Target Cell Type Research Articles

Exosomes for Wound Healing: Purification Optimization and Identification of Bioactive Components.

Human mesenchymal stem cell exosomes have been shown to promote cutaneous wound healing. Their bioactivity is most often attributed to their protein and nucleic acid components, while the function of exosomal lipids remains comparatively unexplored. This work specifically assesses the involvement of lipids and the transmembrane enzyme CD73 in the exosomes’ biological activity in stimulating the cutaneous wound healing process. Since exosome preparation processes are not harmonized yet, certain production and purification parameters are first systematically investigated, enabling the optimization of a standardized protocol delivering high exosome integrity, yield, and purity. An in situ enzymatic assay is introduced to specifically assess the vesicle functionality, and quantitative proteomics is employed to establish the cell culture conditions yielding a stable exosome protein profile. Using a combination of in vitro approaches, CD73 and constitutional lipids of HPV‐16 E6/E7 transformed human bone marrow stromal cell‐derived exosomes are identified as key bioactive components promoting the exosome‐driven acceleration of processes required for wound repair. A pilot wound healing study in mice indeed suggests a role of lipids in the exosomes’ biological activity. Strikingly, the extent of the bioactivity of these exosomal components is found to be dependent on the target cell type.

Single-cell epigenomic analyses implicate candidate causal variants at inherited risk loci for Alzheimer's and Parkinson's diseases.

Genome-wide association studies (GWAS) of neurological diseases have identified thousands of variants associated with disease phenotypes. However, the majority of these variants do not alter coding sequences, making it difficult to assign their function. Here, we present a multi-omic epigenetic atlas of the adult human brain through profiling of single-cell chromatin accessibility landscapes and three-dimensional (3D) chromatin interactions of diverse adult brain regions across a cohort of cognitively healthy individuals. We developed a machine-learning classifier to integrate this multi-omic framework and predict dozens of functional single-nucleotide polymorphisms (SNPs) for Alzheimer’s disease (AD) and Parkinson’s disease (PD), nominating target genes and cell types for previously orphaned GWAS loci. Moreover, we dissected the complex inverted haplotype of the MAPT (encoding tau) PD risk locus, identifying putative ectopic regulatory interactions in neurons that may mediate this disease association. This work expands our understanding of inherited variation and provides a roadmap for the epigenomic dissection of causal regulatory variation in disease.

Analysis of KSHV B lymphocyte lineage tropism in human tonsil reveals efficient infection of CD138+ plasma cells

Despite 25 years of research, the basic virology of Kaposi Sarcoma Herpesviruses (KSHV) in B lymphocytes remains poorly understood. This study seeks to fill critical gaps in our understanding by characterizing the B lymphocyte lineage-specific tropism of KSHV. Here, we use lymphocytes derived from 40 human tonsil specimens to determine the B lymphocyte lineages targeted by KSHV early during de novo infection in our ex vivo model system. We characterize the immunological diversity of our tonsil specimens and determine that overall susceptibility of tonsil lymphocytes to KSHV infection varies substantially between donors. We demonstrate that a variety of B lymphocyte subtypes are susceptible to KSHV infection and identify CD138+ plasma cells as a highly targeted cell type for de novo KSHV infection. We determine that infection of tonsil B cell lineages is primarily latent with few lineages contributing to lytic replication. We explore the use of CD138 and heparin sulfate proteoglycans as attachment factors for the infection of B lymphocytes and conclude that they do not play a substantial role. Finally, we determine that the host T cell microenvironment influences the course of de novo infection in B lymphocytes. These results improve our understanding of KSHV transmission and the biology of early KSHV infection in a naïve human host, and lay a foundation for further characterization of KSHV molecular virology in B lymphocyte lineages.

