Evaluation of a CD206-Targeted Peptide for PET Imaging of Macrophages in Syngeneic Mouse Models of Cancer.

Tumor-associated macrophages (TAMs) are large phagocytic cells that play numerous roles in cancer biology and are an important component of the relationship between immune system response and tumor progression. The peptide, RP832c, targets the Mannose Receptor (CD206) expressed on M2-like macrophages and is cross-reactive to both human and murine CD206. Additionally, it exhibits therapeutic properties through its ability to shift the population of TAMs from an M2-like (protumor) toward an M1-like phenotype (antitumor) and has demonstrated promise in inhibiting tumor resistance in PD-L1 unresponsive melanoma murine models. In addition, it has shown inhibition in bleomycin-induced pulmonary fibrosis through interactions with CD206 macrophages.1,2 Our work aims to develop a novel CD206 positron emission tomography (PET) imaging probe based on RP832c (Kd = 5.64 µM) as a direct, noninvasive method for the assessment of TAMs in mouse models of cancer. We adapted RP832c to incorporate the chelator DOTA to allow for radiolabeling with the PET isotope 68Ga (t1/2 = 68 min; ß+ = 89%). In vitro stability studies were conducted in mouse serum up to 3 h. The in vitro binding characteristics of [68Ga]RP832c to CD206 were determined by a protein plate binding assay and Surface Plasmon Resonance (SPR). PET imaging and biodistribution studies were conducted in syngeneic tumor models. Stability studies in mouse serum demonstrated that 68Ga remained complexed up to 3 h (less than 1% free 68Ga). Binding affinity studies demonstrated high binding of [68Ga]RP832c to mouse CD206 protein and that the binding of the tracer was able to be blocked significantly when incubated with a blocking solution of native RP832c. PET imaging and biodistribution studies in syngeneic tumor models demonstrated uptake in tumor and CD206 expressing organs of [68Ga]RP832c. A significant correlation was found between the percentage of CD206 present in each tumor imaged with [68Ga]RP832c and PET imaging mean standardized uptake values in a CT26 mouse model of cancer. The data shows that [68Ga]RP832c represents a promising candidate for macrophage imaging in cancer and other diseases.

Interleukin-31 promotes pathogenic mechanisms underlying skin and lung fibrosis in scleroderma.

OBJECTIVES: Cytokines released by infiltrating T cells may promote mechanisms leading to fibrosis in scleroderma. The aim of this study was to investigate the role of the Th2 cytokine IL-31, and its receptor IL-31RA, in scleroderma skin and lung fibrosis. METHODS: IL-31 was measured by ELISA of plasma, and by immunochemistry of fibrotic skin and lung tissue of scleroderma patients. The receptor, IL-31RA, was assayed by qPCR of tissue resident cells. Next-generation sequencing was used to profile the responses of normal skin fibroblasts to IL-31. In wild-type Balb/c mice, IL-31 was administered by subcutaneous mini pump, with or without additional TGFß, and the fibrotic reaction measured by histology and ELISA of plasma. RESULTS: IL-31 was present at high levels in plasma and fibrotic skin and lung lesions in a subset of scleroderma patients, and the receptor overexpressed by downstream cells relevant to the disease process, including skin and lung fibroblasts, through loss of epigenetic regulation by miR326. In skin fibroblasts, IL-31 induced next generation sequencing profiles associated with cellular growth and proliferation, anaerobic metabolism and mineralization, and negatively associated with angiogenesis and vascular repair, as well as promoting phenotype changes including migration and collagen protein release via pSTAT3, resembling the activation state in the disease. In mice, IL-31 induced skin and lung fibrosis. No synergy was seen with TGFß, which supressed IL-31RA. CONCLUSION: IL-31/IL-31RA is confirmed as a candidate pro-fibrotic pathway, which may contribute to skin and lung fibrosis in a subset of scleroderma patients.

Phytotoxicity of Extracts of Argemone mexicana and Crotalaria longirostrata on Tomato Seedling Physiology.

