Advances in Immunotherapy for Hepatocellular Carcinoma (HCC).

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related deaths in the world. More than half of patients with HCC present with advanced stage, and highly active systemic therapies are crucial for improving outcomes. Immune checkpoint inhibitor (ICI)-based therapies have emerged as novel therapy options for advanced HCC. Only one third of patients achieve an objective response with ICI-based therapies due to primary resistance or acquired resistance. The liver tumor microenvironment is naturally immunosuppressive, and specific mutations in cell signaling pathways allow the tumor to evade the immune response. Next, gene sequencing of the tumor tissue or circulating tumor DNA may delineate resistance mechanisms to ICI-based therapy and provide a rationale for novel combination therapies. In this review, we discuss the results of key clinical trials that have led to approval of ICI-based therapy options in advanced HCC and summarize the ongoing clinical trials. We review resistance mechanisms to ICIs and discuss how immunotherapies may be optimized based on the emerging research of tumor biomarkers and genomic alterations.

Advanced glycation end products facilitate bacterial adherence in urinary tract infection in diabetic mice.

Diabetic individuals have increased susceptibility to urinary tract infection (UTI), a common, painful condition. During diabetes mellitus, non-enzymatic reactions between reducing sugars and protein amine groups result in excessive production of advanced glycation end products (AGEs) that accumulate in tissues. Since bacteria adhere to cell surfaces by binding to carbohydrates, we hypothesized that adherence of bacteria to the bladder in diabetics may be enhanced by accumulation of AGEs on urothelial surface proteins. Using a murine model of UTI, we observed increased adherence of type 1 fimbriated uropathogenic Escherichia coli (UPEC) to the bladder in streptozotocin-induced diabetic female mice compared with age-matched controls, along with increased concentrations of two common AGEs in superficial urothelial cells from diabetic bladders. Several lectins with different specificities exhibited increased binding to urothelial homogenates from diabetic mice compared with controls, and two of those lectins also bound to AGEs. Furthermore, mannose-binding type 1 fimbriae isolated from UPEC bound to different AGEs, and UPEC adherence to the bladder in diabetic mice, were inhibited by pretreatment of mice with the AGE inhibitor pyridoxamine. These results strongly suggest a role for urothelial AGE accumulation in increased bacterial adherence during UTI in diabetes.

Impaired cytokine expression, neutrophil infiltration and bacterial clearance in response to urinary tract infection in diabetic mice.

Diabetic patients have increased susceptibility to infections, and urinary tract infections (UTI) are the most common type in women with diabetes mellitus. Knowledge of bacterial clearance effectiveness following UTI in diabetics is sparse. In this study, the effects of diabetes on bacterial clearance efficiency and components of the innate immune system in response to UTI in a murine model were investigated. Streptozotocin-induced diabetic and control female C57BL/6J mice were infected with uropathogenic Escherichia coli, and bacterial load, expression of chemokines, and neutrophil infiltration in the bladder over time were investigated. Expression levels of histone deacetylases were also measured to address a potential mechanism underlying the phenotype. Bacterial clearance during UTI was significantly prolonged in diabetic mice relative to controls. Neutrophil infiltration in bladder tissue and urine, and both mRNA and protein expression of chemokines MIP-2, KC, MCP-1 and IL-6 in bladder tissue were diminished at early time points after infection in diabetic mice relative to controls. In addition, mRNA levels of histone deacetylases 1-5 were increased in diabetic mice. This is the first study to show an association of impaired bacterial clearance in diabetic mice with suppression of UTI-induced chemokine expression and neutrophil infiltration in the bladder.

Chronic pelvic allodynia is mediated by CCL2 through mast cells in an experimental autoimmune cystitis model.

The cause of chronic pelvic pain in interstitial cystitis/painful bladder syndrome (IC/PBS) remains unclear; autoimmunity is a possible etiology. We have recently shown that injection of a single immunogenic peptide of uroplakin 3A (UPK3A 65-84) induces experimental autoimmune cystitis (EAC) in female BALB/cJ mice that is unique among experimental models in accurately reflecting both the urinary symptoms and pelvic pain of IC/PBS. The aim of this project was to identify the roles of mast cells and mast cell chemoattractant/activator monocyte chemoattractant protein-1 [chemokine (C-C motif) ligand 2 (CCL2)] in the allodynia in this model. We immunized 6- to 8-wk-old female BALB/cJ mice with UPK3A 65-84 peptide and, 5-40 days later, observed increased responses to stimulation of the suprapubic abdominal and hindpaw surfaces with von Frey monofilaments compared with mice injected with adjuvant alone. Suprapubic and hindpaw tactile allodynia responses by EAC mice were blocked by instillation of lidocaine into the bladder but not by lidocaine in the uterus, confirming the bladder as the source of the hypersensitivity. Markedly increased numbers of activated mast cells and expression of CCL2 were found in the bladder after immunization with UPK3A 65-84. Hypersensitive responses were inhibited by mast cell stabilizer cromolyn sodium and antagonists of histamine receptors 1 and 2. Furthermore, BALB/cJ mice with deletion of the Ccl2 or chemokine (C-C motif) receptor 2 gene exhibited markedly reduced allodynia and accumulation of mast cells after UPK3A 65-84 immunization. These results show that UPK3A 65-84 immunization causes chronic visceral allodynia and suggest that it is mediated by CCL2-driven mast cell accumulation in the bladder.

Assessment of bladder sensation in mice with a novel device.

