Human Mouse Research Articles

Single-sEV profiling identifies the TACSTD2 + sEV subpopulation as a factor of tumor susceptibility in the elderly

BackgroundAging is a very complex physiological phenomenon, and sEVs are involved in the regulation of this mechanism. Serum samples from healthy individuals under 30 and over 60 years of age were collected to analyze differences in sEVs proteomics.ResultsBased on PBA analysis, we found that sEVs from the serum of elderly individuals highly express TACSTD2 and identified a subpopulation marked by TACSTD2. Using ELISA, we verified the upregulation of TACSTD2 in serum from elderly human and aged mouse. In addition, we discovered that TACSTD2 was significantly increased in samples from tumor patients and had better diagnostic value than CEA. Specifically, 9 of the 13 tumor groups exhibited elevated TACSTD2, particularly for cervical cancer, colon cancer, esophageal carcinoma, liver cancer and thyroid carcinoma. Moreover, we found that serum sEVs from the elderly (especially those with high TACSTD2 levels) promoted tumor cell (SW480, HuCCT1 and HeLa) proliferation and migration.ConclusionTACSTD2 was upregulated in the serum of elderly individuals and patients with tumors, and could serve as a dual biomarker for aging and tumors.

The immunoglobulin superfamily ligand B7H6 subjects T cell responses to NK cell surveillance.

Understanding the mechanisms that regulate T cell immunity is critical for the development of effective therapies for diseases associated with T cell dysfunction, including autoimmune diseases, chronic infections, and cancer. Co-inhibitory "checkpoint molecules," such as programmed cell death protein-1, balance excessive or prolonged immune activation by T cell-intrinsic signaling. Here, by screening for mediators of natural killer (NK) cell recognition on T cells, we identified the immunoglobulin superfamily ligand B7H6 to be highly expressed by activated T cells, including patient-infused CD19-targeting chimeric antigen receptor (CAR) T cells. Unlike other checkpoint molecules, B7H6 mediated NKp30-dependent recognition and subsequent cytolysis of activated T cells by NK cells. B7H6+ T cells were prevalent in the tissue and blood of several diseases, and their abundance in tumor tissue positively correlated with clinical response in a cohort of patients with immune checkpoint inhibitor-treated esophageal cancer. In humanized mouse models, NK cell surveillance via B7H6 limited the persistence and antitumor activity of CAR T cells, and its genetic deletion enhanced T cell proliferation and persistence. Together, we provide evidence of B7H6 protein expression by activated T cells and suggest the B7H6-NKp30 axis as a therapeutically actionable NK cell-dependent immune checkpoint that regulates human T cell function.

Abstract PO2-15-11: Kinetic Analysis of [5-11C] glutamine PET tracer data in breast cancer: Preclinical studies in mouse models

Abstract Background: Glutamine is an important metabolic substrate in many aggressive tumors, with comparable importance to glucose metabolism. Utilizing human breast cancer mouse xenograft models, we studied the kinetics of the PET imaging agent, [5-11C] glutamine, a biochemical authentic substrate for glutamine metabolism, to further characterize the metabolism of glutamine and downstream labeled metabolites. Studies were performed with and without inhibition of the enzyme, glutaminase (GLS), the first step in glutamine catabolism that generates glutamate, and key target for therapy directed to glutamine-metabolizing cancers. Methods: The study used xenograft mouse models for two breast cancer cell lines, HCC1806, a highly glutaminolytic triple-negative cell line, and MCF-7, a hormone receptor positive line with only low levels of glutaminolysis. Mice were injected with 5-[11C]L-glutamine, and the contributions of individual metabolites to the total [11C] signal in blood and tumor tissue were estimated at 10, 20, and 30 minutes after injection by assaying the fractional contributions of [5-11C] glutamine and labeled metabolites, specifically [5-11C] glutamate, [11C] CO2 and all other metabolites with [11C], in both blood and tumor tissue. This generated estimated time activity curves for [5-11C] glutamine and downstream metabolites in both HCC1806 and MCF-7 mouse models with and without CB-839 treatment, an inhibitor of GLS. We also investigated a compartment model that describes these data, including model simulations that fit the model to match the measured curves. Results: We found that, out to 30 minutes post-injection, the vast majority of tumor radioactivity was in the form of either 5-11C] glutamine or [5-11C] glutamate, with smaller amounts of radioactivity in the form of more downstream metabolites, including [11C]CO2. Systemic administration of the glutaminase inhibitor alters the arterial input curve by reducing the systemic rate of conversion of [5-11C] glutamine to [5-11C] glutamate. However, in GLS active tumors, [5-11C] glutamate retained in the large cellular glutamate pool represents a greater fraction of total radioactivity than in the blood and leads to a total tumor time-activity curve that is only marginally different than the total tumor time-activity curve after GLS inhibition. Data from blood and tissue analysis were used to test a hypothetical kinetic model for [5-11C] glutamine, which provided estimates of glutamate and glutamate tumor tissue concentrations and tumor GLS activity that agree with direct measurements in the animal models. Conclusion: The study of [ 5-11C] glutamine in breast cancer models leads to kinetic insight that are in line with biochemical studies and direct measurement taken for tumor tissue obtained from the animal models. Retention of metabolized [5-11C] glutamine as [5-11C] glutamate in a large tissue pool in tumors with high GLS activity makes non-invasive inference of GLS activity challenging without additional ex vivo analysis, supporting the use of non-metabolized glutamine analogs such as such as [18F](2S,4R)4-fluoroglutamine ([18F]4F-Gln) to infer in vivo glutamine metabolism. Citation Format: Christopher Hensley, Prashanth Padakanti, Hoon Choi, Christina Dulal, Hsiaojiu Lee, Austin Pantel, Rong Zhou, David Mankoff. Kinetic Analysis of [5-11C] glutamine PET tracer data in breast cancer: Preclinical studies in mouse models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-15-11.