Phytotoxicity caused by secondary metabolites of botanical extracts is a drawback in agriculture. The objective of this study was to evaluate the phytotoxic effects of methanolic extracts of Crotalaria longirostrata and Argemone mexicana on the germination and physiological variables of tomato seedlings. The results indicated that high doses of both extracts (Clong500 and Amex500) inhibited tomato seed germination, while their mixture (Cl50 + Am50) promoted germination by 100%. At 30 days after transplanting (dat), the plant height increased by 15.4% with a high dose of C. longirostrata (Clong500) compared to the control. At 30 dat, the vigor index displayed a notable increase with Cl50 + Am50, reaching 29.5%. The root length increased with the mean dose of A. mexicana (Amex95) at 10, 20, and 30 dat (59.7%, 15.1%, and 22.4%, respectively). The chlorophyll content increased with Amex95 by 66.1% in 10 dat, 22.6% at 20 dat, and 19.6% at 30 dat. On the other hand, Amex95 had a higher nitrogen content throughout the trial. Amex95 produced the greatest increase in root dry weight by 731.5% and 209.4% at 10 and 20 dat. The foliage dry weight increased by 85.7% at 10 dat with Amex95 and up to 209.7% with Amex50 at 30 dat. The present investigation reveals the ability of the extracts to stimulate tomato growth at low and medium doses, though at high doses they exhibit allelopathic effects.

A Novel CD206 Targeting Peptide Inhibits Bleomycin-Induced Pulmonary Fibrosis in Mice.

Activated M2-polarized macrophages are drivers of pulmonary fibrosis in several clinical scenarios, including Idiopathic Pulmonary Fibrosis (IPF). In this study, we investigated the effects of targeting the CD206 receptor in M2-like macrophages with a novel synthetic analogue of a naturally occurring Host Defense Peptide (HDP), RP-832c, to decrease profibrotic cytokines. RP-832c selectively binds to CD206 on M2-polarized bone marrow-derived macrophages (BMDM) in vitro, resulting in a time-dependent decrease in CD206 expression and a transient increase in M1-macrophage marker TNF-α. To elucidate the antifibrotic effects of RP-832c, we used a murine model of bleomycin (BLM)-induced early-stage pulmonary fibrosis. RP-832c significantly reduced fibrosis in a dose-dependent manner, and decreased CD206, TGF-ß1, and α-SMA expression in mouse lungs. Similarly, in an established model of lung fibrosis, RP-832c significantly decreased lung fibrosis and significantly decreased inflammatory cytokines TNF-α, IL-6, IL-10, IFN-γ, CXCL1/2, and fibrosis markers TGF-ß1 and MMP-13. In comparison with the FDA-approved drugs Nintedanib and Pirfenidone, RP-832c exhibited a similar reduction in fibrosis compared to Pirfenidone, and to a greater extent than Nintedanib, with no apparent toxicities observed. In summary, our findings showed that inhibiting the profibrotic alternatively activated M2-like macrophages using a novel peptide, RP-832c, could reduce BLM-induced pulmonary fibrosis in mice, warranting the therapeutic potential of this peptide for patients with pulmonary fibrosis.

Anti-PD-1/L1 lead-in before MAPK inhibitor combination maximizes antitumor immunity and efficacy.

Rationally sequencing and combining PD-1/L1-and MAPK-targeted therapies may overcome innate and acquired resistance. Since increased clinical benefit of MAPK inhibitors (MAPKi) is associated with previous immune checkpoint therapy, we compare the efficacies of sequential and/or combinatorial regimens in subcutaneous murine models of melanoma driven by BrafV600, Nras, or Nf1 mutations as well as colorectal and pancreatic carcinoma driven by KrasG12C. Anti-PD-1/L1 lead-in preceding MAPKi combination optimizes response durability by promoting pro-inflammatory polarization of macrophages and clonal expansion of interferon-γhi, and CD8+ cytotoxic and proliferative (versus CD4+ regulatory) T cells that highly express activation genes. Since therapeutic resistance of melanoma brain metastasis (MBM) limits patient survival, we demonstrate that sequencing anti-PD-1/L1 therapy before MAPKi combination suppresses MBM and improves mouse survival with robust T cell clonal expansion in both intracranial and extracranial metastatic sites. We propose clinically testing brief anti-PD-1/L1 (± anti-CTLA-4) dosing before MAPKi co-treatment to suppress therapeutic resistance.

