Lack Of Immune Response Research Articles

Fusobacterium nucleatum: a novel regulator of antitumor immune checkpoint blockade therapy in colorectal cancer.

Neoadjuvant immune checkpoint blockade (ICB) has achieved significant success in treating various cancers, leading to improved therapeutic responses and survival rates among patients. However, in colorectal cancer (CRC), ICB has yielded poor results in tumors that are mismatch repair proficient, microsatellite-stable, or have low levels of microsatellite instability (MSI-L), which account for up to 95% of CRC cases. The underlying mechanisms behind the lack of immune response in MSI-negative CRC to immune checkpoint inhibitors remain an open conundrum. Consequently, there is an urgent need to explore the intrinsic mechanisms and related biomarkers to enhance the intratumoral immune response and render the tumor "immune-reactive". Intestinal microbes, such as the oral microbiome member Fusobacterium nucleatum (F. nucleatum), have recently been thought to play a crucial role in regulating effective immunotherapeutic responses. Herein, we advocate the idea that a complex interplay involving F. nucleatum, the local immune system, and the tumor microenvironment (TME) significantly influences ICB responses. Several mechanisms have been proposed, including the regulation of immune cell proliferation, inhibition of T lymphocyte, natural killer (NK) cell function, and invariant natural killer T (iNKT) cell function, as well as modification of the TME. This review aims to summarize the latest potential roles and mechanisms of F. nucleatum in antitumor immunotherapies for CRC. Additionally, it discusses the clinical application value of F. nucleatum as a biomarker for CRC and explores novel strategies, such as nano-delivery systems, for modulating F. nucleatum to enhance the efficacy of ICB therapy.

Evaluating hydrophobic recovery of N2 and H2O(g) plasma modified silk fibroin films aged at ambient and elevated temperatures.

Silk fibroin is a naturally derived polymer with great potential for biomedical use due to its strength, lack of immune response, and ability to biodegrade. The relatively hydrophobic nature of silk, however, can cause challenges with cell adhesion in vivo. Therefore, modification must be performed to improve the surface hydrophilicity, enhancing silk utility in the biomedical space. Low-temperature plasma (LTP) treatment is an established method for polymer modification and has the benefits of being a solvent-free, adaptable process. N2 and H2O(g) LTP treatments are both well-documented as strategies to enhance polar functional groups on a polymer's surface. However, many polymers tend to revert to their original hydrophobic state upon aging, reversing the effects of LTP modification. The hydrophobic recovery of N2 and H2O(g) LTP-modified silk has not been previously studied but has important implications for the uses and longevity of silk substrates in biomedical contexts. The goal of this study was to systematically evaluate the hydrophobic recovery of N2 and H2O(g) LTP-treated silk films. Films were LTP-modified using optimized plasma parameters (applied power, pressure, treatment time) and aged under both ambient and elevated temperature conditions up to 6 weeks after the initial treatment. Silk film surface properties were evaluated immediately after treatment and throughout the aging process using both water contact angle goniometry and x-ray photoelectron spectroscopy. LTP-treated silk films demonstrated a significant decrease in hydrophobicity compared to the untreated controls. Remarkably, both N2 and H2O(g) LTP modifications resulted in surfaces that retained hydrophilic properties over the 6 week aging period. Our findings represent a departure from what has been previously demonstrated in most LTP-modified synthetic polymers, suggesting that the secondary structure of silk fibroin plays a critical role in resisting hydrophobic recovery.

High neutralizing antibody mismatch as a possible reason for vaccine failure in two children with severe tick-borne encephalitis

We describe two adolescents (13 and 16 years old) with severe tick-borne encephalitis (TBE) and vaccination breakthrough (VBT). Both suffer from severe persistent neurologic sequelae. Both patients had high TBE-IgG-titers after vaccination at the beginning of the infection and a low or missing TBE-IgM response (Type 2 vaccine failure). Neutralization tests show low titers against the respective infecting TBE virus strain and higher titers against the vaccine strain at the beginning of the infection implying an individual weak or impaired immune response to the respective virus as possible cause of TBE vaccine failure.We do not know of any similar observation or explanation for the phenomenon and at the moment can only speculate of a severe course correlated to highly mismatched IgG. This constellation of high TBE IgGs, the lack of immune response and a severe course strongly resembles the severe TBE courses that occurred in the past after TBE immunoglobulin administration.To our knowledge differentiation between structural and functional antibodies by neutralization tests with a) the affecting TBE virus strain and b) the vaccine virus strain in TBE vaccine failures has never been described before. We conclude (1) to consider a TBE virus infection also in vaccinated children presenting with meningoencephalitis, (2) to perform a broad immunological work-up in severe TBE especially after VBT, (3) to further study if high mismatch IgG's are a possible reason for vaccine failure.