Abstract PO3-26-06: A self-replicating RNA Precision Immunotherapeutic for Overcoming Resistance to Endocrine Therapy in Estrogen Receptor Positive Breast Cancer (ER+BC)

Abstract Advanced or metastatic ER+HER2- breast cancer (ER+BC) is an incurable disease. Standard 1L therapy (SOC) utilizes endocrine blockade with a nonsteroidal aromatase inhibitor (NSAI) combined with a CDK4/6 inhibitor (CDK4/6i). While most patients respond and disease remains controlled for over 2 years (mPFS ~25-28 months), virtually all patients progress over time, requiring more toxic therapies with decreasing periods of disease control. Prolonging the time that the tumor remains under endocrine control is therefore of significant medical value. The most common molecular drivers of endocrine resistance are well characterized and include mutations in ESR1 and bypass mechanisms in select signaling pathways. RBI-1000 is a self-replicating RNA (srRNA) precision immunotherapeutic to generate robust immunity directed against acquired resistance mutations that develop in ER+ BC in response to endocrine blockade by NSAI. RBI-1000 includes 6 on-target mutations within the estrogen receptor ligand binding domain, and bypass mutations either in the form of activating mutations in PI3K kinase or amplifications of HER2/HER3. Precision immunotherapy (PIO), combined with SOC targeted therapy, educates the patient’s immune system to eliminate tumor cells that express acquired resistance mutations. Thus, the tumor is in a lose-lose situation where it is either eliminated by the targeted therapy, or if it develops acquired resistance mutations to the targeted therapy, it is killed by the PIO. Specifically for endocrine resistance, RBI-1000 PIO offers an attractive therapeutic approach to prevent/delay endocrine resistance specifically, by generating an effective immune response against those tumor cells that express the resistance-associated molecules that arise while on NSAI+CDK4/6i. The ability to encode multiple targets in a single therapeutic srRNA molecule, alongside the prolonged expression of srRNA in host cells (>49 days) creates a versatile and powerful cancer drug development platform. srRNA precision immunotherapeutics are delivered intramuscularly (i.e. a jab in the non-dominant deltoid) and generate an effective immune response via the same pathways and mechanisms as a traditional vaccine. We have demonstrated that this srRNA encapsulated in a lipid nanoparticle primes polyfunctional CD4 and CD8 T cells and antibody responses, leading to tumor growth inhibition and improved survival in a mouse model expressing the targeted acquired resistance mutations. Priming of T cells against acquired mutations is also confirmed in human HLA-transgenic mice. The immune cell-mediated elimination of clones expressing the acquired resistance mutations is predicted to prolong endocrine control of ER+BC, in an analogous manner to small molecule or monoclonal antibody targeted therapies, but with a more favorable dosing and adverse event profile due to precise immunologic targeting and no DDI. RBI-1000 is anticipated to enter clinical studies in the first half of 2024. Citation Format: Zelanna Goldberg, Christian Maine, Gabrielle Dailey, Christine Domingo, Gaelle Picarda, Hunter Little, Annie Chou, Jessica Sparks, Darina Spasova, Shiegeki Miyake-Stoner, Zachary C Hartman, Christopher Rabiola, Erika Crosby, Herbert Lyerly, Nathaniel Wang, Parinaz Aliahmad. A self-replicating RNA Precision Immunotherapeutic for Overcoming Resistance to Endocrine Therapy in Estrogen Receptor Positive Breast Cancer (ER+BC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-06.