A Novel CD206 Targeting Peptide Inhibits Bleomycin Induced Pulmonary Fibrosis in Mice.

Activated M2 polarized macrophages are drivers of pulmonary fibrosis in several clinical scenarios such as Acute Respiratory Disease Syndrome (ARDS) and Idiopathic Pulmonary Fibrosis (IPF), through the production of inflammatory and fibrosis-inducing cytokines. In this study, we investigated the effect of targeting the CD206 receptor with a novel fragment of a Host Defense Peptide (HDP), RP-832c to decrease cytokines that cause fibrosis. RP-832c selectively binds to CD206 on M2 polarized bone marrow derived macrophages (BMDM) in vitro , resulting in a time-dependent decrease in CD206 expression, and a transient increase in M1 marker TNFα, which resolves over a 24hr period. To elucidate the antifibrotic effect of RP-832c, we used a murine model of bleomycin (BLM) -induced early-stage pulmonary fibrosis. RP-832c significantly reduced bleomycin-induced fibrosis in a dosage dependent manner, as well as decreased CD206, TGF-ß1 and α-SMA expression in mouse lungs. Interestingly we did not observe any changes in the resident alveolar macrophage marker CD170 expression. Similarly, in an established model of lung fibrosis, RP-832c significantly decreased fibrosis in the lung, as well as significantly decreased inflammatory cytokines TNFα, IL-6, IL-10, INF-γ, CXCL1/2, and fibrosis markers TGF-ß1 and MMP-13. In comparison with FDA approved drugs, Nintedanib and Pirfenidone, RP-832c exhibited a similar reduction in fibrosis compared to Pirfenidone, and to a greater extent than Nintedanib, with no apparent toxicities observed on body weight or blood chemistry. In summary, RP-832c is a potential agent to mitigate the overactivity of M2 macrophages in pathogenesis several pulmonary fibrotic diseases, including SARS-CoV-2 induced lung fibrosis.

Pathogenic Activation of Mesenchymal Stem Cells Is Induced by the Disease Microenvironment in Systemic Sclerosis.

OBJECTIVE: In systemic sclerosis (SSc), a persistent tissue repair process leads to progressive fibrosis of the skin and internal organs. The role of mesenchymal stem cells (MSCs), which characteristically initiate and regulate tissue repair, has not been fully evaluated. We undertook this study to investigate whether dividing metakaryotic MSCs are present in SSc skin and to examine whether exposure to the disease microenvironment activates MSCs and leads to transdifferentiation. METHODS: Skin biopsy material from patients with recent-onset diffuse SSc was examined by collagenase spread of 1-mm-thick surface-parallel sections, in order to identify dividing metakaryotic stem cells in each tissue plane. Adipose-derived MSCs from healthy controls were treated with dermal blister fluid (BF) from patients with diffuse SSc and profiled by next-generation sequencing, or they were evaluated for phenotypic changes relevant to SSc. Differential responses of dermal fibroblasts were studied in parallel. RESULTS: MSC-like cells undergoing active metakaryotic division were identified in SSc sections (but not control sections) most prominently in the deep dermis and adjacent to damaged microvessels, in both clinically involved and uninvolved skin. Furthermore, exposure to SSc BF caused selective MSC activation, inducing a myofibroblast signature, while reducing signatures of vascular repair and adipogenesis and enhancing migration and contractility. Microenvironmental factors implicated in inducing transdifferentiation included the profibrotic transforming growth factor ß, the presence of lactate, and mechanosensing, while the microenvironment Th2 cytokine, interleukin-31, enhanced osteogenic commitment (calcinosis). CONCLUSION: Dividing MSC-like cells are present in the SSc disease microenvironment where multiple factors, likely acting in concert, promote transdifferentiation and lead to a complex and resistant disease state.

Mannose receptor (CD206) activation in tumor-associated macrophages enhances adaptive and innate antitumor immune responses.

