Abstract: Lesioned fascicles (LF) in the sciatic nerves of individuals with diabetic neuropathy (DN) correlate with clinical symptom severity. This study aimed to characterize the structural and molecular composition of these lesions to better understand DN pathogenesis. Sciatic nerves from amputees with and without type 2 diabetes (T2D) were examined using ex vivo magnetic resonance neurography, in vitro imaging, and proteomic analysis. Lesions were only found in T2D donors and exhibited significant structural abnormalities, including axonal degeneration, demyelination, and impaired blood nerve barrier (BNB). While non-lesioned fascicles from T2D donors showed activation of neuroprotective pathways, lesioned fascicles lacked this response and instead displayed increased complement activation via the classical pathway. The detection of liver-derived acute-phase proteins suggests that BNB disruption facilitates harmful inter-organ communication between the liver and nerves. These findings reveal key molecular mechanisms contributing to DN and highlight potential targets for therapeutic intervention.

Induction of tolerance can be achieved with Modified Immune Cells (MIC) by infusion of mononuclear cells challenged with mitomycin C (MMC). Although MIC treatment has been successful in several animal models for autoimmunity and in clinical studies of solid organ transplantation, the mechanism of immunosuppression is not fully elucidated. In lupus nephritis, the glomerular deposition of immune complexes is associated with accumulating immune cells in the kidney. The infiltrating immune cells frequently establish tertiary lymphoid structures (TLS) supporting adaptive autoimmune responses toward locally displayed antigens. Since TLS display a high persistence to peripheral B cell depletion, the destruction of TLS presents an essential treatment goal in lupus nephritis. In this study, we used lupus nephritis prone NZB/W F1 mice to show the destruction of TLS in the kidney after MIC treatment. Independent of treatment, >86% of animals displayed dense lymphocytic aggregates proximal to the pelvic wall of the medulla and the arcuate arteries within the cortex of kidneys. Cell type composition of TLS changed drastically after MIC treatment leading to diminished B-cells, a decreased B-cell/T-cell ratio, and a T cell dominated phenotype. Furthermore, a loss of organization of the TLS was observed after MIC treatment. The strict separation of B-cell and T-cell areas was abrogated, and germinal centers were disintegrated. However, regulatory T-cells remained unchanged indicative of a B-cell centric treatment mechanism. Our data provides a putative in vivomechanism how MIC treatment inhibits progression of active lupus nephritis by the destruction of tertiary lymphoid structures within the kidney. Commercial Support - TolerogenixX GmbH

BACKGROUND AND OBJECTIVES:Antibody response after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is impaired in kidney transplant recipients. Emerging variants, such as B.1.617.2 (δ), are of particular concern because of their higher transmissibility and partial immune escape. Little is known about protection against these variants in immunocompromised patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS:In this prospective two-center study, antispike 1 IgG and surrogate neutralizing antibodies were measured in 173 kidney transplant recipients and 166 healthy controls with different vaccination schedules. In addition, different SARS-CoV-2 epitope antibodies from 135 vaccinated kidney transplant recipients were compared with antibodies in 25 matched healthy controls after second vaccination. In 36 kidney transplant recipients with seroconversion, neutralization against B.1.1.7 (α), B.1.351 (β), and B.1.617.2 (δ) was determined on VeroE6 cells and compared with neutralization in 25 healthy controls. RESULTS:Kidney transplant recipients had significantly lower seroconversion rates compared with healthy controls. After the second vaccination, antispike 1, antireceptor-binding domain, and surrogate neutralizing antibodies were detectable in 30%, 27%, and 24% of kidney transplant recipients, respectively. This compares with 100%, 96%, and 100% in healthy controls, respectively (P<0.001). Neutralization against B.1.1.7 was detectable in all kidney transplant recipients with seroconversion, with a median serum dilution that reduces infection of cells by 50% of 80 (interquartile range, 80-320). In contrast, only 23 of 36 (64%) and 24 of 36 (67%) kidney transplant recipients showed neutralization against B.1.351 and B.1.617.2, respectively, with median serum dilutions that reduce infection of cells by 50% of 20 (interquartile range, 0-40) and 20 (interquartile range, 0-40), respectively. Neutralization against different variants was significantly higher in healthy controls (P<0.001), with all patients showing neutralization against all tested variants. CONCLUSIONS:Seroconverted kidney transplant recipients show impaired neutralization against emerging variants of concern after standard two-dose vaccination. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER:Observational study to assess the SARS-CoV-2 specific immune response in kidney transplant recipients (COVID-19 related immune response), DRKS00024668.

