Functional redundancy of mitochondrial enoyl-CoA isomerases in the oxidation of unsaturated fatty acids.

Mitochondrial enoyl-CoA isomerase (ECI1) is an auxiliary enzyme involved in unsaturated fatty acid oxidation. In contrast to most of the other enzymes involved in fatty acid oxidation, a deficiency of ECI1 has yet to be identified in humans. We used wild-type (WT) and Eci1-deficient knockout (KO) mice to explore a potential presentation of human ECI1 deficiency. Upon food withdrawal, Eci1-deficient mice displayed normal blood ß-hydroxybutyrate levels (WT 1.09 mM vs. KO 1.10 mM), a trend to lower blood glucose levels (WT 4.58 mM vs. KO 3.87 mM, P=0.09) and elevated blood levels of unsaturated acylcarnitines, in particular C12:1 acylcarnitine (WT 0.03 µM vs. KO 0.09 µM, P<0.01). Feeding an olive oil-rich diet induced an even greater increase in C12:1 acylcarnitine levels (WT 0.01 µM vs. KO 0.04 µM, P<0.01). Overall, the phenotypic presentation of Eci1-deficient mice is mild, possibly caused by the presence of a second enoyl-CoA isomerase (Eci2) in mitochondria. Knockdown of Eci2 in Eci1-deficient fibroblasts caused a more pronounced accumulation of C12:1 acylcarnitine on incubation with unsaturated fatty acids (12-fold, P<0.05). We conclude that Eci2 compensates for Eci1 deficiency explaining the mild phenotype of Eci1-deficient mice. Hypoglycemia and accumulation of C12:1 acylcarnitine might be diagnostic markers to identify ECI1 deficiency in humans.

Cutting Edge: Immunological consequences and trafficking of human regulatory macrophages administered to renal transplant recipients.

Regulatory macrophages (M regs) were administered to two living-donor renal transplant recipients. Both patients were minimized to low-dose tacrolimus monotherapy within 24 wk of transplantation and subsequently maintained excellent graft function. After central venous administration, most M regs remained viable and were seen to traffic from the pulmonary vasculature via the blood to liver, spleen, and bone marrow. By 1 y posttransplantation, both patients displayed patterns of peripheral blood gene expression converging upon the IOT-RISET signature. Furthermore, both patients maintained levels of peripheral blood FOXP3 and TOAG-1 mRNA expression within the range consistent with nonrejection. It is concluded that M regs warrant further study as a potential immune-conditioning therapy for use in solid-organ transplantation. The results of this work are being used to inform the design of The ONE Study, a multinational clinical trial of immunomodulatory cell therapy in renal transplantation.

Preclinical safety and efficacy of a therapeutic antibody that targets SARS-CoV-2 at the sotrovimab face but is escaped by Omicron.

The recurrent emerging of novel viral variants of concern (VOCs) with evasion of preexisting antibody immunity upholds severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) case numbers and maintains a persistent demand for updated therapies. We selected the patient-derived antibody CV38-142 based on its potency and breadth against the VOCs Alpha, Beta, Gamma, and Delta for preclinical development into a therapeutic. CV38-142 showed in vivo efficacy in a Syrian hamster VOC infection model after post-exposure and therapeutic application and revealed a favorable safety profile in a human protein library screen and tissue cross-reactivity study. Although CV38-142 targets the same viral surface as sotrovimab, which maintains activity against Omicron, CV38-142 did not neutralize the Omicron lineages BA.1 and BA.2. These results highlight the contingencies of developing antibody therapeutics in the context of antigenic drift and reinforce the need to develop broadly neutralizing variant-proof antibodies against SARS-CoV-2.

Characterization of immunostimulatory components of orf virus (parapoxvirus ovis).

Inactivated orf virus (ORFV, parapoxvirus ovis) induces antiviral activity in animal models of acute and chronic viral infections and exerts strong effects on human immune cells. ORFV activates antigen presenting cells (APC) via CD14 and, probably, Toll-like receptor signalling, and triggers the release of IFN-γ that has been identified as the key mediator of the antiviral activity. After delineating virus proteins as being the most likely active constituent, we aimed to characterize the ORFV proteins responsible for the therapeutic effect. By using a vaccinia virus/ORFV expression library we identified several multi-gene DNA fragments with strong immunomodulatory activity. Together these fragments contain 27 ORFs. The encoded proteins are related to virion structure and transcription but are otherwise unrelated. Two proteins were separately expressed and purified, and demonstrated immunostimulatory activity. Gene expression profiles induced by ORFV and the identified fragments were investigated by microarray analysis. Interestingly, all active fragments induced a similar gene-expression pattern, differing only in quantitative aspects. Obviously, several proteins of ORFV activate similar cellular pathways, modulating APC to generate a strong T-helper 1-dominated immune response. This was balanced by additional induction of immune dampening mechanisms, suggesting regulatory differences compared to single cytokine therapies. We conclude that ORFV may have the potential to enrich the armamentarium of antiviral therapies.

