Multivariate Analysis of Cellular Uptake Characteristics for a (Co)polymer Particle Library.

Controlling cellular responses to nanoparticles so far is predominantly empirical, typically requiring multiple rounds of optimization of particulate carriers. In this study, a systematic model-assisted approach should lead to the identification of key parameters that account for particle properties and their cellular recognition. A copolymer particle library was synthesized by a combinatorial approach in soap free emulsion copolymerization of styrene and methyl methacrylate, leading to a broad compositional as well as constitutional spectrum. The proposed structure-property relationships could be elucidated by multivariate analysis of the obtained experimental data, including physicochemical characteristics such as molar composition, molecular weight, particle diameter, and particle charge as well as the cellular uptake pattern of nanoparticles. It was found that the main contributors for particle size were the polymers' molecular weight and the zeta potential, while particle uptake is mainly directed by the particles' composition. This knowledge and the reported model-assisted procedure to identify relevant parameters affecting particle engulfment of particulate carriers by nonphagocytic and phagocytic cells can be of high relevance for the rational design of pharmaceutical nanocarriers and assessment of biodistribution and nanotoxicity, respectively.

Differential Expression of microRNAs in Serum of Patients with Chronic Painful Polyneuropathy and Healthy Age-Matched Controls.

Polyneuropathies (PNP) are the most common type of disorder of the peripheral nervous system in adults. However, information on microRNA expression in PNP is lacking. Following microRNA sequencing, we compared the expression of microRNAs in the serum of patients experiencing chronic painful PNP with healthy age-matched controls. We have been able to identify four microRNAs (hsa-miR-3135b, hsa-miR-584-5p, hsa-miR-12136, and hsa-miR-550a-3p) that provide possible molecular links between degenerative processes, blood flow regulation, and signal transduction, that eventually lead to PNP. In addition, these microRNAs are discussed regarding the targeting of proteins that are involved in high blood flow/pressure and neural activity dysregulations/disbalances, presumably resulting in PNP-typical symptoms such as chronical numbness/pain. Within our study, we have identified four microRNAs that may serve as potential novel biomarkers of chronic painful PNP, and that may potentially bear therapeutic implications.

Toll-like receptors control the accumulation of neutrophils in lymph nodes that expand CD4+ T cells during experimental autoimmune encephalomyelitis.

Toll-like receptors (TLR) control the activation of dendritic cells that prime CD4+ T cells in draining lymph nodes, where these T cells then undergo massive clonal expansion. The mechanisms controlling this clonal T cell expansion are poorly defined. Using the CD4+ T cell-mediated disease experimental autoimmune encephalomyelitis (EAE), we show here that this process is markedly suppressed when TLR9 signaling is increased, without noticeably affecting the transcriptome of primed T cells, indicating a purely quantitative effect on CD4+ T cell expansion. Addressing the underpinning mechanisms revealed that CD4+ T cell expansion was preceded and depended on the accumulation of neutrophils in lymph nodes a few days after immunization. Underlying the importance of this immune regulation pathway, blocking neutrophil accumulation in lymph nodes by treating mice with a TLR9 agonist inhibited EAE progression in mice with defects in regulatory T cells or regulatory B cells, which otherwise developed a severe chronic disease. Collectively, this study demonstrates the key role of neutrophils in the quantitative regulation of antigen-specific CD4+ T cell expansion in lymph nodes, and the counter-regulatory role of TLR signaling in this process.

Regulation of the apoptosis/autophagy switch by propionic acid in ventromedial hypothalamus of rats with type 2 diabetes mellitus.