Despite limited data on safety and immunogenicity, heterologous prime-boost vaccination is currently recommended for individuals with ChAdOx1 nCoV-19 prime immunization in certain age groups. In this prospective, single-center study we included 166 health care workers from Heidelberg University Hospital who received either heterologous ChAdOx1 nCoV-19/BNT162b2, homologous BNT162b2 or homologous ChAdOx1 nCoV-19 vaccination between December 2020 and May 2021. We measured anti-S1 IgG, SARS-CoV-2 specific neutralizing antibodies, and antibodies against different SARS-CoV-2 fragments 0–3 days before and 19–21 days after boost vaccination. Before boost, 55/70 (79%) ChAdOx1 nCoV-19-primed compared with 44/45 (98%) BNT162b2-primed individuals showed positive anti-S1 IgG with a median (IQR) anti-S1 IgG index of 1.95 (1.05–2.99) compared to 9.38 (6.26–17.12). SARS-CoV-2 neutralizing antibodies exceeded the threshold in 24/70 (34%) of ChAdOx1 nCoV-19-primed and 43/45 (96%) of BNT162b2-primed individuals. After boosting dose, median (IQR) anti-S1 IgG index in heterologous ChAdOx1 nCoV-19/BNT162b2 vaccinees was 116.2 (61.84–170), compared to 13.09 (7.03–29.02) in homologous ChAdOx1 nCoV-19 and 145.5 (100–291.1) in homologous BNT162b2 vaccinees. All boosted vaccinees exceeded the threshold for neutralization, irrespective of their vaccination scheme. Vaccination was well-tolerated overall. We show that heterologous ChAdOx1 nCoV-19/BNT162b2 vaccination is safe and induces a strong and broad humoral response in healthy individuals.

Visual Abstract Background and objectives Patients receiving hemodialysis are at high risk for both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and severe coronavirus disease 2019. A lifesaving vaccine is available, but sensitivity to vaccines is generally lower in patients on dialysis. Little is yet known about antibody responses after coronavirus disease 2019 (COVID-19) vaccination in this vulnerable group. Design, setting, participants, and measurements In this prospective single-center study, we included 22 patients on dialysis and 46 healthy controls from Heidelberg University Hospital between December 2020 and February 2021. We measured anti-S1 IgG with a threshold index for detection greater than one, neutralizing antibodies with a threshold for viral neutralization of ≥30%, and antibodies against different SARS-CoV2 fragments 17–22 days after the first dose and 18–22 days after the second dose of the mRNA vaccine BNT162b2. Results After the first vaccine dose, four of 22 (18%) patients on dialysis compared with 43 of 46 (93%) healthy controls developed positive anti-S1 IgG, with a median anti-S1 IgG index of 0.2 (interquartile range, 0.1–0.7) compared with nine (interquartile range, 4–16), respectively. SARS-CoV2 neutralizing antibodies exceeded the threshold for neutralization in four of 22 (18%) patients on dialysis compared with 43 of 46 (93%) healthy controls, with a median percent inhibition of 11 (interquartile range, 3–24) compared with 65 (interquartile range, 49–75), respectively. After the second dose, 14 of 17 (82%) patients on dialysis developed neutralizing antibodies exceeding the threshold for viral neutralization and antibodies against the receptor binding S1 domain of the spike protein, compared with 46 of 46 (100%) healthy controls, respectively. The median percent inhibition was 51 (interquartile range, 32–86) compared with 98 (interquartile range, 97–98) in healthy controls. Conclusions Patients receiving long-term hemodialysis show a reduced antibody response to the first and second doses of the mRNA vaccine BNT162b2. The majority (82%) develop neutralizing antibodies after the second dose but at lower levels than healthy controls.

In light of the limited treatment options of diabetic polyneuropathy (DPN) available, suitable animal models are essential to investigate pathophysiological mechanisms and to identify potential therapeutic targets. In vivo evaluation with current techniques, however, often provides only restricted information about disease evolution. In the study of patients with DPN, magnetic resonance neurography (MRN) has been introduced as an innovative diagnostic tool detecting characteristic lesions within peripheral nerves. We developed a novel multicontrast ultra high field MRN strategy to examine major peripheral nerve segments in diabetic mice non-invasively. It was first validated in a cross-platform approach on human nerve tissue and then applied to the popular streptozotocin(STZ)-induced mouse model of DPN. In the absence of gross morphologic alterations, a distinct MR-signature within the sciatic nerve was observed mirroring subtle changes of the nerves’ fibre composition and ultrastructure, potentially indicating early re-arrangements of DPN. Interestingly, these signal alterations differed from previously reported typical nerve lesions of patients with DPN. The capacity of our approach to non-invasively assess sciatic nerve tissue structure and function within a given mouse model provides a powerful tool for direct translational comparison to human disease hallmarks not only in diabetes but also in other peripheral neuropathic conditions.

