Lysosomal diacylglycerol pyrophosphate phosphatase is not essential in Trypanosoma brucei.

Mg2+-independent phosphatidic acid phosphatase (PAP2), diacylglycerol pyrophosphate phosphatase 1 (Dpp1) is a membrane-associated enzyme in Saccharomyces cerevisiae. The enzyme is responsible for inducing the breakdown of ß-phosphate from diacylglycerol pyrophosphate (DGPP) into phosphatidate (PA) and then removes the phosphate from PA to give diacylglycerol (DAG). In this study through RNAi suppression, we have demonstrated that Trypanosoma brucei diacylglycerol pyrophosphate phosphatase 1 (TbDpp1) procyclic form production is not required for parasite survival in culture. The steady-state levels of triacylglycerol (TAG), the number of lipid droplets, and the PA content are all maintained constant through the inducible down-regulation of TbDpp1. Furthermore, the localization of C-terminally tagged variants of TbDpp1 in the lysosome was demonstrated by immunofluorescence microscopy.

Mast cells in meningiomas.

Meningioma represents the most frequent tumor of the central nervous system (CNS). Correlations between the presence of mast cells (MCs) and grade or other histological features of meningioma are still debated. Our study aimed to better understand the relationship between mast cells and meningiomas and to compare our results based on specific histological subtypes and novel 2021 CNS WHO grading system. We observed some differences as regards the number of MCs and meningioma grade. In low-grade (grade 1) meningiomas, MCs were observed in 7/22 cases, while they were consistently present in all eight high-grade cases (grade 2 and grade  3). Among the grade 1 meningiomas, we observed two "low-positive", two "intermediate-positive", and three "high-positive" cases. Among the group of high-grade meningiomas, the six cases grade 2 were considered as "low-positive", while the two grade 3 cases showed a higher number of MCs and were included in the "intermediate-positive" group. Even though with no statistical significance, due to the low number of cases, our results seem to confirm a sort of relationship between meningioma grading and the number of MCs, as demonstrated by the higher percentage of high-grade meningiomas showing MCs infiltrates, compared to low-grade meningiomas.

Macrophages and angiogenesis in human lymphomas.

A link exists between chronic inflammation and cancer and immune cells, angiogenesis, and tumor progression. In hematologic malignancies, tumor-associated macrophages (TAMs) are a significant part of the tumor microenvironment. Macrophages are classified into M1/classically activated and M2/alternatively activated. In tumors, TAMs are mainly constituted by M2 subtype, which promotes angiogenesis, lymphangiogenesis, repair, and remodeling, suppressing adaptive immunity, increasing tumor cell proliferation, drug resistance, histological malignancy, and poor clinical prognosis. The aim of our review article is to define the role of TAMs and their relationship with the angiogenesis in patients with lymphoma reporting both an analysis of main published data and those emerging from our studies. Finally, we have discussed the anti-angiogenic approach in the treatment of lymphomas.

Vascular co-option in resistance to anti-angiogenic therapy.

Three different mechanisms of neovascularization have been described in tumor growth, including sprouting angiogenesis, intussusceptive microvascular growth and glomeruloid vascular proliferation. Tumors can also grow by means of alternative mechanisms including vascular co-option, vasculogenic mimicry, angiotropism, and recruitment of endothelial precursor cells. Vascular co-option occurs in tumors independently of sprouting angiogenesis and the non-angiogenic cancer cells are described as exploiting pre-existing vessels. Vascular co-option is more frequently observed in tumors of densely vascularized organs, including the brain, lung and liver, and vascular co-option represents one of the main mechanisms involved in metastasis, as occurs in liver and lung, and resistance to anti-angiogenic therapy. The aim of this review article is to analyze the role of vascular co-option as mechanism through which tumors develop resistance to anti-angiogenic conventional therapeutic approaches and how blocking co-option can suppress tumor growth.

PTX3 shapes profibrotic immune cells and epithelial/fibroblast repair and regeneration in a murine model of pulmonary fibrosis.