Background: Hypothalamic dysregulation may cause abnormal glucose metabolism and type 2 diabetes mellitus (T2DM). The balance between autophagy and apoptosis is important for maintaining cellular/tissue homeostasis and may be disrupted in T2DM. Objectives: Since propionic acid (PA) exerts neuroprotective effects, the aim was to investigate its effects on apoptosis/autophagy switch in the ventromedial hypothalamus (VMH) of T2DM rats. Materials and methods: Male Wistar rats were divided: 1) control; 2) T2DM; groups that received (14 days, orally): 3) metformin (60 mg/kg); 4) sodium salt of PA (60 mg/kg); 5) PA + metformin. Western blotting (Bax, Bcl-xl, LC3, Beclin-1, caspase-3), RT-PCR (Bax, Bcl-xl, LC3, Beclin-1), transmission electron microscopy and immunohistochemical staining (Bax, Bcl-xl) were performed on the VMH samples. Results: T2DM-induced apoptosis and mitoptosis, enlarged endoplasmic reticulum (ER) tubules/cisterns were observed in VMH, and accompanied by an imbalance of pro- and anti-apoptotic factors: elevation of pro-apoptotic markers Bax and caspase-3, decrease in autophagy protein LC3 and anti-apoptotic Bcl-xl. Metformin and PA administration partially improved VMH ultrastructural changes by reducing mitochondrial swelling and diminishing the number of apoptotic neurons. Metformin inhibited neuronal apoptosis, however, caused reactive astrogliosis and accumulation of lipofuscin granules. Elevated number of autophagosomes was associated with the LC3, Beclin-1 and Bcl-xl increase and decrease in Bax and caspase-3 vs. T2DM. PA switched cell fate from apoptosis to autophagy by elevating LC3 and Beclin-1 levels, increasing Bcl-xl content that altogether may represent adaptive response to T2DM-induced apoptosis. PA + metformin administration lowered relative area of ER membranes/cisterns vs. control, T2DM and metformin, and was optimal considering ratio between the pro-apoptotic, anti-apoptotic and autophagy markers. Conclusion: T2DM was associated with apoptosis activation leading to impairments in VMH. PA in combination with metformin may be effective against diabetes-induced cell death by switching apoptosis to autophagy in VMH.

In vitro and in vivo evidence that the switch from calcineurin to mTOR inhibitors may be a strategy for immunosuppression in Epstein-Barr virus-associated post-transplant lymphoproliferative disorder.

Post-transplant lymphoproliferative disorder is a life-threatening complication of immunosuppression following transplantation mediated by failure of T cells to control Epstein-Barr virus (EBV)-infected and transformed B cells. Typically, a modification or reduction of immunosuppression is recommended, but insufficiently defined thus far. In order to help delineate this, we characterized EBV-antigen-specific T cells and lymphoblastoid cell lines from healthy donors and in patients with a kidney transplant in the absence or presence of the standard immunosuppressants tacrolimus, cyclosporin A, prednisolone, rapamycin, and mycophenolic acid. Phenotypes of lymphoblastoid cell-lines and T cells, T cell-receptor-repertoire diversity, and T-cell reactivity upon co-culture with autologous lymphoblastoid cell lines were analyzed. Rapamycin and mycophenolic acid inhibited lymphoblastoid cell-line proliferation. T cells treated with prednisolone and rapamycin showed nearly normal cytokine production. Proliferation and the viability of T cells were decreased by mycophenolic acid, while tacrolimus and cyclosporin A were strong suppressors of T-cell function including their killing activity. Overall, our study provides a basis for the clinical decision for the modification and reduction of immunosuppression and adds information to the complex balance of maintaining anti-viral immunity while preventing acute rejection. Thus, an immunosuppressive regime based on mTOR inhibition and reduced or withdrawn calcineurin inhibitors could be a promising strategy for patients with increased risk of or manifested EBV-associated post-transplant lymphoproliferative disorder.

Severe Acute Respiratory Syndrome Coronavirus 2 Cross-Reactive B and T Cell Responses in Kidney Transplant Patients.

BACKGROUND: Immune responses to seasonal endemic coronaviruses might have a pivotal role in protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Those SARS-CoV-2-crossreactive T cells were recently described in immunocompetent individuals. Still, data on cross-reactive humoral and cellular immunity in kidney transplant recipients is currently lacking. METHODS: The pre-existing, cross-reactive antibody B and T cell immune responses against SARS-CoV-2 in unexposed adults with kidney transplantation (Tx, n = 14) and without (non-Tx, n = 12) sampled before the pandemic were compared with 22 convalescent patients with COVID-19 (Cp) applying enzyme-linked immunosorbent assay and flow cytometry. RESULTS: In both unexposed groups, SARS-CoV-2 IgG antibodies were not detectable. Memory B cells binding spike (S) protein SARS-CoV-2 were detected in unexposed individuals (64% among Tx; 50% among non-Tx) and higher frequencies after infection (80% Cp). The numbers of SARS-CoV-2-reactive T cells were comparable between patients who had undergone Tx and those who had not. SARS-CoV-2-reactive follicular T helper cells were present in 61% of the unexposed cohort in both patients who had undergone Tx and those who had not. CONCLUSIONS: Cross-reactive memory B and T cells against SARS-CoV-2 exist also in transplanted adults, suggesting a primed adaptive immunity. The effect on the disease course may depend on the concomitant immunosuppressive drugs.