Abstract PO5-04-02: Combination of trastuzumab competing and non-competing fully human internalizing anti-Her2 monoclonal antibodies drug conjugates increases their inhibitory effect on the growth of HER2 positive metastatic breast cancer cells

Abstract The human epidermal growth factor receptor (HER) family of receptors plays a key role in proliferation stimulator for several human cancers. In particular, HER2 is overexpressed in 15-30% of breast cancers with or without gene amplification with prognostic and predictive implications. HER2 overexpression is associated with shorter disease-free and overall survivals, resistance to hormonal agents and increased risk of brain metastasis. Trastuzumab is a humanized anti-HER2 monoclonal antibody that binds to domain IV of HER2 extracellular domain and blocks its signaling. More recently, two antibody drug conjugates using trastuzumab have been approved: Ado-Trastuzumab-Emtansine (Kadcyla) consisting of trastuzumab conjugated to the drug mertansine DM1 and fam-trastuzumab-deruxtecan-nhki (Enhertu to deliver the topoisomerase inhibitor deruxtecan. We are reporting here the development of fully human internalizing anti-HER2 antibodies with distinct epitopes on the HER2protein which compete or not with trastuzumab for binding to HER2. These monoclonal antibodies have been developed by immunizing fully human (TC-Mab mouse) mice with recombinant HER2 protein. After production of hybridoma secreting fully human immunoglobulins, the screening process included competition with trastuzumab for binding to Her2 by enzyme linked immunoassay and Octet epitope binning and affinity determination as well as internalization assay. Several internalizing antibodies able to compete or not with trastuzumab and with high affinity (Kd ranging from 10−9 M to 10−12 M) were selected. Their ability to deliver a cytotoxic payload in HER2 overexpressing breast cancer cells such as SKBR3, BT474 and AU565 was next investigated. Two groups of antibodies: one competing with trastuzumab and the other one non-competing were further characterized. Data related to these antibodies’ biochemical characteristics as well as their ability to inhibit proliferation in vitro and in vivo in mouse xenografts studies will be presented here. In conclusion, the use of fully human mice to develop monoclonal antibodies provides a powerful and attractive approach to develop fully human monoclonal antibodies against cancer targets by-passing the need for humanization and affinity maturation of antibodies. This work is supported by 5R44CA224718 SBIR grant from the National Cancer Institute. Citation Format: Ginette Serrero, Binbin Yue, Jianping Dong, Jun Hayashi. Combination of trastuzumab competing and non-competing fully human internalizing anti-Her2 monoclonal antibodies drug conjugates increases their inhibitory effect on the growth of HER2 positive metastatic breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-04-02.

Inhibition of caspase pathways limits CD4+ T cell loss and restores host anti-retroviral function in HIV-1 infected humanized mice with augmented lymphoid tissue.

The study of HIV infection and pathogenicity in physical reservoirs requires a biologically relevant model. The human immune system (HIS) mouse is an established model of HIV infection, but defects in immune tissue reconstitution remain a challenge for examining pathology in tissues. We utilized exogenous injection of the human recombinant FMS-like tyrosine kinase 3 ligand (rFLT-3L) into the hematopoietic stem cell (HSC) cord blood HIS mouse model to significantly expand the total area of lymph node (LN) and the number of circulating human T cells. The results enabled visualization and quantification of HIV infectivity, CD4 T cell depletion and other measures of pathogenesis in the secondary lymphoid tissues of the spleen and LN. Treatment with the Caspase-1/4 inhibitor VX-765 limited CD4+ T cell loss in the spleen and reduced viral load in both the spleen and axillary LN. In situ hybridization further demonstrated a decrease in viral RNA in both the spleen and LN. Transcriptomic analysis revealed that in vivo inhibition of caspase-1/4 led to an upregulation in host HIV restriction factors including SAMHD1 and APOBEC3A. These findings highlight the use of rFLT-3L to augment human immune system characteristics in HIS mice to support investigations of HIV pathogenesis and test host directed therapies, though further refinements are needed to further augment LN architecture and cellular populations. The results further provide in vivo evidence of the potential to target inflammasome pathways as an avenue of host-directed therapy to limit immune dysfunction and virus replication in tissue compartments of HIV+ persons.

Abstract PO4-06-11: Development of fully human anti-progranulin monoclonal antibodies as inhibitors of triple negative breast cancer cell growth and tumor formation

