Remodeling of extracellular matrix by normal and tumor-associated fibroblasts promotes cervical cancer progression

Background: Comparison of tissue microarray results of 29 cervical cancer and 27 normal cervix tissue samples using immunohistochemistry revealed considerable reorganization of the fibrillar stroma of these tumors. Preliminary densitometry analysis of laminin-1, α -smooth muscle actin (SMA) and fibronectin immunostaining demonstrated 3.8-fold upregulation of laminin-1 and 5.2-fold increase of SMA in the interstitial stroma, indicating that these proteins and the activated fibroblasts play important role in the pathogenesis of cervical cancer. In the present work we investigated the role of normal and tumor-associated fibroblasts. Methods: In vitro models were used to throw light on the multifactorial process of tumor-stroma interaction, by means of studying the cooperation between tumor cells and fibroblasts. Fibroblasts from normal cervix and cervical cancers were grown either separately or in co-culture with CSCC7 cervical cancer cell line. Changes manifest in secreted glycoproteins, integrins and matrix metallo-proteases (MMPs) were explored. Results: While normal fibroblasts produced components of interstitial matrix and TGF- β 1 that promoted cell proliferation, cancer-associated fibroblasts (CAFs) synthesized ample amounts of laminin-1. The following results support the significance of laminin-1 in the invasion of CSCC7 cells: 1.) Tumor-associated fibroblasts produced more laminin-1 and less components of fibrillar ECM than normal cells; 2.) The production of laminin chains was further increased when CSCC7 cells were grown in co-culture with fibroblasts; 3.) CSCC7 cells were capable of increasing their laminin production; 4.) Tumor cells predominantly expressed integrin α 6 β 4 laminin receptors and migrated towards laminin. The integrin profile of both normal and tumor-associated fibroblasts was similar, expressing receptors for fibronectin, vitronectin and osteopontin. MMP-7 secreted by CSCC7 cells was upregulated by the presence of normal fibroblasts, whereas MMP-2 produced mainly by fibroblasts was activated in the presence of CSCC7 cells. Conclusions: Our results indicate that in addition to degradation of the basement membrane, invasion of cervical cancer is accomplished by the remodeling of the interstitial stroma, which process includes decrease and partial replacement of fibronectin and collagens by a laminin-rich matrix

Early farmers from across Europe directly descended from Neolithic Aegeans

Farming and sedentism first appeared in southwestern Asia during the early Holocene and later spread to neighboring regions, including Europe, along multiple dispersal routes. Conspicuous uncertainties remain about the relative roles of migration, cultural diffusion, and admixture with local foragers in the early Neolithization of Europe. Here we present paleogenomic data for five Neolithic individuals from northern Greece and northwestern Turkey spanning the time and region of the earliest spread of farming into Europe. We use a novel approach to recalibrate raw reads and call genotypes from ancient DNA and observe striking genetic similarity both among Aegean early farmers and with those from across Europe. Our study demonstrates a direct genetic link between Mediterranean and Central European early farmers and those of Greece and Anatolia, extending the European Neolithic migratory chain all the way back to southwestern Asia

Laser‐facilitated epicutaneous immunotherapy with depigmented house dust mite extract alleviates allergic responses in a mouse model of allergic lung inflammation

Background Skin-based immunotherapy of type 1 allergies has recently been re-investigated as an alternative for subcutaneous injections. In the current study, we employed a mouse model of house dust mite (HDM)-induced lung inflammation to explore the potential of laser-facilitated epicutaneous allergen-specific treatment. Methods Mice were sensitized against native Dermatophagoides pteronyssinus extract and repeatedly treated by application of depigmented D pteronyssinus extract via laser-generated skin micropores or by subcutaneous injection with or without alum. Following aerosol challenges, lung function was determined by whole-body plethysmography and bronchoalveolar lavage fluid was analyzed for cellular composition and cytokine levels. HDM-specific IgG subclass antibodies were determined by ELISA. Serum as well as cell-bound IgE was measured by ELISA, rat basophil leukemia cell assay, and ex vivo using a basophil activation test, respectively. Cultured lymphocytes were analyzed for cytokine secretion profiles and cellular polarization by flow cytometry. Results Immunization of mice by subcutaneous injection or epicutaneous laser microporation induced comparable IgG antibody levels, but the latter preferentially induced regulatory T cells and in general downregulated T cell cytokine production. This effect was found to be a result of the laser treatment itself, independent from extract application. Epicutaneous treatment of sensitized animals led to induction of blocking IgG, and improvement of lung function, superior compared to the effects of subcutaneous therapy. During the whole therapy schedule, no local or systemic side effects occurred. Conclusion Allergen-specific immunotherapy with depigmented HDM extract via laser-generated skin micropores offers a safe and effective treatment option for HDM-induced allergy and lung inflammation

