4-Phenylbutyric Acid Reduces Endoplasmic Reticulum Stress, Trypsin Activation, and Acinar Cell Apoptosis While Increasing Secretion in Rat Pancreatic Acini

Objectives: Endoplasmic reticulum (ER) stress leads to misfolded proteins inside the ER and initiates unfolded protein response (UPR). Unfolded protein response components are involved in pancreatic function and activated during pancreatitis. However, the exact role of ER stress in the exocrine pancreas is unclear. The present study examined the effects of 4-phenylbutyric acid (4-PBA), an ER chaperone, on acini and UPR components. Methods: Rat acini were stimulated with cholecystokinin (10 pmol/L to 10 nmol/L) with or without preincubation of 4-PBA. The UPR components were analyzed, including chaperone-binding protein, protein kinaselike ER kinase, X-box-binding protein 1, c-Jun NH2-terminal kinase, CCAAT/enhancer-binding protein homologous protein, caspase 3, and apoptosis. Effects of 4-PBA were measured on secretion, calcium, and trypsin activation. Results: 4-Phenylbutyric acid led to an increase of secretion, whereas trypsin activation with supraphysiological cholecystokinin was significantly reduced. 4-Phenylbutyric acid prevented chaperone-binding protein up-regulation, diminished protein kinaselike ER kinase, and c-Jun NH2-terminal kinase phosphorylation, prohibited X-box-binding protein 1 splicing and CCAAT/enhancer-binding protein homologous protein expression, caspase 3 activation, and apoptosis caused by supraphysiological cholecystokinin. Conclusion: By incubation with 4-PBA, beneficial in urea cycle deficiency, it was possible to enhance enzyme secretion to suppress trypsin activation, UPR activation, and proapoptotic pathways. The data hint new perspectives for the use of chemical chaperones in pancreatic diseases

4-Phenylbutyric Acid Reduces Endoplasmic Reticulum Stress, Trypsin Activation, and Acinar Cell Apoptosis While Increasing Secretion in Rat Pancreatic Acini

Objectives: Endoplasmic reticulum (ER) stress leads to misfolded proteins inside the ER and initiates unfolded protein response (UPR). Unfolded protein response components are involved in pancreatic function and activated during pancreatitis. However, the exact role of ER stress in the exocrine pancreas is unclear. The present study examined the effects of 4-phenylbutyric acid (4-PBA), an ER chaperone, on acini and UPR components. Methods: Rat acini were stimulated with cholecystokinin (10 pmol/L to 10 nmol/L) with or without preincubation of 4-PBA. The UPR components were analyzed, including chaperone-binding protein, protein kinaselike ER kinase, X-box-binding protein 1, c-Jun NH2-terminal kinase, CCAAT/enhancer-binding protein homologous protein, caspase 3, and apoptosis. Effects of 4-PBA were measured on secretion, calcium, and trypsin activation. Results: 4-Phenylbutyric acid led to an increase of secretion, whereas trypsin activation with supraphysiological cholecystokinin was significantly reduced. 4-Phenylbutyric acid prevented chaperone-binding protein up-regulation, diminished protein kinaselike ER kinase, and c-Jun NH2-terminal kinase phosphorylation, prohibited X-box-binding protein 1 splicing and CCAAT/enhancer-binding protein homologous protein expression, caspase 3 activation, and apoptosis caused by supraphysiological cholecystokinin. Conclusion: By incubation with 4-PBA, beneficial in urea cycle deficiency, it was possible to enhance enzyme secretion to suppress trypsin activation, UPR activation, and proapoptotic pathways. The data hint new perspectives for the use of chemical chaperones in pancreatic diseases

Do red deer stags (Cervus elaphus) use roar fundamental frequency (F0) to assess rivals?