Abstract Triple negative breast cancer (TNBC) is characterized by invasiveness and poor survival. Identifying novel TNBC targeted therapies to potentiate standard of care (SOC) therapy, is an unmet need. In the past 4 years, several targeted therapies for TNBC have been approved by the Food and Drug Administration (FDA). However, the identification of targeted therapies against other biological drivers of TNBC remains important. Progranulin (PGRN/GP88) is a biological driver of tumorigenesis, survival, and drug resistance in breast cancer. PGRN/GP88 tissue expression is an independent prognostic factor of recurrence while elevated serum PGRN/GP88 level is associated with poor outcomes such as progression of disease and shortened survival. PGRN/GP88 expression is elevated in 30% TNBC. The importance of inhibiting PGRN/GP88 effect on the proliferation and tumor growth of triple negative breast cancer cells was previously established. In the current study, in association with Precision Antibody, we developed anti-PGRN/GP88 fully human monoclonal antibodies by immunizing TC transgenic humanized mice with recombinant human progranulin. Within 60 days since the start of immunization, several fully human monoclonal antibodies were successfully developed, characterized by several functional assays including neutralization, inhibition of PGRN/GP88 cell surface binding, proliferation, migration in MDA-MB-231 cells in vitro and in vivo. Results: The inhibition of PGRN/GP88 action by fully human monoclonal antibodies inhibited proliferation and migration in a dose-and time-dependent fashion in TNBC cells. Octet analysis determined Kd in the range of 10−11 M to 10−10 M. Transwell assay showed that anti-PGRN treatment inhibited migration In vivo xenograft studies with MDA-MB-231 cells injected in athymic nude mice showed that several fully human anti-PGRN antibodies inhibited tumor growth when compared to antibody control treated mice. In vivo dose response of AG01 in MDAMB-231 tumor bearing mice will be provided. Conclusion: PGRN/GP88 represents a therapeutic target for TNBC with two companion diagnostics (tissue test and ELISA to measure GP88 circulating levels). The use of the TC transgenic mice allows for the rapid development of fully human high affinity monoclonal antibodies bypassing the need for chimerization or humanization of mouse monoclonal antibodies. The development of fully human anti-progranulin antibodies provides novel targeted therapeutic option for TNBC. This work is supported by a an SBIR grant 5R44 CA 224718 from the National Cancer Institute to GS. Citation Format: Ginette Serrero, Jianping Dong, Jun Hayashi, Chun Dong. Development of fully human anti-progranulin monoclonal antibodies as inhibitors of triple negative breast cancer cell growth and tumor formation [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-11.

Development and therapeutic evaluation of 5D3(CC-MLN8237)3.2 antibody-theranostic conjugates for PSMA-positive prostate cancer therapy

Prostate cancer (PC) is an aggressive cancer that can progress rapidly and eventually become castrate-resistant prostate cancer (CRPC). Stage IV metastatic castrate-resistant prostate cancer (mCRPC) is an incurable late-stage cancer type with a low 5-year overall survival rate. Targeted therapeutics such as antibody-drug conjugates (ADCs) based on high-affinity monoclonal antibodies and potent drugs conjugated via smart linkers are being developed for PC management. Conjugating further with in vitro or in vivo imaging agents, ADCs can be used as antibody-theranostic conjugates (ATCs) for diagnostic and image-guided drug delivery. In this study, we have developed a novel ATC for PSMA (+) PC therapy utilizing (a) anti-PSMA 5D3 mAb, (b) Aurora A kinase inhibitor, MLN8237, and (c) for the first time using tetrazine (Tz) and trans-cyclooctene (TCO) click chemistry-based conjugation linker (CC linker) in ADC development. The resulting 5D3(CC-MLN8237)3.2 was labeled with suitable fluorophores for in vitro and in vivo imaging. The products were characterized by SDS-PAGE, MALDI-TOF, and DLS and evaluated in vitro by optical imaging, flow cytometry, and WST-8 assay for cytotoxicity in PSMA (+/−) cells. Therapeutic efficacy was determined in human PC xenograft mouse models following a designed treatment schedule. After the treatment study animals were euthanized, and toxicological studies, complete blood count (CBC), blood clinical chemistry analysis, and H&E staining of vital organs were conducted to determine side effects and systemic toxicities. The IC50 values of 5D3(CC-MLN8237)3.2-AF488 in PSMA (+) PC3-PIP and PMSA (−) PC3-Flu cells are 8.17 nM and 161.9 nM, respectively. Pure MLN8237 shows 736.9 nM and 873.4 nM IC50 values for PC3-PIP and PC3-Flu cells, respectively. In vivo study in human xenograft mouse models confirmed high therapeutic efficacy of 5D3(CC-MLN8237)3.2-CF750 with significant control of PSMA (+) tumor growth with minimal systemic toxicity in the treated group compared to PSMA (−) treated and untreated groups. Approximately 70% of PSMA (+) PC3-PIP tumors did not exceed the threshold of the tumor size in the surrogate Kaplan-Meyer analysis. The novel ATC successfully controlled the growth of PSMA (+) tumors in preclinical settings with minimal systemic toxicities. The therapeutic efficacy and favorable safety profile of novel 5D3(CC-MLN8237)3.2 ATC demonstrates their potential use as a theranostic against aggressive PC.

