Empowering pancreatic tumor homing with augmented anti-tumor potency of CXCR2-tethered CAR-NK cells.

Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells are a promising immunotherapy for solid cancers; however, their effectiveness against pancreatic cancer is limited by the immunosuppressive tumor microenvironment. In particular, low NK cell infiltration poses a major obstacle that reduces cytotoxicity. The current study aimed to enhance the tumor-homing capacity of CAR-NK cells by targeting the chemokine-chemokine receptor axis between NK and pancreatic cancer cells. To this end, data from a chemokine array and The Cancer Genome Atlas pan-cancer cohort were analyzed. Pancreatic cancer cells were found to secrete high levels of ligands for C-X-C motif receptor 1 (CXCR1) and CXCR2. Subsequently, we generated anti-mesothelin CAR-NK cells incorporating CXCR1 or CXCR2 and evaluated their tumor-killing abilities in 2D cancer cell co-culture and 3D tumor-mimetic organoid models. CAR-NK cells engineered with CXCR2 demonstrated enhanced tumor killing and strong infiltration of tumor sites. Collectively, these findings highlight the potential of CXCR2-augmented CAR-NK cells as a clinically relevant modality for effective pancreatic cancer treatment. By improving their infiltration and tumor-killing capabilities, these CXCR2-augmented CAR-NK cells have the potential to overcome the challenges posed by the immunosuppressive tumor microenvironment, providing improved therapeutic outcomes.

Serum Caffeine Concentration at the Time of Traumatic Brain Injury and Its Long-Term Clinical Outcomes.

Caffeine is one of the most widely consumed psychoactive drugs in the general population. It has a neuroprotective effect in degenerative neurological disorders; however, the association between caffeine and traumatic brain injury (TBI) outcomes is contradictory. The objective of this study was to evaluate the association between serum caffeine concentration at the time of injury and long-term functional outcomes of patients with TBI visiting the emergency department (ED). This was a prospective multi-center cohort study including adult patients with intracranial injury confirmed by radiological examination, who visited five participating EDs within 72 h after TBI. The main exposure was the serum caffeine level within 4 h after injury, and the study outcome was a favorable functional recovery at 6 months after injury. Multi-variable logistic regression analysis adjusted for potential confounders was performed to calculate adjusted odds ratios (AORs) with 95% confidence intervals (CIs). Among the 334 study participants, caffeine was not detected in 102 patients (30.5 %). In patients with identifiable caffeine level, serum caffeine level was categorized into tercile groups; low (0.01-0.58 µg/mL), intermediate (0.59-1.66 µg/mL), and high (1.67-10.00 µg/mL). The proportions of patients with a 6-month favorable functional recovery were 56.9% in the no-caffeine group, 79.2% in the low-caffeine group, 75.3% in the intermediate-caffeine group, and 66.7% in the high-caffeine group (p = 0.006). In multi-variable logistic regression analysis, the low- and intermediate-caffeine groups were significantly associated with a higher probability of 6-month favorable functional recovery compared with the no-caffeine group [AORs (95% CI): 2.82 (1.32-6.02) and 2.18 (1.06-4.47], respectively. This study showed a significant association between a serum caffeine concentration of 0.01 to 1.66 µg/mL and good functional recovery at 6 months after injury compared with the no-caffeine group of patients with TBI with intracranial injury. These results suggest the possibility of using serum caffeine level as a potential biomarker for TBI outcome prediction and of using caffeine as a therapeutic agent in the clinical care of patients with TBI.

Synergistic therapeutic combination with a CAF inhibitor enhances CAR-NK-mediated cytotoxicity via reduction of CAF-released IL-6.