Objective The underlying causes of type 2 diabetes (T2DM) remain poorly understood. Adipose tissue dysfunction with high leptin, inflammation, and increased oxidative stress may play a pivotal role in T2DM development in obese patients. Little is known about the changes in the adipose tissue after Roux-Y gastric bypass (RYGB) in non-severely obese patients (BMI < 35 kg/m(2)) and since these patients have more T2DM-associated complications than obese patients ("obesity paradox"), we investigated changes in adipose tissue function in a cohort of BMI < 35 kg/m(2) with insulin-dependent T2DM after RYGB surgery which resolves T2DM.Methods Twenty patients with insulin-dependent T2DM and BMI < 35 kg/m(2) underwent RYGB. Insulin-resistance, leptin, oxidative stress, and cytokines were determined over 24 months. Expression of cytokines and NF-kappaB pathway genes were measured in leukocytes (PBMC). Adipose tissue inflammation was examined histologically preoperatively and 24 months after RGYB in subcutaneous adipose tissue.Results Insulin-resistance, leptin, oxidative stress as well as adipose tissue inflammation decreased significantly after RYGB. Similarly, systemic inflammation was reduced and peripheral blood mononuclear cells (PBMCs) were reprogrammed towards an M2-type inflammation. Loss of BMI correlated with leptin levels (r = 0.891, p < 0.0001), insulin resistance (r = 0.527, p = 0.003), and oxidative stress (r = 0.592, p = 0.016). Leptin correlated with improved insulin resistance (r = 0.449, p = 0.032) while reduced leptin showed a strong association with improved oxidative stress (r = 0.809, p = 0.001). Lastly, reduced oxidative stress correlated strongly with improved insulin-resistance (r = 0.776, p = 0.001).Conclusions RYGB improves adipose tissue function and inflammation. Leptin as marker for adipose tissue dysfunction may be the mediating factor between insulin resistance and oxidative stress and thereby likely improving T2DM.

Streptozotocin (STZ) treatment, a common model for inducing diabetes in rodent models, induces thermal hyperalgesia and neuronal toxicity independently of hyperglycemia by oxidizing and activating TRPA1 and TRPV1. Following treatment with STZ, CD45+ immune cells were found to be depleted in sciatic nerve (SN) and DRG in mice, prior to hyperglycemia. Macrophages were also lost in DRG and NFκB-p65-activation was increased in SN macrophages. Immune cells were significantly reduced in both SN and DRG up to three weeks, post-treatment. Loss of PNS-resident macrophages in response to STZ-mediated toxicity may affect the regenerative capacity of the nerve in response to further injury caused by diabetes.

Purpose: External electric muscle stimulation (EMS) of the thigh muscles was found to reduce pain resulting from diabetic neuropathy (DN), a vascular complication of diabetes. This study investigated circulating hematopoietic stem cells (HSCs) after EMS treatment. Impaired function of HSCs and the subpopulation endothelial progenitor cells (EPCs), important for neovascularization and endothelial repair, has been associated with DN.Methods: Twenty-four patients with painful DN were treated 3 times with EMS over a period of 1 week. Blood samples were collected before and after the first EMS treatment. Before a fourth treatment, neuropathic pain was evaluated and a third blood sample was collected. Cells were used for flow cytometry.Findings: Patients with painful DN reported that the pain decreased after 3 times of 1-hour treatments with EMS (Neuropathy Symptom Score: from 8 to 6, P = 0.001; Neuropathy Disability Score: from 5.5 to 5, P = 0.027, n = 24). At the end of the study, diastolic blood pressure had decreased from 80 to 70 mm Hg (P = 0.043), and plasma adrenaline and noradrenaline metabolites metanephrine and normetanephrine were reduced (both P <= 0.01; n = 21). A single EMS treatment caused an immediate and transient decrease in the frequency of CD34(+) HSCs in circulation (-20%; P < 0.001; n = 27). In 9 of the patients with DN, the proportion of HSCs expressing vascular endothelial growth factor receptor 2 (VEGFR2; defining the HSCs as EPCs) increased by 36% (P = 0.011) after EMS treatment. Proteins required for binding to endothelium (junctional adhesion molecule A and CD31), homing toward hypoxic tissue (C-X-C chemokine receptor type 4), and endothelial differentiation (CD31) were increased on HSCs immediately after EMS treatment. An increased frequency of VEGFR2 expression was also observed on HSCs of 6 healthy control volunteers (34%; P = 0.046) after EMS treatment, but not after sham treatment.Implications: Three EMS treatments decreased symptoms of pain caused by DN and reduced diastolic blood pressure and biomarkers of stress. A single EMS treatment increased molecules mediating attachment and differentiation on the surface of HSCs in circulation. We hypothesize that the EMS-induced increase in surface attachment molecules on the HSCs caused the HSCs to leave circulation and that EMS treatment improves the function of HSCs and EPCs in vivo. (C) 2017 The Authors. Published by Elsevier HS Journals, Inc.