Development of a dual channel detection system for pan-genotypic simultaneous quantification of hepatitis B and delta viruses

Hepatitis B virus (HBV) infection remains a major public health problem and, in associated co-infection with hepatitis delta virus (HDV), causes the most severe viral hepatitis and accelerated liver disease progression. As a defective satellite RNA virus, HDV can only propagate in the presence of HBV infection, which makes HBV DNA and HDV RNA the standard biomarkers for monitoring the virological response upon antiviral therapy, in co-infected patients. Although assays have been described to quantify these viral nucleic acids in circulation independently, a method for monitoring both viruses simultaneously is not available, thus hampering characterization of their complex dynamic interactions. Here, we describe the development of a dual fluorescence channel detection system for pan-genotypic, simultaneous quantification of HBV DNA and HDV RNA through a one-step quantitative PCR. The sensitivity for both HBV and HDV is about 10 copies per microliter without significant interference between these two detection targets. This assay provides reliable detection for HBV and HDV basic research in vitro and in human liver chimeric mice. Preclinical validation of this system on serum samples from patient on or off antiviral therapy also illustrates a promising application that is rapid and cost-effective in monitoring HBV and HDV viral loads simultaneously.

Extravascular administration of IGF1R antagonists protects against aortic aneurysm in rodent and porcine models.

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease. We identified plasma insulin-like growth factor 1 (IGF1) as an independent risk factor in patients with AAA by correlating plasma IGF1 with risk. Smooth muscle cell- or fibroblast-specific knockout of Igf1r, the gene encoding the IGF1 receptor (IGF1R), attenuated AAA formation in two mouse models of AAA induced by angiotensin II infusion or CaCl2 treatment. IGF1R was activated in aortic aneurysm samples from human patients and mice with AAA. Systemic administration of IGF1C, a peptide fragment of IGF1, 2 weeks after disease development inhibited AAA progression in mice. Decreased AAA formation was linked to competitive inhibition of IGF1 binding to its receptor by IGF1C and modulation of downstream alpha serine/threonine protein kinase (AKT)/mammalian target of rapamycin signaling. Localized application of an IGF1C-loaded hydrogel was developed to reduce the side effects observed after systemic administration of IGF1C or IGF1R antagonists in the CaCl2-induced AAA mouse model. The inhibitory effect of the IGF1C-loaded hydrogel administered at disease onset on AAA formation was further evaluated in a guinea pig-to-rat xenograft model and in a sheep-to-minipig xenograft model of AAA formation. The therapeutic efficacy of IGF1C for treating AAA was tested through extravascular delivery in the sheep-to-minipig model with AAA established for 2 weeks. Percutaneous injection of the IGF1C-loaded hydrogel around the AAA resulted in improved vessel flow dynamics in the minipig aorta. These findings suggest that extravascular administration of IGF1R antagonists may have translational potential for treating AAA.

Neoadjuvant radiotherapy in ER+, HER2+, and triple-negative -specific breast cancer based humanized tumor mice enhances anti-PD-L1 treatment efficacy

Pre-operative radiation therapy is not currently integrated into the treatment protocols for breast cancer. However, transforming immunological “cold” breast cancers by neoadjuvant irradiation into their “hot” variants is supposed to elicit an endogenous tumor immune defense and, thus, enhance immunotherapy efficiency. We investigated cellular and immunological effects of sub-lethal, neoadjuvant irradiation of ER pos., HER2 pos., and triple-negative breast cancer subtypes in-vitro and in-vivo in humanized tumor mice (HTM). This mouse model is characterized by a human-like immune system and therefore facilitates detailed analysis of the mechanisms and efficiency of neoadjuvant, irradiation-induced “in-situ vaccination”, especially in the context of concurrently applied checkpoint therapy. Similar to clinical appearances, we observed a gradually increased immunogenicity from the luminal over the HER2-pos. to the triple negative subtype in HTM indicated by an increasing immune cell infiltration into the tumor tissue. Anti-PD-L1 therapy divided the HER2-pos. and triple negative HTM groups into responder and non-responder, while the luminal HTMs were basically irresponsive. Irradiation alone was effective in the HER2-pos. and luminal subtype-specific HTM and was supportive for overcoming irresponsiveness to single anti-PD-L1 treatment. The treatment success correlated with a significantly increased T cell proportion and PD-1 expression in the spleen. In all subtype-specific HTM combination therapy proved most effective in diminishing tumor growth, enhancing the immune response, and converted non-responder into responder during anti-PD-L1 therapy. In HTM, neoadjuvant irradiation reinforced anti-PD-L1 checkpoint treatment of breast cancer in a subtype –specific manner. According to the “bench to bedside” principle, this study offers a vital foundation for clinical translating the use of neoadjuvant irradiation in the context of checkpoint therapy.