It is well established that in humans, male voices are disproportionately lower pitched than female voices, and recent studies suggest that this dimorphism in fundamental frequency (F0) results from both intrasexual (male competition) and intersexual (female mate choice) selection for lower pitched voices in men. However, comparative investigations indicate that sexual dimorphism in F0 is not universal in terrestrial mammals. In the highly polygynous and sexually dimorphic Scottish red deer Cervus elaphus scoticus, more successful males give sexually-selected calls (roars) with higher minimum F0s, suggesting that high, rather than low F0s advertise quality in this subspecies. While playback experiments demonstrated that oestrous females prefer higher pitched roars, the potential role of roar F0 in male competition remains untested. Here we examined the response of rutting red deer stags to playbacks of re-synthesized male roars with different median F0s. Our results show that stags’ responses (latencies and durations of attention, vocal and approach responses) were not affected by the F0 of the roar. This suggests that intrasexual selection is unlikely to strongly influence the evolution of roar F0 in Scottish red deer stags, and illustrates how the F0 of terrestrial mammal vocal sexual signals may be subject to different selection pressures across species. Further investigations on species characterized by different F0 profiles are needed to provide a comparative background for evolutionary interpretations of sex differences in mammalian vocalizations

Targeted T cell receptor gene editing provides predictable T cell product function for immunotherapy.

Adoptive transfer of T cells expressing a transgenic T cell receptor (TCR) has the potential to revolutionize immunotherapy of infectious diseases and cancer. However, the generation of defined TCR-transgenic T cell medicinal products with predictable in vivo function still poses a major challenge and limits broader and more successful application of this "living drug." Here, by studying 51 different TCRs, we show that conventional genetic engineering by viral transduction leads to variable TCR expression and functionality as a result of variable transgene copy numbers and untargeted transgene integration. In contrast, CRISPR/Cas9-mediated TCR replacement enables defined, targeted TCR transgene insertion into the TCR gene locus. Thereby, T cell products display more homogeneous TCR expression similar to physiological T cells. Importantly, increased T cell product homogeneity after targeted TCR gene editing correlates with predictable in vivo T cell responses, which represents a crucial aspect for clinical application in adoptive T cell immunotherapy

Interferon-gamma and Tumor Necrosis Factor-alpha Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis

BACKGROUND & AIMS: Viral clearance involves immune cell cytolysis of infected cells. However, studies of hepatitis B virus (HBV) infection in chimpanzees have indicated that cytokines released by T cells also can promote viral clearance via non-cytolytic processes. We investigated the noncytolytic mechanisms by which T cells eliminate HBV from infected hepatocytes. METHODS: We performed a cytokine enzyme-linked immunosorbent assay of serum samples from patients with acute and chronic hepatitis B. Liver biopsy specimens were analyzed by in situ hybridization. HepG2-H1.3 cells, HBV-infected HepaRG cells, and primary human hepatocytes were incubated with interferon-gamma (IFN gamma) or tumor necrosis factor-a (TNF-alpha), or co-cultured with T cells. We measured markers of HBV replication, including the covalently closed circular DNA (cccDNA). RESULTS: Levels of IFN gamma and TNF-alpha were increased in serum samples from patients with acute vs chronic hepatitis B and controls. In human hepatocytes with stably replicating HBV, as well as in HBV-infected primary human hepatocytes or HepaRG cells, IFN gamma and TNF-alpha each induced deamination of cccDNA and interfered with its stability; their effects were additive. HBV-specific T cells, through secretion of IFN gamma and TNF-alpha, inhibited HBV replication and reduced cccDNA in infected cells without the direct contact required for cytolysis. Blocking IFN gamma and TNF-alpha after T-cell stimulation prevented the loss of cccDNA. Deprivation of cccDNA required activation of nuclear APOBEC3 deaminases by the cytokines. In liver biopsy specimens from patients with acute hepatitis B, but not chronic hepatitis B or controls, hepatocytes expressed APOBEC3A and APO-BEC3B. CONCLUSIONS: IFN gamma and TNF-alpha, produced by T cells, reduce levels of HBV cccDNA in hepatocytes by inducing deamination and subsequent cccDNA decay