Calcium Dobesilate Is Protective against Inflammation and Oxidative/Nitrosative Stress in the Retina of a Type 1 Diabetic Rat Model.

Calcium dobesilate (CaD) has been prescribed to some patients in the early stages of diabetic retinopathy to delay its progression. We previously reported that the treatment of diabetic animals (4 weeks of diabetes) with CaD, during the last 10 days of diabetes, prevents blood-retinal barrier breakdown. Here, we aimed to investigate whether later treatment of diabetic rats with CaD would reverse inflammatory processes in the retina. Diabetes was induced with streptozotocin, and 6 weeks after diabetes onset, CaD (100 mg/kg/day) was administered for 2 weeks. The treatment with CaD significantly increased glial fibrillary acidic protein (GFAP) levels in the retina of nondiabetic animals (138.6 ± 12.8% of control) and enhanced the diabetes-induced increase in GFAP levels (174.8 ± 5.6% of control). In addition, CaD prevented the increase in mRNA and protein expression of tumor necrosis factor and interleukin-1ß, as well as the formation of oxidized carbonyl residues and the increase in nitrotyrosine immunoreactivity, particularly in the ganglion cell layer of diabetic animals. We demonstrate that the treatment of diabetic animals with CaD can reverse the established proinflammatory processes in the retina. These beneficial effects appear to be attributed, at least partially, to the antioxidant properties of CaD.

Global mass spectrometry and transcriptomics array based drug profiling provides novel insight into glucosamine induced endoplasmic reticulum stress.

We investigated the molecular effects of glucosamine supplements, a popular and safe alternative to nonsteroidal anti-inflammatory drugs, for decreasing pain, inflammation, and maintaining healthy joints. Numerous studies have reported an array of molecular effects after glucosamine treatment. We questioned whether the differences in the effects observed in previous studies were associated with the focus on a specific subproteome or with the use of specific cell lines or tissues. To address this question, global mass spectrometry- and transcription array-based glucosamine drug profiling was performed on malignant cell lines from different stages of lymphocyte development. We combined global label-free MS-based protein quantitation with an open search for modifications to obtain the best possible proteome coverage. Our data were largely consistent with previous studies in a variety of cellular models. We mainly observed glucosamine induced O-GlcNAcylation/O-GalNAcylation (O-HexNAcylation); however, we also observed global and local changes in acetylation, methylation, and phosphorylation. For example, our data provides two additional examples of "yin-yang" between phosphorylation and O-HexNAcylation. Furthermore, we mapped novel O-HexNAc sites on GLU2B and calnexin. GLU2B and calnexin are known to be located in the endoplasmic reticulum (ER) and involved in protein folding and quality control. The O-HexNAc sites were regulated by glucosamine treatment and correlated with the up-regulation of the ER stress marker GRP78. The occupancy of O-HexNAc on GLU2B and calnexin sites differed between the cytosolic and nuclear fractions with a higher occupancy in the cytosolic fraction. Based on our data we propose the hypothesis that O-HexNAc either inactivates calnexin and/or targets it to the cytosolic fraction. Further, we hypothesize that O-HexNAcylation induced by glucosamine treatment enhances protein trafficking.

Protocol for ex vivo culture of patient-derived tumor fragments.

The lack of suitable models currently hampers our understanding of how the tumor microenvironment responds to immunotherapy treatment. Here, we present a protocol for ex vivo culture of patient-derived tumor fragments (PDTFs). We describe the steps for tumor collection, generation and cryopreservation of PDTFs, and their subsequent thawing. We detail culture of PDTFs and their preparation for analysis. This protocol preserves the tumor microenvironment's composition, architecture, and cellular interactions, which can be perturbed by ex vivo treatment. For complete details on the use and execution of this protocol, please refer to Voabil et al. (2021).1.

Addition of interleukin-2 overcomes resistance to neoadjuvant CTLA4 and PD1 blockade in ex vivo patient tumors.

Neoadjuvant immunotherapy with anti-cytotoxic T lymphocyte-associated protein 4 (CTLA4) + anti-programmed cell death protein 1 (PD1) monoclonal antibodies has demonstrated remarkable pathological responses and relapse-free survival in ~80% of patients with clinically detectable stage III melanoma. However, about 20% of the treated patients do not respond. In pretreatment biopsies of patients with melanoma, we found that resistance to neoadjuvant CTLA4 + PD1 blockade was associated with a low CD4/interleukin-2 (IL-2) gene signature. Ex vivo, addition of IL-2 to CTLA4 + PD1 blockade induced T cell activation and deep immunological responses in anti-CTLA4 + anti-PD1-resistant human tumor specimens. In the 4T1.2 breast cancer mouse model of neoadjuvant immunotherapy, triple combination of anti-CTLA4 + anti-PD1 + IL-2 cured almost twice as many mice as compared with dual checkpoint inhibitor therapy. This improved efficacy was due to the expansion of tumor-specific CD8+ T cells and improved proinflammatory cytokine polyfunctionality of both CD4+ and CD8+ T effector cells and regulatory T cells. Depletion studies suggested that CD4+ T cells were critical for priming of CD8+ T cell immunity against 4T1.2 and helped in the expansion of tumor-specific CD8+ T cells early after neoadjuvant triple immunotherapy. Our results suggest that the addition of IL-2 can overcome resistance to neoadjuvant anti-CTLA4 + anti-PD1, providing the rationale for testing this combination as a neoadjuvant therapy in patients with early-stage cancer.

An ex vivo tumor fragment platform to dissect response to PD-1 blockade in cancer.

