Novel bispecific nanobody mitigates experimental intestinal inflammation in mice by targeting TNF-α and IL-23p19 bioactivities.

BACKGROUND: Inflammatory bowel diseases (IBDs) pose significant challenges in terms of treatment non-response, necessitating the development of novel therapeutic approaches. Although biological medicines that target TNF-α (tumour necrosis factor-α) have shown clinical success in some IBD patients, a substantial proportion still fails to respond. METHODS: We designed bispecific nanobodies (BsNbs) with the ability to simultaneously target human macrophage-expressed membrane TNF-α (hmTNF-α) and IL-23. Additionally, we fused the constant region of human IgG1 Fc (hIgG1 Fc) to BsNb to create BsNb-Fc.  Our study encompassed in vitro and in vivo characterization of BsNb and BsNb-Fc. RESULTS: BsNb-Fc exhibited an improved serum half-life, targeting capability and effector function than BsNb. It's demonstrated that BsNb-Fc exhibited superior anti-inflammatory effects compared to the anti-TNF-α mAb (infliximab, IFX) combined with anti-IL-12/IL-23p40 mAb (ustekinumab, UST) by Transwell co-culture assays. Notably, in murine models of acute colitis brought on by 2,4,6-trinitrobenzene sulfonic acid（TNBS) and dextran sulphate sodium (DSS), BsNb-Fc effectively alleviated colitis severity. Additionally, BsNb-Fc outperformed the IFX&UST combination in TNBS-induced colitis, significantly reducing colon inflammation in mice with colitis produced by TNBS and DSS. CONCLUSION: These findings highlight an enhanced efficacy and improved biostability of BsNb-Fc, suggesting its potential as a promising therapeutic option for IBD patients with insufficient response to TNF-α inhibition. KEY POINTS: A bispecific nanobody (BsNb) was created to target TNF-α and IL-23p19, exhibiting high affinity and remarkable stability. BsNb-Fc inhibited the release of cytokines in CD4+T cells during co-culture experiments. BsNb-Fc effectively alleviated colitis severity in mouse model with acute colitis induced by DSS or TNBS, outperforming the IFX&UST combination.

MCPIP1 restrains mucosal inflammation by orchestrating the intestinal monocyte to macrophage maturation via an ATF3-AP1S2 axis.

OBJECTIVE: Monocyte chemotactic protein-1-induced protein 1 (MCPIP1) is highly expressed in inflamed mucosa of inflammatory bowel disease (IBD) and negatively regulates immune response, while the underlying mechanisms regulating mucosal macrophage functions remain unknown. Here, we investigated the roles of MCPIP1 in modulating the differentiation and functions of intestinal macrophages in the pathogenesis of IBD. DESIGN: ScRNA-seq was used to cluster the monocyte/macrophage lineage from macrophage-specific Mcpip1-deficient (Mcpip1 ∆Mye) mice and Mcpip1 fl/fl littermates. The differentially expressed genes were confirmed by RNA-seq, luciferase assay, CUT&Tag assay and Western blotting. Effects of MCPIP1 and the activating transcription factor 3 (ATF3)-AP1S2 axis were assessed in patients with IBD. RESULTS: Mcpip1 ∆Mye mice developed more severe dextran sulfate sodium (DSS)-induced colitis characterised by an increase in macrophage migratory capacity and M1 macrophage polarisation but a decrease in the monocyte-to-macrophage maturation in gut mucosa compared with their littermates. ScRNA-seq unravelled a proinflammatory population (Ccr2+Il-1ß+Tlr2+Cx3cr1-Cd163-Mrc1-Ly6c+) of the monocyte/macrophage lineage from lamina propria CD11b+ cells and an arrest of Mcpip1 ∆Mye monocyte-to-macrophage maturation in an Atf3-Ap1s2 axis-dependent manner. Silencing of Ap1s2 or Atf3 markedly suppressed Mcpip1 ∆Mye macrophage migration, M1-like polarisation, and production of proinflammatory cytokines and chemokines. Notably, in vivo blockage of Ap1s2 ameliorated DSS-induced colitis in Mcpip1 ΔMye mice through enhancing intestinal macrophage maturation. Furthermore, MCPIP1, ATF3 and AP1S2 were highly expressed in inflamed mucosa of active patients with IBD and blockage of ATF3 or AP1S2 significantly suppressed IBD CD14+-derived M1-like macrophage polarisation and proinflammatory cytokine production. CONCLUSIONS: Macrophage-specific Mcpip1 deficiency polarises macrophages towards M1-like phenotype, arrests macrophage maturation and exacerbates intestinal inflammation in an Atf3-Ap1s2-dependent manner, thus providing novel mechanistic insight into intestinal macrophage functions during IBD.