Analysis of the peripheral T-cell repertoire in kidney transplant patients.

The long-term stability of renal grafts depends on the absence of chronic rejection. As T cells play a key role in rejection processes, analyzing the T-cell repertoire may be useful for understanding graft function outcomes. We have therefore investigated the power of a new statistical tool, used to analyze the peripheral blood TCR repertoire, for determining immunological differences in a group of 229 stable renal transplant patients undergoing immunosuppression. Despite selecting the patients according to stringent criteria, the patients displayed heterogeneous T-cell repertoire usage, ranging from unbiased to highly selected TCR repertoires; a skewed TCR repertoire correlating with an increase in the CD8(+) /CD4(+) T-cell ratio. T-cell repertoire patterns were compared in patients with clinically opposing outcomes i.e. stable drug-free operationally tolerant recipients and patients with the "suspicious" form of humoral chronic rejection and were found significantly different, from polyclonal to highly selected TCR repertoires, respectively. Moreover, a selected TCR repertoire was found to positively correlate with the Banff score grade. Collectively, these data suggest that TCR repertoire categorization might be included in the calculation of a composite score for the follow-up of patients after kidney transplantation.

Gene expression profiling of individual hypothalamic nuclei from single animals using laser capture microdissection and microarrays.

In order to identify novel genes involved in appetite and body weight regulation we have developed a microarray based method suitable for detecting small changes in gene expression in discrete groups of hypothalamic neurons. The method is based on a combination of stereological sampling, laser capture microdissection (LCM), PCR based amplification (SuperAmp), and one-color cDNA microarray analysis. To validate the method we assessed and compared fasting induced changes in mRNA levels of Neuropeptide Y (NPY) and proopiomelanocortin (POMC) in the hypothalamic arcuate nucleus (ARC) of diet-induced obese rats using cDNA microarrays, quantitative PCR and in situ hybridization. All methods revealed statistically significant fasting-induced changes in NPY and POMC expression. An additional 3480 differentially expressed probes (fold change >1.22, t-test p=0.05) were identified in the microarray analysis. Our findings demonstrate a consistent gene expression pattern across three different gene expression detection methods and strongly suggest that LCM coupled microarray analysis combined with SuperAmp can be used as a semi-quantitative mRNA profiling tool. Importantly, the sensitivity of the method greatly improves the usefulness of the microarray technology for gene expression profiling in non-homogeneous tissues such as the brain.

Heme oxygenase-1 ameliorates ischemia/reperfusion injury by targeting dendritic cell maturation and migration.

Ischemia/reperfusion injury (IRI) has a major impact on short- and long-term renal allograft survival by increasing graft immunogenicity. Donor preconditioning by inducing heme oxygenase 1 (HO-1) has been proven to exert cytoprotective and antiinflammatory effects on the graft, thus resulting in reduced graft immunogenicity. The study analyzed the effects and mechanisms of HO-1-mediated cytoprotection in rat kidney transplants exposed to cold preservation. We studied the differential gene-expression patterns of allografts after either short or long cold ischemia using a customized cDNA microarray. Prolonged cold ischemia led, 12 h after engraftment, to enhanced levels of adhesion molecules, heat-shock proteins, chemokines (CXCL10), and a remarkable upregulation of immunoproteasomes. Next we addressed the question whether induction of HO-1 or its byproduct carbon monoxide (CO) in organ donors targets these candidate markers related to enhanced immunogenicity. Induction of HO-1 or CO in organ donors 24 h before organ harvesting resulted in reduced mRNA levels of immunoproteasomes, MHC class II expression, and co-stimulatory molecules in the recipient's spleen, suggesting diminished migration and activation of donor dendritic cells. This observation suggests that HO-1/CO induction protects marginal allografts by inhibiting the immunogenicity of donor-derived dendritic cells.

Long-term cell monitoring of kidney recipients after an antilymphocyte globulin induction with and without steroids.