OBJECTIVE: To develop and test the efficacy of an implantable bladder electrode device that can be used with the Neurometer electrodiagnostic stimulator to assess fiber-specific afferent bladder sensation in the mouse. METHODS: We constructed a ball-tipped platinum electrode and surgically implanted it into the mouse bladder. The Neurometer was connected to the electrode to apply selective nerve fiber stimuli (250 Hz for Aδ fibers and 5 Hz for C fibers) of increasing intensities to the bladder mucosa in the mouse to determine bladder sensory threshold (BST) values. Using 58 female C57BL/6J mice, we measured the temporal and interobserver consistency of BST measurements, the effects of intravesical administration of lidocaine and resiniferatoxin on the BST, and the effects of our device on voiding behavior and bladder mucosal integrity. RESULTS: BST values at 250 and 5 Hz did not vary significantly when measured 2, 4, and 6 days after device implantation, or when obtained by 2 blinded independent observers. Intravesical lidocaine yielded a transient increase in BST values at both 250 Hz and 5 Hz, whereas resiniferatoxin yielded a significant increase only at the 5 Hz stimulus frequency after 24 hours. Moderately increased micturition frequency and decreased volume per void were observed 4 and 6 days after device implantation. Histology revealed mild inflammatory changes in the area of the bladder adjacent to the implanted BST device. CONCLUSION: Assessment of neuroselective bladder sensation in mice is feasible with our device, which provides reproducible BST values for autonomic bladder afferent nerve fibers.

Uroplakin peptide-specific autoimmunity initiates interstitial cystitis/painful bladder syndrome in mice.

The pathophysiology of interstitial cystitis/painful bladder syndrome (IC/PBS) is enigmatic. Autoimmunity and impaired urothelium might lead the underlying pathology. A major shortcoming in IC/PBS research has been the lack of an appropriate animal model. In this study, we show that the bladder specific uroplakin 3A-derived immunogenic peptide UPK3A 65-84, which contains the binding motif for IA(d) MHC class II molecules expressed in BALB/c mice, is capable of inducing experimental autoimmune cystitis in female mice of that strain. A highly antigen-specific recall proliferative response of lymph node cells to UPK3A 65-84 was observed, characterized by selectively activated CD4+ T cells with a proinflammatory Th1-like phenotype, including enhanced production of interferon γ and interleukin-2. T cell infiltration of the bladder and bladder-specific increased gene expression of inflammatory cytokines were observed. Either active immunization with UPK3A 65-84 or adoptive transfer of peptide-activated CD4+ T cells induced all of the predominant IC/PBS phenotypic characteristics, including increased micturition frequency, decreased urine output per micturition, and increased pelvic pain responses to stimulation with von Frey filaments. Our study demonstrates the creation of a more specific experimental autoimmune cystitis model that is the first inducible model for IC/PBS that manifests all of the major symptoms of this debilitating condition.

A novel murine model of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) induced by immunization with a spermine binding protein (p25) peptide.

The pathophysiology of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is poorly understood. Inflammatory and autoimmune mechanisms may play a role. We developed a murine model of experimental autoimmune prostatitis (EAP) that mimics the human phenotype of CP/CPPS. Eight-week-old mice were immunized subcutaneously with prostate-specific peptides in an emulsion of complete Freund's adjuvant. Mice were euthanized 10 days after immunization, and lymph node cells were isolated and assessed for recall proliferation to each peptide. P25 99-118 was the most immunogenic peptide. T-cell and B-cell immunity and serum levels of C-reactive protein and nitrate/nitrite levels were evaluated over a 9-wk period. Morphometric studies of prostate, 24-h micturition frequencies, and urine volume per void were evaluated. Tactile referred hyperalgesia was measured using von Frey filaments to the pelvic region. The unpaired Student's t-test was used to analyze differences between EAP and control groups. Prostates from p25 99-118-immunized mice demonstrated elevated gene expression levels of TNF-α, IL-17A, IFN-γ, and IL-1ß, not observed in control mice. Compared with controls, p25 99-118-immunized mice had significantly higher micturition frequency and decreased urine output per void, and they demonstrated elevated pelvic pain response. p25 99-118 immunization of male SWXJ mice induced prostate-specific autoimmunity characterized by prostate-confined inflammation, increased micturition frequency, and pelvic pain. This autoimmune prostatitis model provides a useful tool for exploring the pathophysiology and new treatments.

Connective tissue and its growth factor CTGF distinguish the morphometric and molecular remodeling of the bladder in a model of neurogenic bladder.

We previously reported that mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), develop profound urinary bladder dysfunction. Because neurogenic bladder in MS patients causes marked bladder remodeling, we next examined morphometric and molecular alterations of the bladder in EAE mice. EAE was created in female SJL/J mice by immunization with the p139-151 encephalitogenic peptide of myelin proteolipid protein in complete Freund's adjuvant, along with intraperitoneal injections of Bordetella pertussis toxin. Seventy days after immunization, mice were scored for the level of neurological impairment and then killed. Spinal cord sections were assessed for demyelination, inflammation, and T cell infiltration; the composition of the bladder tissue was measured quantitatively; and gene expression of markers of tissue remodeling and fibrosis was assessed. A significant increase in the bladder weight-to-body weight ratio was observed with increasing neurological impairment, and morphometric analysis showed marked bladder remodeling with increased luminal area and tissue hypertrophy. Despite increased amounts of all tissue components (urothelium, smooth muscle, and connective tissue), the ratio of connective tissue to muscle increased significantly in EAE mice compared with control mice. Marked increases in mRNA expression of collagen type I α(2), tropoelastin, transforming growth factor-ß3, and connective tissue growth factor (CTGF) were observed in EAE mice, as were decreased levels of mRNAs for smooth muscle myosin heavy chain, nerve growth factors, and muscarinic and purinergic receptors. Our results suggest that bladder remodeling corresponding to EAE severity may be due to enhanced expression of CTGF and increased growth of connective tissue.