ETS-1 facilitates Th1 cell-mediated mucosal inflammation in inflammatory bowel diseases through upregulating CIRBP.

BACKGROUND & AIMS: As a susceptibility gene for human inflammatory bowel diseases (IBD), how avian erythroblastosis virus E26 oncogene homolog-1 (ETS-1) modulates intestinal mucosal immune response remains unclear. Here we studied the potential roles of ETS-1 in the pathogenesis of IBD. METHODS: ETS-1 expression was examined in IBD patients. CD45RBhighCD4+ T cell-transfer colitis, dextran sulfate sodium (DSS)-induced colitis, and azomethane (AOM)/DSS-induced colitis-associated cancer (CAC) models were constructed to probe the function of ETS-1 in vivo. RNA-sequencing of CD4+ T cells from Ets-1 transgenic (Tg) mice was performed to decipher the key differentially expressed genes. Adenovirus transduction was conducted to verify the therapeutic potentials of ETS-1 in vivo. RESULTS: ETS-1 expression was significantly increased in CD4+ T cells from active IBD patients compared with healthy controls, which was upregulated by TNF-α but markedly suppressed by anti-TNF-α mAb therapy. More severe colitis was observed in Rag1-/- mice reconstituted with Ets-1TgCD45RBhighCD4+ T cells or in Ets-1 Tg mice after DSS exposure compared with controls, characterized by higher TNF-α and IFN-γ expression in inflamed colon. Ets-1 Tg mice were more prone to develop AOM/DSS-induced CAC, and bone marrow chimeras further proved that lamina propria immune cells but not intestinal epithelial cells contributed to the development of colitis. RNA-sequencing and luciferase analysis revealed cold-inducible RNA-binding protein (CIRBP) as a functional target of ETS-1 to promote Th1 cell-driven immune response. Consistently, intraperitoneal administration of adenovirus-m-cirbp-shRNA ameliorated trinitrobenzene sulfonic acid (TNBS)-induced colitis of Ets-1 Tg mice. CONCLUSIONS: Our data identify that ETS-1 is highly expressed in IBD patients and promotes Th1-driven mucosal inflammation through CIRBP. CIRBP may serve as a novel therapeutic target for treatment of human IBD.

Effects of Super-Activated Platelet Lysate on Early Healing of Tooth Extraction Sockets in Rats.