BACKGROUND: Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) contribute to an impaired functionality of natural killer (NK) cells that have emerged as a promising therapeutic modality. The interaction between CAFs and NK cells within the TME exerts major inhibitory effects on immune responses, indicating CAF-targeted therapies as potential targets for effective NK-mediated cancer killing. METHODS: To overcome CAF-induced NK dysfunction, we selected an antifibrotic drug, nintedanib, for synergistic therapeutic combination. To evaluate synergistic therapeutic efficacy, we established an in vitro 3D Capan2/patient-derived CAF spheroid model or in vivo mixed Capan2/CAF tumor xenograft model. The molecular mechanism of NK-mediated synergistic therapeutic combination with nintedanib was revealed through in vitro experiments. In vivo therapeutic combination efficacy was subsequently evaluated. Additionally, the expression score of target proteins was measured in patient-derived tumor sections by the immunohistochemical method. RESULTS: Nintedanib blocked the platelet-derived growth factor receptor ß (PDGFRß) signaling pathway and diminished the activation and growth of CAFs, markedly reducing CAF-secreted IL-6. Moreover, coadministration of nintedanib improved the mesothelin (MSLN) targeting chimeric antigen receptor-NK-mediated tumor killing abilities in CAF/tumor spheroids or a xenograft model. The synergistic combination resulted in intense NK infiltration in vivo. Nintedanib alone exerted no effects, whereas blockade of IL-6 trans-signaling ameliorated the function of NK cells. The combination of the expression of MSLN and the PDGFRß+-CAF population area, a potential prognostic/therapeutic marker, was associated with inferior clinical outcomes. CONCLUSION: Our strategy against PDGFRß+-CAF-containing pancreatic cancer allows improvements in the therapy of pancreatic ductal adenocarcinoma.

Sex Disparities in Prehospital Advanced Cardiac Life Support in Out-of-Hospital Cardiac Arrest in South Korea.

OBJECTIVE: Sex disparities have been reported in the prehospital and in-hospital care among patients with out-of-hospital cardiac arrest (OHCA). The aim of this study was to investigate the association between sex and prehospital advanced cardiac life support (ACLS) interventions provided by emergency medical services (EMS). METHODS: This was a cross-sectional observational study using a nationwide OHCA registry in South Korea. The study included adult OHCAs with presumed cardiac etiology from January 2016 to December 2019. The main exposure was the sex of the victim, and the primary outcomes were prehospital ACLS interventions, including advanced airway management (AAM), intravenous access (IV), and epinephrine (EPI) administration. Multivariable logistic regression analysis accounted for age group, health insurance, comorbidities, place of arrest, urbanization level, witness status, bystander CPR and initial rhythm was performed to calculate adjusted odds ratios (AORs) with 95% confidence intervals (95% CIs). RESULTS: Among 71,154 eligible patients, females with OHCA received less prehospital ACLS interventions than males: risk difference, (95% CIs) -2.76 (-3.41;-2.11) for AAM, -6.03 (-6.79;-5.27) for IV, and -3.81 (-4.37;-3.25) for EPI. In multivariable logistic regression analysis, female sex was significantly associated with a lower probability of prehospital ACLS provision: AOR, (95% CIs) 0.87 (0.84-0.91) for AAM, 0.85 (0.82-0.88) for IV, and 0.81 (0.77-0.84) for EPI. CONCLUSION: Compared to male patients, female patients were less likely to receive prehospital ACLS. This offers opportunities for EMS systems to reduce disparities and to improve compliance with OHCA resuscitation guidelines and outcomes through quality improvement and educational interventions.

Serum Acylcarnitine and Long-Term Functional Prognosis after Traumatic Brain Injury with Intracranial Injury: A Multi-Center Prospective Study.

Serum biomarkers have potential to help predict prognosis of traumatic brain injury (TBI). The objective of this study was to evaluate the association between serum acylcarnitine levels and functional outcomes at 1 month/6 months after injury for TBI patients with intracranial hemorrhage or diffuse axonal injury. This study is a multi-center prospective cohort study in which adult TBI patients with intracranial injury visiting the emergency departments (EDs) from December 2018 to June 2020 were enrolled. Serum acylcarnitine levels at the time of ED arrival were categorized into four groups: low (1.2-5.5 µmol/L), low-normal (5.6-10.0 µmol/L), high-normal (10.1-14.5 µmol/L), and high (1.4.6-56.6 µmol/L). The study outcome was set as poor functional recovery at 1 month/6 months after injury (Glasgow Outcome Scale score, 1-3). Multi-level logistic regression analyses were conducted to estimate association between serum acylcarnitine and functional outcomes. Among total of 549 patients, poor functional recovery at 1 month and 6 months after injury were observed in 29.1% (160/549) and 29.1% (158/543, follow-up loss n = 6). The odds for 1-month poor functional outcome increased in the high-normal and the high groups [adjusted odds ratios, AORs (95% confidence intervals, CIs): 1.56 (1.09-2.23) and 2.47 (1.63-3.75)], compared with the low-normal group) and also as a continuous variable [1.05 (1.03-1.07) for each 1 µmol/L]. Regarding 6-month mortality, the high group had significantly higher odds when compared with the low-normal group [AOR (95% CI): 2.16 (1.37-3.40)]. Higher serum acylcarnitine levels are associated with poor functional outcomes at 1 month/6 months after injury for TBI patients with intracranial injury.