Identifying mechanisms of acquired immune escape from sequential, paired biopsies.

2519 Background: Resistance to immune checkpoint blockade (ICB) can manifest as disease progression either at the initiation of treatment (primary resistance) or after some initial response (acquired resistance). To better understand the mechanisms underlying acquired resistance, the imCORE Network is conducting a study (NCT03333655) to examine changes in tumor biology from pre-treatment to disease progression across cancer types. Methods: Eligible patients included those experiencing clinical benefit on ICB (defined as objective response or stable disease longer than 6 months) and had evaluable tissue samples from both pre-treatment and within 30 days of progression. Samples were subjected to whole exome sequencing (WES), RNA sequencing (RNA-Seq), and immunohistochemistry (IHC). Whole blood samples or adjacent normal tissue were used as a reference for tumor variant calling. Results: As of December 3rd, 2021, 24 enrolled patients have complete sample pairs. Of those, melanoma, bladder, and lung were most common (n = 7, 6 and 5 pairs, respectively), but our cohort also included patients with breast cancer, squamous head & neck, and renal cell carcinoma. IHC data unexpectedly showed a modest, but consistent increase in tumor infiltrating CD8+ T cells at progression. In addition, IHC evidence of a decrease in MHC-I proteins (HLA-A and B2M) suggest that in 5 out of 24 cases, key proteins needed for antigen presentation are lost. Global differential gene expression analysis showed that immune gene expression was significantly increased at progression, including numerous chemokines and significant enrichment in gene sets responsible for antigen presentation machinery. Protein-altering mutations at progression appeared in B2M in one patient, and CXCL9 in another. However, in most cases it is unclear what genetic alterations are responsible. We do not observe evidence of consistent IFNγ and Jak/stat signaling loss or antigen presentation loss. Conclusions: While ICB resistance is thought to be associated with a lack of immune response within the TME, we found that acquired resistance is usually associated with either maintenance or an increase in immune infiltration. Multiple alterations that are unique to individual patients also continue to emerge. Our data shows ICB resistance is multifactorial and associated with dynamic changes to markers amid immune activation and inhibition. Clinical trial information: NCT03333655.

Tumor-specific T cells support chemokine-driven spatial organization of intratumoral immune microaggregates needed for long survival

BackgroundThe composition of the tumor immune microenvironment (TIME) associated with good prognosis generally also predicts the success of immunotherapy, and both entail the presence of pre-existing tumor-specific T cells. Here,...

Sex-dependent pain trajectories induced by prolactin require an inflammatory response for pain resolution

Pain development and resolution patterns in many diseases are sex-dependent. This study aimed to develop pain models with sex-dependent resolution trajectories, and identify factors linked to resolution of pain in females and males. Using different intra-plantar (i.pl.) treatment protocols with prolactin (PRL), we established models with distinct, sex-dependent patterns for development and resolution of pain. An acute PRL-evoked pain trajectory, in which hypersensitivity is fully resolved within 1 day, showed substantial transcriptional changes after pain-resolution in female and male hindpaws and in the dorsal root ganglia (DRG). This finding supports the notion that pain resolution is an active process. Prolonged treatment with PRL high dose (1 μg) evoked mechanical hypersensitivity that resolved within 5–7 days in mice of both sexes and exhibited a pro-inflammatory transcriptional response in the hindpaw, but not DRG, at the time point preceding resolution. Flow cytometry analysis linked pro-inflammatory responses in female hindpaws to macrophages/monocytes, especially CD11b+/CD64+/MHCII+ cell accumulation. Prolonged low dose PRL (0.1 μg) treatment caused non-resolving mechanical hypersensitivity only in females. This effect was independent of sensory neuronal PRLR and was associated with a lack of immune response in the hindpaw, although many genes underlying tissue damage were affected. We conclude that different i.pl. PRL treatment protocols generates distinct, sex-specific pain hypersensitivity resolution patterns. PRL-induced pain resolution is preceded by a pro-inflammatory macrophage/monocyte-associated response in the hindpaws of mice of both sexes. On the other hand, the absence of a peripheral inflammatory response creates a permissive condition for PRL-induced pain persistency in females.

Oral Consumption of Bread from an RNAi Wheat Line with Strongly Silenced Gliadins Elicits No Immunogenic Response in a Pilot Study with Celiac Disease Patients.