Effects of mild steel welding fume particles on pulmonary epithelial inflammation and endothelial activation

Welders have an increased risk for cardiovascular disease (CVD) following exposure to welding fumes. The underlying mechanisms are largely unknown; however, oxidative stress, systemic inflammation, and endothelial dysfunction have been suggested as contributing factors to particle-induced CVD. We investigated effects of mild steel welding fume (MSWF) on three target cell types: macrophages, pulmonary epithelial, and vascular endothelial cells. Cells were exposed to MSWF at nontoxic doses for 6 h/day, for five consecutive days. The expression of 40 genes involved in inflammation, fibrosis, and endothelial activation was analyzed. Moreover, changes in the reactive oxygen species production and migration capacity of cells were assessed. The expression of matrix metallopeptidase 1 (MMP1) was induced in both epithelial and endothelial cells following repeated exposure to MSWF. Although MMP1 is important in inflammatory responses in vivo, this effect was not concurrent with changes in the inflammatory status, cell proliferation, and migration capacities, nor did it induce oxidative stress in the cells. Thus, repeated exposure with low doses of MSWF was sufficient neither for inducing inflammatory stress in epithelial cells and macrophages nor for endothelial activation, and higher concentrations of MSWF or the nonparticle fraction of MSWF may be critical in causing the increased risk of CVD observed among welders.

Novel 199 base pair NEFH promoter drives expression in retinal ganglion cells

Retinal ganglion cells (RGCs) are known to be involved in several ocular disorders, including glaucoma and Leber hereditary optic neuropathy (LHON), and hence represent target cells for gene therapies directed towards these diseases. Restricting gene therapeutics to the target cell type in many situations may be preferable compared to ubiquitous transgene expression, stimulating researchers to identify RGC-specific promoters, particularly promoter sequences that may also be appropriate in size to fit readily into recombinant adeno associated viral (AAV) vectors, the vector of choice for many ocular gene therapies. In the current study we analysed EGFP expression driven by various sequences of the putative human NEFH promoter in order to define sequences required for preferential expression in RGCs. EGFP expression profiles from four different potential NEFH promoter constructs were compared in vivo in mice using retinal histology and mRNA expression analysis. Notably, two efficient promoter sequences, one comprising just 199 bp, are presented in the study.

The Role of Escherichia coli Shiga Toxins in STEC Colonization of Cattle.

Many cattle are persistently colonized with Shiga toxin-producing Escherichia coli (STEC) and represent a major source of human infections with human-pathogenic STEC strains (syn. enterohemorrhagic E. coli (EHEC)). Intervention strategies most effectively protecting humans best aim at the limitation of bovine STEC shedding. Mechanisms enabling STEC to persist in cattle are only partialy understood. Cattle were long believed to resist the detrimental effects of Shiga toxins (Stxs), potent cytotoxins acting as principal virulence factors in the pathogenesis of human EHEC-associated diseases. However, work by different groups, summarized in this review, has provided substantial evidence that different types of target cells for Stxs exist in cattle. Peripheral and intestinal lymphocytes express the Stx receptor globotriaosylceramide (Gb3 syn. CD77) in vitro and in vivo in an activation-dependent fashion with Stx-binding isoforms expressed predominantly at early stages of the activation process. Subpopulations of colonic epithelial cells and macrophage-like cells, residing in the bovine mucosa in proximity to STEC colonies, are also targeted by Stxs. STEC-inoculated calves are depressed in mounting appropriate cellular immune responses which can be overcome by vaccination of the animals against Stxs early in life before encountering STEC. Considering Stx target cells and the resulting effects of Stxs in cattle, which significantly differ from effects implicated in human disease, may open promising opportunities to improve existing yet insufficient measures to limit STEC carriage and shedding by the principal reservoir host.

Deconstructing Stepwise Fate Conversion of Human Fibroblasts to Neurons by MicroRNAs.

Cell-fate conversion generally requires reprogramming effectors to both introduce fate programs of the target cell type and erase the identity of starting cell population. Here, we reveal insights into the activity of microRNAs miR-9/9∗ and miR-124 (miR-9/9∗-124) as reprogramming agents that orchestrate direct conversion of human fibroblasts into motor neurons by first eradicating fibroblast identity and promoting uniform transition to a neuronal state in sequence. We identify KLF-family transcription factors as direct target genes for miR-9/9∗-124 and show their repression is critical for erasing fibroblast fate. Subsequent gain of neuronal identity requires upregulation of a small nuclear RNA, RN7SK, which induces accessibilities of chromatin regions and neuronal gene activation to push cells to a neuronal state. Our study defines deterministic components in the microRNA-mediated reprogramming cascade.

Gene Therapy for Hemophilia A: Where We Stand.

