ABSTRACT
Immunoproteasomes are a special class of proteasomes, which can be induced with IFN-γ in an inflammatory environment. In recent years, it became evident that certain immune cell types constitutively express high levels of immunoproteasomes. However, information regarding the basal expression of proteolytically active immunoproteasome subunits in different types of immune cells is still rare. Hence, we quantified standard proteasome subunits (ß1c, ß2c, ß5c) and immunoproteasome subunits (LMP2, MECL-1, LMP7) in the major murine (CD4+ T cells, CD8+ T cells, CD19+ B cells, CD11c+ dendritic cells, CD49d+ natural killer cells, Ly-6G+ neutrophils) and human immune cell (CD4+ T cells, CD8+ T cells, CD19+ B cells, CD1c+CD141+ myeloid dendritic cells, CD56+ natural killer cells, granulocytes) subsets. The different human immune cell types were isolated from peripheral blood and the murine immune cell subsets from spleen. We found that proteasomes of most immune cell subsets mainly consist of immunoproteasome subunits. Our data will serve as a reference and guideline for immunoproteasome expression and imply a special role of immunoproteasomes in immune cells.
Subject(s)
CD8-Positive T-Lymphocytes , Proteasome Endopeptidase Complex , Animals , Mice , Humans , CD8-Positive T-Lymphocytes/metabolismABSTRACT
The prime function of proteasomes is the control of protein homeostasis in cells (i.e., the removal of proteins that are not properly folded, damaged by stress conditions like reactive oxygen species formation, or degraded on the basis of regular protein turnover). During viral infection, the standard proteasome is replaced by the so-called immunoproteasome (IP) in an IFN-γ-dependent manner. It has been proposed that the IP is required to protect cell viability under conditions of IFN-induced oxidative stress. In this study, we investigated the requirement for IP to cope with the enhanced need for protein degradation during lymphocytic choriomeningitis virus (LCMV) infection in mice lacking the IP subunit LMP7. We found that IP are upregulated in the liver but not in the spleen during LCMV infection, although the total proteasome content was not altered. The expression of standard proteasome subunits is not induced in LMP7-deficient mice, indicating that enhanced proteasomal activity is not required during viral infection. Furthermore, ubiquitin accumulation, apoptosis induction, and viral titers were similar in LCMV-infected mice lacking LMP7 compared with wild-type mice. Taken together, these data indicate that the IP is not required to regulate protein homeostasis during LCMV infection.
Subject(s)
Lymphocytic Choriomeningitis/immunology , Lymphocytic choriomeningitis virus/physiology , Proteasome Endopeptidase Complex/metabolism , T-Lymphocytes/immunology , Animals , Cells, Cultured , Homeostasis , Interferon-gamma/metabolism , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Knockout , Proteasome Endopeptidase Complex/genetics , Proteolysis , Up-RegulationABSTRACT
MECL-1 (ß2i), LMP2 (ß1i), and LMP7 (ß5i) are the proteolytically active subunits of the immunoproteasome (IP), a special type of proteasome mainly expressed in hematopoietic cells. Targeting the IP in autoimmune diseases proved to be therapeutically effective in preclinical mouse models. In endotoxin-stimulated human PBMCs, IP inhibition reduces the secretion of several proinflammatory cytokines, with the suppression of IL-23 being the most prominent. In this study, we investigated why the production of IL-23, a key mediator of inflammation in autoimmunity, is blocked when the IP is inhibited in LPS-stimulated human PBMCs. CD14+ monocytes could be identified as the main producers of IL-23 in LPS-stimulated PBMCs. We found that IP inhibition with the irreversible LMP7/LMP2 inhibitor ONX 0914 induced apoptosis in CD14+ monocytes, whereas CD4+, CD3+, CD19+, and CD56+ cells remained unaffected. A high expression of IPs renders monocytes susceptible to IP inhibition, leading to an accumulation of polyubiquitylated proteins and the induction of the unfolded protein response. Similar to IP inhibition, inducers of the unfolded protein response selectively kill CD14+ monocytes in human PBMCs. The blockage of the translation in CD14+ monocytes protects these cells from ONX 0914-induced cell death, indicating that the IP is required to maintain protein turnover in monocytes. Taken together, our data reveal why IP inhibition is particularly effective in the suppression of IL-23-driven autoimmunity.