Mechanisms of particles in sensitization, effector function and therapy of allergic disease

Humans have always been in contact with natural airborne particles from many sources including biologic particulate matter (PM) which can exhibit allergenic properties. With industrialization, anthropogenic and combustion-derived particles have become a major fraction. Currently, an ever-growing number of diverse and innovative materials containing engineered nanoparticles (NPs) are being developed with great expectations in technology and medicine. Nanomaterials have entered everyday products including cosmetics, textiles, electronics, sports equipment, as well as food, and food packaging. As part of natural evolution humans have adapted to the exposure to particulate matter, aiming to protect the individual's integrity and health. At the respiratory barrier, complications can arise, when allergic sensitization and pulmonary diseases occur in response to particle exposure. Particulate matter in the form of plant pollen, dust mites feces, animal dander, but also aerosols arising from industrial processes in occupational settings including diverse mixtures thereof can exert such effects. This review article gives an overview of the allergic immune response and addresses specifically the mechanisms of particulates in the context of allergic sensitization, effector function and therapy. In regard of the first theme (i), an overview on exposure to particulates and the functionalities of the relevant immune cells involved in allergic sensitization as well as their interactions in innate and adaptive responses are described. As relevant for human disease, we aim to outline (ii) the potential effector mechanisms that lead to the aggravation of an ongoing immune deviation (such as asthma, chronic obstructive pulmonary disease, etc.) by inhaled particulates, including NPs. Even though adverse effects can be exerted by (nano)particles, leading to allergic sensitization, and the exacerbation of allergic symptoms, promising potential has been shown for their use in (iii) therapeutic approaches of allergic disease, for example as adjuvants. Hence, allergen-specific immunotherapy (AIT) is introduced and the role of adjuvants such as alum as well as the current understanding of their mechanisms of action is reviewed. Finally, future prospects of nanomedicines in allergy treatment are described, which involve modern platform technologies combining immunomodulatory effects at several (immuno-)functional levels

Laser‐facilitated epicutaneous immunotherapy with hypoallergenic beta‐glucan neoglycoconjugates suppresses lung inflammation and avoids local side effects in a mouse model of allergic asthma

Background Allergen-specific immunotherapy via the skin targets a tissue rich in antigen-presenting cells, but can be associated with local and systemic side effects. Allergen-polysaccharide neoglycogonjugates increase immunization efficacy by targeting and activating dendritic cells via C-type lectin receptors and reduce side effects. Objective We investigated the immunogenicity, allergenicity, and therapeutic efficacy of laminarin-ovalbumin neoglycoconjugates (LamOVA). Methods The biological activity of LamOVA was characterized in vitro using bone marrow-derived dendritic cells. Immunogenicity and therapeutic efficacy were analyzed in BALB/c mice. Epicutaneous immunotherapy (EPIT) was performed using fractional infrared laser ablation to generate micropores in the skin, and the effects of LamOVA on blocking IgG, IgE, cellular composition of BAL, lung, and spleen, lung function, and T-cell polarization were assessed. Results Conjugation of laminarin to ovalbumin reduced its IgE binding capacity fivefold and increased its immunogenicity threefold in terms of IgG generation. EPIT with LamOVA induced significantly higher IgG levels than OVA, matching the levels induced by s.c. injection of OVA/alum (SCIT). EPIT was equally effective as SCIT in terms of blocking IgG induction and suppression of lung inflammation and airway hyperresponsiveness, but SCIT was associated with higher levels of therapy-induced IgE and TH2 cytokines. EPIT with LamOVA induced significantly lower local skin reactions during therapy compared to unconjugated OVA. Conclusion Conjugation of ovalbumin to laminarin increased its immunogenicity while at the same time reducing local side effects. LamOVA EPIT via laser-generated micropores is safe and equally effective compared to SCIT with alum, without the need for adjuvant

Preclinical Efficacy of Cabazitaxel Loaded Poly (2-alkyl cyanoacrylate) Nanoparticle Variants