Hemophilia A (HA) is a hereditary hemorrhagic disease caused by a deficiency of coagulation factor VIII (FVIII) in blood plasma. Patients with HA usually suffer from spontaneous and recurrent bleeding in joints and muscles, or even intracerebral hemorrhage, which might lead to disability or death. Although the disease is currently manageable via delivery of plasma-derived or recombinant FVIII, this approach is costly, and neutralizing antibodies may be generated in a large portion of patients, which render the regimens ineffective and inaccessible. Given the monogenic nature of HA and that a slight increase in FVIII can remarkably alleviate the phenotypes, HA has been considered to be a suitable target disease for gene therapy. Consequently, the introduction of a functional F8 gene copy into the appropriate target cells via viral or nonviral delivery vectors, including gene correction through genome editing approaches, could ultimately provide an effective therapeutic method for HA patients. In this review, we discuss the recent progress of gene therapy for HA with viral and nonviral delivery vectors, including piggyBac, lentiviral and adeno-associated viral vectors, as well as new raising issues involving liver toxicity, pre-existing neutralizing antibodies of viral approach, and the selection of the target cell type for nonviral delivery.

Hepatocyte-like cells derived from human induced pluripotent stem cells using small molecules: implications of a transcriptomic study

BackgroundHepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (iPSCs) hold great promise in toxicological applications as well as in regenerative medicine. Previous efforts on hepatocyte differentiation have mostly relied on the use of growth factors (GFs) to recapitulate developmental signals under in vitro conditions. Recently, the use of small molecules (SMs) has emerged as an attractive tool to induce cell fate transition due to its superiority in terms of both quality and cost. However, HLCs derived using SMs have not been well characterized, especially on the transcriptome level.MethodsHLCs were differentiated from human iPSCs using a protocol that only involves SMs and characterized by transcriptomic analysis using whole genome microarrays.ResultsHLCs derived using the SM protocol (HLC_SM) displayed specific hepatic marker expression and demonstrated key hepatic functions. Transcriptomic analysis of the SM-driven differentiation defined a hepatocyte differentiation track and characterized the expression of some key marker genes in major stages of hepatocyte differentiation. In addition, HLC_SM were scored with CellNet, a bioinformatics tool quantifying how closely engineered cell populations resemble their target cell type, and compared to primary human hepatocytes (PHHs), adult liver tissue, fetal liver tissue, HLCs differentiated using GFs (HLC_GF), and commercially available HLCs. Similar to HLC_GF, HLC_SM displayed a mixed phenotype of fetal and adult hepatocytes and had relatively low expression of metabolic enzymes, transporters, and nuclear receptors compared to PHHs. Finally, the differentially expressed genes in HLC_SM compared to HLC_GF and to PHHs were analyzed to identify pathways and upstream transcription regulators which could potentially be manipulated to improve the differentiation of HLCs.ConclusionsOverall, the present study demonstrated the usefulness of the SM-based hepatocyte differentiation method, offered new insights into the molecular basis of hepatogenesis and associated gene regulation, and suggested ways for further improvements in hepatocyte differentiation in order to obtain more mature HLCs that could be used in toxicological studies.

The Anti-apoptotic Murine Cytomegalovirus Protein vMIA-m38.5 Induces Mast Cell Degranulation.

Mast cells (MC) represent “inbetweeners” of the immune system in that they are part of innate immunity by acting as first-line sentinels for environmental antigens but also provide a link to adaptive immunity by secretion of chemokines that recruit CD8 T cells to organ sites of infection. An interrelationship between MC and cytomegalovirus (CMV) has been a blank area in science until recently when the murine model revealed a role for MC in the resolution of pulmonary infection by murine CMV (mCMV). As to the mechanism, MC were identified as a target cell type of mCMV. Infected MC degranulate and synthesize the CC-chemokine ligand-5 (CCL-5), which is released to attract protective virus-specific CD8 T cells to infected host tissue for confining and eventually resolving the productive, cytopathogenic infection. In a step forward in our understanding of how mCMV infection of MC triggers their degranulation, we document here a critical role for the mCMV m38.5 gene product, a mitochondria-localized inhibitor of apoptosis (vMIA). We show an involvement of mCMV vMIA-m38.5 in MC degranulation by two reciprocal approaches: first, by enhanced degranulation after m38.5 gene transfection of bone marrow-derived cell culture-grown MC (BMMC) and, second, by reduced degranulation of MC in peritoneal exudate cell populations infected ex corpore or in corpore with mutant virus mCMV-Δm38.5. These studies thus reveal a so far unknown function of mCMV vMIA-m38.5 and offer a previously unconsidered but biologically relevant cell system for further analyzing functional analogies between vMIAs of different CMV species.