Celiac disease (CD) is a genetically predisposed, T cell-mediated and autoimmune-like disorder caused by dietary exposure to the storage proteins of wheat and related cereals. A gluten-free diet (GFD) is the only treatment available for CD. The celiac immune response mediated by CD4+ T-cells can be assessed with a short-term oral gluten challenge. This study aimed to determine whether the consumption of bread made using flour from a low-gluten RNAi wheat line (named E82) can activate the immune response in DQ2.5-positive patients with CD after a blind crossover challenge. The experimental protocol included assessing IFN-γ production by peripheral blood mononuclear cells (PBMCs), evaluating gastrointestinal symptoms, and measuring gluten immunogenic peptides (GIP) in stool samples. The response of PBMCs was not significant to gliadin and the 33-mer peptide after E82 bread consumption. In contrast, PBMCs reacted significantly to Standard bread. This lack of immune response is correlated with the fact that, after E82 bread consumption, stool samples from patients with CD showed very low levels of GIP, and the symptoms were comparable to those of the GFD. This pilot study provides evidence that bread from RNAi E82 flour does not elicit an immune response after a short-term oral challenge and could help manage GFD in patients with CD.

Immunograph-based analysis of the influenza a(H1N1)pdm09 vaccine strain immunogenicity in the pandemic and post-pandemic period (2009–2014)

Currently, the assessment of the immunogenic properties of influenza viruses as a part of influenza vaccines is carried out by using seroprotection, seroconversion as well as the rate of increases in post-vaccination antibodies. At the same time, significant differences in the immunogenicity of vaccines related to dynamic formation of high antibody titers responsible for long-term protection of the vaccinated, are neglected. Influenza viruses such as A(H1N1)pdm09 that caused 2009–2010 pandemic continue to circulate in the population, therefore, the assessment of the immunogenic activity of vaccine viruses prepared during the pandemic period is interesting in for the methodology to prepare pandemic vaccines to be used in various groups (adults, children, elderly people). Analyzing immunogenicity of influenza vaccines used during the 2009–2010 swine influenza pandemic and the post-pandemic period up to the year 2014 was carried out by applying the graphical method for assessing immunogenicity (immunographs) measured as follows: for each group of vaccinated subjects (depending on the vaccine used), an increased rate in antibody level was calculated and the graphs of immunogenicity were plotted. An increased rate of serum antibodies magnitude from vaccinated subjects and the number of sera (in %) with a given fold increase rate in antibody level from 1 to the maximum magnitude were plotted on the x- and y-axis, respectively. The proposed method for assessing immunogenicity allows to plot immunogenicity graphs regardless of the serum antibodies level found in volunteers. The assessment described above revealed a several features for developing immune response to the pandemic virus A(H1N1)pdm09 such as the lack of immune response in a substantial number of adult volunteers (25–27%) and young children (60–70%) after monovaccine administration. The reason for such immune response can be both an insufficient dose of vaccine-containing viral antigen and suppressed immune response caused by the influenza A(H1N1)pdm09. A study on the immunogenic properties for seasonal influenza vaccines containing the influenza A(H1N1)pdm09 virus antigen in the years 2010–2014 revealed a variety in emerging humoral immunity ranging from a short-term, low-frequency increase in antibodies from vaccinated children to the formation of high antibody titers in elderly. Practically, immunographic analysis of influenza vaccines particularly those derived from the influenza A(H1N1)pdm09 virus, may result in proposing recommendations to increase an antigenic load at the beginning of a pandemic cycle and/or block the suppressive properties of vaccine-contained viruses in pediatric vaccines, because escalating virus dose in the vaccine may not always be achievable in this case.

ERAP, KIR, and HLA-C Profile in Recurrent Implantation Failure.