Serum vitamin E level and functional prognosis after traumatic brain injury with intracranial injury: A multicenter prospective study.

Background: Traumatic brain injury (TBI) is a major public health problem with high mortality and disability. Vitamin E, one of the antioxidants for treatment of TBI, has not been sufficiently evaluated for predicting prognosis of TBI. This study aimed to evaluate the prognostic value of vitamin E on functional outcomes of TBI patients with intracranial injury. Methods: A multi-center prospective cohort study was conducted in five university hospitals between 2018 and 2020. Adult TBI patients who visited the emergency department (ED) with intracranial hemorrhage or diffuse axonal injury confirmed by radiological examination were eligible. Serum vitamin E levels (mg/dL) were categorized into 4 groups: low (0.0-5.4), low-normal (5.5-10.9), high-normal (11.0-16.9), and high (17.0-). Study outcomes were set as 1- and 6-month disability (Glasgow outcome scale (GOS) 1-4). Multilevel logistic regression analysis was conducted to calculate the adjusted odds ratios (AORs) of vitamin E for related outcomes. Results: Among 550 eligible TBI patients with intracranial injury, the median (IQR) of serum vitamin E was 10.0 (8.0-12.3) mg/dL; 204/550 (37.1%) had 1-month disability and 197/544 (36.1%) had 6-month disability of GOS 1-4. Compared with the high-normal group, the odds of 1-month disability and 6-month disability increased in the low and low-normal group (AORs (95% CIs): 3.66 (1.62-8.27) and 2.60 (1.15-5.85) for the low group and 1.63 (1.08-2.48) and 1.60 (1.04-2.43) for the low-normal group, respectively). Conclusion: Low serum vitamin E level was associated with poor prognosis at 1 and 6 months after TBI with intracranial injury.

Serum Zinc and Long-Term Prognosis after Acute Traumatic Brain Injury with Intracranial Injury: A Multicenter Prospective Study.

Serum zinc levels in the acute stages after traumatic brain injury (TBI) may be capable of predicting cinical and functional prognoses. This study aimed to evaluate the association between serum zinc levels and long-term survival and neurological outcomes in TBI patients with intracranial injury. This multicenter prospective cohort study enrolled adult TBI patients with intracranial injury who visited emergency departments between December 2018 and June 2020. Serum zinc levels drawn within 24 h after injury were categorized into four groups: low (<80.0 mcg/dL), low−normal (80.0−100.0 mcg/dL), high−normal (100.1−120.0 mcg/dL), and high (>120.0 mcg/dL). The study outcomes were 6-month mortality and disability (Glasgow Outcome Scale, 1−3). A multilevel multivariable logistic regression analysis was conducted to estimate associations between serum zinc and study outcomes. From the eligible TBI patients (N = 487), the median (interquartile range) serum zinc level was 112.0 mcg/dL (95.0−142.0). Six-month mortality and disability were 21.1% (103/487) and 29.6% (144/487), respectively. Compared to the high−normal zinc group, there were significant associations with 6-month mortality and disability observed in the low zinc group (aORs (95% CIs): 1.91 (1.60−2.28) and 1.95 (1.62−2.36) for the low group; 1.14 (0.67−1.94) and 1.15 (0.91−1.46) for the low−normal group; and 0.72 (0.44−1.16) and 0.88 (0.61−1.27) for the high group, respectively). Among the 122 TBI patients with diabetes mellitus, the low zinc group showed a higher incidence of 6-month mortality (aOR (95% CI): 9.13 (4.01−20.81)) compared to the high−normal zinc group. Moreover, the low and low−normal groups had higher odds for 6-month disability (aORs (95% CIs): 6.63 (3.61−12.15) for the low group and 2.37 (1.38−4.07) for the low−normal group). Serum zinc deficiency is associated with a higher incidence of 6-month mortality and disability after injury for TBI patients with intracranial injury.