Preclinical imaging evaluation of a bispecific antibody targeting hPD1/CTLA4 using humanized mice

BackgroundThe lack of an efficient way to screen patients who are responsive to immunotherapy challenges PD1/CTLA4-targeting cancer treatment. Immunotherapeutic efficacy cannot be clearly determined by peripheral blood analyses, tissue gene markers or CT/MR value. Here, we used a radionuclide and imaging techniques to investigate the novel dual targeted antibody cadonilimab (AK104) in PD1/CTLA4-positive cells in vivo. MethodsFirst, humanized PD1/CTLA4 mice were purchased from Biocytogen Pharmaceuticals (Beijing) Co., Ltd. to express hPD1/CTLA4 in T-cells. Then, mouse colon cancer MC38-hPD-L1 cell xenografts were established in humanized mice. A bispecific antibody targeting PD1/CTLA4 (AK104) was labeled with radio-nuclide iodine isotopes. Immuno-PET/CT imaging was performed using a bispecific monoclonal antibody (mAb) probe 124I-AK104, developed in-house, to locate PD1+/CTLA4+ tumor-infiltrating T cells and monitor their distribution in mice to evaluate the therapeutic effect. ResultsThe 124I-AK104 dual-antibody was successfully constructed with ideal radiochemical characteristics, in vitro stability and specificity. The results of immuno-PET showed that 124I-AK104 revealed strong hPD1/CTLA4-positive responses with high specificity in humanized mice. High uptake of 124I-AK104 was observed not only at the tumor site but also in the spleen. Compared with PD1- or CTLA4-targeting mAb imaging, 124I-AK104 imaging had excellent standard uptake values at the tumor site and higher tumor to nontumor (T/NT) ratios. ConclusionsThe results demonstrated the potential of translating 124I-AK104 into a method for screening patients who benefit from immunotherapy and the efficacy, as well as the feasibility, of this method was verified by immuno-PET imaging of humanized mice.

Lineage tracing reveals a novel PDGFRβ+ satellite cell subset that contributes to myo-regeneration of chronically injured rotator cuff muscle

Massive rotator cuff (RC) tendon tears are associated with progressive fibro-adipogenesis and muscle atrophy that altogether cause shoulder muscle wasting. Platelet derived growth factor β (PDGFRβ) lineage cells, that co-express PDGFRα have previously been shown to directly contribute to scar formation and fat accumulation in a mouse model of irreversible tendon and nerve transection (TTDN). Conversely, PDGFRβ+ lineage cells have also been  shown to be myogenic in cultures and in other models of skeletal muscle injury. We therefore hypothesized that PDGFRβ demarcates two distinct RC residing subpopulations, fibro-adipogenic and myogenic, and aimed to elucidate the identity of the PDGFRβ myogenic precursors and evaluate their contribution, if any, to RC myo-regeneration. Lineage tracing revealed increasing contribution of PDGFRβ+ myo-progenitors to the formation of GFP+ myofibers, which were the most abundant myofiber type in regenerated muscle at 2 weeks post-TTDN. Muscle regeneration preceded muscle atrophy and both advanced from the lateral site of tendon transection to the farthest medial region. GFP+/PDGFRβ+Sca-1−lin−CXCR4+Integrin-β1+ marked a novel subset of satellite cells with confirmed myogenic properties. Further studies are warranted to identify the existence of PDGFRβ+ satellite cells in human and other mouse muscles and to define their myo-regenerative potential following acute and chronic muscle injury.

Targeted depletion of PD-1-expressing cells induces immune tolerance through peripheral clonal deletion.

Thymic negative selection of the T cell receptor (TCR) repertoire is essential for establishing self-tolerance and acquired allograft tolerance following organ transplantation. However, it is unclear whether and how peripheral clonal deletion of alloreactive T cells induces transplantation tolerance. Here, we establish that programmed cell death protein 1 (PD-1) is a hallmark of alloreactive T cells and is associated with clonal expansion after alloantigen encounter. Moreover, we found that diphtheria toxin receptor (DTR)-mediated ablation of PD-1+ cells reshaped the TCR repertoire through peripheral clonal deletion of alloreactive T cells and promoted tolerance in mouse transplantation models. In addition, by using PD-1-specific depleting antibodies, we found that antibody-mediated depletion of PD-1+ cells prevented heart transplant rejection and the development of experimental autoimmune encephalomyelitis (EAE) in humanized PD-1 mice. Thus, these data suggest that PD-1 is an attractive target for peripheral clonal deletion and induction of immune tolerance.

Lithium-Doped Titanium Dioxide-Based Multilayer Hierarchical Structure for Accelerating Nerve-Induced Bone Regeneration.

Despite considerable advances in artificial bone tissues, the absence of neural network reconstruction in their design often leads to delayed or ineffective bone healing. Hence, we propose a multilayer hierarchical lithium (Li)-doped titanium dioxide structure, constructed through microarc oxidation combined with alkaline heat treatment. This structure can induce the sustained release of Li ions, mimicking the environment of neurogenic osteogenesis characterized by high brain-derived neurotrophic factor (BDNF) expression. During in vitro experiments, the structure enhanced the differentiation of Schwann cells (SCs) and the growth of human umbilical vein endothelial cells (HUVECs) and mouse embryo osteoblast progenitor cells (MC3T3-E1). Additionally, in a coculture system, the SC-conditioned media markedly increased alkaline phosphatase expression and the formation of calcium nodules, demonstrating the excellent potential of the material for nerve-induced bone regeneration. In an in vivo experiment based on a rat distal femoral lesion model, the structure substantially enhanced bone healing by increasing the density of the neural network in the tissue around the implant. In conclusion, this study elucidates the neuromodulatory pathways involved in bone regeneration, providing a promising method for addressing bone deformities.