Purpose: To evaluate the effect of super-activated platelet lysate (sPL) on wound healing of tooth extraction sockets in rats. Methods: Rat models of the tooth extraction socket were established. Thirty-six rats were divided into control and sPL groups and sacrificed on days 7, 14, and 28 after tooth extraction. Bone formation in tooth extraction sockets were observed by microscopic computed tomography (micro-CT) and hematoxylin and eosin (HE) staining; osteoprotegerin (OPG), receptor activator of nuclear factor kappa-Β ligand (RANKL), interleukin 6(IL-6), and tumor necrosis factor-alpha (TNF-α) proteins were detected by immunohistochemistry; and chemokine and osteogenic gene expressions were detected by polymerase chain reaction (PCR). Results: sPL accelerated soft tissue wound healing in the extraction socket of rats. Micro-CT showed that the amount of bone formation and bone volume fraction were higher in the sPL group than the control 14 days after extraction. HE staining showed promotion of the formation of bony trabeculae by sPL in the apical third of the extraction socket 7 days after extraction and more mature and organized bony trabeculae in the sPL group than the control 14 days after extraction; mature bony trabeculae filling most of the fossa with lesser bone porosity in the socket in the sPL group than the control 28 days after extraction. Immunohistochemistry showed that sPL induced OPG expressions 7 and 14 days after tooth extraction but did not affect the RANKL expression while transiently promoting the IL-6 expression 7 days after extraction. PCR showed that sPL promoted chemokine expressions 7 and 14 days after extraction. The expressions of osteogenesis-related factors were higher in the sPL group than the control 7 and 28 days after extraction, while the opposite trend was observed 14 days after extraction. Conclusion: sPL has a transient pro-inflammatory effect and promotes soft tissue healing and bone formation during early wound healing of extraction sockets in rats.

Macrophage-derived EDA-A2 inhibits intestinal stem cells by targeting miR-494/EDA2R/ß-catenin signaling in mice.

The mucosa microenvironment is critical for intestinal stem cell self-renewal and reconstruction of the epithelial barrier in inflammatory bowel disease (IBD), where the mechanisms underlying cross-talk between intestinal crypts and the microenvironment remain unclear. Here, we firstly identified miR-494-3p as an important protector in colitis. miR-494-3p levels were decreased and negatively correlated with the severity in human IBD samples, as well as in colitis mice. In colitis crypts, a notable cytokine-cytokine receptor, miR-494-3p-targeted EDA2R and the ligand EDA-A2, suppressed colonic stemness and epithelial repair by inhibiting ß-catenin/c-Myc. In differentiated IECs, miR-494-3p inhibits macrophage recruitment, M1 activation and EDA-A2 secretion by targeting IKKß/NF-κB in colitis. A miR-494-3p agomir system notably ameliorated the severity of colonic colitis in vivo. Collectively, our findings uncover a miR-494-3p-mediated cross-talk mechanism by which macrophage-induced intestinal stem cell impairment aggravates intestinal inflammation.

The DNA controllable peroxidase mimetic activity of MoS2 nanosheets for constructing a robust colorimetric biosensor.

The low activity of nanozymes, which work as an alternative to natural enzymes, limits their applications in the fabrication of biosensors, drawing increasing attention aimed at improving their catalytic capacity. In this work, the peroxidase-like activity of MoS2 nanosheets (NSs) was dramatically enhanced through DNA modification, and was 4.3-times higher than that of bare MoS2 NSs. Such an enhancement of catalytic activity was mainly ascribed to the increased affinity of the DNA/MoS2 NSs toward the substrate, TMB, further accelerating electron transfer from TMB to H2O2. On the basis of DNA-tuned MoS2 NS nanozyme activity, a colorimetric sensing platform was developed for the facile detection of carcinoembryonic antigen (CEA) in a sensitive manner. Interestingly, a convenient, affordable, and instrument-free portable test kit was fabricated to visually monitor CEA via rooting the aptamer/MoS2 NS system into an agarose hydrogel. Importantly, our work illuminates the feasibility of using DNA to enhance the catalysis of nanozymes and their application potential in the label-free, portable, and visual detection of aptamer-targeted biomolecules.

Monocyte Chemotactic Protein 1-Induced Protein 1 Is Highly Expressed in Inflammatory Bowel Disease and Negatively Regulates Neutrophil Activities.