Subject(s)
Gene Expression Regulation, Enzymologic/drug effects , Interleukin-23/immunology , Lipopolysaccharide Receptors/immunology , Monocytes/immunology , Oligopeptides/pharmacology , Proteasome Endopeptidase Complex/immunology , Proteasome Inhibitors/pharmacology , Protein Biosynthesis/drug effects , Cell Death/drug effects , Cell Death/immunology , Gene Expression Regulation, Enzymologic/immunology , Humans , Lipopolysaccharides/toxicity , Monocytes/pathology , Protein Biosynthesis/immunology , Proteolysis/drug effectsABSTRACT
OBJECTIVE: To identify prenatal, perinatal, and postnatal risk factors for dialysis within the first year of life in children with autosomal recessive polycystic kidney disease (ARPKD) as a basis for parental counseling after prenatal and perinatal diagnosis. STUDY DESIGN: A dataset comprising 385 patients from the ARegPKD international registry study was analyzed for potential risk markers for dialysis during the first year of life. RESULTS: Thirty-six out of 385 children (9.4%) commenced dialysis in the first year of life. According to multivariable Cox regression analysis, the presence of oligohydramnios or anhydramnios, prenatal kidney enlargement, a low Apgar score, and the need for postnatal breathing support were independently associated with an increased hazard ratio for requiring dialysis within the first year of life. The increased risk associated with Apgar score and perinatal assisted breathing was time-dependent and vanished after 5 and 8 months of life, respectively. The predicted probabilities for early dialysis varied from 1.5% (95% CI, 0.5%-4.1%) for patients with ARPKD with no prenatal sonographic abnormalities to 32.3% (95% CI, 22.2%-44.5%) in cases of documented oligohydramnios or anhydramnios, renal cysts, and enlarged kidneys. CONCLUSIONS: This study, which identified risk factors associated with onset of dialysis in ARPKD in the first year of life, may be helpful in prenatal parental counseling in cases of suspected ARPKD.
Subject(s)
Polycystic Kidney, Autosomal Recessive/therapy , Renal Dialysis , Risk Assessment , Female , Follow-Up Studies , Humans , Infant , Infant, Newborn , Male , Polycystic Kidney, Autosomal Recessive/diagnosis , Pregnancy , Prospective Studies , Retrospective Studies , Risk Factors , Time Factors , Ultrasonography, PrenatalABSTRACT
BACKGROUND: Autosomal recessive polycystic kidney disease (ARPKD) is a rare but frequently severe disorder that is typically characterized by cystic kidneys and congenital hepatic fibrosis but displays pronounced phenotypic heterogeneity. ARPKD is among the most important causes for pediatric end stage renal disease and a leading reason for liver-, kidney- or combined liver kidney transplantation in childhood. The underlying pathophysiology, the mechanisms resulting in the observed clinical heterogeneity and the long-term clinical evolution of patients remain poorly understood. Current treatment approaches continue to be largely symptomatic and opinion-based even in most-advanced medical centers. While large clinical trials for the frequent and mostly adult onset autosomal dominant polycystic kidney diseases have recently been conducted, therapeutic initiatives for ARPKD are facing the challenge of small and clinically variable cohorts for which reliable end points are hard to establish. METHODS/DESIGN: ARegPKD is an international, mostly European, observational study to deeply phenotype ARPKD patients in a pro- and retrospective fashion. This registry study is conducted with the support of the German Society for Pediatric Nephrology (GPN) and the European Study Consortium for Chronic Kidney Disorders Affecting Pediatric Patients (ESCAPE Network). ARegPKD clinically characterizes long-term ARPKD courses by a web-based approach that uses detailed basic data questionnaires in combination with yearly follow-up visits. Clinical data collection is accompanied by associated biobanking and reference histology, thus setting roots for future translational research. DISCUSSION: The novel registry study ARegPKD aims to characterize miscellaneous subcohorts and to compare the applied treatment options in a large cohort of deeply characterized patients. ARegPKD will thus provide evidence base for clinical treatment decisions and contribute to the pathophysiological understanding of this severe inherited disorder.