\ua9 2024 Valsalakumari et al. This work is published and licensed by Dove Medical Press Limited.Background: Biodegradable poly(alkyl cyanoacrylate) (PACA) nanoparticles (NPs) are receiving increasing attention in anti-cancer nanomedicine development not only for targeted cancer chemotherapy, but also for modulation of the tumor microenvironment. We previously reported promising results with cabazitaxel (CBZ) loaded poly(2-ethylbutyl cyanoacrylate) NPs (PEBCA-CBZ NPs) in a patient derived xenograft (PDX) model of triple-negative breast cancer, and this was associated with a decrease in M2 macrophages. The present study aims at comparing two endotoxin-free PACA NP variants (PEBCA and poly(2-ethylhexyl cyanoacrylate); PEHCA), loaded with CBZ and test whether conjugation with folate would improve their effect. Methods: Cytotoxicity assays and cellular uptake of NPs by flow cytometry were performed in different breast cancer cells. Biodistribution and efficacy studies were performed in PDX models of breast cancer. Tumor associated immune cells were analyzed by multiparametric flow cytometry. Results: In vitro studies showed similar NP-induced cytotoxicity patterns despite difference in early NP internalization. On intravenous injection, the liver cleared the majority of NPs. Efficacy studies in the HBCx39 PDX model demonstrated an enhanced effect of drug-loaded PEBCA variants compared with free drug and PEHCA NPs. Furthermore, the folate conjugated PEBCA variant did not show any enhanced effects compared with the unconjugated counterpart which might be due to unfavorable orientation of folate on the NPs. Finally, analyses of the immune cell populations in tumors revealed that treatment with drug loaded PEBCA variants affected the myeloid cells, especially macrophages, contributing to an inflammatory, immune activated tumor microenvironment. Conclusion: We report for the first time, comparative efficacy of PEBCA and PEHCA NP variants in triple negative breast cancer models and show that CBZ-loaded PEBCA NPs exhibit a combined effect on tumor cells and on the tumor associated myeloid compartment, which may boost the anti-tumor response

In silico design of Phl p 6 variants with altered Fold-Stability significantly impacts antigen processing, immunogenicity and immune polarization

Introduction: Understanding, which factors determine the immunogenicity and immune polarizing properties of proteins, is an important prerequisite for designing better vaccines and immunotherapeutics. While extrinsic immune modulatory factors such as pathogen associated molecular patterns are well-understood, far less is known about the contribution of protein inherent features. Protein fold-stability represents such an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization. Methods: MAESTRO software was used for in silico prediction of stabilizing or destabilizing point mutations. Mutated proteins were expressed in E. coli, and their thermal stability and resistance to endolysosomal proteases was determined. Resulting peptides were analyzed by mass spectrometry. The structure of the most stable mutant protein was assessed by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation in vitro, as well as antibody responses and T cell polarization in vivo in an adjuvant-free BALB/c mouse model. Results: In comparison to wild-type protein, stabilized or destabilized mutants displayed changes in thermal stability ranging from −5 to +14°. While highly stabilized mutants were degraded very slowly, destabilization led to faster proteolytic processing in vitro. This was confirmed in BMDCs, which processed and presented the immunodominant epitope from a destabilized mutant more efficiently compared to a highly stable mutant. In vivo, stabilization resulted in a shift in immune polarization from TH2 to TH1/TH17 as indicated by higher levels of IgG2a and increased secretion of TNF-α, IFN-γ, IL-17, and IL-21. Conclusion: MAESTRO software was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with the speed of proteolytic degradation and presentation of peptides on the surface of dendritic cells in vitro. This change in processing kinetics significantly influenced the polarization of T cell responses in vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines

Early farmers from across Europe directly descended from Neolithic Aegeans

Farming and sedentism first appeared in southwestern Asia during the early Holocene and later spread to neighboring regions, including Europe, along multiple dispersal routes. Conspicuous uncertainties remain about the relative roles of migration, cultural diffusion, and admixture with local foragers in the early Neolithization of Europe. Here we present paleogenomic data for five Neolithic individuals from northern Greece and northwestern Turkey spanning the time and region of the earliest spread of farming into Europe. We use a novel approach to recalibrate raw reads and call genotypes from ancient DNA and observe striking genetic similarity both among Aegean early farmers and with those from across Europe. Our study demonstrates a direct genetic link between Mediterranean and Central European early farmers and those of Greece and Anatolia, extending the European Neolithic migratory chain all the way back to southwestern Asia

Author Correction: Amplitude of travelling front as inferred from 14C predicts levels of genetic admixture among European early farmers.

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