The mother’s uterine immune system is dominated by uterine natural killer (NK) cells during the first trimester of pregnancy. These cells express killer cell immunoglobulin-like receptors (KIRs) of inhibitory or activating function. Invading extravillous trophoblast cells express HLA-C molecules, and both maternal and paternal HLA-C allotypes are presented to KIRs. Endoplasmic reticulum aminopeptidase 1 (ERAP1) and 2 (ERAP2) shape the HLA class I immunopeptidome. The ERAPs remove N-terminal residues from antigenic precursor peptides and generate optimal-length peptides to fit into the HLA class I groove. The inability to form the correct HLA class I complexes with the appropriate peptides may result in a lack of immune response by NK cells. The aim of this study was to investigate the role of ERAP1 and ERAP2 polymorphisms in the context of KIR and HLA-C genes in recurrent implantation failure (RIF). In addition, for the first time, we showed the results of ERAP1 and ERAP2 secretion into the peripheral blood of patients and fertile women. We tested a total of 881 women. Four hundred ninety-six females were patients who, together with their partners, participated in in vitro fertilization (IVF). A group of 385 fertile women constituted the control group. Women positive for KIR genes in the Tel AA region and HLA-C2C2 were more prevalent in the RIF group than in fertile women (p/pcorr. = 0.004/0.012, OR = 2.321). Of the ERAP polymorphisms studied, two of them (rs26653 and rs26618) appear to affect RIF susceptibility in HLA-C2-positive patients. Moreover, fertile women who gave birth in the past secreted significantly more ERAP1 than IVF women and control pregnant women (p < 0.0001 and p = 0.0005, respectively). In the case of ERAP2, the opposite result was observed; i.e., fertile women secreted far less ERAP2 than IVF patients (p = 0.0098). Patients who became pregnant after in vitro fertilization embryo transfer (IVF-ET) released far less ERAP2 than patients who miscarried (p = 0.0032). Receiver operating characteristic (ROC) analyses indicate a value of about 2.9 ng/ml of ERAP2 as a point of differentiation between patients who miscarried and those who gave birth to a healthy child. Our study indicates that both ERAP1 and ERAP2 may be involved in processes related to reproduction.

Severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) detection pitfalls

The Severe Acute Respiratory Syndrome (CoVID 19) provoked by Coronavirus 2 (SARS CoV 2) require science-based responses. The aim of this work is to assess pitfalls found during the search of viral genomes due to sampling timing, swabbing, storage, heat-infectivity inactivation and further sample processing. According to several meta-analysis, on the day of symptom onset, the median false-negative rate is estimated to be 38% and decreased to 20% on day 8 (3 days after symptom onset) then increased to 66% on day 21 suggesting that rRT-PCRs adds little information immediately after exposure. RNA isolation from samples requires cautious handling using RNase-free solutions, pipet tips and glassware. The rRT PCR detection limits are estimated between 39 and 779 copies/mL but 3000 to 20.000 copies/ml for the antigen test. External cross contamination by imperceptible splatting requires risk management integrating the Pharmacopoeias by processing at least 10 negative contiguous to 10 positive controls in each sennries of 100 tests. . For Ct &gt;34 it was suggested no transmissible disease. The detection of antibodies one month or later after clinical signs may confirm positivity. Lack of immune response in non-immune compromised asymptomatic people may invalidate positivity. False positive disrupts efficiency for containing infections and leads to societal anxiety undermining health workforce. Because spurious methods create confusion, each step of diagnosis requires quality-control and risk assessment, knowing that rRT PCRs amplify more than 10.000 million times the signal of 1 viral element

Lack of immune response after mRNA vaccination to SARS‐CoV‐2 in a solid organ transplant patient

The recent approval and distribution of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) have been a major development in the fight against the current coronavirus disease 2019 (COVID‐19) pandemic. The first two vaccines approved in the United States, mRNA‐1273, and BNT162b2, are both messenger RNA (mRNA) based and highly effective in immunocompetent persons, but efficacy in patients on immunosuppressants has not been established. Additionally, data suggests these patients are less likely than immunocompetent people to develop neutralizing antibodies after COVID‐19 infection. Given the high risk of poor outcomes in organ transplant and immunosuppressed patients, effective vaccination is paramount in this group. We present the first reported case of a solid organ transplant patient who failed to achieve seroconversion after two doses of mRNA vaccine. This case has significant implications about how immunosuppressed patients should be counseled about SARS‐CoV‐2 vaccination and the protection provided. Physicians should remain clinically suspicious for infection with SARS‐CoV‐2 despite vaccination status in solid organ transplant patients.

Transgene Delivery to Human Induced Pluripotent Stem Cells Using Nanoparticles.

Human induced pluripotent stem cells (hiPSCs) and hiPSCs-derived cells have the potential to revolutionize regenerative and precision medicine. Genetically reprograming somatic cells to generate hiPSCs and genetic modification of hiPSCs are considered the key procedures for the study and application of hiPSCs. However, there are significant technical challenges for transgene delivery into somatic cells and hiPSCs since these cells are known to be difficult to transfect. The existing methods, such as viral transduction and chemical transfection, may introduce significant alternations to hiPSC culture which affect the potency, purity, consistency, safety, and functional capacity of hiPSCs. Therefore, generation and genetic modification of hiPSCs through non-viral approaches are necessary and desirable. Nanotechnology has revolutionized fields from astrophysics to biology over the past two decades. Increasingly, nanoparticles have been used in biomedicine as powerful tools for transgene and drug delivery, imaging, diagnostics, and therapeutics. The most successful example is the recent development of SARS-CoV-2 vaccines at warp speed to combat the 2019 coronavirus disease (COVID-19), which brought nanoparticles to the center stage of biomedicine and demonstrated the efficient nanoparticle-mediated transgene delivery into human body. Nanoparticles have the potential to facilitate the transgene delivery into the hiPSCs and offer a simple and robust approach. Nanoparticle-mediated transgene delivery has significant advantages over other methods, such as high efficiency, low cytotoxicity, biodegradability, low cost, directional and distal controllability, efficient in vivo applications, and lack of immune responses. Our recent study using magnetic nanoparticles for transfection of hiPSCs provided an example of the successful applications, supporting the potential roles of nanoparticles in hiPSC biology. This review discusses the principle, applications, and significance of nanoparticles in the transgene delivery to hiPSCs and their successful application in the development of COVID-19 vaccines.