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated ≥ 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Introduction: External electric muscle stimulation (EMS) can reduce pain resulting from diabetic neuropathy (DN), a vascular complication of diabetes. It was hypothesized that EMS-treatment activated circulating CD34+ hematopoietic stem cells (HSC), which can participate in vessel repair by differentiating into endothelial progenitor cells (EPC).

Introduction: Streptozotocin (STZ) treatment is an accepted pharmacological method for the induction of type 1 diabetes in rodent models. Recent publications have shown that STZ induces thermal hyperalgesia in mice, independently of hyperglycemia, and that STZ is neurotoxic in vitro. These findings bring into question the limitations of STZ-induced diabetes as a model for diabetic neuropathy. The aim of this study was to investigate effects of STZ treatment on nerve physiology in vivo.

Introduction: Pyruvate kinase (PK) catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate as the last step of glycolysis. The two major PK isoforms, PKM1 and PKM2, show different activity levels, with PKM2 being highly expressed in cancer. PK isoforms mRNA expression was investigated in control and diabetic material from human and mouse in order to investigate whether PKM isoforms show differential expression in diabetes and how this balance affects late diabetic complications.

The clinical symptoms of diabetic neuropathy (DN) manifest in a time dependent manner as a positive symptoms (i. e. pain, hypersensitivity, tingling, cramps, cold feet etc.) during its early stages and by a loss of function (i. e. loss of sensory perception, delayed wound healing etc.) predominating in the later stages. Elevated blood glucose alone cannot explain the development and progression of DN and the lowering of blood glucose is insufficient in preventing and/or reversing neuropathy in patients with type 2 diabetes. Recently it has been shown that the endogenous reactive metabolite methylglyoxal (MG) can contribute to the gain of function via post-translational modification in DN of neuronal ion channels involved in chemosensing and action potential generation in nociceptive nerve endings. Dicarbonyls, such as MG, that are elevated in diabetic patients, modify DNA as well as extra- and intracellular proteins, leading to the formation of advanced glycation endproducts (AGEs). Increased formation of AGEs leads to increased cellular stress, dysfunction and ultimately cell death. The interaction of AGE-modified proteins through cell surface receptors, such as RAGE, can lead to increased cellular activation and sustained inflammatory responses, which are the molecular hallmarks of the later, degenerative, stages of DN. The direct and indirect effects of dicarbonyls on nerves or neuronal microvascular network provides a unifying mechanism for the development and progression of DN. Targeting the accumulation of MG and/or prevention of RAGE interactions may therefore provide new, more effective, therapeutic approaches for the treatment of DN.