Subject(s)
Biological Specimen Banks/organization & administration , Kidney Failure, Chronic/diagnosis , Polycystic Kidney, Autosomal Recessive/diagnosis , Polycystic Kidney, Autosomal Recessive/therapy , Registries , Adult , Child , Disease Progression , Europe , Female , Genetic Diseases, Inborn/diagnosis , Genetic Diseases, Inborn/epidemiology , Genetic Diseases, Inborn/therapy , Humans , Internationality , Internet , Kidney Failure, Chronic/epidemiology , Kidney Failure, Chronic/therapy , Liver Cirrhosis/diagnosis , Liver Cirrhosis/epidemiology , Liver Cirrhosis/therapy , Male , Polycystic Kidney, Autosomal Recessive/epidemiology , Quality Control , Research Design , Sensitivity and Specificity , Severity of Illness IndexABSTRACT
This study is based on our attempts to further explore the structure-activity relationship (SAR) of VX-148 (3) in an attempt to identify inosine 5'-mono-phosphate dehydrogenase (IMPDH) inhibitors superior to mycophenolic acid. A five-point pharmacophore developed using structurally diverse, known IMPDH inhibitors guided further design of novel analogs of 3. Several conventional as well as novel medicinal chemistry strategies were tried. The combined structure- and ligand-based approaches culminated in a few analogs with either retained or slightly higher potency. The compounds which retained the potency were also checked for their ability to inhibit human peripheral blood mononuclear cells proliferation. This study illuminates the stringent structural requirements and strict SAR for IMPDH II inhibition.
Subject(s)
Enzyme Inhibitors/chemical synthesis , IMP Dehydrogenase/antagonists & inhibitors , IMP Dehydrogenase/chemistry , Cell Proliferation/drug effects , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Mycophenolic Acid/chemistry , Mycophenolic Acid/pharmacology , Phenylurea Compounds/chemistry , Phenylurea Compounds/pharmacology , Structure-Activity RelationshipABSTRACT
In common variable immunodeficiency (CVID) defects in early stages of B-cell development, bone marrow (BM) plasma cells and T lymphocytes have not been studied systematically. Here we report the first morphologic and flow cytometric study of B- and T-cell populations in CVID BM biopsies and aspirates. Whereas the hematopoietic compartment showed no major lineage abnormalities, analysis of the lymphoid compartment exhibited major pathologic alterations. In 94% of the patients, BM plasma cells were either absent or significantly reduced and correlated with serum immunoglobulin G levels. Biopsies from CVID patients had significantly more diffuse and nodular CD3(+) T lymphocyte infiltrates than biopsies from controls. These infiltrates correlated with autoimmune cytopenia but not with other clinical symptoms or with disease duration and peripheral B-cell counts. Nodular T-cell infiltrates correlated significantly with circulating CD4(+)CD45R0(+) memory T cells, elevated soluble IL2-receptor and neopterin serum levels indicating an activated T-cell compartment in most patients. Nine of 25 patients had a partial block in B-cell development at the pre-B-I to pre-B-II stage. Because the developmental block correlates with lower transitional and mature B-cell counts in the periphery, we propose that these patients might form a new subgroup of CVID patients.
Subject(s)
B-Lymphocytes/pathology , Bone Marrow Cells/pathology , Common Variable Immunodeficiency/immunology , Common Variable Immunodeficiency/pathology , T-Lymphocytes/pathology , Adolescent , Adult , Aged , Biopsy , Cohort Studies , Female , Humans , Lymphocyte Count , Male , Middle Aged , Young AdultABSTRACT
Ubiquitin-independent protein degradation via the 20S proteasome without the 19S regulatory particle has gained increasing attention over the last years. The degradation of the ubiquitin-like modifier FAT10 by the 20S proteasome was investigated in this study. We found that FAT10 was rapidly degraded by purified 20S proteasomes in vitro, which was attributed to the weak folding of FAT10 and the N-terminally disordered tail. To confirm our results in cellulo, we established an inducible RNA interference system in which the AAA-ATPase Rpt2 of the 19S regulatory particle is knocked down to impair the function of the 26S proteasome. Using this system, degradation of FAT10 in cellulo was strongly dependent on functional 26S proteasome. Our data indicate that in vitro degradation studies with purified proteins do not necessarily reflect biological degradation mechanisms occurring in cells and, therefore, cautious data interpretation is required when 20S proteasome function is studied in vitro.