The long pentraxin 3 (PTX3) is protective in different pathologies but was not analyzed in-depth in Idiopathic Pulmonary Fibrosis (IPF). Here, we have explored the influence of PTX3 in the bleomycin (BLM)-induced murine model of IPF by looking at immune cells (macrophages, mast cells, T cells) and stemness/regenerative markers of lung epithelium (SOX2) and fibro-blasts/myofibroblasts (CD44) at different time points that retrace the progression of the disease from onset at day 14, to full-blown disease at day 21, to incomplete regression at day 28. We took advantage of transgenic PTX3 overexpressing mice (Tie2-PTX3) and Ptx3 null ones (PTX3-KO) in which pulmonary fibrosis was induced. Our data have shown that PTX3 overexpression in Tie2-PTX3 compared to WT or PTX3-KO: reduced CD68+ and CD163+ macrophages and the Tryptase+ mast cells during the whole experimental time; on the contrary, CD4+ T cells are consistently present on day 14 and dramatically decreased on day 21; CD8+ T cells do not show significant differences on day 14, but are significantly reduced on day 21; SOX2 is reduced on days 14 and 21; CD44 is reduced on day 21. Therefore, PTX3 could act on the proimmune and fibrogenic microenvironment to prevent fibrosis in BLM-treated mice.

Different patterns of mast cell distribution in B-cell non-Hodgkin lymphomas.

Tumor growth, progression, and metastatic capability in non-Hodgkin lymphomas (NHLs) are influenced by different component of tumor microenvironment, including inflammatory cells. Among these latter, mast cells play a crucial role. The spatial distribution of mast cells inside the tumor stroma of different types of B-cell NHLs has not yet been investigated. The aim of this study is to analyze the pattern of distribution of mast cells in biopsy samples obtained from three different types of B-cell NHLs by utilizing an image analysis system and a mathematical model to allow a quantitative estimation to characterize their spatial distribution. As concerns the spatial distributions exhibited by mast cells in diffuse large B cell lymphoma (DLBCL), some clustering was detected in both activated B-like (ABC) and germinal center B-like (GBC) groups. In follicular lymphoma (FL), mast cell spatial distribution tends to uniformly fill the tissue space as far as the grade of the pathology increases. Finally, in marginal lymphoma tissue (MALT) lymphoma, mast cells maintain a significantly clustered spatial distribution, suggesting a lower tendency of the cells to fill the tissue space in this pathological condition. Overall, the data of this study confirm that the analysis of the spatial distribution of the tumor cells is of particular significance for the knowledge of the biological processes occurring in tumor stroma and for the development of parameters to characterize the morphologic organization of the cellular patterns in different types of tumors.

Autophagy increase in Merosin-Deficient Congenital Muscular Dystrophy type 1A.

The autophagy process recycles dysfunctional cellular components and protein aggregates by sequestering them in autophagosomes directed to lysosomes for enzymatic degradation. A basal level of autophagy is essential for skeletal muscle maintenance. Increased autophagy occurs in several forms of muscular dystrophy and in the merosin-deficient congenital muscular dystrophy 1A mouse model (dy3k/dy3k) lacking the laminin-α2 chain. This pilot study aimed to compare autophagy marker expression and autophagosomes presence using light and electron microscopes and western blotting in diagnostic muscle biopsies from newborns affected by different congenital muscular myopathies and dystrophies. Morphological examination showed dystrophic muscle features, predominance of type 2A myofibers, accumulation of autophagosomes in the subsarcolemmal areas, increased number of autophagosomes overexpressing LC3b, Beclin-1 and ATG5, in the merosin-deficient newborn suggesting an increased autophagy. In Duchenne muscular dystrophy, nemaline myopathy, and spinal muscular atrophy the predominant accumulation of p62+ puncta rather suggests an autophagy impairment.

Vascular Growth in Lymphomas: Angiogenesis and Alternative Ways.

The formation of new blood vessels is a critical process for tumor growth and may be achieved through different mechanisms. Angiogenesis represents the first described and most studied mode of vessel formation, but tumors may also use alternative ways to secure blood supply and eventually acquire resistance to anti-angiogenic treatments. These non-angiogenic mechanisms have been described more recently, including intussusceptive microvascular growth (IMG), vascular co-option, and vasculogenic mimicry. Like solid tumors, angiogenic and non-angiogenic pathways in lymphomas play a fundamental role in tumor growth and progression. In view of the relevant prognostic and therapeutic implications, a comprehensive understanding of these mechanisms is of paramount importance for improving the efficacy of treatment in patients with lymphoma. In this review, we summarize the current knowledge on angiogenic and non-angiogenic mechanisms involved in the formation of new blood vessels in Hodgkin's and non-Hodgkin's lymphomas.