Interactive effect of multi-tier response and advanced airway management on clinical outcomes after out-of-hospital cardiac arrest: a nationwide population-based observational study.

OBJECTIVE: We hypothesized that a multi-tier response (MTR) will provide high-quality cardiopulmonary resuscitation including airway management. However, the type of tier response system and airway management will have different interactive effects resulting in varying outcomes following out-of-hospital cardiac arrest (OHCA). This study aimed to determine whether the advanced airway management method has an effect on OHCA outcomes and to compare the size of the effect across MTR types. METHODS: This is a retrospective population-based observational study using the Korea OHCA Registry. Airway management methods were categorized into endotracheal intubation (ETI) and supraglottic airway (SGA) groups. The tier system was categorized into single-tier response (STR) or two types of MTR: ambulance-ambulance MTR or fire engine-ambulance MTR. RESULTS: In total, 45,264 patients were analyzed among the 89,087 emergency medical service assessed OHCAs. The SGA group was significantly associated with a lower prehospital return of spontaneous circulation (ROSC) rate compared to the ETI group (adjusted odds ratio [aOR], 0.79; 95% confidence interval [CI], 0.72-0.88). Both MTR with an ambulance or fire engine were significantly associated with higher prehospital ROSC rates compared to STR (STR vs. MTR with an ambulance: aOR, 1.33; 95% CI, 1.21-1.47; STR vs. MTR with a fire engine: aOR, 1.43; 95% CI, 1.20-1.71). Prehospital SGA was significantly associated with poor neurological outcomes in MTR with fire engine (aOR, 0.71; 95% CI, 0.53-0.96). CONCLUSION: In this nationwide observational study, we observed that MTR was associated with higher prehospital ROSC than STR. Moreover, SGA is associated with a lower prehospital ROSC rate regardless of tier response type compared to ETI.

Effects of pre-hospital re-arrest on outcomes based on transfer to a heart attack centre in patients with out-of-hospital cardiac arrest.

AIM: We aimed to investigate the interaction effects between transfer to a heart attack centre [HAC] and prehospital re-arrest on the clinical outcomes of patients with out-of-hospital cardiac arrest [OHCA]. METHODS: We included adult patients with OHCA of presumed cardiac aetiology from January 2012 to December 2018. The main exposure variable was prehospital re-arrest, defined as recurrence of cardiac arrest with a loss of palpable pulse upon hospital arrival. The other exposure variable was the resuscitation capacity of the receiving hospital [HAC or Non-HAC]. The outcome variable was neurological recovery. A multivariable logistic regression was performed to determine the interaction effects. RESULTS: The final analysis included 6935 patients. Of these, 21.9% (n = 1521) experienced prehospital re-arrest, whereas 41.3% (n = 2866) were transferred to a non-HAC. The prehospital re-arrest group associated with poor neurological recovery (adjusted odds ratio [AOR], 0.25; 95% confidence interval [CI], 0.21-0.29;). Transfer to an HAC had beneficial effects on neurological recovery (AOR, 3.40 [95% CI, 3.04-3.85]. In the interaction model, wherein prehospital re-arrest patients who were transferred to a non-HAC were used as reference, the AOR of prehospital re-arrest patients who were transferred to an HAC, non-re-arrest patients who were transferred to a non-HAC, and non-re-arrest patients who were transferred to a non-HAC was 2.41 (95% CI, 1.73-3.35), 3.09 (95% CI, 2.33-4.10), and 11.07 (95% CI, 8.40-14.59) respectively (interaction p = 0.001). CONCLUSION: Transport to a heart attack centre was beneficial to the clinical outcomes of patients who achieved prehospital ROSC after OHCA. The magnitude of that benefit was significantly modified by whether prehospital re-arrest had occurred.