Tacrolimus-resistant SARS-CoV-2-specific T cell products to prevent and treat severe COVID-19 in immunosuppressed patients.

Solid organ transplant (SOT) recipients receive therapeutic immunosuppression that compromises their immune response to infections and vaccines. For this reason, SOT patients have a high risk of developing severe coronavirus disease 2019 (COVID-19) and an increased risk of death from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Moreover, the efficiency of immunotherapies and vaccines is reduced due to the constant immunosuppression in this patient group. Here, we propose adoptive transfer of SARS-CoV-2-specific T cells made resistant to a common immunosuppressant, tacrolimus, for optimized performance in the immunosuppressed patient. Using a ribonucleoprotein approach of CRISPR-Cas9 technology, we have generated tacrolimus-resistant SARS-CoV-2-specific T cell products from convalescent donors and demonstrate their specificity and function through characterizations at the single-cell level, including flow cytometry, single-cell RNA (scRNA) Cellular Indexing of Transcriptomes and Epitopes (CITE), and T cell receptor (TCR) sequencing analyses. Based on the promising results, we aim for clinical validation of this approach in transplant recipients. Additionally, we propose a combinatory approach with tacrolimus, to prevent an overshooting immune response manifested as bystander T cell activation in the setting of severe COVID-19 immunopathology, and tacrolimus-resistant SARS-CoV-2-specific T cell products, allowing for efficient clearance of viral infection. Our strategy has the potential to prevent severe COVID-19 courses in SOT or autoimmunity settings and to prevent immunopathology while providing viral clearance in severe non-transplant COVID-19 cases.

Early prediction of renal graft function: Analysis of a multi-center, multi-level data set.

PURPOSE OF THE STUDY: Long-term graft survival rates after renal transplantation are still poor. We aimed to build an early predictor of an established long-term outcomes marker, the estimated glomerular filtration rate (eGFR) one year post-transplant (eGFR-1y). MATERIALS AND METHODS: A large cohort of 376 patients was characterized for a multi-level bio-marker panel including gene expression, cytokines, metabolomics and antibody reactivity profiles. Almost one thousand samples from the pre-transplant and early post-transplant period were analysed and employed for machine learning-assisted prediction. RESULTS: Pre-transplant data led to a prediction achieving a Pearson's correlation coefficient of r=0.38 between measured and predicted eGFR-1y. Two weeks post-transplant, the correlation was improved to r=0.63, and at the third month, to r=0.76. eGFR values were stable throughout the first post-transplant year. Several characteristics were predictive for eGFR, including age of donor and recipient, body mass index, HLA mismatch, cytomegalovirus mismatch and valganciclovir prophylaxis. Additionally, a subset of 19 nuclear magnetic resonance bins of the urine metabolome data was shown to have potential applications in non-invasive eGFR monitoring. Importantly, we identified the expression of the genes TMEM176B and HMMR as potential prognostic markers for changes in the eGFR after the second post-transplantation week. CONCLUSIONS: Our multi-center, multi-level data set represents a milestone in the efforts to predict transplant outcome. While an acceptable predictive capacity was achieved, we are still far from predicting changes in the eGFR precisely. Additional studies employing further marker panels are needed to establish predictors of eGFR-1y for clinical application; herein, gene expression markers seem to hold the most promise.

Immune Response in Moderate to Critical Breakthrough COVID-19 Infection After mRNA Vaccination.