Abstract 6705: An integrated in vitro and in vivo approach providing insights in immune modulation

Abstract In order to exploit the complex tumour microenvironment, immunoreactivity in a multitude of cell-based assays is required to predict mode of action (MOA), pharmacodynamics, and efficacy downstream. We have developed an integrated approach starting with single cell and co-culture cell-based assays in 2D and 3D formats, followed by an in vivo set-up based on humanized mice to monitor an immunotherapeutic's interaction with specific cell types such as T cells including CAR-T cells, natural killer cells, macrophages, dendritic cells, neutrophils, and fibroblasts. Rituximab, a monoclonal antibody against CD20, is mainstay in the therapy for a broad variety of B-cell malignancies. Its MOA is not fully understood as direct signalling, complement dependent cellular cytotoxicity and antibody dependent cellular cytotoxicity (ADCC) all seem to have an impact. To elucidate the share and effect each of those mechanism has, we tested Rituximab in our immuno-oncology translational platform. Furthermore, our immuno-oncology platform was utilized to assess efficacy, bystander effects and off-target toxicity of a CD19 CAR-T cell therapy. In vitro assays for efficacy and bystander effect involved co-culture of the CAR-T cells with a mixture of non-target expressing cells and mid to high target antigen expressing cells, where T cell activation and cell death of both target cell types was quantified. Beyond efficacy the assay platforms were used to examine the influence of the therapy, antibodies or CAR-T cells, on normal human cells. These in vitro assays utilizing primary human cells from healthy tissue and/or differentiated iPCS-derived cells were used to assess cytotoxicity. The primary tissues were selected based on their potential safety risk by either expressing low levels of the target antigen or being major organs to de-risk for off-target effects. The results from our in vitro screen were validated in a xenograft in vivo system in the presence and absence of human immune cells. Four cell line derived xenografts were tested in vivo in immunocompromised mice subcutaneously and disseminated. The presence of human immune cells enhanced the antitumoral activity of Rituximab markedly, although the targeted effect of Rituximab induced already a significant prolongation in the overall survival (p< 0.005; Log-rank (Mantel-Cox) test).Over the last 30 years IO has progressed considerably with approvals for the use of various IO therapeutics including vaccines, cytokines, tumor-directed monoclonal antibodies, immune checkpoint inhibitors as well as Chimeric antigen receptor (CAR) and T cell receptor (TCR) engineered T cell therapies. Comprehensive characterization of novel IO compounds using a multitude of such in vitro and in vivo assays is essential to provide insights and understanding of the detailed mechanism of action and identify potential toxicity issues to ultimately improve translation to the clinic. Citation Format: Shilina Roman, Julia Schüler, Sanne Holt, Jeroen DeGroot, Omar Aziz, Ian Waddell. An integrated in vitro and in vivo approach providing insights in immune modulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6705.

Abstract 6257: DPX™ platform for drug delivery: Evaluation of lymph node-targeted delivery of immunomodulatory agents in DPX

