597, p<0.01) and total ET excretion (r=-0.560, p<0.01) and positively correlated with ET clearance (r=0.559, p<0.05). Urinary ET-1 levels were negatively correlated with umbilical artery BE levels (r=-0.612, p<0.05). To our study, elevated urinary ET-1 levels were observed during perinatal asphyxia and urinary ET-1 levels were negatively correlated with 5th minute Apgar score and cord blood base excess levels. For this reason urinary ET-1 levels could be a marker of perinatal asphyxia as cord blood ET-1 levels. With investigations showing renal production is independent from plasma and increased urinary ET-1/Cr levels in newborn with perinatal asphyxia and also negative correlation between the total ET excretion and FeNa, urinary ET-1 levels could be served as a useful marker to detecting also impaired renal functions in infants with perinatal asphyxia.     

MACF1 gene structure: a hybrid of plectin and dystrophin

Mammalian MACF1 (Macrophin1; previously named ACF7) is a giant cytoskeletal linker protein with three known isoforms that arise by alternative splicing. We isolated a 19.1-kb cDNA encoding a fourth isoform (MACF1-4) with a unique N-terminus. Instead of an N-terminal actin-binding domain found in the other three isoforms, MACF1-4 has eight plectin repeats. The MACF1 gene is located on human Chr 1p32, contains at least 102 exons, spans over 270 kb, and gives rise to four major isoforms with different N-termini. The genomic organization of the actin-binding domain is highly conserved in mammalian genes for both plectin and BPAG1. All eight plectin repeats are encoded by one large exon; this feature is similar to the genomic structure of plectin. The intron positions within spectrin repeats in MACF1 are very similar to those in the dystrophin gene. This demonstrates that MACF1 has characteristic features of genes for two classes of cytoskeletal proteins, i.e., plectin and dystrophin. Received: 24 April 2001 / Accepted: 29 June 2001     

Myrtucommulone‐A Induces both Extrinsic and Intrinsic Apoptotic Pathways in Cancer Cells

Myrtucommulone‐A is the active compound derived from Myrtus communis. The molecular targets of myrtucommulone‐A is widely unknown, which impedes its potential therapeutic use. In this study, we demonstrated the cytotoxicity of MC‐A and its potential to induce apoptosis in cancer cells. Myrtucommulone‐A was also found to be antiproliferative and strongly inhibited cancer cell migration. Eighty four apoptotic pathway genes were used to assess the effect of myrtucommulone‐A on cancer cells. Myrtucommulone‐A mediated an increase in apoptotic genes including Fas, FasL, Gadd45a, Tnf, Tnfsf12, Trp53, and caspase 4. The increase in myrtucommulone‐A dose (25 μM versus 6.25 μM) also upregulated the expression of genes, which are involved mainly in apoptosis, regulation of apoptosis, role of mitochondria in apoptotic signaling, cytokine activity, and tumor necrosis factor signaling. Our data indicate that myrtucommulone‐A could be utilized as a potential therapeutic compound with its molecular targets in apoptotic pathways.