Inhibitors of the PD-1-PD-L1 axis have been approved as therapy for many human cancers. In spite of the evidence for their widespread clinical activity, little is known about the immunological alterations that occur in human cancer tissue after PD-1 blockade. We developed and employed a patient-derived tumor fragment platform to dissect the early immunological response of human tumor tissue to ex vivo PD-1 blockade. We observed that the capacity of immune cells to be reactivated ex vivo was predictive of clinical response, and perturbation analyses identified tumor-resident T cells as a key component of this immunological response. In addition, through combined analysis of baseline properties and immune response capacity, we identified a new subgroup of infiltrated tumors that lacks the capacity to respond to PD-1 blockade. Finally, the baseline presence of tertiary lymphoid structures and their components correlated with the capacity of cancers to undergo intratumoral immune cell reactivation.

Genomics- and Transcriptomics-Based Patient Selection for Cancer Treatment With Immune Checkpoint Inhibitors: A Review.

IMPORTANCE: Checkpoint blockade therapy targeting cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and the programmed cell death protein 1 pathways (PD-1/PD-L1) have achieved success in treating a number of malignancies. However, only a subset of patients responds to these therapies, and optimization of patient selection for treatment is imperative to avoid adverse effects without clinical benefit and keep costs manageable. OBSERVATIONS: The past few years have witnessed checkpoint inhibition becoming a first-line treatment option with US Food and Drug Administration approvals for various tumor types. Genomic analyses (whole genome, exome, and transcriptome) have been instrumental in identifying a genetic profile associated with sensitivity to checkpoint inhibitors. Therapy outcome is determined at various levels: (1) the degree of tumor "foreignness," as reflected by mutational burden and expression of viral genes, (2) the composition and activity of a preexisting immune infiltrate, and (3) mechanisms of tumor escape from immune surveillance. In addition, there are opportunities for genomic analyses of genetic polymorphisms and the gut microbiome that may be associated with clinical response to therapy. CONCLUSIONS AND RELEVANCE: Genomics provides powerful tools for the identification of biomarkers for response to immune checkpoint blockade, given their potential to analyze multiple parameters simultaneously in an unbiased manner. This offers the opportunity for genomics- and transcriptomics-based selection of patients for rationally designed therapy with immune checkpoint inhibitors.

Tools for protein posttranslational modifications analysis: FAK, a case study.

Recent advances in mass spectrometry have resulted in an exponential increase in annotation of posttranslational modifications (PTMs). Just in the Swiss-Prot Knowledgebase, there are 89,931 of a total of 27 characterized PTM types reported experimentally. A single protein can be dynamically modified during its lifetime for regulation of its function. Considering a PTM can occur at different levels and the number of different PTMs described, the number of possibilities for a single protein is unthinkable. Narrowing the study to a single PTM can be rather unmerited considering that most proteins are heavily modified. Currently crosstalk between PTMs is plentifully reported in the literature. The example of amino acids serine and threonine on one hand and lysine on the other hand, as targets of different modifications, demand a more global analysis approach of a protein. Besides the direct competition for the same amino acid, a PTM can directly or indirectly influence other PTMs in the same protein molecule by for example steric hindrance due to close proximity between the modifications or creation of a binding site such as an SH2 binding domain for protein recruitment and further modifications. Given the complexity of PTMs a number of tools have been developed to archive, analyze, and visualize modifications. VISUALPROT is presented here to demonstrate the usefulness of visualizing all annotated protein features such as amino acid content, domains, amino acid modification sites and single amino acid polymorphisms in a single image. VISUALPROT application is demonstrated for the protein focal adhesion kinase (FAK) as an example. FAK is a highly phosphorylated cytoplasmatic tyrosine kinase comprising different domains and regions. FAK is crucial for integrating signals from integrins and receptor tyrosine kinases in processes such as cell survival, proliferation, and motility.

Calcium dobesilate inhibits the alterations in tight junction proteins and leukocyte adhesion to retinal endothelial cells induced by diabetes.

OBJECTIVE: Calcium dobesilate (CaD) has been used in the treatment of diabetic retinopathy in the last decades, but its mechanisms of action are not elucidated. CaD is able to correct the excessive vascular permeability in the retina of diabetic patients and in experimental diabetes. We investigated the molecular and cellular mechanisms underlying the protective effects of CaD against the increase in blood-retinal barrier (BRB) permeability induced by diabetes. RESEARCH DESIGN AND METHODS: Wistar rats were divided into three groups: controls, streptozotocin-induced diabetic rats, and diabetic rats treated with CaD. The BRB breakdown was evaluated using Evans blue. The content or distribution of tight junction proteins (occludin, claudin-5, and zonula occluden-1 [ZO-1]), intercellular adhesion molecule-1 (ICAM-1), and p38 mitogen-activated protein kinase (p38 MAPK) was evaluated by Western blotting and immunohistochemistry. Leukocyte adhesion was evaluated in retinal vessels and in vitro. Oxidative stress was evaluated by the detection of oxidized carbonyls and tyrosine nitration. NF-κB activation was measured by enzyme-linked immunosorbent assay. RESULTS: Diabetes increased the BRB permeability and retinal thickness. Diabetes also decreased occludin and claudin-5 levels and altered the distribution of ZO-1 and occludin in retinal vessels. These changes were inhibited by CaD treatment. CaD also inhibited the increase in leukocyte adhesion to retinal vessels or endothelial cells and in ICAM-1 levels, induced by diabetes or elevated glucose. Moreover, CaD decreased oxidative stress and p38 MAPK and NF-κB activation caused by diabetes. CONCLUSIONS: CaD prevents the BRB breakdown induced by diabetes, by restoring tight junction protein levels and organization and decreasing leukocyte adhesion to retinal vessels. The protective effects of CaD are likely to involve the inhibition of p38 MAPK and NF-κB activation, possibly through the inhibition of oxidative/nitrosative stress.