Abstract Small molecule drugs and biological macromolecules are currently being investigated as therapies for various types of disorders such as cancer and infectious disease. Generally, these agents are systemically administered. However, many immunomodulatory agents are active primarily on specific immune cells such as dendritic cells or T cells. The purpose of this study was to evaluate the efficacy of the novel delivery platform, DPX, for targeted delivery of immunomodulatory agents to the lymph node. As the lymph node is the primary site for priming and activation of immune cells, this approach may improve delivery over systemic administration resulting in increased drug efficacy, the use of less drug, and reduced off-target toxicity. To evaluate the efficacy of the DPX platform for lymph node-targeted drug delivery, we used the C57Bl/6 murine model. DPX formulation of small molecule dyes allowed us to visualize the kinetics of lymph node-specific delivery by visual assessments and by flow cytometry. DPX-dye formulations demonstrated a controlled and sustained uptake of molecules from the site of injection, travelling to the regional lymph node, without significant systemic exposure. To test impacts on tumor directed immune responses, the HPV16 E7 expressing C3 tumor model was used. Novel formulations were tested in conjunction with DPX-FP (a C3 tumor-specific antigen HPV16 E749-57 conjugated to a T helper epitope). DPX-mediated delivery of low doses of cyclophosphamide (CPA) were delivered subcutaneously as a single injection. This low dose of CPA has shown to act as an immunomodulatory agent when delivered systemically over a week. Using DPX to deliver CPA resulted in a significant reduction in the number of lymph node cells. This was evident after a single administration, showing equivalent therapeutic benefit was achieved with significantly less active agent. This was observed irrespective of whether CPA was administered alone or with DPX-FP. We also evaluated DPX delivery of the checkpoint inhibitor anti-CTLA-4. We found that administration in DPX was equivalent to systemic administration by intraperitoneal injection in terms of overall survival of tumor-bearing mice and in the proportion of CD3+ T cells bound by anti-CTLA-4, with fewer administrations of antibody. This demonstrates maintenance of biological functionality of macromolecules in the DPX platform. In conclusion, DPX holds promise in terms of lymph node-targeted delivery of functional agents. This delivery is facilitated by a sustained uptake from the site of injection and can offer reduced dosing and off-target toxicity as compared to systemic delivery. Many approaches to targeted therapies have failed to provide a therapeutic index commensurate with their promise through systemic delivery, with a challenge in achieving selectivity for the target cell type. Here, we show the DPX drug delivery platform has the potential to overcome this challenge and thereby increase the efficacy of immune and cancer therapies. Citation Format: Heather Torrey, Ava Vila-Leahey, Yuchen Cen, Danielle MacKay, Alecia MacKay, Leanne Lucas, Brittany Myers, Arthvan Sharma, Douglas Vieira, Andrea Penwell, Rajkannan Rajagopalan, Marianne Stanford. DPX™ platform for drug delivery: Evaluation of lymph node-targeted delivery of immunomodulatory agents in DPX [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6257.

Passive controlled flow for Parkinson's disease neuronal cell culture in 3D microfluidic devices

Controlled flow within a lab-on-a-chip is a critical element of successfully implementing culture protocols for differentiation and maintenance of stem cell derived neurons in microfluidic devices. There have been a multitude of passive pumping technologies that have been successfully used to control the flow within a lab-on-a-chip. However, most of which were only able to generate flow for very few minutes, while the most successful ones were able to achieve around an hour of flow. This is not convenient for culture protocols requiring constant flow, as hourly media changes will have to be conducted. Herein, we present a design technique adapted for the OrganoPlate, a cell culture plate fully compatible with laboratory automation, which allows its redimension to achieve over 24 h of flow. This technique uses a similarity model of a target cell type and a simple fluid flow mathematical prediction model to iterate to the optimum dimensions within some manufacturing constraints. This technique has the potential to be applied to many cell types to generate optimum design for their culture. We applied this technique to design a 3D microfluidic device, dynamically optimised for neuronal cell culture. • Design and construction of a new form of microfluidics plate for cell culture. • Compatibility of the design with cell culture automation. • Continues flow of medium for 24 h culture period. • Optimisation of neuronal differentiation of human stem cells.

Integration of GWAS Summary Statistics and Gene Expression Reveals Target Cell Types Underlying Kidney Function Traits

Genetic variants identified in genome-wide association studies (GWAS) are often not specific enough to reveal complex underlying physiology. By integrating RNA-seq data and GWAS summary statistics, novel computational methods allow unbiased identification of trait-relevant tissues and cell types. The CKDGen consortium provided GWAS summary data for eGFR, urinary albumin-creatinine ratio (UACR), BUN, and serum urate. Genotype-Tissue Expression Project (GTEx) RNA-seq data were used to construct the top 10% specifically expressed genes for each of 53 tissues followed by linkage disequilibrium (LD) score-based enrichment testing for each trait. Similar procedures were performed for five kidney single-cell RNA-seq datasets from humans and mice and for a microdissected tubule RNA-seq dataset from rat. Gene set enrichment analyses were also conducted for genes implicated in Mendelian kidney diseases. Across 53 tissues, genes in kidney function-associated GWAS loci were enriched in kidney (P=9.1E-8 for eGFR; P=1.2E-5 for urate) and liver (P=6.8·10-5 for eGFR). In the kidney, proximal tubule was enriched in humans (P=8.5E-5 for eGFR; P=7.8E-6 for urate) and mice (P=0.0003 for eGFR; P=0.0002 for urate) and confirmed as the primary cell type in microdissected tubules and organoids. Gene set enrichment analysis supported this and showed enrichment of genes implicated in monogenic glomerular diseases in podocytes. A systematic approach generated a comprehensive list of GWAS genes prioritized by cell type-specific expression. Integration of GWAS statistics of kidney function traits and gene expression data identified relevant tissues and cell types, as a basis for further mechanistic studies to understand GWAS loci.