Monocyte chemotactic protein 1-induced protein 1 (MCPIP-1) is highly expressed in activated immune cells and plays an important role in negatively regulating immune responses. However, its role in regulating neutrophil functions in the pathogenesis of inflammatory bowel disease (IBD) is still unclear. Here, we found that MCPIP-1 was markedly increased at both the transcriptional and translational levels in inflamed mucosa of IBD patients compared with healthy controls, which was mainly expressed in neutrophils. Interestingly, MG-132, a proteasome inhibitor reducing the degradation of MCPIP-1, further facilitated neutrophils to express MCPIP-1 in vitro. Importantly, MCPIP-1 markedly downregulated the production of ROS, MPO, and proinflammatory cytokines (e.g., interleukin-1ß, interleukin-6, tumor necrosis factor-α, interleukin-8, and interferon-γ) and suppressed the migration of IBD neutrophils. Consistently, the same functional changes were observed in neutrophils from mice with myeloid-targeted overexpression of MCPIP-1 as MG-132 did. Altogether, these findings suggest that MCPIP-1 plays a negative role in regulating neutrophil activities through suppressing the production of ROS, MPO, and proinflammatory cytokines and inhibiting the migration. MG-132 may partially modulate the function of neutrophils via the induction of MCPIP-1. Therefore, targeting MCPIP-1 or exogenous supplementation of MG-132 may provide a therapeutic approach in the treatment of IBD.

Plant-Inspired Layer-by-Layer Self-Assembly of Super-Hydrophobic Coating for Oil Spill Cleanup.

A versatile, facile, energy-saving, low-cost and plant-inspired self-assembly strategy was used to prepare super-hydrophobic coating in this study. Concretely, an appealing super-hydrophobicity surface was obtained by designing a molecular building block phytic acid (PA)-Fe (III) complex to anchor the substrate and hydrophobic thiol groups (HT). The facile and green modification method can be applied to variety of substrates. The as-prepared PA-Fe (III)-HT coated melamine composite sponge possesses both super-hydrophobic and superlipophilicity property. Moreover, it displays superior efficiency to separate the oil-water mixture and splendid oil spill cleanup.

Realizing the Control of Electronic Energy Level Structure and Gas-Sensing Selectivity over Heteroatom-Doped In2O3 Spheres with an Inverse Opal Microstructure.

Understanding the effect of substitutional doping on gas-sensing performances is essential for designing high-activity sensing nanomaterials. Herein, formaldehyde sensors based on gallium-doped In2O3 inverse opal (IO-(Ga xIn1- x)2O3) microspheres were purposefully prepared by a simple ultrasonic spray pyrolysis method combined with self-assembled sulfonated polystyrene sphere templates. The well-aligned inverse opal structure, with three different-sized pores, plays the dual role of accelerating the diffusion of gas molecules and providing more active sites. The Ga substitutional doping can alter the electronic energy level structure of (Ga xIn1- x)2O3, leading to the elevation of the Fermi level and the modulation of the band gap close to a suitable value (3.90 eV), hence, effectively optimizing the oxidative catalytic activity for preferential CH2O oxidation and increasing the amount of adsorbed oxygen. More importantly, the gas selectivity could be controlled by varying the energy level of adsorbed oxygen. Accordingly, the IO-(Ga0.2In0.8)2O3 microsphere sensor showed a high response toward formaldehyde with fast response and recovery speeds, and ultralow detection limit (50 ppb). Our findings finally offer implications for designing Fermi level-tailorable semiconductor nanomaterials for the control of selectivity and monitoring indoor air pollutants.

Novel Self-Assembly Route Assisted Ultra-Fast Trace Volatile Organic Compounds Gas Sensing Based on Three-Dimensional Opal Microspheres Composites for Diabetes Diagnosis.