BACKGROUND: Because of several side effects, the corticosteroid usage has been minimized in kidney transplantation. The increased acute rejection episodes associated with their withdrawal may counterbalance with induction treatment using polyclonal antilymphocyte globulin (ALG). The effects of ALG on blood cell phenotype have already been the subject of several reports. However, to date, no data are available concerning the comparison of blood phenotype when ALG is given with or without steroids and no gene profiling study has been performed. METHODS: We report here on a longitudinal blood cell analysis of a selected cohort of kidney recipients enrolled in a randomized study of steroid avoidance or withdrawal (during 6 months) during ALG induction. RESULTS: In the two groups, ALG quickly and massively depleted all the T cells and natural killer cells, but not B cells. Interestingly, the lymphopenia-driven homeostatic proliferation of CD4 and CD8T cells strongly differed with persistent low CD4 (including CD25CD4) T-cell counts. Effector memory CD8T cells reappeared rapidly. ALG induced apoptosis-associated molecules and increased myeloid cell genes. However, few genes were found differentially expressed with a low fold ratio between the two groups during and at distance of corticotherapy. CONCLUSION: Thus initial steroid avoidance or withdrawal associated with ALG induction has a weak influence on phenotype and transcriptional pattern of blood leukocytes. In contrast, ALG therapy induces an early and strong depletion of all T-cell subsets with contrasted long-lasting homeostatic regulation.

Sequential Targeting of CD52 and TNF Allows Early Minimization Therapy in Kidney Transplantation: From a Biomarker to Targeting in a Proof-Of-Concept Trial.

BACKGROUND: There is high medical need for safe long-term immunosuppression monotherapy in kidney transplantation. Selective targeting of post-transplant alloantigen-(re)activated effector-T cells by anti-TNF antibodies after global T cell depletion may allow safe drug minimization, however, it is unsolved what might be the best maintenance monotherapy. METHODS: In this open, prospective observational single-centre trial, 20 primary deceased donor kidney transplant recipients received 2x20 mg Alemtuzumab (d0/d1) followed by 5 mg/kg Infliximab (d2). For 14 days all patients received only tacrolimus, then they were allocated to either receive tacrolimus (TAC, n = 13) or sirolimus (SIR, n = 7) monotherapy, respectively. Protocol biopsies and extensive immune monitoring were performed and patients were followed-up for 60 months. RESULTS: TAC-monotherapy resulted in excellent graft survival (5yr 92%, 95%CI: 56.6-98.9) and function, normal histology, and no proteinuria. Immune monitoring revealed low intragraft inflammation (urinary IP-10) and hints for the development of operational tolerance signature in the TAC- but not SIR-group. Remarkably, the TAC-monotherapy was successful in all five presensitized (ELISPOT+) patients. However, recruitment into SIR-arm was stopped (after n = 7) because of high incidence of proteinuria and acute/chronic rejection in biopsies. No opportunistic infections occurred during follow-up. CONCLUSIONS: In conclusion, our novel fast-track TAC-monotherapy protocol is likely to be safe and preliminary results indicated an excellent 5-year outcome, however, a full-scale study will be needed to confirm our findings. TRIAL REGISTRATION: EudraCT Number: 2006-003110-18.

The HOPE-technique permits Northern blot and microarray analyses in paraffin-embedded tissues.

There is an increasing demand for tissue samples that, after having been used for conventional histologic examination, are also suited for molecular analyses. As to formalin-fixed, paraffin embedded (FFPE) tissue, the latter applications are very limited. The HOPE (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) technique comprises a new protection-solution with an organic buffer, with acetone as the only dehydrating agent, and pure paraffin of 52-54 degrees C melting temperature, allowing for all pathologic routine investigations. In contrast to FFPE tissue, the HOPE-technique allows for the application of molecular methods, such as high molecular DNA and RNA isolation, which can be used for PCR and reverse transcription PCR (RT-PCR). In this study, we investigated whether RNA from HOPE-fixed tissue samples is suitable for Northern blot and microarray analyses. RNAs of two HOPE-fixed breast cancer specimens of different histologic grade were used to carry out an array experiment. It turned out that RNA from HOPE-fixed tissue is of high quality and can be successfully used for array experiments. In addition, by detecting GAPDH and high mobility group protein gene B1 (HMGB1)-specific transcripts, we were able to demonstrate that RNA from HOPE-fixed tissue can also be used for Northern blot hybridization.

Gene expression profiling of immunomagnetically separated cells directly from stabilized whole blood for multicenter clinical trials.