Elevated blood glucose alone cannot explain the development and progression of diabetic neuropathy (DN) and the lowering of blood glucose is insufficient in preventing and/or reversing neuropathy in patients with type 2 diabetes. Dicarbonyls, such as methylglyoxal (MG), are endogenous by-products of glycolysis, which are elevated in diabetic patients. Reactive metabolite such as MG can modify DNA as well as extra- and intracellular proteins, leading to the formation of advanced glycation endproducts (AGEs). MG can contribute to the development of DN via post-translational modification of neuronal ion channels involved in chemosensing and action potential generation in nociceptive nerve endings. Increased formation of AGEs leads to increased cellular stress, dysfunction and ultimately cell death. The interaction of AGE-modified proteins through cell surface receptors, such as RAGE, can lead to increased cellular activation and sustained inflammatory responses. An inflammatory immune response is associated with later, degenerative, stages of DN. The direct and indirect effects of dicarbonyls on nerves provides a unifying mechanism for the development and progression of DN. Preventing the accumulation of MG during diabetes and/or inhibiting of RAGE signalling may therefore provide new, more effective, therapeutic approaches for the treatment of DN. Cuvinte cheie: neuropatie diabetică, mecanism, metabolism Rezumat: Doar valorile crescute ale glucozei in sânge nu pot explica dezvoltarea si avansarea neuropatiei diabetice, iar scăderea valorilor glucozei in sânge nu este suficientă pentru a preveni neuropatia la pacien ţii cu diabet zaharat de tip 2. Dicarboxili cum ar fi metilglioxalul sunt produsi endogeni secundari glicolizei, inregistrând valori crescute la pacienţii diabetici. Metabolitul reactiv, de exemplu metilglioxalul poate modifica ADN-ul, precum si proteinele extracelulare si intracelulare, conducând la formarea de produ si finali de glicozilare avansată. Metilglioxalul poate contribui la dezvoltarea neuropatiei diabetice prin modificarea post - transla țională a canalelor ionice neuronale implicate in chemosensibilitate și generarea de acțiune potenţială in terminațiile nervoase nociceptive. Formarea crescută de produsi finali de glicozilare avansată duce la cre sterea stresului celular, disfuncție și in cele din urmă la moartea celulei. Interacțiunea proteinelor modificate ca urmare a produsilor finali de glicozilare avansată prin receptori celulari de suprafață, cum ar fi receptorii pentru produsi finali de glicozilare avansată, poate duce la cre șterea activării celulare și la r ăspunsuri inflamatorii susținute. Un răspuns imun inflamator este asociat cu etapele ulterioare, degenerative ale neuropatiei diabetice. Efectele directe și indirecte ale dicarbonililor asupra nervilor oferă un mecanism unificator pentru dezvoltarea si avansarea neuropatiei diabetice. Prevenirea acumulării de metilglioxal in caz de diabet și / sau inhibarea semnalizării receptorilor pentru produsi finali de glicozilare avansată (RAGE) poate astfel oferi abordări terapeutice noi, mai eficiente privind tratamentul neuropatiei diabetice.

Background Endothelial progenitor cells (EPC) are bone marrow-derived cells which can undergo differentiation into endothelial cells and participate in endothelial repair and angiogenesis. Insulin facilitates this in vitro mediated by the IGF-1 receptor. Clinical trials showed that the number of circulating EPCs is influenced by glucose control and EPC are a predictor of cardiovascular death. To study direct effects of insulin treatment on EPCs in type 2 diabetes patients, add-on basal insulin treatment was compared to an escalation of oral medication aiming at similar glucose control between the groups. Methods 55 patients with type 2 diabetes (61.6±5.9 years) on oral diabetes medication were randomized in a 2:2:1 ratio in 3 groups. Patients were treated additionally with insulin glargine (n=20), NPH insulin (n=22) or escalated with oral medication (n=13). Number of circulating EPC, EPC-outgrowth, intima media thickness, skin microvascular function and HbA1c were documented at baseline and/or after 4 weeks and 4 months. Results HbA1c at baseline was, 7.3+/-0.7% in the oral group, 7.3+/-0.9% and 7.5+/-0.7% in the glargine and NPH insulin respectively (p=0.713). HbA1c after 4 months decreased to 6.8+/-0.8%, 6.6+/-0.7% and 6.7+/-0.6%, in the oral, glargine and NPH insulin group respectively (p=0.61). FACS analysis showed no difference in number of circulating EPC between the groups after 4 weeks and 4 months. However, the outgrowth of EPCs as detected by colony forming assay was increased in the NPH insulin and glargine groups (29.2+/-6.4 and 29.4+/- 6.7 units respectively) compared to the group on oral medication (23.2+/-6.3, p=0.013) after 4 months of treatment. A significant decrease of IMT from 0.80mm (+/-0.14) at baseline to 0.76mm (+/-0.12) after 4 months could be observed in all patients only (p=0.03) with a trend towards a reduction of IMT after 4 months when all patients on insulin treatment were compared to the oral treatment group (p=0.06). Skin microvascular function revealed no differences between the groups (p=0.74). Conclusion The study shows that a 4-month treatment with add-on insulin significantly increases the outgrowth of EPC in patients with type 2 diabetes mellitus. Trial registration (Clinical Trials Identifier: NCT00523393).