The development of ultra-fast response semiconductor gas sensors for high-accuracy detection of trace volatile organic compounds in human exhaled breath still remains a challenge. Herein, we propose a novel self-assembly synthesis concept for preparing intricate three-dimensional (3D) opal porous (OP) SnO2-ZnO hollow microspheres (HM), by employing sulfonated polystyrene (S-PS) spheres template-assisted ultrasonic spray pyrolysis. The high gas accessibility of the unique opal hollow structures resulted in the existence of 3D interconnection and bimodal (mesoscale and macroscale) pores, and the n-n heterojunction-induced change in oxygen adsorption. The 3D OP SnO2-ZnO HM sensor exhibited high response and ultra-fast dynamic process (response time ∼4 s and recovery time ∼17 s) to 1.8 ppm acetone under highly humid ambient condition (98% relative humidity), and it could rapidly identify the states of the exhaled breath of healthy people and simulated diabetics. In addition, the rational structure design of the 3D OP SnO2 HM enables the ultra-fast detection (within 1 s) of ethanol in simulation drunk driving testing. Our results obtained in this work provided not only a facile self-assembly approach to fabricate metal oxides with 3D OP HM structures but also a new methodology for achieving noninvasive real-time exhaled breath detection.

High-response and low-temperature nitrogen dioxide gas sensor based on gold-loaded mesoporous indium trioxide.

Nitrogen dioxide (NO2), as a typical threatening atmospheric pollutant, is hazardous to the environment and human health. Thus, the development of a gas sensor with high response and low detection limit for NO2 detection is highly important. The highly ordered mesoporous indium trioxide (In2O3) prepared by simple nanocasting method using mesoporous silica as template and decorated with Au nanoparticles was investigated for NO2 detection. The prepared materials were characterized by X-ray diffraction, transmission electron microscopy, and X-ray photoelectron spectroscopy. Characterization results showed that the samples exhibited ordered mesostructure and were successfully decorated with Au. The gas sensing performance of the sensors based on a series of Au-loaded mesoporous In2O3 were systematically investigated. The Au loading level strongly affected the sensing performance toward NO2. The optimal sensor, which was based on 0.5 wt% Au-loaded In2O3, displayed high sensor response and low detection limit of 10 ppb at low operating temperature of 65 °C. The excellent sensing properties were mainly attributed to the ordered mesoporous structure and the catalytic performance of Au. We believe that the Au-loaded mesoporous In2O3 can provide a promising platform for NO2 gas sensors with excellent performance.

Critical Role of p53 and K-ras in the Diagnosis of Early Colorectal Cancer: a One-year, Single-center Analysis.

Background: Colorectal cancer (CRC) is strongly associated with colorectal polyps, which has become the third most common cancer in China. In the present study, we revealed the susceptible population and risk factors of colorectal polyps, and analyzed the expression of Ki-67, p53 and K-ras in the intestinal mucosa of patients with colorectal polyps in order to explore their significance in the detection and prognosis of CRC at an early stage. Materials and Methods: Total 801 cases of colorectal polyps were collected during endoscopic resection including endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD). Expression of Ki-67, p53 and K-ras in the intestinal mucosa was detected by immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Histological analysis was performed by Hematoxylin and eosin (HE) staining. Categorical variables were compared by one-way ANOVA, Pearson test, Spearman test, Kruskal-Wallis test and analysis of regression. Results: Of all patients with colorectal polyps, 90.76% of patients (n = 727) were ≥ 50 years old. 530 cases (66.17%) were males compared with 271 females (33.83%) in all 801 cases. More importantly, 1.03% patients (n = 7) underwent polypectomy and histological examination was confirmed to be the early stage of CRC. The expression of p53 was found to be significantly decreased, while K-ras was increased in tumor tissues of CRC compared with that in hyperplastic polyps and healthy controls. Conclusions: 1.03% patients (n = 7) underwent polypectomy was confirmed to be the early stage of CRC. Histological analysis for expression of p53 and K-ras can guarantee to screen the early stage of CRC.

Vitamin D-deficiency induces eosinophil spontaneous activation.