Subject(s)
Proteasome Endopeptidase Complex , Ubiquitin , Proteasome Endopeptidase Complex/metabolism , Ubiquitin/metabolism , Ubiquitins/genetics , Ubiquitins/metabolism , RNA InterferenceABSTRACT
PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is currently one of the leading causes of cancer death worldwide. Therefore, building further subgroups as well as enabling individual patient therapy and diagnostics are needed. X-linked inhibitor of apoptosis protein (XIAP) is known to modulate apoptotic and inflammatory pathways. Its expression was found to correlate with patients' survival in other tumor entities. This study aims to examine the role of XIAP in patients with PDAC in relation to the inflammatory microenvironment. METHODS: The PANCALYZE multicenter study group included 257 patients with PDAC. Paraffin-embedded tumor samples were stained immunohistochemically for CD3, CD20, CD38, CD56, CD66b, CD117, and CD163 and XIAP. These stainings were further analyzed digitally with QuPath and survival analyses were done. RESULTS: XIAP-positive patients with T-cell, respectively, neutrophil enriched tumors survived significantly longer compared to XIAP-negative patients (CD3: 37.6 vs. 24.6 months, p = 0.028; CD66b: 34.1 vs. 14.9 months, p = 0.027). Additionally, XIAP-positive patients showed better survival in the lymph node-negative population (48.4 vs. 24.2 months, p = 0.019). Regarding the total population, our findings did not show a correlation between XIAP expression and survival. In multivariate cox regression analyzes XIAP proves to be an independent factor for better survival in the identified subgroups (CD3: p = 0.043; CD66b: p = 0.012, N0: p = 0.040). CONCLUSION: We found XIAP-positive subgroups with significantly better survival in patients with PDAC in T-cell-rich, neutrophil-rich, or lymph node-negative cohorts. This could lead to further individualized cancer treatment with less aggressive therapy protocols for XIAP-positive tumors or more intensive follow-up for XIAP-negative tumors.
Subject(s)
Adenocarcinoma , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , X-Linked Inhibitor of Apoptosis Protein/metabolism , Prognosis , Adenocarcinoma/pathology , Pancreas/metabolism , Pancreas/pathology , Pancreatic Neoplasms/surgery , Pancreatic Neoplasms/metabolism , Carcinoma, Pancreatic Ductal/surgery , Carcinoma, Pancreatic Ductal/metabolism , Biomarkers, Tumor/metabolism , Tumor Microenvironment , Pancreatic NeoplasmsABSTRACT
The novel coronavirus pandemic, first reported in December 2019, was caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infection leads to a strong immune response and activation of antigen-presenting cells, which can elicit acute respiratory distress syndrome (ARDS) characterized by the rapid onset of widespread inflammation, the so-called cytokine storm. In response to viral infections, monocytes are recruited into the lung and subsequently differentiate into dendritic cells (DCs). DCs are critical players in the development of the acute lung inflammation that causes ARDS. Here we focus on the interaction of a specific SARS-CoV-2 open reading frame protein, ORF8, with DCs. We show that ORF8 binds to DCs, causes a pre-maturation of differentiating DCs, and induces the secretion of multiple proinflammatory cytokines by these cells. In addition, we identified DC-SIGN as a possible interaction partner of ORF8 on DCs. Blockade of ORF8 leads to reduced production of IL-1ß, IL-6, IL-12p70, TNF-α, MCP-1 (also named CCL2), and IL-10 by DCs. Therefore, a neutralizing antibody blocking the ORF8-mediated cytokine and chemokine response could be an improved therapeutical strategy against severe SARS-CoV-2.
