Chimeric TIM-4 receptor-modified T cells targeting phosphatidylserine mediates both cytotoxic anti-tumor responses and phagocytic uptake of tumor-associated antigen for T cell cross-presentation.

To leverage complementary mechanisms for cancer cell removal, we developed a novel cell engineering and therapeutic strategy co-opting phagocytic clearance and antigen presentation activity into T cells. We engineered a chimeric engulfment receptor (CER)-1236, which combines the extracellular domain of TIM-4, a phagocytic receptor recognizing the "eat me" signal phosphatidylserine, with intracellular signaling domains (TLR2/TIR, CD28, and CD3ζ) to enhance both TIM-4-mediated phagocytosis and T cell cytotoxic function. CER-1236 T cells demonstrate target-dependent phagocytic function and induce transcriptional signatures of key regulators responsible for phagocytic recognition and uptake, along with cytotoxic mediators. Pre-clinical models of mantle cell lymphoma (MCL) and EGFR mutation-positive non-small cell lung cancer (NSCLC) demonstrate collaborative innate-adaptive anti-tumor immune responses both in vitro and in vivo. Treatment with BTK (MCL) and EGFR (NSCLC) inhibitors increased target ligand, conditionally driving CER-1236 function to augment anti-tumor responses. We also show that activated CER-1236 T cells exhibit superior cross-presentation ability compared with conventional T cells, triggering E7-specific TCR T responses in an HLA class I- and TLR-2-dependent manner, thereby overcoming the limited antigen presentation capacity of conventional T cells. Therefore, CER-1236 T cells have the potential to achieve tumor control by eliciting both direct cytotoxic effects and indirect-mediated cross-priming.

Novel therapeutic approach for neurogenic erectile dysfunction: effect of neurotrophic tyrosine kinase receptor type 1 monoclonal antibody.

BACKGROUND: Erectile dysfunction (ED) is a major health issue in aged populations, and neurogenic ED is particularly difficult to treat. Novel therapeutic approaches are needed for treatment of neurogenic ED of peripheral origin. OBJECTIVE: To investigate the therapeutic effects of a neurotrophic tyrosine kinase receptor type 1 monoclonal antibody (TrkA-mAb) on erectile function and sexual behavior in a rat model of cavernous nerve injury (CNI). DESIGN, SETTING, AND PARTICIPANTS: In one experiment, 84 male rats were randomly assigned to seven groups. The groups underwent either CNI or sham surgery, subsequent injection into the major pelvic ganglion (IMPG) of phosphate-buffered saline (PBS), an immunoglobulin G (IgG) control, or TrkA-mAb, and then intracavernosal (IC) injection of either PBS or varying TrkA-mAb concentrations immediately after surgery and then 1 wk later. Erectile function was assessed and histologic/molecular analyses were performed at 6 wk after surgery. In a second experiment, 36 male rats were randomly divided into three groups. The groups underwent CNI or sham surgery and then IC injection of PBS, IgG, or TrkA-mAb immediately after surgery and for 5 wk thereafter. At 6 wk after surgery, the performance of the rats in sexual behavior tests was videotaped. INTERVENTION: CNI or sham surgery; IMPG of PBS, IgG, or TrkA-mAb; IC injection of PBS or TrkA-mAb. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The intracavernous pressure response to cavernous nerve electrostimulation was measured and midpenile cross-sections were histologically examined. Western blotting (WB) of cavernous tissue protein was performed. Rats were assessed for chasing, mounting, intromission, and ejaculation behaviors during sexual behavior tests. The data were analyzed using one-way analysis of variance followed by the Tukey-Kramer t test. RESULTS AND LIMITATIONS: Recovery of erectile function of varying degrees was observed in the TrkA-mAb groups. TrkA-mAb treatment significantly suppressed tyrosine hydroxylase-positive nerve fibers in the corpus cavernosum and enhanced neuronal nitric oxide synthase-positive fibers in the dorsal nerve. The ratio of smooth muscle to collagen in the corpus cavernosum was significantly improved in TrkA-mAb treatment groups compared to PBS vehicle and IgG control groups. WB confirmed these biological changes. There was a nonsignificant increase in the average number of intromissions and ejaculations in the TrkA-mAb group. The study limitations include small sample size, variability in sexual behavior, lack of data on the neuromuscular mechanism involved, and lack of information of the role of neurotrophins or cytokines in regeneration. CONCLUSIONS: TrkA-mAb successfully inhibits sympathetic nerve regeneration, leads to parasympathetic nerve regeneration, and has therapeutic effects on ED and sexual behavior disorder in a rat model of CNI. PATIENT SUMMARY: This report provides strong evidence that a neurotrophic tyrosine kinase receptor type 1 monoclonal antibody (TrkA-mAb) inhibits sympathetic nerve regeneration, leads to parasympathetic nerve regeneration, and has therapeutic effects on erectile dysfunction and sexual behavior disorder in a rat model of cavernous nerve injury. The results raise the possibility that human patients with neurogenic erectile dysfunction may respond to TrkA-mAb in a manner that parallels the response seen in our rodent study.