Expression of neurotrophic factor genes by human adipose stem cells post-induction by deprenyl.

Human adipose stem cells (hASCs) were introduced as appropriate candidate due to advantages like ease of isolation, in vitro expansion and lack of immune response. Deprenyl (Dep) was used to induce bone marrow stem cells into neuron-like cells. We investigated the Dep effect on neurotrophin genes expression in hASCs and their differentiation into neuron-like cells. The cells were isolated from small pieces of abdominal adipose tissue and subjected to flow cytometry to confirm purification. The osteogenic and adipogenic differentiation were identified. The proliferation rate and neurotrophin genes expression of treated cells were evaluated by MTT, TH immunostaining and RT-PCR. hASCs had positive response to CD44, CD73, CD90, CD105 markers and negative response to CD34 and CD45 markers and differentiated into adipocytes and osteocytes. Exposure to 10–7 M of Dep for 24 hours caused a significant increase of viable cells and BDNF, NTF-3 genes expression as compared to cultured cells in serum free medium and had no effect on the expression of NGF and GDNF genes. Based on our results, Dep is able to induce BDNF, NTF-3 and NTF-4 genes expression and neroun-like morphology in hASCs.

Efficiency of complex pig therapy with pathology of the distal department of the limbs

Studies have been conducted to establish the dynamics of the course of treatment of purulent-inflammatory process of distal limb in pigs. The treatment algorithm was presented by two schemes: classic (I – experimental screw) and experimental (II – experimental group). Among the morphological parameters investigated: hemoglobin level, erythrocyte sedimentation rate (ESR), hematocrit, leukograms, total erythrocytes and leukocytes in the blood. To determine the immunobiological activity of the body of sick pigs, serum T: lymphocytes (CD2, CD3), T-helper cells (CD4), T-suppressors/killers (CD8), IRI (T-help/T-sup.), NK cells (CD16), B-lymphocytes (CD22), immunoglobulins A, M, G, CEC and phagocytic index. Research objectives – determining the effectiveness of treatment regimens algorithm for morphological and immunological composition of the blood in different forms of lymphoma disease distal extremities in pigs. Manifestation effector function was observed a decrease in lymphocytes, proliferation and differentiation of corresponding antibodies of the immune system of pigs. Inhibition of regulatory function in the inflammatory process reduces the mechanisms of formation of biologically active compounds “chemotaxis factor”, “transfer factor”, “phagocytosis factor”, migration of neutrophils and macrophages. The decrease in the concentration of NK cells in 2 times, with the pathology open wound, indicates a decrease in the process of opsonization (antibody-dependent cellular cytotoxicity) due to the lack of immune response to pathogens in the inflammatory cell. In abscesses, purulent pododermatitis and laminitis, NK cells decrease 1.5 times with the indirect manifestation of specific cytotoxic cells (B-lymphocytes, macrophages). It is determined that the drug “Trifuzol 1%” activates effector and reparative function of specific components of immune activity (T and B lymphocytes, population of T cells, NK cells). The results of these studies in the article thoroughly show the dynamics of changes in morphological and immunological status of pigs for diseases of the musculoskeletal system of the distal extremity. The results will facilitate the work of veterinary practitioners in the treatment of nosological forms of distal limb diseases in pigs.