Keratin 8/18 Regulate the Akt Signaling Pathway.

Keratin 8 and keratin 18 (K8/K18) are intermediate filament proteins that form the obligate heteropolymers in hepatocytes and protect the liver against toxins. The mechanisms of protection include the regulation of signaling pathway associated with cell survival. Previous studies show K8/K18 binding with Akt, which is a well-known protein kinase involved in the cell survival signaling pathway. However, the role of K8/K18 in the Akt signaling pathway is unclear. In this study, we found that K8/K18-Akt binding is downregulated by K8/K18 phosphorylation, specifically phosphorylation of K18 ser7/34/53 residues, whereas the binding is upregulated by K8 gly-62-cys mutation. K8/K18 expression in cultured cell system tends to enhance the stability of the Akt protein. A comparison of the Akt signaling pathway in a mouse system with liver damage shows that the pathway is downregulated in K18-null mice compared with nontransgenic mice. K18-null mice with Fas-induced liver damage show enhanced apoptosis combined with the downregulation of the Akt signaling pathway, i.e., lower phosphorylation levels of GSK3ß and NFκB, which are the downstream signaling factors in the Akt signaling pathway, in K18-null mice compared with the control mice. Our study indicates that K8/K18 expression protects mice from liver damage by participating in enhancing the Akt signaling pathway.

Keratin 8 mutations in transgenic mice predispose to lung injury.

Keratin 8 (K8) is the cytoskeletal intermediate filament protein of simple-type epithelia. Mutations in K8 predispose the affected individual and transgenic mouse to liver disease. However, the role of K8 in the lung has not been reported in mutant transgenic mouse models. Here, we investigated the susceptibility of two different transgenic mice expressing K8 Gly62-Cys (Gly62 replaced with Cys) or Ser74-Ala (Ser74 replaced with Ala) to lung injury. The mutant transgenic mice were highly susceptible to two independent acute and chronic lung injuries compared with control mice. Both K8 Gly62-Cys mice and K8 Ser74-Ala mice showed markedly increased mouse lethality (∼74% mutant mice versus ∼34% control mice) and more severe lung damage, with increased inflammation and apoptosis, under L-arginine-mediated acute lung injury. Moreover, the K8 Ser74-Ala mice had more severe lung damage, with extensive hemorrhage and prominent fibrosis, under bleomycin-induced chronic lung injury. Our study provides the first direct evidence that K8 mutations predispose to lung injury in transgenic mice.

Keratins regulate Hsp70-mediated nuclear localization of p38 mitogen-activated protein kinase.

Intermediate filament protein keratin 8 (K8) binds to heat shock protein 70 (Hsp70) and p38 MAPK, and is phosphorylated at Ser74 by p38α (MAPK14, hereafter p38). However, a p38 binding site on K8 and the molecular mechanism of K8-p38 interaction related to Hsp70 are unknown. Here, we identify a p38 docking site on K8 (Arg148/149 and Leu159/161) that is highly conserved in other intermediate filaments. A docking-deficient K8 mutation caused increased p38-Hsp70 interaction and enhanced p38 nuclear localization, indicating that the p38 dissociated from mutant K8 makes a complex with Hsp70, which is known as a potential chaperone for p38 nuclear translocation. Comparison of p38 MAPK binding with keratin variants associated with liver disease showed that the K18 I150V variant dramatically reduced binding with p38, which is similar to the effect of the p38 docking-deficient mutation on K8. Because the p38 docking site on K8 (Arg148/149 and Leu159/161) and the K18 Ile150 residue are closely localized in the parallel K8/K18 heterodimer, the K18 I150V mutation might interfere with K8-p38 interaction. These findings show that keratins, functioning as cytoplasmic anchors for p38, modulate p38 nuclear localization and thereby might affect a number of p38-mediated signal transduction pathways.

Liver disease-associated keratin 8 and 18 mutations modulate keratin acetylation and methylation.