Recruitment of intracavernously injected adipose-derived stem cells to the major pelvic ganglion improves erectile function in a rat model of cavernous nerve injury.

BACKGROUND: Intracavernous (IC) injection of stem cells has been shown to ameliorate cavernous-nerve (CN) injury-induced erectile dysfunction (ED). However, the mechanisms of action of adipose-derived stem cells (ADSC) remain unclear. OBJECTIVES: To investigate the mechanism of action and fate of IC injected ADSC in a rat model of CN crush injury. DESIGN, SETTING, AND PARTICIPANTS: Sprague-Dawley rats (n=110) were randomly divided into five groups. Thirty-five rats underwent sham surgery and IC injection of ADSC (n=25) or vehicle (n=10). Another 75 rats underwent bilateral CN crush injury and were treated with vehicle or ADSC injected either IC or in the dorsal penile perineural space. At 1, 3, 7 (n=5), and 28 d (n=10) postsurgery, penile tissues and major pelvic ganglia (MPG) were harvested for histology. ADSC were labeled with 5-ethynyl-2-deoxyuridine (EdU) before treatment. Rats in the 28-d groups were examined for erectile function prior to tissue harvest. MEASUREMENTS: IC pressure recording on CN electrostimulation, immunohistochemistry of the penis and the MPG, and number of EdU-positive (EdU+) cells in the injection site and the MPG. RESULTS AND LIMITATIONS: IC, but not perineural, injection of ADSC resulted in significantly improved erectile function. Significantly more EdU+ ADSC appeared in the MPG of animals with CN injury and IC injection of ADSC compared with those injected perineurally and those in the sham group. One day after crush injury, stromal cell-derived factor-1 (SDF-1) was upregulated in the MPG, providing an incentive for ADSC recruitment toward the MPG. Neuroregeneration was observed in the group that underwent IC injection of ADSC, and IC ADSC treatment had beneficial effects on the smooth muscle/collagen ratio in the corpus cavernosum. CONCLUSIONS: CN injury upregulates SDF-1 expression in the MPG and thereby attracts intracavernously injected ADSC. At the MPG, ADSC exert neuroregenerative effects on the cell bodies of injured nerves, resulting in enhanced erectile response.

The effect of intracavernous injection of adipose tissue-derived stem cells on hyperlipidemia-associated erectile dysfunction in a rat model.