Abstract 2714: The metastatic potential of circulating cancer spheroids: Diagnostic assay development using embryonic circulation models

Abstract Introduction: Progression into metastatic disease is a critical step in most malignancies. Cancer cells may enter circulation very early (circulating tumor cells or CTCs), but distant metastases are disseminated only by cancer stem cells (CSCs) that survive circulation. CSCs are known to form cell clusters in vitro (spheroids/tumorspheres). We reported that tumorspheres also circulate in blood in vivo. We found that they carry cancer stem cells and strongly correlate with metastatic disease. We collected tumorspheres from blood by filtration. This project aims to test the metastatic potential of tumorspheres by developing an assay using in vivo circulation models. The goal is to establish a novel prognostic test for metastatic predisposition in cancer patients. Methods: To develop an in vivo assay for metastasis we use the chorioallantoic circulation in the chicken embryo, with several advantages: easy access to circulation, lack of immune response, rapid turnaround time (5 to 10 days) and inexpensive and simple technique. For clinical applications we introduced three technical innovations: (i) to make fertilized chicken embryos easily available we worked out a method to store viable embryos beyond 30 days; (ii) we developed a new coverglass chamber technique for micromanipulations; and (iii) for microscopic analysis new over-drained chorioallantois preparations can accommodate high resolution objectives. To test tumorspheres in embryonic circulation we used both highly metastatic (e.g. MDA-MB-436) and non-metastatic (e.g. MCF7) cell lines with GFP label, DiI membrane dye and CFDA-SA dye. For molecular studies DNA was extracted 5 days after cancer spheroid implantation and used qPCR to detect human sequences. Results: In traditional storage few embryos survive beyond 6 days. As fertilized eggs carry pluripotent stem cells, we optimized temperature, gas and humidity combinations to match known stem cell optimums. By suppressing metabolism, blocking embryo adhesion and controlling gas and humidity exchange we extended viable embryo storage beyond 30 days. We modified the traditional window method by forming visually clear microscopic coverglass chambers, and the new over-draining approach lowered the chorioallantois membranes and allowed high resolution imaging. The results showed invasive and hematogenic dissemination of the cancer spheroids. Molecular studies of human markers detected metastatic progression and generated quantitative data. Conclusions: We optimized the in vivo embryonic metastasis assay. We show that it has the potential for a sensitive and quantitative method to diagnose metastatic predisposition in cancer patients. The molecular approach (DNA extraction, PCR markers) is easily up-scalable, the instrumentation is available in most hospitals and offers cost effective and critical early diagnosis of cancer progression. Citation Format: Peter Geck, James Kubilus, Andrew Makarovskiy, Kathleen Marinelli, Christine Kyritsis, Viktoria Denes. The metastatic potential of circulating cancer spheroids: Diagnostic assay development using embryonic circulation models [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 2714.

Elaboration and Biocompatibility of an Eggshell-Derived Hydroxyapatite Material Modified with Si/PLGA for Bone Regeneration in Dentistry.

Hydroxyapatite (HAp) is the most commonly used biomaterial in modern bone regeneration studies because of its chemical similarity to bone, biocompatibility with different polymers, osteoconductivity, low cost, and lack of immune response. However, to overcome the disadvantages of HAp, which include fragility and low mechanical strength, current studies typically focus on property modification through the addition of other materials. Objective. To develop and evaluate the biocompatibility of a HAp material extracted from eggshells and modified with silicon (Si) and poly(lactic-co-glycolic) acid (PLGA). Materials and Methods. An in vitro experimental study in which a HAp material prepared from eggshells was synthesized by wet chemical and conventional chemical precipitation. Subsequently, this material was reinforced with Si/PLGA using the freezing/lyophilization method, and then osteoblast cells were seeded on the experimental material (HAp/Si/PLGA). To analyse the biocompatibility of this composite material, scanning electron microscopy (SEM) and fluorescence confocal microscopy (FCM) techniques were used. PLGA, bovine bone/PLGA (BB/PLGA), and HAp/PLGA were used as controls. Results. A cellular viability of 96% was observed for the experimental HAp/Si/PLGA material as well as for the PLGA. The viability for the BB/PLGA material was 90%, and the viability for the HAp/PLGA was 86%. Cell adhesion was observed on the exterior surface of all materials. However, a continuous monolayer and the presence of filopodia were observed over both external and internal surface of the experimental materials. Conclusions. The HAp/Si/PLGA material is highly biocompatible with osteoblastic cells and can be considered promising for the construction of three-dimensional scaffolds for bone regeneration in dentistry.