The Tumor Microenvironment in Classic Hodgkin's Lymphoma in Responder and No-Responder Patients to First Line ABVD Therapy.

Although classical Hodgkin lymphoma (CHL) is typically curable, 15-25% of individuals eventually experience a relapse and pass away from their disease. In CHL, the cellular microenvironment is constituted by few percent of H/RS (Hodgkin/Reed-Sternberg) tumor cells surrounded from a heterogeneous infiltration of inflammatory cells. The interplay of H/RS cells with other immune cells in the microenvironment may provide novel strategies for targeted immunotherapies. In this paper we analyzed the microenvironment content in CHL patients with responsive disease (RESP) and patients with relapsed/refractory disease to treatment (REL). Our results indicate the increase of CD68+ and CD163+ macrophages, the increase of PDL-1+ cells and of CD34+ microvessels in REL patients respective to RESP patients. In contrast we also found the decrease of CD3+ and of CD8+ lymphocytes in REL patients respective to RESP patients. Finally, in REL patients our results show the positive correlation between CD68+ macrophages and PDL-1+ cells as well as a negative correlation between CD163+ and CD3+.

Chick embryo in experimental embryology and more.

Chicken remains an undisputed, powerful, useful, and practical model in developmental research. Chick embryos have been used as model systems for studies in experimental embryology and teratology. As the chicken embryo develops outside the mother, effects of external stresses on cardiovascular development can be studied without interferences of maternal hormonal, metabolic, or hemodynamic alterations. In 2004, the first draft sequence of the complete chicken genome was released, allowing broad genetic analysis and comparison to humans, and enabling expansion of transgenic techniques within the chick model. The chick embryo is relatively simple, quick, and low-cost model. The main advantages of the chick as a model system for experimental embryology are the ease with cells and tissues can be labeled, transplanted, and cultured, along with its similarity to mammalian systems.

IKOSA® CAM Assay Application to Quantify Blood Vessels on Chick Chorioallantoic Membrane (CAM).

Among in vivo angiogenesis assays, chick chorioallantoic membrane (CAM) assay has allowed meaningful progress in elucidating the mechanism of the vasoproliferative response to several pro- and antiangiogenic factors, thanks to its low costs, relatively easy management, and lower ethical concerns compared to other animal in vivo models.Here, we present a method to quantify angiogenesis and antiangiogenesis processes in the CAM based on the segmentation of microscopic images of blood vessels. Images captured from in vivo CAM samples can be analyzed for their vascularization with IKOSA CAM Assay to measure their total area, length, mean thickness, and the number of branching points. This chapter presents a detailed protocol to perform a CAM assay and analysis, and the IKOSA CAM Assay output is described.

Double Immunohistochemical Staining on Formalin-Fixed Paraffin-Embedded Tissue Samples to Study Vascular Co-option.

Vascular co-option is a non-angiogenic mechanism whereby tumor growth and progression move on by hijacking the pre-existing and nonmalignant blood vessels and is employed by various tumors to grow and metastasize.The histopathological identification of co-opted blood vessels is complex, and no specific markers were defined, but it is critical to develop new and possibly more effective therapeutic strategies. Here, in glioblastoma, we show that the co-opted blood vessels can be identified, by double immunohistochemical staining, as weak CD31+ vessels with reduced P-gp expression and proliferation and surrounded by highly proliferating and P-gp- or S100A10-expressing tumor cells. Results can be quantified by the Aperio Colocalization algorithm, which is a valid and robust method to handle and investigate large data sets.

Differential P-Glycoprotein/CD31 Expression as Markers of Vascular Co-Option in Primary Central Nervous System Tumors.

BACKGROUND: Vascular co-option is one of the main features of brain tumor progression. It is identified using histopathological analysis, but no antibody-specific markers were found, and no universally accepted histological features were defined. METHODS: We employed double immunohistochemical stainings for CD31, P-gp, S100A10, and mitochondria on formalin-fixed, paraffin-embedded human samples of IDH-WT glioblastoma, IDH-mutant astrocytoma, and meningioma to study vascular co-option across different brain tumors and across normal, peritumoral, and intratumoral areas using the Aperio colocalization algorithm, which is a valid and robust method to handle and investigate large data sets. RESULTS: The results have shown that (i) co-opted vessels could be recognized by the presence of metabolically overactive (evaluated as mitochondria expression) and P-gp+ or S100A10+ tumor cells surrounding CD31+ endothelial cells; (ii) vascular co-option occurs in the intratumoral area of meningioma and astrocytoma; and (iii) vascular co-option is prevalent in peritumoral glioblastoma area. CONCLUSIONS: The described approach identifies new markers for cellular components of the vessel wall and techniques that uncover the order and localization of vascularization mechanisms, which may contribute to developing new and possibly more effective therapeutic strategies.