Solid tumors elicit a detectable immune response including the infiltration of tumor-associated macrophages (TAMs). Unfortunately, this immune response is co-opted into contributing toward tumor growth instead of preventing its progression. We seek to reestablish an antitumor immune response by selectively targeting surface receptors and endogenous signaling processes of the macrophage subtypes driving cancer progression. RP-182 is a synthetic 10-mer amphipathic analog of host defense peptides that selectively induces a conformational switch of the mannose receptor CD206 expressed on TAMs displaying an M2-like phenotype. RP-182-mediated activation of this receptor in human and murine M2-like macrophages elicits a program of endocytosis, phagosome-lysosome formation, and autophagy and reprograms M2-like TAMs to an antitumor M1-like phenotype. In syngeneic and autochthonous murine cancer models, RP-182 suppressed tumor growth, extended survival, and was an effective combination partner with chemo- or immune checkpoint therapy. Antitumor activity of RP-182 was also observed in CD206high patient-derived xenotransplantation models. Mechanistically, via selective reduction of immunosuppressive M2-like TAMs, RP-182 improved adaptive and innate antitumor immune responses, including increased cancer cell phagocytosis by reprogrammed TAMs.

Use of Patterned Collagen Coated Slides to Study Normal and Scleroderma Lung Fibroblast Migration.

Systemic sclerosis (SSc) is a spreading fibrotic disease affecting the skin and internal organs. We aimed to model pathogenic fibroblast migration in SSc in order to identify enhancing factors, measure the effect of migrating cells on underlying extracellular matrix (ECM) and test possible therapeutic inhibitors. Novel patterned collagen substrates were used to investigate alignment and migration of skin and lung fibroblasts from SSc patients and healthy controls. Normal lung but not skin fibroblasts consistently elongated and aligned with underlying collagen and migrated dependent on PDGF or serum. SSc lung fibroblasts remained growth factor dependent, did not migrate more rapidly and were less restricted to alignment of the collagen. Multiple collagen proline and lysine-modifying enzymes were identified in SSc but not control fibroblast extracellular matrix preparations, indicating differential levels of ECM modification by the diseased cells. Profiling of migrating cells revealed a possible SCF/c-Kit paracrine mechanism contributing to migration via a subpopulation of cells. Heparin, which binds ligands including PDGF and SCF, and imatininib which blocks downstream tyrosine kinase receptors, both inhibited lung fibroblast migration individually but showed synergy in SSc cells. Pathologic lung fibroblasts from SSc patients modify ECM during migration but remain growth factor dependent and sensitive to inhibitors.

Multiplex cytokine analysis of dermal interstitial blister fluid defines local disease mechanisms in systemic sclerosis.

INTRODUCTION: Clinical diversity in systemic sclerosis (SSc) reflects multifaceted pathogenesis and the effect of key growth factors or cytokines operating within a disease-specific microenvironment. Dermal interstitial fluid sampling offers the potential to examine local mechanisms and identify proteins expressed within lesional tissue. We used multiplex cytokine analysis to profile the inflammatory and immune activity in the lesions of SSc patients. METHODS: Dermal interstitial fluid sample from the involved forearm skin, and synchronous plasma samples were collected from SSc patients (n = 26, diffuse cutaneous SSc (DcSSc) n = 20, limited cutaneous SSc (LcSSc) n = 6), and healthy controls (HC) (n = 10) and profiled by Luminex® array for inflammatory cytokines, chemokines, and growth factors. RESULTS: Luminex® profiling of the dermal blister fluid showed increased inflammatory cytokines (median interleukin ( IL)-6 in SSc 39.78 pg/ml, HC 5.51 pg/ml, p = 0.01, median IL-15 in SSc 6.27 pg/ml, HC 4.38 pg/ml, p = 0.03), chemokines (monocyte chemotactic protein (MCP)-3 9.81 pg/ml in SSc, 7.18 pg/ml HC, p = 0.04), and profibrotic growth factors (platelet derived growth factor (PDGF)-AA 10.38 pg/ml versus 6.94 pg/ml in HC, p = 0.03). In general dermal fluid and plasma cytokine levels did not correlate, consistent with predominantly local production of these factors within the dermal lesions, rather than leakage from the serum. In hierarchical clustering and network analysis IL-6 emerged as a key central mediator. CONCLUSIONS: Our data confirm that an immuno-inflammatory environment and aberrant vascular repair are intimately linked to fibroblast activation in lesional skin in SSc. This non-invasive method could be used to profile disease activity in the clinic, and identifies key inflammatory or pro-fibrotic proteins that might be targeted therapeutically. Distinct subgroups of SSc may be defined that show innate or adaptive immune cytokine signatures.