Diversity and function of corticopetal and corticofugal GABAergic projection neurons.

It is still widely thought that cortical projections to distant brain areas derive by and large from glutamatergic neurons. However, an increasing number of reports provide evidence that cortical GABAergic neurons comprise a smaller population of 'projection neurons' in addition to the well-known and much-studied interneurons. GABAergic long-range axons that derive from, or project to, cortical areas are thought to entrain distant brain areas for efficient information transfer and processing. Research conducted over the past 10 years has revealed that cortical GABAergic projection neurons are highly diverse in terms of molecular marker expression, synaptic targeting (identity of targeted cell types), activity pattern during distinct behavioural states and precise temporal recruitment relative to ongoing neuronal network oscillations. As GABAergic projection neurons connect many cortical areas unidirectionally or bidirectionally, it is safe to assume that they participate in the modulation of a whole series of behavioural and cognitive functions. We expect future research to examine how long-range GABAergic projections fine-tune activity in distinct distant networks and how their recruitment alters the behaviours that are supported by these networks.

Growth-factor-mediated coupling between lineage size and cell fate choice underlies robustness of mammalian development.

Precise control and maintenance of population size is fundamental for organismal development and homeostasis. The three cell types of the mammalian blastocyst are generated in precise proportions over a short time, suggesting a mechanism to ensure a reproducible outcome. We developed a minimal mathematical model demonstrating growth factor signaling is sufficient to guarantee this robustness and which anticipates an embryo's response to perturbations in lineage composition. Addition of lineage-restricted cells both in vivo and in silico, causes a shift of the fate of progenitors away from the supernumerary cell type, while eliminating cells using laser ablation biases the specification of progenitors toward the targeted cell type. Finally, FGF4 couples fate decisions to lineage composition through changes in local growth factor concentration, providing a basis for the regulative abilities of the early mammalian embryo whereby fate decisions are coordinated at the population level to robustly generate tissues in the right proportions.

Strategies for the Synthesis of 19-nor-Vitamin D Analogs.

1α,25-Dihydroxyvitamin D3 [1α,25-(OH)2-D3], the hormonally active form of vitamin D3, classically regulates bone formation, calcium, and phosphate homeostasis. In addition, this hormone also exerts non-classical effects in a wide variety of target tissues and cell types, such as inhibition of the proliferation and stimulation of the differentiation of normal and malignant cells. However, to produce these actions, supraphysiological doses are required resulting in calcemic effects that limit the use of this natural hormone. During the past 30 years, many structurally modified analogs of the 1α,25-(OH)2-D3 have been synthesized in order to find derivatives that can dissociate the beneficial antiproliferative effects from undesired calcemic effects. Among these candidates, 1α,25-(OH)2-19-nor-D3 analogs have shown promise as good derivatives since they show equal or better activity relative to the parent hormone but with reduced calcemic effects. In this review, we describe the synthetic strategies to obtain the 19-nor-D3 derivatives and briefly describe their physiological activities.

Neutrophil-Mediated Cardiac Damage After Acute Myocardial Infarction: Significance of Defining a New Target Cell Type for Developing Cardioprotective Drugs.