Keratin 8 (K8) and keratin 18 (K18) are the intermediate filament proteins whose phosphorylation/transamidation associate with their aggregation in Mallory-Denk bodies found in patients with various liver diseases. However, the functions of other post-translational modifications in keratins related to liver diseases have not been fully elucidated. Here, using a site-specific mutation assay combined with nano-liquid chromatography-tandem mass spectrometry, we identified K8-Lys108 and K18-Lys187/426 as acetylation sites, and K8-Arg47 and K18-Arg55 as methylation sites. Keratin mutation (Arg-to-Lys/Ala) at the methylation sites, but not the acetylation sites, led to decreased stability of the keratin protein. We compared keratin acetylation/methylation in liver disease-associated keratin variants. The acetylation of K8 variants increased or decreased to various extents, whereas the methylation of K18-del65-72 and K18-I150V variants increased. Notably, the highly acetylated/methylated K18-I150V variant was less soluble and exhibited unusually prolonged protein stability, which suggests that additional acetylation of highly methylated keratins has a synergistic effect on prolonged stability. Therefore, the different levels of acetylation/methylation of the liver disease-associated variants regulate keratin protein stability. These findings extend our understanding of how disease-associated mutations in keratins modulate keratin acetylation and methylation, which may contribute to disease pathogenesis.-Jang, K.-H., Yoon, H.-N., Lee, J., Yi, H., Park, S.-Y., Lee, S.-Y., Lim, Y., Lee, H.-J., Cho, J.-W., Paik, Y.-K., Hancock, W. S., Ku, N.-O. Liver disease-associated keratin 8 and 18 mutations modulate keratin acetylation and methylation.

The role of keratins in the digestive system: lessons from transgenic mouse models.

Keratins are the largest subfamily of intermediate filament proteins. They are either type I acidic or type II basic keratins. Keratins form obligate heteropolymer in epithelial cells and their expression patterns are tissue-specific. Studies have shown that keratin mutations are the cause of many diseases in humans or predispose humans to acquiring them. Using mouse models to study keratin-associated human diseases is critical, because they allow researchers to get a better understanding of these diseases and their progressions, and so many such studies have been conducted. Acknowledging the importance, researches with genetically modified mice expressing human disease-associated keratin mutants have been widely done. Numerous studies using keratin knockout mice, keratin-overexpressed mice, or transgenic mice expressing keratin mutants have been conducted. This review summarizes the mouse models that have been used to study type I and type II keratin expression in the digestive organs, namely, the liver, pancreas, and colon.

A mutation in keratin 18 that causes caspase-digestion resistance protects homozygous transgenic mice from hepatic apoptosis and injury.

Cytoskeletal keratin 18 (K18) undergoes caspase-mediated digestion during apoptosis, which leads to dramatic disassembly of keratin filaments. We studied the significance of K18 caspase digestion in a mouse model and generated transgenic mice expressing the human K18 caspase digestion-resistant double-mutant K18-D238/397E in a mouse (m) K18-null background, and compared their response to injury mediated by administration of antibody against tumor necrosis factor receptor superfamily member 6 (Fas), anti-FasAb. Notably, K18-D238/397E;mK18-null mice were significantly more resistant to anti-FasAb-induced injury as compared with K18-WT;mK18-null mice (23% vs 57% lethality, respectively; P<0.001). The same applied when the toxin microcystin-LR (MLR) was used to induce liver injury, i.e. lethality of K18-D238/397E;mK18-null mice in response to MLR treatment was reduced compared with the control mouse strain. The lesser rate of apoptosis in K18-D238/397E;mK18-null livers is associated with delayed degradation and, thus, sustained activation of cell-survival-related protein kinases, including stress-activated protein kinases and the NF-κB transcription factor, up to 6-8 h after administration of anti-FasAb. However, activation of the kinases and NF-κB in K18-WT-reconstituted livers decreases dramatically 8 h after anti-FasAb administration. In addition, the D238/397E double-mutation results in prolonged stability of K18 protein in transfected cells and transgenic livers. Therefore, our results show that the caspase digestion-resistant K18 helps to maintain keratin filament organization and delays apoptosis, thereby resulting in protection from liver injury.