SARS-CoV-2 variants of concern (VOCs) can trigger severe endemic waves and vaccine breakthrough infections (VBI). We analyzed the cellular and humoral immune response in 8 patients infected with the alpha variant, resulting in moderate to fatal COVID-19 disease manifestation, after double mRNA-based anti-SARS-CoV-2 vaccination. In contrast to the uninfected vaccinated control cohort, the diseased individuals had no detectable high-avidity spike (S)-reactive CD4+ and CD8+ T cells against the alpha variant and wild type (WT) at disease onset, whereas a robust CD4+ T-cell response against the N- and M-proteins was generated. Furthermore, a delayed alpha S-reactive high-avidity CD4+ T-cell response was mounted during disease progression. Compared to the vaccinated control donors, these patients also had lower neutralizing antibody titers against the alpha variant at disease onset. The delayed development of alpha S-specific cellular and humoral immunity upon VBI indicates reduced immunogenicity against the S-protein of the alpha VOC, while there was a higher and earlier N- and M-reactive T-cell response. Our findings do not undermine the current vaccination strategies but underline a potential need for the inclusion of VBI patients in alternative vaccination strategies and additional antigenic targets in next-generation SARS-CoV-2 vaccines.

Chemical modification of uridine modulates mRNA-mediated proinflammatory and antiviral response in primary human macrophages.

In vitro transcribed (IVT)-mRNA has been accepted as a promising therapeutic modality. Advances in facile and rapid production technologies make IVT-mRNA an appealing alternative to protein- or virus-based medicines. Robust expression levels, lack of genotoxicity, and their manageable immunogenicity benefit its clinical applicability. We postulated that innate immune responses of therapeutically relevant human cells can be tailored or abrogated by combinations of 5'-end and internal IVT-mRNA modifications. Using primary human macrophages as targets, our data show the particular importance of uridine modifications for IVT-mRNA performance. Among five nucleotide modification schemes tested, 5-methoxy-uridine outperformed other modifications up to 4-fold increased transgene expression, triggering moderate proinflammatory and non-detectable antiviral responses. Macrophage responses against IVT-mRNAs exhibiting high immunogenicity (e.g., pseudouridine) could be minimized upon HPLC purification. Conversely, 5'-end modifications had only modest effects on mRNA expression and immune responses. Our results revealed how the uptake of chemically modified IVT-mRNA impacts human macrophages, responding with distinct patterns of innate immune responses concomitant with increased transient transgene expression. We anticipate our findings are instrumental to predictively address specific cell responses required for a wide range of therapeutic applications from eliciting controlled immunogenicity in mRNA vaccines to, e.g., completely abrogating cell activation in protein replacement therapies.

In-depth analysis of T cell immunity and antibody responses in heterologous prime-boost-boost vaccine regimens against SARS-CoV-2 and Omicron variant.

With the emergence of novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Variants of Concern (VOCs), vaccination studies that elucidate the efficiency and effectiveness of a vaccination campaign are critical to assess the durability and the protective immunity provided by vaccines. SARS-CoV-2 vaccines have been found to induce robust humoral and cell-mediated immunity in individuals vaccinated with homologous vaccination regimens. Recent studies also suggest improved immune response against SARS-CoV-2 when heterologous vaccination strategies are employed. Yet, few data exist on the extent to which heterologous prime-boost-boost vaccinations with two different vaccine platforms have an impact on the T cell-mediated immune responses with a special emphasis on the currently dominantly circulating Omicron strain. In this study, we collected serum and peripheral blood mononuclear cells (PBMCs) from 57 study participants of median 35-year old's working in the health care field, who have received different vaccination regimens. Neutralization assays revealed robust but decreased neutralization of Omicron VOC, including BA.1 and BA.4/5, compared to WT SARS-CoV-2 in all vaccine groups and increased WT SARS-CoV-2 binding and neutralizing antibodies titers in homologous mRNA prime-boost-boost study participants. By investigating cytokine production, we found that homologous and heterologous prime-boost-boost-vaccination induces a robust cytokine response of CD4+ and CD8+ T cells. Collectively, our results indicate robust humoral and T cell mediated immunity against Omicron in homologous and heterologous prime-boost-boost vaccinated study participants, which might serve as a guide for policy decisions.