Oral Sex as a Potential Route for Toxoplasma Gondii Transmission: Experiment with Human Semen and Laboratory Mice Model.

Toxoplasma gondii is one of the most widespread parasites in the human population globally. Several modes of its transmission have been proposed: some are well-researched and confirmed, others remain unconfirmed. One unconfirmed hypothesis pertains to potential transmission of Toxoplasma gondii via oral sex (fellatio) in humans. A recent study found tissue cysts in the semen of men with latent toxoplasmosis. Therefore, we aimed to test the hypothesis of Toxoplasma gondii transmission through oral sex experimentally. Eighty-two laboratory mice were orally administered semen samples from 41 men with latent toxoplasmosis. These semen samples were examined for the presence of Toxoplasma gondii DNA using PCR. We detected Toxoplasma gondii DNA in three of the 41 semen samples from men with latent toxoplasmosis. Oral administration of semen samples to laboratory mice did not result in parasite transmission. We have not demonstrated the transmission of Toxoplasma to mice by oral exposure to semen from infected men. While this does not conclusively rule out the possibility of such transmission in humans, the results suggest that, if it does occur, this mode of transmission is likely infrequent.

Downregulation of HMGCS2 mediated AECIIs lipid metabolic alteration promotes pulmonary fibrosis by activating fibroblasts

BackgroundAbnormal lipid metabolism has recently been reported as a crucial signature of idiopathic pulmonary fibrosis (IPF). However, the origin and biological function of the lipid and possible mechanisms of increased lipid content in the pathogenesis of IPF remains undetermined.MethodsOil-red staining and immunofluorescence analysis were used to detect lipid accumulation in mouse lung fibrosis frozen sections, Bleomycin-treated human type II alveolar epithelial cells (AECIIs) and lung fibroblast. Untargeted Lipid omics analysis was applied to investigate differential lipid species and identified LysoPC was utilized to treat human lung fibroblasts and mice. Microarray and single-cell RNA expression data sets identified lipid metabolism-related differentially expressed genes. Gain of function experiment was used to study the function of 3-hydroxy-3-methylglutaryl-Coa Synthase 2 (HMGCS2) in regulating AECIIs lipid metabolism. Mice with AECII-HMGCS2 high were established by intratracheally delivering HBAAV2/6-SFTPC- HMGCS2 adeno-associated virus. Western blot, Co-immunoprecipitation, immunofluorescence, site-directed mutation and flow cytometry were utilized to investigate the mechanisms of HMGCS2-mediated lipid metabolism in AECIIs.ResultsInjured AECIIs were the primary source of accumulated lipids in response to Bleomycin stimulation. LysoPCs released by injured AECIIs could activate lung fibroblasts, thus promoting the progression of pulmonary fibrosis. Mechanistically, HMGCS2 was decreased explicitly in AECIIs and ectopic expression of HMGCS2 in AECIIs using the AAV system significantly alleviated experimental mouse lung fibrosis progression via modulating lipid degradation in AECIIs through promoting CPT1A and CPT2 expression by interacting with PPARα.ConclusionsThese data unveiled a novel etiological mechanism of HMGCS2-mediated AECII lipid metabolism in the genesis and development of pulmonary fibrosis and provided a novel target for clinical intervention.

Chondrocyte Invasion May Be a Mechanism for Persistent Staphylococcus Aureus Infection In Vitro.

Recurrent bone and joint infection with Staphylococcus aureus is common. S. aureus can invade and persist in osteoblasts and fibroblasts, but little is known about this mechanism in chondrocytes. If S. aureus were able to invade and persist within chondrocytes, this could be a difficult compartment to treat. Can S. aureus infiltrate and persist intracellularly within chondrocytes in vitro? Cell lines were cultured in vitro and infected with S. aureus. Human chondrocytes (C20A4) were compared with positive controls of human osteoblasts (MG63) and mouse fibroblasts (NIH3T3), which have previously demonstrated S. aureus invasion and persistence (human fibroblasts were not available to us). Six replicates per cell type were followed for 6 days after infection. Cells were treated daily with antibiotic media for extracellular killing. To determine whether S. aureus can infiltrate chondrocytes, fluorescence microscopy was performed to qualitatively assess the presence of intracellular bacteria, and intracellular colony-forming units (CFU) were enumerated 2 hours after infection. To determine whether S. aureus can persist within chondrocytes, intracellular CFUs were enumerated from infected host cells each day postinfection. S. aureus invaded human chondrocytes (C20A4) at a level (2.8 x 105 ± 5.5 x 104 CFUs/mL) greater than positive controls of human osteoblasts (MG63) (9.5 x 102 ± 2.5 x 102 CFUs/mL; p = 0.01) and mouse fibroblasts (NIH3T3) (9.1 x 104 ± 2.5 x 104 CFUs/mL; p = 0.02). S. aureus also persisted within human chondrocytes (C20A4) for 6 days at a level (1.4 x 103 ± 5.3 x 102 CFUs/mL) greater than that of human osteoblasts (MG63) (4.3 x 102 ± 3.5 x 101 CFUs/mL; p = 0.02) and mouse fibroblasts (NIH3T3) (0 CFUs/mL; p < 0.01). S. aureus was undetectable within mouse fibroblasts (NIH3T3) after 4 days. There were 0 CFUs yielded from cell media, confirming extracellular antibiotic treatment was effective. S. aureus readily invaded human chondrocytes (C20A4) in vitro and persisted viably for 6 days after infection, evading extracellular antibiotics. Chondrocytes demonstrated a greater level of intracellular invasion and persistence by S. aureus than positive control human osteoblast (MG63) and mouse fibroblast (NIH3T3) cell lines. Chondrocyte invasion and persistence may contribute to recurrent bone and joint infections. Additional research should assess longer periods of persistence and whether this mechanism is present in vivo.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A promotes the inclusion of amyloid precursor protein exon 7