Predisposition to apoptosis in keratin 8-null liver is related to inactivation of NF-κB and SAPKs but not decreased c-Flip.

Keratin 8 and 18 (K8/K18) are major intermediate filament proteins of liver hepatocytes. They provide mechanical and nonmechanical stability, thereby protecting cells from stress. Hence, K8-null mice are highly sensitive to Fas-mediated liver cell apoptosis. However, the role of c-Flip protein in K8-null related susceptibility to liver injury is controversial. Here we analyzed c-Flip protein expression in various K8 or K18 null/mutant transgenic livers and show that they are similar in all analyzed transgenic livers and that previously reported c-Flip protein changes are due to antibody cross-reaction with mouse K18. Furthermore, analysis of various apoptosis- or cell survival-related proteins demonstrated that inhibition of phosphorylation of NF-κB and various stress activated protein kinases (SAPKs), such as p38 MAPK, p44/42 MAPK and JNK1/2, is related to the higher sensitivity of K8-null hepatocytes whose nuclear NF-κB is rapidly depleted through Fas-mediated apoptosis. Notably, we found that NF-κB and the studied protein kinases are associated with the K8/K18 complex and are released upon phosphorylation. Therefore, interaction of keratins with cell survival-related protein kinases and transcription factors is another important factor for hepatocyte survival.

Enhanced cellular immune responses elicited by an engineered HIV-1 subtype B consensus-based envelope DNA vaccine.

An important goal for human immunodeficiency virus (HIV) vaccines is to develop immunogens that induce broader and more potent cellular immune responses. In this study of DNA vaccine potency, we constructed a novel subtype B env gene (EY2E1-B) with the goal of increasing vaccine antigen immune potency. The vaccine cassette was designed based on subtype B-specific consensus sequence with several modifications, including codon optimization, RNA optimization, the addition of a Kozak sequence, and a substituted immunoglobulin E leader sequence. The V1 and V2 loops were shortened and the cytoplasmic tail was truncated to prevent envelope recycling. Three different strains of mice (BALB/c, C57BL/6, and HLA-A2 transgenic mice) were immunized three times with pEY2E1-B or the primary DNA immunogen pEK2P-B alone. The analysis of specific antibody responses suggested that EY2E1-B could induce a moderate subtype B-specific antibody response. Moreover, this construct was up to four times more potent at driving cellular immune responses. Epitope mapping results indicated that there is an increase in the breadth and magnitude of cross-reactive cellular responses induced by the EY2E1-B immunogen. These properties suggest that such a synthetic immunogen deserves further examination for its potential to serve as a component antigen in an HIV vaccine cocktail.

Immunogenicity testing of a novel engineered HIV-1 envelope gp140 DNA vaccine construct.

DNA vaccines expressing the envelope (env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating strong immune responses. In this study, we described the development of a recombinant plasmid DNA (pEK2P-B) expressing an engineered codon-optimized envelope gp140 gene of primary (nonrecombinant) HIV-1 subtype B isolate 6101. Codon usage and RNA optimization of HIV-1 structural genes has been shown to increase protein expression in vitro as well as in the context of DNA vaccines in vivo. To further increase the expression, a synthetic IgE leader with kozak sequences were fused into the env gene. The cytoplasmic tail of the gene was also truncated to prevent recycling. The expression of env by the recombinant pEK2P-B was evaluated using T7 coupled transcription/translation. The construct demonstrated high expression of the HIV-1 env gene in eukaryotic cells as demonstrated in transfected 293-T and RD cells. Immunogenicity of pEK2P-B was evaluated in mice using IFN-gamma ELISpot assay, and the construct was found to be highly immunogenic and crossreactive with HIV-1 clade C env peptides. Three immunodominant peptides were also mapped out. Furthermore, by performing a CFSE flow cytometry-based proliferation assay, 2.4 and 1.5% proliferation was observed in CD4+, CD8+, and CCR+ memory T cells, respectively. Therefore, this engineered synthetic optimized env DNA vaccine may be useful in DNA vaccine and other studies of HIV-1 immunogenicity.