BACKGROUND: Clinically useful biomarkers for patient stratification and monitoring of disease progression and drug response are in big demand in drug development and for addressing potential safety concerns. Many diseases influence the frequency and phenotype of cells found in the peripheral blood and the transcriptome of blood cells. Changes in cell type composition influence whole blood gene expression analysis results and thus the discovery of true transcript level changes remains a challenge. We propose a robust and reproducible procedure, which includes whole transcriptome gene expression profiling of major subsets of immune cell cells directly sorted from whole blood. METHODS: Target cells were enriched using magnetic microbeads and an autoMACS® Pro Separator (Miltenyi Biotec). Flow cytometric analysis for purity was performed before and after magnetic cell sorting. Total RNA was hybridized on HGU133 Plus 2.0 expression microarrays (Affymetrix, USA). CEL files signal intensity values were condensed using RMA and a custom CDF file (EntrezGene-based). RESULTS: Positive selection by use of MACS® Technology coupled to transcriptomics was assessed for eight different peripheral blood cell types, CD14+ monocytes, CD3+, CD4+, or CD8+ T cells, CD15+ granulocytes, CD19+ B cells, CD56+ NK cells, and CD45+ pan leukocytes. RNA quality from enriched cells was above a RIN of eight. GeneChip analysis confirmed cell type specific transcriptome profiles. Storing whole blood collected in an EDTA Vacutainer® tube at 4°C followed by MACS does not activate sorted cells. Gene expression analysis supports cell enrichment measurements by MACS. CONCLUSIONS: The proposed workflow generates reproducible cell-type specific transcriptome data which can be translated to clinical settings and used to identify clinically relevant gene expression biomarkers from whole blood samples. This procedure enables the integration of transcriptomics of relevant immune cell subsets sorted directly from whole blood in clinical trial protocols.

Immunological Outcome in Haploidentical-HSC Transplanted Patients Treated with IL-10-Anergized Donor T Cells.

T-cell therapy after hematopoietic stem cell transplantation (HSCT) has been used alone or in combination with immunosuppression to cure hematologic malignancies and to prevent disease recurrence. Here, we describe the outcome of patients with high-risk/advanced stage hematologic malignancies, who received T-cell depleted (TCD) haploidentical-HSCT (haplo-HSCT) combined with donor T lymphocytes pretreated with IL-10 (ALT-TEN trial). IL-10-anergized donor T cells (IL-10-DLI) contained T regulatory type 1 (Tr1) cells specific for the host alloantigens, limiting donor-vs.-host-reactivity, and memory T cells able to respond to pathogens. IL-10-DLI were infused in 12 patients with the goal of improving immune reconstitution after haplo-HSCT without increasing the risk of graft-versus-host-disease (GvHD). IL-10-DLI led to fast immune reconstitution in five patients. In four out of the five patients, total T-cell counts, TCR-Vß repertoire and T-cell functions progressively normalized after IL-10-DLI. These four patients are alive, in complete disease remission and immunosuppression-free at 7.2 years (median follow-up) after haplo-HSCT. Transient GvHD was observed in the immune reconstituted (IR) patients, despite persistent host-specific hypo-responsiveness of donor T cells in vitro and enrichment of cells with Tr1-specific biomarkers in vivo. Gene-expression profiles of IR patients showed a common signature of tolerance. This study provides the first indication of the feasibility of Tr1 cell-based therapy and paves way for the use of these Tr1 cells as adjuvant treatment for malignancies and immune-mediated disorders.

Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans.

Identifying transplant recipients in whom immunological tolerance is established or is developing would allow an individually tailored approach to their posttransplantation management. In this study, we aimed to develop reliable and reproducible in vitro assays capable of detecting tolerance in renal transplant recipients. Several biomarkers and bioassays were screened on a training set that included 11 operationally tolerant renal transplant recipients, recipient groups following different immunosuppressive regimes, recipients undergoing chronic rejection, and healthy controls. Highly predictive assays were repeated on an independent test set that included 24 tolerant renal transplant recipients. Tolerant patients displayed an expansion of peripheral blood B and NK lymphocytes, fewer activated CD4+ T cells, a lack of donor-specific antibodies, donor-specific hyporesponsiveness of CD4+ T cells, and a high ratio of forkhead box P3 to alpha-1,2-mannosidase gene expression. Microarray analysis further revealed in tolerant recipients a bias toward differential expression of B cell-related genes and their associated molecular pathways. By combining these indices of tolerance as a cross-platform biomarker signature, we were able to identify tolerant recipients in both the training set and the test set. This study provides an immunological profile of the tolerant state that, with further validation, should inform and shape drug-weaning protocols in renal transplant recipients.