ABSTRACT
Inflammation conveys the development of glomerular injury and is a major cause of progressive kidney disease. NF-κB signaling is among the most important regulators of proinflammatory signaling. Its role in podocytes, the epithelial cells at the kidney filtration barrier, is poorly understood. Here, we inhibited NF-κB signaling in podocytes by specific ablation of the NF-κB essential modulator (NEMO, IKKγ). Podocyte-specific NEMO-deficient mice (NEMO(pko)) were viable and did not show proteinuria or overt changes in kidney morphology. After induction of glomerulonephritis, both NEMO(pko) and control mice developed significant proteinuria. However, NEMO(pko) mice recovered much faster, showing rapid remission of proteinuria and restoration of podocyte morphology. Interestingly, quantification of infiltrating macrophages, T-lymphocytes, and granulocytes at day 7 revealed no significant difference between wild-type and NEMO(pko). To further investigate the underlying mechanisms, we created a stable NEMO knockdown mouse podocyte cell line. Again, no overt changes in morphology were observed. Translocation of NF-κB to the nucleus after stimulation with TNFα or IL-1 was sufficiently inhibited. Moreover, secretion of proinflammatory chemokines from podocytes after stimulation with TNFα or IL-1 was significantly reduced in NEMO-deficient podocytes and in glomerular samples obtained at day 7 after induction of nephrotoxic nephritis. Collectively, these results show that proinflammatory activity of NF-κB in podocytes aggravates proteinuria in experimental glomerulonephritis in mice. Based on these data, it may be speculated that immunosuppressive drugs may not only target professional immune cells but also podocytes directly to convey their beneficial effects in various types of glomerulonephritis.
Subject(s)
Glomerulonephritis/metabolism , NF-kappa B/metabolism , Podocytes/metabolism , Proteinuria/metabolism , Signal Transduction/physiology , Animals , Disease Models, Animal , Glomerulonephritis/pathology , HEK293 Cells , Humans , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Inflammation/metabolism , Inflammation/pathology , Interleukin-1/pharmacology , Macrophages/metabolism , Macrophages/pathology , Mice , Mice, Knockout , Podocytes/drug effects , Podocytes/pathology , Proteinuria/pathology , RNA Interference , Signal Transduction/drug effects , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
BACKGROUND: The infantile form of primary hyperoxaluria type I (PHI) is the most devastating PH subtype leading to early end-stage renal failure and severe systemic oxalosis. Combined or sequential liver-kidney transplantation (LKTx) is the only curative option but it involves substantial risks, especially in critically ill infants. The procedure also requires resources that are simply not available to many children suffering from PHI worldwide. Less invasive and less complex therapeutic interventions allowing a better timing are clearly needed. Liver cell transplantation (LCT) may expand the narrow spectrum of auxiliary measures to buy time until LKTx for infants can be performed more safely. METHODS: We performed LCT (male neonate donor) in a 15-month-old female in reduced general condition suffering from systemic oxalosis. Renal replacement therapy, initiated at the age of 3 months, was complicated by continuous haemodialysis access problems. Living donor liver transplantation was not available for this patient. Plasma oxalate (Pox) was used as the primary outcome measure. RESULTS: Pox decreased from 104.3±8.4 prior to 70.0±15.0 µmol/L from Day 14 to Day 56 after LCT. A significant persistent Pox reduction (P<0.001) comparing mean levels prior to (103.8 µmol/L) and after Day 14 of LCT until LKTx (77.3 µmol/L) was seen, although a secondary increase and wider range of Pox was also observed. In parallel, the patient's clinical situation markedly improved and the girl received a cadaveric LKTx 12 months after LCT. However, biopsy specimens taken from the explanted liver did not show male donor cells by amelogenin polymerase chain reaction. CONCLUSIONS: With due caution, our pilot data indicate that LCT in infantile oxalosis warrants further investigation. Improvement of protocol and methodology is clearly needed in order to develop a procedure that could assist in the cure of PHI.