Abstract 3766: Embryonal metastasis assay, an in vivo test of circulating tumorspheres to detect metastatic predisposition

Abstract INTRODUCTION One of the unsolved problems of cancer is its progression into systemic, metastatic disease. Malignant cells are released into circulation very early (circulating tumor cells or CTCs), but distant metastases are disseminated only by cancer stem cells (CSCs) that survive circulation. We investigated a unique feature of CSCs, formation of cell clusters (spheroids or tumorspheres) in vitro. We found that it also takes place in vivo, cancer tumorspheres circulate in blood and we showed that they carry cancer stem cells and strongly correlate with metastatic disease. In this project we developed a filtration method to collect live circulating cancer stem cell in tumorspheres and searched for a method to establish a prognostic assay to predict their metastatic potential. METHODS The conventional approaches use knockout mice to assay metastasis (NOD-SCID mice, etc.), but he high cost, the complex procedure and the lengthy assay (5 weeks) ruled them out as a routine clinical assay. Our goal was to establish an in vivo translational assay to detect metastatic predisposition early, with a chance to prevent systemic progression. We found that the rapidly progressing environment in the chicken embryo offers critical advantages to test the metastatic potential of a patient’s cancer. Easy access to chorioallantois circulation and to the embryo, the lack of immune response, the rapid turnaround time (5 days) and the technical simplicity rendered the new embryonal metastatic assay a promising and cost effective translational method. We used tumorspheres of both highly metastatic and non-metastatic cell lines. For image analysis a highly metastatic cell line (MDA-MB-436) was labeled by Green Fluorescent Protein. To model the technique of labeling real life patient spheroids (DiI membrane dye and protein tracing by CFDA-SA), we used the highly metastatic MDA-MB-231 and the non metastatic MCF7 and T47D cell lines. For quantitative analysis of embryonal metastasis we used methods applied in microchimerism detection. Embryonal DNA/RNA was extracted 5-9 days after cancer spheroid implantation and used qPCR to detect human sequences. RESULTS In the embryonal metastatic assays, fluorescent and confocal imaging detected metastatic cells in the embryo. DiI and CFDA-SA labeling of native tumorspheres indicated a level of toxicity and needed to be optimized. Molecular detection of human microchimerism in the embryos was extremely sensitive and generated quantitative metastatic data. CONCLUSIONS The results indicate that the embryonal metastasis assay using molecular microchimerism detection has the potential for a sensitive and quantitative method to assess the metastatic predisposition of a cancer patient. The method needs only an incubator, DNA extraction and a thermocycler, easily available in most hospitals and offers cost effective, relevant in vivo prognostic data within days. Citation Format: Peter Geck, James Kubilus, Andrew Makarovskiy, Viktoria Denes. Embryonal metastasis assay, an in vivo test of circulating tumorspheres to detect metastatic predisposition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3766.

EVALUATION OF THE CYTOKINE STATUS IN PATIENTS WITH DIFFERENT VARIANTS OF THE COURSE OF HPV INFECTION

Aim.To evaluate the performance of some pro-inflammatory and anti-inflammatory cytokines in patients with various forms of papillomavirus infection (PVI), to conduct a clinical evaluation of the effective application of the drug containing a complex of sodium deoxyribonucleate with iron.Materials and methods. The examination of women with a latent course of PVI and a mild case of intraepithelial lesions of the cervix L-SIL (Lowgrade Squamous Intraepitelial Lesions) was conducted using a liquid-based Cytology ThinPrepR, colposcopy, biopsy of cervix, detection of HPV 16 and 18 in the cervical smear by DNA techniques (PCR in real time). Also, cytokines (IFN-gamma, IL-2, IL- 10, TNF-alpha) were quantified at the systemic level by the immunoenzyme method. The repeated examination was carried out after a course of treatment with a drug containing a complex of sodium deoxyribonucleate with iron in 3 and 6 months.Results.After 6 months of HPV therapy PCR was not detected in 94% of patients with a latent course of PVI and 75% of patients with L-SIL. Patients with a latent course of PVI showed a significant increase in the levels of IFNγ, TNF-α and IL-10 before the treatment. Patients with L-SIL showed less significant increase in IFNγ and TNF-α before the treatment, the levels of IL-10 and IL-2 had no significant deviations. After 3 months of the treatment patients with a latent course of PVI showed a decrease in the concentration of IFNγ, TNF-α and IL-10 to the indicators of conditionally healthy women. Patients with L-SIL showed a slight decrease in the concentration of TNF-α and a decrease in the concentration of IL-10 to the indicators of conditionally healthy women. The level of IFNγ and IL-2 remained almost at the same level as before the treatment.Conclusion.The use of a drug containing a complex of sodium deoxyribonucleate with iron during a latent course of PVI can help to prevent its transition to subclinical and clinical forms. The clinical efficacy of the drug Ferrovir was confirmed, manifested in the fact that 6 months after the treatment the virus DNA ceased to be determined in 94% of cases in patients from the group with a latent course of papillomavirus infection. There was a lack of immune response to HPV in L-SIL, as well as less pronounced clinical efficacy of Ferrovir in this group. It can be assumed that a more pronounced clinical effect in the treatment of patients with L-SIL can be obtained by using the complex therapy, including the appointment of the drug Ferrovir and destructive methods of treatment of the cervix or an additional prescription of the targeted therapy, aimed at the treatment of already affected and altered cells of the cervical epithelium.