Mosaicismo sexual / Sexual mosaicism

RESUMEN Los trastornos del desarrollo sexual son estados congénitos en los cuales el desarrollo del sexo cromosómico, gonadal o anatómico es atípico. Por tratarse de un caso sumamente raro consideramos de interés su presentación. Se presenta adolescente masculino de 15 años, con antecedentes de genitales atípicos al nacer, desarrollo de baja talla y estigmas turnerianos, pubertad espontánea y normal. Los estudios genéticos determinaron como sexo cromosómico un mosaico 45,X/46,XY/47XYY, y sexo molecular varón. Se inscribió socialmente como varón, se le realizó cirugía de reconstrucción genital y utilizó tratamiento con hormona de crecimiento biosintética que mantiene actualmente. La evolución clínica ha sido favorable con adecuada integración social. Ante la presencia de genitales atípicos al nacer se necesita de un manejo multidisciplinario. El diagnóstico etiológico de los trastornos de la diferenciación sexual requiere de una alta pericia médica. Un tratamiento integral en estos pacientes les garantiza una buena calidad de vida(AU)

ABSTRACT Sexual development´s disorders are congenital states in which the development of the chromosomal, anatomic or gonadal sex is atypical. Since this is a very rare case, we consider it as of interests for presentation. It is presented a teenager, 15-years-old male, with a history of atypical genitalia at birth, development of short height and Turner's stigmas, and spontaneous and normal puberty. The genetic studies identified as chromosomal sex a mosaic 45,X/46,XY/47XYY and male as molecular sex. He was socially registered as a male, he had a genital reconstruction surgery and he was under treatment with biosynthetic growth hormone that he currently maintains. The clinical evolution has been favourable with adequate social integration. In the presence of atypical genitalia at birth, it is needed a multidisciplinary management. The etiological diagnosis of disorders of sexual differentiation requires a high level of medical expertise. A comprehensive treatment in these patients guarantees them a good quality of life(AU)

Diet-induced obesity and hepatic gene expression alterations in C57BL/6J and ICAM-1-deficient mice.

The effects of high-fat feeding on the development of obesity were evaluated in intercellular adhesion molecule-1 (ICAM-1) knockout and C57BL/6J (B6) male mice fed a high-fat diet for < or =50 days. Serum and tissues were collected at baseline and after 1, 11, and 50 days on the diet. After 11 days on the diet, ICAM-1-deficient, but not B6, mice developed fatty livers and showed a significant increase in inguinal fat pad weight. At day 50, ICAM-1-deficient mice weighed less, and their adiposity index and circulating leptin levels were significantly lower than those of B6 controls. To better understand the early differential response to the diet, liver gene expression was analyzed at three time points by use of Affymetrix GeneChips. In both strains, a similar pattern of gene expression was detected in response to the high-fat diet. However, sterol regulatory element-binding protein-1, apolipoprotein A4, and adipsin mRNAs were significantly induced in ICAM-1-deficient livers, suggesting that these genes and their associated pathways may be involved in the acute diet response observed in the knockout mice.

Factores de riesgo y protectores en el medio universitario costarricense: investigación cualitativa-grupos focales / Risk and protective factors in the Costa Rican university environment

El informe representa representa el esfuerzo pionero de un grupo de profesionales que se enfrentan por primera vez a coordinar e implementar un estudio interuniversitario e interdisciplinario, bajo la modalidad de investigación cualitativa, utilizando la metodología de "grupos focales". El estudio constituye una aproximación y un análisis "del sentir" y de las "expresiones" de un grupo de estudiantes universitarios (seleccionados bajo las características que demanda un grupo focal), que con sus respuestas y reflexiones, llegan a sistematizar y complementar los datos cuantitativos (encuestas y entrevistas) existentes sobre el tema en cuestion.