Eosinophils (Eo) play a critical role in immunity and immune inflammation. The maintenance of Eo homeostasis is not fully understood yet. Vitamin D (VitD) is involved in the regulation of a large number of biochemical reactions. This study tests a hypothesis that VitD receptor (VDR) contributes to the homeostasis of Eos. In this study, EoL-1 cells (an Eo cell line) were cultured in the presence or absence of calcitriol. The Eo-mediators, including major basic protein (MBP), Eo peroxidase (EPX), Eo cationic protein (ECP) and Eo-derived neurotoxin (EDN), were assessed in the culture supernatant and in EoL-1 cells. We observed that, in a VitD deficient environment, EoL-1 cells produced high levels of the Eo-mediators, including MBP, EPX, ECP and EDN, which could be suppressed by the addition of calcitriol to the culture. EoL-1 cells expressed VitD receptor (VDR), which was up regulated by exposure to calcitriol. VDR formed complexes with the transcription factors of the Eo-mediators, which prevented the transcription factors to bind to the promoters of the Eo-mediators, and therefore prevented the Eo-mediated gene transcription. The Eo spontaneous activation was also found in the intestinal mucosa of VDR-deficient mice, in which the intestinal epithelial barrier dysfunction was observed. In conclusion, VDR contributes to the maintenance of the homeostasis of Eos by regulating the gene transcription of the Eo mediators. The VDR-deficiency is one of the causative factors inducing Eo spontaneous activation. This phenomenon may be taken into account in the management of the Eo-related diseases.

Clinicopathological and Ileocolonoscopic Characteristics in Patients with Nodular Lymphoid Hyperplasia in the Terminal Ileum.

Nodular lymphoid hyperplasia (NLH) in the small intestine is a rare benign lesion, characterized by the presence of multiple small nodules on the surface of the intestine. To define the clinicopathological and colonoscopic characteristics in Chinese patients with ileal NLH, we collected 65 patients with NLH in the terminal ileum from the endoscopic database in our hospital and clinical data from medical records. Histology and immunohistochemical staining were performed in the biopsies. The results demonstrated that the main symptoms included diarrhea (70.8%), abdominal pain (60.0%), hematochezia (46.2%), anemia (40.0%), and hypoproteinemia (21.5%). Enteroscopy revealed multiple, sporadic, granular or round-shaped nodules with diameters between 2 and 5 mm in the terminal ileum. The histology revealed the nodules consisted of mass lymphoid follicles in the lamina propria and submucosa of the terminal ileum. The follicles contained mitotically active germinal centers surrounded by well-defined lymphocyte mantles and composed predominantly of CD20+ B cells. The diseases found in patients with NLH included chronic diarrhea, Crohn's disease, ischemic enterocolitis and allergic purpura. The level of hemoglobin in NLH patients who had diarrhea and hematochezia remarkably decreased as compared with those in patients with chronic diarrhea. In conclusion, ileocolonoscopic screening is an important step to find the NLH in terminal ileum patients with diarrhea, abdominal pain, hematochezia, and hypoproteinemia. Histological examination is necessary for the exclusion of malignancy and chronic inflammation.

Pediatric eosinophilic esophagitis: single-center experience in northwestern USA.

BACKGROUND: Eosinophilic esophagitis (EE) is an emerging disease. Its incidence and prevalence have been exponentially increasing in the last decade. The clinical manifestations of EE vary especially in children. Allergic reaction is strongly implicated in the disease. The aim of the present study was to retrospectively examine the demographic, clinical, laboratory, and endoscopic features of pediatric patients with EE, in order to better understand the diversity of the disease, thereby improving clinical diagnosis and treatment. METHODS: The data were retrospectively collected from 20 pediatric patients with biopsy-proven EE. Demographic information, clinical symptoms and duration, as well as endoscopic findings were correlated and statistically analyzed. RESULTS: Median age at diagnosis was 11 years. Male patients had higher prevalence of EE. Thirty-five percent of the children had food allergy on either skin prick test or IgE radioallergosorbent test, and atopic diseases were common in the group. Gastroesophageal reflux appeared to be the major symptom in younger children, whereas dysphagia and food impaction were more common in older patients. The referral and final diagnosis were often delayed. Esophageal white exudates were the most prominent feature in the younger age group, whereas esophageal ring-like lesion occurred more often in older children. CONCLUSION: EE is more common in male subjects. The clinical and endoscopic features depend upon the age. Allergic processes are strongly involved in pathogenesis. The present results will improve understanding of the characteristics of the disease in the pediatric population and enhance clinicians' vigilance for the diagnosis of EE.