Bacterial RNA (bRNA) can induce cytokine production in macrophages and dendritic cells (DCs) through a previously unidentified receptor. Gene expression analysis of murine DCs showed that bRNA induced gene regulation similar to that induced by stimulation of TLR7 with R848. Although TLR7 was dispensable for cytokine induction by bRNA, TLR-associated proteins MyD88 and UNC93B were required. TLR13 is an endosomal murine TLR that has been described to interact with UNC93B with, so far, no characterized ligand. Small interfering RNA against TLR13 reduced cytokine induction by bRNA in DCs. Moreover, Chinese hamster ovary cells transfected with TLR13, but not with TLR7 or 8, could activate NF-κB in response to bRNA or Streptococcus pyogenes in an RNA-specific manner. TLR7 antagonist IRS661 could, in addition, inhibit TLR13 signaling and reduced recognition of whole Gram-positive bacteria by DCs, also in the absence of TLR7. The results identify TLR13 as a receptor for bRNA.

The bank vole (Myodes glareolus) is a common small mammal in Europe and a natural host for several important emerging zoonotic viruses, e.g. Puumala hantavirus (PUUV) that causes hemorrhagic fever with renal syndrome (HFRS). Hantaviruses are known to interfere with several signaling pathways in infected human cells, and HFRS is considered an immune-mediated disease. There is no in vitro-model available for infectious experiments in bank vole cells, nor tools for analyses of bank vole immune activation and responses. Consequently, it is not known if there are any differences in the regulation of virus induced responses in humans compared to natural hosts during infection. We here present an in vitro-model for studies of bank vole borne viruses and their interactions with natural host cell innate immune responses. Bank vole embryonic fibroblasts (VEFs) were isolated and shown to be susceptible for PUUV-infection, including a wild-type PUUV strain (only passaged in bank voles). The significance of VEFs as a model system for bank vole associated viruses was further established by infection studies showing that these cells are also susceptible to tick borne encephalitis, cowpox and Ljungan virus. The genes encoding bank vole IFN-β and Mx2 were partially sequenced and protocols for semi-quantitative RT-PCR were developed. Interestingly, PUUV did not induce an increased IFN-β or Mx2 mRNA expression. Corresponding infections with CPXV and LV induced IFN-β but not Mx2, while TBEV induced both IFN-β and Mx2. In conclusion, VEFs together with protocols developed for detection of bank vole innate immune activation provide valuable tools for future studies of how PUUV and other zoonotic viruses affect cells derived from bank voles compared to human cells. Notably, wild-type PUUV which has been difficult to cultivate in vitro readily infected VEFs, suggesting that embryonic fibroblasts from natural hosts might be valuable for isolation of wild-type hantaviruses.

ABSTRACT We analyzed the defensive role of the cytosolic innate recognition receptor nucleotide oligomerization domain 1 (NOD1) during infection with Listeria monocytogenes . Mice lacking NOD1 showed increased susceptibility to systemic intraperitoneal and intravenous infection with high or low doses of L. monocytogenes , as measured by the bacterial load and survival. NOD1 also controlled dissemination of L. monocytogenes into the brain. The increased susceptibility to reinfection of NOD1 −/− mice was not associated with impaired triggering of listeria-specific T cells, and similar levels of costimulatory molecules or activation of dendritic cells was observed. Higher numbers of F480 + Gr1 + inflammatory monocytes and lower numbers of F480 − Gr1 + neutrophils were recruited into the peritoneum of infected WT mice than into the peritoneum of infected NOD1 −/− mice. We determined that nonhematopoietic cells accounted for NOD1-mediated resistance to L. monocytogenes in bone marrow radiation chimeras. The levels of NOD1 mRNA in fibroblasts and bone marrow-derived macrophages (BMM) were upregulated after infection with L. monocytogenes or stimulation with different Toll-like receptor ligands. NOD1 −/− BMM, astrocytes, and fibroblasts all showed enhanced intracellular growth of L monocytogenes compared to WT controls. Gamma interferon-mediated nitric oxide production and inhibition of L. monocytogenes growth were hampered in NOD1 −/− BMM. Thus, NOD1 confers nonhematopoietic cell-mediated resistance to infection with L. monocytogenes and controls intracellular bacterial growth in different cell populations in vitro.