Significance: Acute myocardial infarction (AMI) is a leading cause of death worldwide. Post-AMI survival rates have increased with the introduction of angioplasty as a primary coronary intervention. However, reperfusion after angioplasty represents a clinical paradox, restoring blood flow to the ischemic myocardium while simultaneously inducing ion and metabolic imbalances that stimulate immune cell recruitment and activation, mitochondrial dysfunction and damaging oxidant production. Recent Advances: Preclinical data indicate that these metabolic imbalances contribute to subsequent heart failure through sustaining local recruitment of inflammatory leukocytes and oxidative stress, cardiomyocyte death, and coronary microvascular disturbances, which enhance adverse cardiac remodeling. Both left ventricular dysfunction and heart failure are strongly linked to inflammation and immune cell recruitment to the damaged myocardium. Critical Issues: Overall, therapeutic anti-inflammatory and antioxidant agents identified in preclinical trials have failed in clinical trials. Future Directions: The versatile neutrophil-derived heme enzyme, myeloperoxidase (MPO), is gaining attention as an important oxidative mediator of reperfusion injury, vascular dysfunction, adverse ventricular remodeling, and atrial fibrillation. Accordingly, there is interest in therapeutically targeting neutrophils and MPO activity in the setting of heart failure. Herein, we discuss the role of post-AMI inflammation linked to myocardial damage and heart failure, describe previous trials targeting inflammation and oxidative stress post-AMI, highlight the potential adverse impact of neutrophil and MPO, and detail therapeutic options available to target MPO clinically in AMI patients.

Spatial proteome profiling by immunohistochemistry-based laser capture microdissection and data-independent acquisition proteomics

Understanding the tumor heterogeneity through spatially resolved proteome profiling is important for biomedical research and clinical application. Laser capture microdissection (LCM) is a powerful technology for exploring local cell populations without losing spatial information. Conventionally, tissue sections are stained with hematoxylin and eosin (H&E) for cell-type identification before LCM. However, it generally requires experienced pathologists to distinguish different cell types, which limits the application of LCM to broad cancer research field. Here, we designed an immunohistochemistry (IHC)-based workflow for cell type-resolved proteome analysis of tissue samples. Firstly, targeted cell type was marked by IHC using antibody targeting cell-type specific marker to improve accuracy and efficiency of LCM. Secondly, to increase protein recovery from chemically crosslinked IHC tissues, we optimized a decrosslinking procedure to seamlessly combine with the integrated spintip-based sample preparation technology SISPROT. This newly developed approach, termed IHC-SISPROT, has comparable performance as H&E staining-based proteomic analysis. High sensitivity and reproducibility of IHC-SISPROT were achieved by combining with data independent acquisition proteomics. More than 3500 proteins were identified from only 0.2 mm2 and 12 μm thickness of hepatocellular carcinoma (HCC) tissue section. Furthermore, using 5 mm2 and 12 μm thickness of HCC tissue section, 6660 and 6052 protein groups were quantified from cancer cells and cancer-associated fibroblasts (CAFs) by the IHC-SISPROT workflow. Bioinformatic analysis revealed the enrichment of cell type-specific ligands and receptors and potentially new communications between cancer cells and CAFs by these signaling proteins. Therefore, IHC-SISPROT is a sensitive and accurate proteomic approach for spatial profiling of cell type-specific proteome from tissues.

Intracellular aggregation of peptide-reprogrammed small molecule nanoassemblies enhances cancer chemotherapy and combinatorial immunotherapy

The intracellular retention of nanotherapeutics is essential for their therapeutic activity. The immobilization of nanotherapeutics inside target cell types can regulate various cell behaviors. However, strategies for the intracellular immobilization of nanoparticles are limited. Herein, a cisplatin prodrug was synthesized and utilized as a glutathione (GSH)-activated linker to induce aggregation of the cisplatin prodrug/IR820/docetaxel nanoassembly. The nanoassembly has been reprogrammed with peptide-containing moieties for tumor-targeting and PD-1/PD-L1 blockade. The aggregation of the nanoassemblies is dependent on GSH concentration. Evaluations in vitro and in vivo revealed that GSH-induced intracellular aggregation of the nanoassemblies enhances therapeutic activity in primary tumors by enhancing the accumulation and prolonging the retention of the chemotherapeutics in the tumor site and inducing reactive oxygen species (ROS) generation and immunogenic cell death. Moreover, the nanoassemblies reinvigorate the immunocytes, especially the systemic immunocytes, and thereby alleviate pulmonary metastasis, even though the population of immunocytes in the primary tumor site is suppressed due to the enhanced accumulation of chemotherapeutics. This strategy provides a promising option for the intracellular immobilization of nanoparticles in vitro and in vivo.