The NOD2 defect in Blau syndrome does not result in excess interleukin-1 activity.

OBJECTIVE: Blau syndrome is a rare, autosomal-dominant, autoinflammatory disorder characterized by granulomatous arthritis, uveitis, and dermatitis. Genetics studies have shown that the disease is caused by single nonsynonymous substitutions in NOD-2, a member of the NOD-like receptor or NACHT-leucine-rich repeat (NLR) family of intracellular proteins. Several NLRs function in the innate immune system as sensors of pathogen components and participate in immune-mediated cellular responses via the caspase 1 inflammasome. Mutations in a gene related to NOD-2, NLRP3, are responsible for excess caspase 1-dependent interleukin-1beta (IL-1beta) in cryopyrinopathies such as Muckle-Wells syndrome. Furthermore, functional studies demonstrate that caspase 1-mediated release of IL-1beta also involves NOD-2. The aim of this study was to test the hypothesis that IL-1beta may mediate the inflammation seen in patients with Blau syndrome. METHODS: IL-1beta release was measured in peripheral blood mononuclear cells cultured in vitro, obtained from 5 Blau syndrome individuals with a NOD2 (CARD15) mutation. RESULTS: We observed no evidence for increased IL-1beta production in cells obtained from subjects with Blau syndrome compared with healthy control subjects. Furthermore, we presented 2 cases of Blau syndrome in which recombinant human IL-1 receptor antagonist (anakinra) was ineffective treatment. CONCLUSION: Taken together, these data suggest that in contrast to related IL-1beta-dependent autoinflammatory cryopyrinopathies, Blau syndrome is not mediated by excess IL-1beta or other IL-1 activity.

Adenovirus induced acute hepatitis in non-human primates after liver-directed gene therapy.

OBJECTIVE: To define the mechanism of acute hepatitis in non-human primates after liver directed gene therapy. METHODS: Differences in immune response exhibited by 8 rhesus monkeys receiving adenovirus (Ad) or lipofectamine-mediated gene transfer by various routes, the time course, and the nature of the specific immune responses to both adenoviral vectors and transgene products were studied using HE staining (H&E) and immunohistochemical staining. RESULTS: The monkeys developed mild to moderate acute hepatitis 1 to 3 weeks after intravenous or intrabiliary injection of first generation replication-defective adenoviruses carrying the Escherichia coli lacZ gene. This was accompanied by adenovirus-mediated T-cell proliferation and neutralizing antibodies to the adenovirus. Increased numbers of CD3(+), CD4(+) and CD8(+) T-lymphocytes were detected in the diseased livers, while B-lymphocytes were absent. Hepatocytes demonstrated increased expression of beta 2-microglobulins (beta 2-MG) and HLA-DR antigens in the plasma membranes. The development of acute hepatitis and the accompanying immune abnormalities were delayed in immunosuppressed monkeys until after the discontinuation of immunosuppressive therapy. The monkeys infused with Ad. CMVluc showed more significant and longer durations of hepatitis than the monkeys infused with adenoviruses carrying the lacZ gene. Lipofectamine-mediated gene transfer was inefficient. There was neither lacZ expression nor significant immune response in the liver of monkeys infused with lipofectamine via the portal vein or the common bile duct. CONCLUSION: Immune response to the hepatocytes in liver directed gene therapy is MHC class I restricted and T-cell mediated. Both adenoviral vectors and foreign genes are related to the liver damage. Mild to moderate hepatic inflammation seen with the E-1 deleted vector is reversible. Immunosuppression regimens may prolong transgene expression and delay the development of acute adenoviral hepatitis.