INTRODUCTION: Hyperlipidemia has been associated with erectile dysfunction (ED) via damage to the cavernous endothelium and nerves. Adipose tissue-derived stem cells (ADSC) have been shown to differentiate into endothelial cells and secrete vasculotrophic and neurotrophic factors. AIM: To assess whether ADSC have therapeutic effects on hyperlipidemia-associated ED. METHODS: Twenty-eight male rats were induced to develop hyperlipidemia with a high-fat diet (hyperlipidemic rats, HR). Ten additional male rats were fed a normal diet to serve as controls (normal rats, NR). Five months later, all rats were subjected to ADSC isolation from paragonadal fat. The cells were cultured for 1 week, labeled with 5-ethynyl-2'-deoxyuridine (EdU), and then injected autologously into the corpus cavernosum of 18 HR. The remaining 10 HR rats were injected with phosphate buffered saline (PBS). At 2 and 14 days post-transplantation, four rats in the HR + ADSC group were sacrificed for tracking of the transplanted cells. At 28 days post-transplantation, all remaining rats were analyzed for serum biochemistry, erectile function, and penile histology. MAIN OUTCOME MEASURES: Erectile function was assessed by intracavernous pressure (ICP) measurement during electrostimulation of the cavernous nerve. Cavernous nerves, endothelium, and smooth muscle were assessed by immunohistochemistry. RESULTS: Serum total cholesterol and low-density lipoprotein levels were significantly higher in HR than in NR. High-density lipoprotein level was significantly lower in HR than in NR. Mean ICP/mean arterial pressure ratio was significantly lower in HR + PBS than in NR + PBS or HR + ADSC. Neuronal nitric oxide synthase (nNOS)-positive nerve fibers and endothelial cells were fewer in HR + PBS than in HR + ADSC. Smooth muscle content was significantly higher in both HR groups than in NR. CONCLUSIONS: Hyperlipidemia is associated with abnormalities in both the nerves and endothelium. Treatment with ADSC ameliorates these adverse effects and holds promise as a potential new therapy for ED.

Effects of icariin on phosphodiesterase-5 activity in vitro and cyclic guanosine monophosphate level in cavernous smooth muscle cells.

OBJECTIVES: To investigate the effect of icariin on the cyclic guanosine monophosphate (cGMP)-hydrolytic activity of phosphodiesterase-5 (PDE5) isoforms and the cGMP levels in cavernous smooth muscle cells treated with sodium nitroprusside (SNP). METHODS: PDE5 isoforms (PDE5A1, A2, and A3) were isolated from sf9 insect cells infected with baculoviruses carrying PDE5 isoform cDNA. Icariin was isolated from Epimedii herba. Varying amounts (10(-6) to 10(-11) M) of icariin or zaprinast were added to reaction mixtures containing PDE5 isoforms and cGMP. The inhibitory effects of icariin and zaprinast were analyzed by GraphPad Software and are expressed as concentration that inhibits 50% (IC50) values. Cavernous smooth muscle cells were isolated from 3-month-old rats, treated with icariin (100 and 200 microM) or zaprinast (200 microM) for 15 minutes, and then with 10 microM SNP for 30, 60, 120, 240, and 360 minutes. The cells were then analyzed for the cGMP concentration using an enzyme immunoassay system. RESULTS: Icariin inhibited PDE5A1, A2, and A3 with an IC50 value of 1.0, 0.75, and 1.1 microM, respectively. The corresponding IC50 values for zaprinast were 0.33, 0.23, and 0.32 microM. Icariin consistently outperformed the control (SNP-only treatment) in maintaining greater cGMP levels, particularly at the greater concentration of 200 microM. In contrast, zaprinast at 200 microM did better than the control only at 60 and 360 minutes. CONCLUSIONS: Icariin was inhibitory to all three PDE5 isoforms with similar IC50 values, which were approximately three times greater than those for zaprinast. Icariin was able to enhance cGMP levels in SNP-treated cavernous smooth muscle cells.

[Establishment of high-throughput drug screening cell models based on JAK-STAT signal pathway].

AIM: To discover new drugs which may be applied to diseases of the immune system, hemogenesis system diseases and tumors, several high-throughput drug screening cell models based on JAK-STAT signal pathway have been established. METHODS: Four repeats of STAT DNA binding conserved sequences were synthesized, subcloned into pGL-Luc reporter vector and stably transfected into cell lines in vitro. Cell clones with high copy numbers of STAT binding sites and reporter genes were chosen as high-throughput drug screening cell models. The cell models were tested with known anti-allergic drugs and anti-tumor drugs by determining luciferase activity. The reaction was performed in 96 well micro-plates with a final volume of 50 microL. RESULTS: The cell models by performing rapid fluorescence assay were shown to be highly sensitive and stable after testing with cytokine and drugs. The modification of the expression plasmid simplified this method and made it more practical. It also provided good linear correlation, wide range of assay, highly sensitive and good reproducibility. CONCLUSION: The method can be performed by high-throughput drug screening for effective extraction of Chinese traditional herbs.