Inhibition of dendritic cell maturation and function is independent of heme oxygenase 1 but requires the activation of STAT3.

The induction of heme oxygenase 1 (HO-1) by a single treatment with cobalt protoporphyrin (CoPPIX) protects against inflammatory liver failure and ischemia reperfusion injury after allotransplantation. In this context, the HO-1-mediated inhibition of donor-derived dendritic cell maturation and migration is discussed as one of the key events of graft protection. To investigate the poorly understood mechanism of CoPPIX-induced HO-1 activity in more detail, we performed gene expression analysis in murine liver, revealing the up-regulation of STAT3 after CoPPIX treatment. By using wild-type and HO-1-deficient dendritic cells we demonstrated that LPS-induced maturation is dependent on STAT3 phosphorylation and independent of HO-1 activity. In summary, our observations revise our understanding of the anti-inflammatory properties of HO-1 and highlight the immunomodulatory capacity of STAT3, which might be of further interest for targeting undesired immune responses, including ischemia reperfusion injury.

Disruption of mitochondrial beta -oxidation of unsaturated fatty acids in the 3,2-trans-enoyl-CoA isomerase-deficient mouse.

Cellular energy metabolism is largely sustained by mitochondrial beta-oxidation of saturated and unsaturated fatty acids. To study the role of unsaturated fatty acids in cellular lipid and energy metabolism we generated a null allelic mouse, deficient in 3,2-trans-enoyl-CoA isomerase (ECI) (eci(-/-) mouse). ECI is the link in mitochondrial beta-oxidation of unsaturated and saturated fatty acids and essential for the complete degradation and for maximal energy yield. Mitochondrial beta-oxidation of unsaturated fatty acids is interrupted in eci(-/-)mice at the level of their respective 3-cis- or 3-trans-enoyl-CoA intermediates. Fasting eci(-/-) mice accumulate unsaturated fatty acyl groups in ester lipids and deposit large amounts of triglycerides in hepatocytes (steatosis). Gene expression studies revealed the induction of peroxisome proliferator-activated receptor activation in eci(-/-) mice together with peroxisomal beta- and microsomal omega-oxidation enzymes. Combined peroxisomal beta- and microsomal omega-oxidation of the 3-enoyl-CoA intermediates leads to a specific pattern of medium chain unsaturated dicarboxylic acids excreted in the urine in high concentration (dicarboxylic aciduria). The urinary dicarboxylate pattern is a reliable diagnostic marker of the ECI genetic defect. The eci(-/-) mouse might be a model of a yet undefined inborn mitochondrial beta-oxidation disorder lacking the enzyme link that channels the intermediates of unsaturated fatty acids into the beta-oxidation spiral of saturated fatty acids.

Kinetics of gene expression profiling in Swiss 3T3 cells exposed to aqueous extracts of cigarette smoke.

Previous studies from different laboratories have demonstrated that cigarette smoke (CS) harbours a strong oxidative stress potential, which broadly impacts exposed cells. Many of these studies have been devoted to identifying differentially expressed genes in exposed cells. Emerging DNA microarray techniques provide a sophisticated tool to characterize gene expression on a more comprehensive basis. Here, we report on kinetic studies performed to characterize gene expression profiles in Swiss 3T3 cells exposed to aqueous extracts of CS ('smoke-bubbled phosphate-buffered saline') up to 24 h through glass chips containing 513 different cDNA probes. The results obtained display a distinct expression pattern of up regulated and repressed genes, which was most evident after 4-8 h of exposure. The CS-related stress response involves mainly antioxidant response genes coding for, e.g. haem oxygenase-1 (HO-1), metallothionein 1/2 (MT1/2) and heat shock proteins (HSPs); genes coding for transcription factors, e.g. JunB and CAAT/enhancer binding protein (C/EBP); cell cycle-related genes, e.g. gadd34 and gadd45; and notably, genes described as mediators of an inflammatory/immune-regulatory response, e.g. st2, kc and id3. From a kinetic perspective, the stress response is characterized by the synchronized up regulation of antioxidant pathways, e.g. as reflected by the co-ordinated expression of ho-1 and ferritin. This expression pattern is obviously orchestrated by stress-responsive transcription factors, as exemplified by the early and strong expression of junB and c/ebp. Interestingly, among the 10 most up regulated genes are five which are known to counteract stress brought about by peroxynitrite. Altogether, these results demonstrate that CS induces a distinct signature of differential gene expression in exposed cells.