Subject(s)
Hepatocytes/transplantation , Hyperoxaluria, Primary/surgery , Kidney Failure, Chronic/etiology , Kidney Transplantation , Liver Transplantation , Cells, Cultured , Child, Preschool , Female , Follow-Up Studies , Hepatocytes/cytology , Humans , Hyperoxaluria, Primary/complications , Infant , Male , Oxalates/metabolism , Pilot Projects , Prognosis , Risk Factors , Tissue DonorsABSTRACT
IMPDH (Inosine 5'-monophosphate dehydrogenase) catalyzes a rate-limiting step in the de novo biosynthesis of guanine nucleotides. IMPDH inhibition in sensitive cell types (e.g., lymphocytes) blocks proliferation (by blocking RNA and DNA synthesis as a result of decreased cellular levels of guanine nucleotides). This makes it an interesting target for cancer and autoimmune disorders. Currently available IMPDH inhibitors such as mycophenolic acid (MPA, uncompetitive inhibitor) and nucleoside analogs (e.g., ribavirin, competitive inhibitor after intracellular activation by phosphorylation) have unfavorable tolerability profiles which limit their use. Hence, the quest for novel IMPDH inhibitors continues. In the present study, a ligand-based virtual screening using IMPDH inhibitor pharmacophore models was performed on in-house compound collection. A total of 50,000 virtual hits were selected for primary screen using in vitro IMPDH II inhibition up to 10 µM. The list of 2,500 hits (with >70 % inhibition) was further subjected to hit confirmation for the determination of IC(50) values. The hits obtained were further clustered using maximum common substructure based formalism resulting in 90 classes and 7 singletons. A thorough inspection of these yielded 7 interesting classes in terms of mini-SAR with IC(50) values ranging from 0.163 µM to little over 25 µM. The average ligand efficiency was found to be 0.3 for the best class. The classes thus discovered represent structurally novel chemotypes which can be taken up for further development.
Subject(s)
Enzyme Inhibitors/chemistry , High-Throughput Screening Assays , IMP Dehydrogenase/metabolism , Pharmaceutical Preparations/chemistry , Cloning, Molecular , Enzyme Inhibitors/pharmacology , Humans , IMP Dehydrogenase/genetics , Models, Chemical , Molecular Structure , Protein ConformationABSTRACT
In December 2019, the first case of COVID-19 was reported and since then several groups have already published that the virus can be present in the testis. To study the influence of SARS-CoV-2 which cause a dysregulation of the androgen receptor (AR) level, thereby leading to fertility problems and inducing germ cell testicular changes in patients after the infection. Formalin-Fixed-Paraffin-Embedded (FFPE) testicular samples from patients who died with or as a result of COVID-19 (n = 32) with controls (n = 6), inflammatory changes (n = 9), seminoma with/without metastasis (n = 11) compared with healthy biopsy samples (n = 3) were analyzed and compared via qRT-PCR for the expression of miR-371a-3p. An immunohistochemical analysis (IHC) and ELISA were performed in order to highlight the miR-371a-3p targeting the AR. Serum samples of patients with mild or severe COVID-19 symptoms (n = 34) were analyzed for miR-371a-3p expression. In 70% of the analyzed postmortem testicular tissue samples, a significant upregulation of the miR-371a-3p was detected, and 75% of the samples showed a reduced spermatogenesis. In serum samples, the upregulation of the miR-371a-3p was also detectable. The upregulation of the miR-371a-3p is responsible for the downregulation of the AR in SARS-CoV-2-positive patients, resulting in decreased spermatogenesis. Since the dysregulation of the AR is associated with infertility, further studies have to confirm if the identified dysregulation is regressive after a declining infection.
Subject(s)
Anuria/etiology , Glomerulosclerosis, Focal Segmental/surgery , Graft Rejection/prevention & control , Immunosuppressive Agents/therapeutic use , Kidney Failure, Chronic/surgery , Kidney Transplantation , Kidney/pathology , Postoperative Complications/etiology , Adolescent , Cyclosporine/therapeutic use , Female , Glomerulosclerosis, Focal Segmental/complications , Glomerulosclerosis, Focal Segmental/pathology , Glomerulosclerosis, Focal Segmental/therapy , Humans , Immunosorbent Techniques , Kidney/ultrastructure , Mycophenolic Acid/therapeutic use , Plasmapheresis , Postoperative Complications/therapy , Prednisolone/therapeutic use , Recurrence , Renal DialysisABSTRACT
We have developed a syringolin-based chemical probe and explored its utility for the profiling of metabolite extracts as potent inhibitors of the 20S proteasome. Activity-guided fractionation by competitive labeling allowed us to isolate and identify glidobactin A and C as well as luminmycin A from a Burkholderiales strain. The natural products exhibited unique subunit specificities for the proteolytic subunits of human and mouse constitutive and immunoproteasome in the lower nanomolar range. In particular, glidobactin C displayed an unprecedented ß2/ß5 coinhibition profile with single-digit nanomolar potency in combination with sufficiently high cell permeability. These properties render glidobactin C a promising live cell proteasome inhibitor with potent activity against human breast cancer cell lines and comparably low immunotoxicity.