Abstract 2792: The combination of immune checkpoint blockers with the anti-ICOS KY1044 antibody results in a strong tumor response

Abstract The last few years have shown a strong paradigm shift in cancer therapies with the approval of antibodies targeting immune checkpoints (CTLA-4, PD-1 and PD-L1). These immune checkpoint blockers (ICBs) are associated with a strong and long-lasting response in patients suffering from different malignancies. However, there is still a high proportion of patients showing intrinsic or acquired resistance to ICBs. Although the mechanisms associated with the lack of immune response are multiple, the presence of highly anti-inflammatory regulatory T cells (TRegs) in the tumour microenvironment (TME) is known to negatively affect the response to these therapies. Similarly, high TReg levels in the TME have been associated with poor prognosis in several cancers. Together, this highlights the potential of targeting and depleting TRegs to enhance anti-tumour responses. The Inducible T-cell costimulator (ICOS/CD278) is related to the CD28 superfamily and is induced when T cells get activated. ICOS expression levels vary in different immune cell subtypes and in different tissues. In preclinical mouse tumour models, TRegs (CD4+/FOXP3+) constitutively express ICOS on their surface and the expression of ICOS on TRegs is significantly higher than that on effector T cells (TEffs). In addition, ICOS expression on TRegs is higher in the TME than in the blood or spleen, which makes it a strong candidate for preferential depletion of TRegs in tumours. By immunizing Kymice™ in which the endogenous Icos gene has been knocked out, we identified a novel, fully human antibody called KY1044 that cross-reacts with mouse ICOS. KY1044 is an anti-ICOS subclass G1 kappa monoclonal antibody that selectively binds to dimeric ICOS (Fc fusion) with an affinity of less than 2nM. Using in vitro and in vivo approaches we demonstrate that KY1044 has a dual mechanism of action: (1) it promotes the preferential depletion of intratumoural ICOShigh TRegs resulting in an increase in the TEff:TReg ratio in the TME; and (2) it stimulates ICOSLow TEff cells. Using the mouse effector enabled version of KY1044 (mIgG2a) we confirm, using syngeneic models, a strong anti-tumour efficacy as monotherapy or in combination with surrogates of ICBs. We also demonstrated a tumour antigen specific immunity, as highlighted by the rejection of the original tumour cells in animals cured of the disease and re-challenged by the same cell line. Noteworthy, Pharmacodynamic studies demonstrate long-term depletion of TRegs and a significant increase in the TEff:TReg ratio in response to KY1044. In summary, our data demonstrate that targeting ICOS with KY1044 is a valid approach for manipulating the immune system and for inducing a strong anti-tumour response in several indications. The data presented here also warrant the assessment of KY1044 in cancer patients in a clinical trial. Citation Format: Richard C. Sainson, Matthew McCourt, Anil Thotakura, Nahida Parveen, Miha Kismac, Gwenoline Borhis, Joana Carvalho, Tracey Myers, Robert Rowlands, Hanif Ali, Hannah Craig, Vivian Wong, Qi Liang, Volker Germaschewski. The combination of immune checkpoint blockers with the anti-ICOS KY1044 antibody results in a strong tumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2792.

Diminished oligomerization in the synthesis of new anti-angiogenic cyclic peptide using solution instead of solid-phase cyclization.

The design and synthesis of novel peptides that inhibit angiogenesis is an important area for anti-angiogenic drug development. Cyclic and small peptides present several advantages for therapeutic application, including stability, solubility, increased bio-availability and lack of immune response in the host cell. We describe here the synthesis and biological evaluations of a new cyclic peptide analog of CBO-P11: cyclo(RIKPHE), designated herein as CBO-P23M, a hexamer peptide encompassing residues 82 to 86 of VEGF which are involved in the interaction with VEGF receptor-2. CBO-P23M was prepared using in solution cyclization, therefore reducing the peptide cyclodimerization occurred during solid-phase cyclization. The cyclic dimer of CBO-P23M, which was obtained as the main side product during synthesis of the corresponding monomer, was also isolated and investigated. Both peptides markedly reduce VEGF-A-induced phosphorylation of VEGFR-2 and Erk1/2. Moreover, they exhibit anti-angiogenic activity in an in vitro morphogenesis study. Therefore CBO-P23M and CBO-P23M dimer appear as attractive candidates for the development of novel angiogenesis inhibitors for the treatment of cancer and other angiogenesis-related diseases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 368-375, 2016.