Inflammatory microenvironment in classical Hodgkin's lymphoma with special stress on mast cells.

Classical Hodgkin's lymphoma (CHL) accounts for 10% of all lymphomas. Nodular sclerosis and mixed cellularity accounts for nearly 80% of all CHL cases. The number of mast cells in CHL correlates with poor prognosis, is significantly higher in nodular sclerosis than in other CHL subtypes, and an association between the degree of angiogenesis and the number of intratumoral mast cells has been demonstrated in CHL. Even with the best available treatment, a significant percentage of CHL patients progress or relapse after first-line therapy. 50% of patients with disease relapse achieve subsequent long-term disease control with salvage therapies. In this context, new potential therapeutic opportunities are required, and mast cells may be regarded as a new target for adjuvant treatment of CHL through the inhibition of angiogenesis and tissue remodeling and allowing the secretion of cytotoxic cytokines.

Microglia-derived CCL2 has a prime role in neocortex neuroinflammation.

BACKGROUND: In myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), several areas of demyelination are detectable in mouse cerebral cortex, where neuroinflammation events are associated with scarce inflammatory infiltrates and blood-brain barrier (BBB) impairment. In this condition, the administration of mesenchymal stem cells (MSCs) controls neuroinflammation, attenuating astrogliosis and promoting the acquisition of stem cell traits by astrocytes. To contribute to the understanding of the mechanisms involved in the pathogenesis of EAE in gray matter and in the reverting effects of MSC treatment, the neocortex of EAE-affected mice was investigated by analyzing the cellular source(s) of chemokine CCL2, a molecule involved in immune cell recruitment and BBB-microvessel leakage. METHODS: The study was carried out by immunohistochemistry (IHC) and dual RNAscope IHC/in situ hybridization methods, using astrocyte, NG2-glia, macrophage/microglia, and microglia elective markers combined with CCL2. RESULTS: The results showed that in EAE-affected mice, hypertrophic microglia are the primary source of CCL2, surround the cortex neurons and the damaged BBB microvessels. In EAE-affected mice treated with MSCs, microgliosis appeared diminished very soon (6 h) after treatment, an observation that was long-lasting (tested after 10 days). This was associated with a reduced CCL2 expression and with apparently preserved/restored BBB features. In conclusion, the hallmark of EAE in the mouse neocortex is a condition of microgliosis characterized by high levels of CCL2 expression. CONCLUSIONS: This finding supports relevant pathogenetic and clinical aspects of the human disease, while the demonstrated early control of neuroinflammation and BBB permeability exerted by treatment with MSCs may have important therapeutic implications.

Update in TIGIT Immune-Checkpoint Role in Cancer.

The in-depth characterization of cross-talk between tumor cells and T cells in solid and hematological malignancies will have to be considered to develop new therapeutical strategies concerning the reactivation and maintenance of patient-specific antitumor responses within the patient tumor microenvironment. Activation of immune cells depends on a delicate balance between activating and inhibitory signals mediated by different receptors. T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) is an inhibitory receptor expressed by regulatory T cells (Tregs), activated T cells, and natural killer (NK) cells. TIGIT pathway regulates T cell-mediated tumor recognition in vivo and in vitro and represents an exciting target for checkpoint blockade immunotherapy. TIGIT blockade as monotherapy or in combination with other inhibitor receptors or drugs is emerging in clinical trials in patients with cancer. The purpose of this review is to update the role of TIGIT in cancer progression, looking at TIGIT pathways that are often upregulated in immune cells and at possible therapeutic strategies to avoid tumor aggressiveness, drug resistance, and treatment side effects. However, in the first part, we overviewed the role of immune checkpoints in immunoediting, the TIGIT structure and ligands, and summarized the key immune cells that express TIGIT.