Extracellular amyloid plaques made of Amyloid-β (Aβ) derived from amyloid precursor protein (APP) is one of the major neuropathological hallmarks of Alzheimer’s disease (AD). There are three major isoforms of APP, APP770, APP751, and APP695 generated by alternative splicing of exons 7 and 8. Exon 7 encodes the Kunitz protease inhibitor (KPI) domain. Its inclusion generates APP isoforms containing KPI, APPKPI+, which is elevated in AD and Down syndrome (DS) brains and associated with increased Aβ deposition. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) phosphorylates many splicing factors and regulates the alternative splicing of pre-mRNA. It is upregulated in DS and AD brain. However, it is not yet clear whether Dyrk1A could regulate APP alternative splicing. In the present study, we overexpressed or knocked down Dyrk1A in cultured cells and observed that Dyrk1A promoted the inclusion of both APP exons 7 and 8. Moreover, a significant increase in APP exon7 inclusion was also detected in the forebrain and hippocampus of human Dyrk1A transgenic mice – Tg/Dyrk1A. Screening for splicing factors regulated by Dyrk1A revealed that serine/arginine-rich protein 9G8 inhibited APP exon7 inclusion and interacted with APP pre-mRNA. In vitro, expression of exon 7 facilitated APP cleavage. In human Dyrk1A transgenic mice, we also found an increase in Aβ production. These findings suggest that Dyrk1A inhibits the splicing factor 9G8 and promotes APP exon 7 inclusion, leading to more APPKPI+ expression and APP cleavage and potentially contributing to Aβ production in vivo.

Ischemia does not provoke the full immune training repertoire in human cardiac fibroblasts.

Trained immunity of monocytes, endothelial, and smooth muscle cells augments the cytokine response to secondary stimuli. Immune training is characterized by stabilization of hypoxia-inducible factor (HIF)-1α, mTOR activation, and aerobic glycolysis. Cardiac fibroblast (CF)-myofibroblast transition upon myocardial ischemia/reperfusion (I/R) features epigenetic and metabolic adaptations reminiscent of trained immunity. We assessed the impact of I/R on characteristics of immune training in human CF and mouse myocardium. I/R was simulated in vitro with transient metabolic inhibition. CF primed with simulated I/R or control buffer were 5days later re-stimulated with Pam3CSK for 24h. Mice underwent transient left anterior descending artery occlusion or sham operation with reperfusion for up to 5days. HIF-regulated metabolic targets and cytokines were assessed by qPCR, immunoblot, and ELISA and glucose consumption, lactate release, and lactate dehydrogenase (LDH) by chromogenic assay. Simulated I/R increased HIF-1α stabilization, mTOR phosphorylation, glucose consumption, lactate production, and transcription of PFKB3 and F2RL3, a HIF-regulated target gene, in human CF. PGK1 and LDH mRNAs were suppressed. Intracellular LDH transiently increased after simulated I/R, and extracellular LDH showed sustained elevation. I/R priming increased abundance of pro-caspase-1, auto-cleaved active caspase-1, and the expression and secretion of interleukin (IL)-1β, but did not augment Pam3CSK-stimulated cytokine transcription or secretion. Myocardial I/R in vivo increased abundance of HIF-1 and the precursor and cleaved forms of caspase-1, caspase-11, and caspase-8, but not of LDH-A or phospho-mTOR. I/R partially reproduces features of immune training in human CF, specifically HIF-1α stabilization, aerobic glycolysis, mTOR phosphorylation, and PFKB3 transcription. I/R does not augment PGK1 or LDH expression or the cytokine response to Pam3CSK. Regulation of PAR4 and inflammasome caspases likely occurs independently of an immune training repertoire.