RNAscope for VEGF-A Detection in Human Tumor Bioptic Specimens.

Different pro-angiogenic factors, such as vascular endothelial growth factor-A (VEGF-A), have been related to microvascular density, clinicopathologic factors, and poor prognosis in many tumors. VEGF-A binds its receptor 2 (VEGFR2) to induce neo-angiogenesis, a constant hallmark of tumor initiation and progression. Based on VEGF-A/VEGFR2 relevance in tumor angiogenesis, several inhibitors were developed. However, the clinical benefits of anti-angiogenic therapies are limited because tumors activate different mechanisms of drug resistance.The need for understanding tumor biology, limitation or failure of anti-angiogenic therapies, and the demand for a personalized therapeutic approach has boosted the search for robust biomarkers for patient stratification as responder or non-responder to anti-VEGF therapies.This chapter presents a detailed protocol to perform chromogenic VEGF-A mRNA detection and quantification in human tumor bioptic specimens using RNAscope technology and RNA-in situ hybridization (ISH) algorithm. RNAscope for VEGF-A detection, even for small amounts, is compatible with precious clinical samples and diagnostic laboratory workflows.

Tumor Microenvironment and Microvascular Density in Follicular Lymphoma.

Follicular lymphoma (FL) is a slowly progressive disease and constitutes the second most common non-Hodgkin lymphoma. Biological factors, such as the tumor microenvironment and the host response, are determinants in the outcome of FL but the experimental data about microenvironment and tumor cells in FL are variable and contradictory. In this morphometric study, we analyzed by immunohistochemistry the cellular components of the tumor microenvironment and correlated these data with the microvascular vascular density in three different grades of FL lymph node biopsies, comparing the results to healthy lymph node controls. The results indicated a significant increase in the number of CD68+ and CD163+ macrophages in all three analyzed FL grades. Tryptase+ mast cells resulted in an increase only in grade 1. PDL-1+ cells, CD4- and CD8-lymphocytes number results were reduced in FL samples. The higher number of CD34+ microvessels in the FL grades 1 and 2 of samples positively correlated with CD68+ and CD163+ cells, underlining the important angiogenic potential of this subset of macrophages.

Preferentially Expressed Antigen in Melanoma (PRAME) and Human Malignant Melanoma: A Retrospective Study.

BACKGROUND: Preferentially expressed antigen in melanoma (PRAME) is a cancer testis antigen (CTA) identified in 1997 through analysis of the specificity of tumor-reactive T-cell clones derived from a patient with metastatic cutaneous melanoma. Although at first it seemed even more specific, various studies have shown that PRAME can also be expressed in the context of atypical lesions that do not correspond solely to the definition of malignant melanoma. METHODS: A systematic review of English articles was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. RESULTS: 126 records were identified in the literature search, of which 9 were duplicates. After screening for eligibility and inclusion criteria, 53 publications were included. CONCLUSIONS: The advent of a new marker such as PRAME is surely a step forward not only in the diagnostic approach, but also in the immunotherapeutic approach to MM. However, various studies have shown that PRAME can also be expressed in the context of atypical lesions apart from MM and, for this reason, the diagnostic sensitivity and specificity (hence accuracy) are clearly lower. Further studies with larger case series will be necessary to understand better what possibilities are offered in terms of diagnostic reliability by PRAME.

The Effect of the Tumor Microenvironment on Lymphoid Neoplasms Derived from B Cells.

Lymphomas are characteristic tumors surrounded by an inflammatory microenvironment. The cells of the microenvironment are essential for the growth and survival of neoplastic cells and are recruited through the effect of cytokines/chemokines. Lymphomas include heterogeneous groups of neoplasms infiltrating various lymphoid structures which may arise from B lymphocytes, T lymphocytes, and natural killer (NK) cells at various stages of their differentiation state. In this review article, we analyze the literature data concerning the involvement of the tumor microenvironment (TME) in the progression of lymphomas and the recent advances in the analysis of microenvironment components in the most common forms: some mature B cell lymphoma neoplasms and classic Hodgkin lymphomas. The complex crosstalk between the TME and tumor cells led to the discovery of many mechanisms usable as molecular-targeted therapy through the control of diverse elements of the TME, varying from inhibitors of angiogenic cytokines and their receptors to the regulation of cells' activities and the novel immune checkpoint inhibitors.