ABSTRACT
OBJECTIVE: Dilatation of the aorta at the landing zone site may be exaggerated by the radial force of stent grafts potentially limiting long-term results of endovascular therapy. We evaluated growth patterns and morphology of the thoracic aorta in young piglets after thoracic stent-graft placement. METHODS: Eight domestic piglets (37+/-2 kg) had an endovascular stent graft placed in the proximal descending thoracic aorta using retroperitoneal access. At implantation, the stent was oversized by 10%. Aortic size was documented after thoracotomy by intraoperative measurement and angiography. Subsequently the piglets were grown to adult size (181+/-42 kg). At explantation 6-15 months later, CT scan and surgical evaluation for endoleaks, defined as perigraft flow, was performed. Histopathological assessment of the explanted aorta was performed in stented and non-stented segments and compared to five normal porcine aortas. RESULTS: No endoleak (perigraft flow) or stent migration occurred even in 230kg pigs. The stent grafts expanded to full size, but there was no further growth in the stented area. The aortic diameter increased significantly by 32+/-9% 1cm proximal to the stents (p=0.0012) and by 45+/-13% 1cm distal to the stents (p=0.0033). The stented area grew less than the proximal (p=0.0011) and distal aorta (p<0.0001). In all pigs, the distal aorta was larger than the proximal overstented segment. Histology of the stented aorta showed significant thickening of the intima (p=0.018) and media (p=0.006) with neointimal formation and segmental fibrosis of the inner 1/3 of the media with loss of smooth muscle cells and compression of the elastic fibers but normal architecture in the outer 2/3 of the media. CONCLUSIONS: Endovascular stent grafting may inhibit growth of the nonatherosclerotic normal aorta and lead to intimal hyperplasia and focal fibrosis in the inner media part adjacent to the stent. Stent-graft interaction with aortic tissue over time is important and should receive more detailed evaluation. Testing this interaction in an animal model of nonatherosclerotic dilative aortic disease could be of great interest.
Subject(s)
Aorta, Thoracic/growth & development , Aorta, Thoracic/surgery , Blood Vessel Prosthesis Implantation/methods , Stents , Animals , Aorta, Thoracic/diagnostic imaging , Aorta, Thoracic/pathology , Aortic Aneurysm, Thoracic/surgery , Aortography , Disease Models, Animal , Fibrosis/etiology , Fibrosis/pathology , Stents/adverse effects , Sus scrofa , Tomography, X-Ray ComputedABSTRACT
Since Tydén's description of ABO-incompatible (ABOi) kidney transplantations based on antigen-specific immunoadsorption (IA) and rituximab (Tydén et al., Am J Transplant 2005;5:145-148), this technique has been successfully adopted by many transplant centers worldwide. The majority of centers strictly adhere to the Swedish protocol and perform IAs with a target volume of 1.5-2 plasma volumes on preoperative days -6, -5, -2, and -1, and postoperative days +3, +6, and +9, respectively. Patients who initially present with an IgG anti-A/B titer higher than 1:128 are not considered suitable candidates for ABOi transplantation by the Swedish protocol. Our center has gone beyond these suggestions and follows a slightly different strategy: We do not exclude patients with initial IgG anti-A/B titers higher than 1:128 and we perform as many preoperative antigen-specific extracorporeal treatments as needed to reach a threshold isoagglutinine titer of 1:4 or less. To intensify isoagglutinine clearance preoperatively, the total target volume per treatment was increased to 2.5-3 plasma volumes. Preconditioning IAs are performed every other day, instead of daily. Postoperatively we perform IAs only, if titers mandate us to do so (Wilpert et al., Nephrol Dial Transplant 2007;22:3048-3051). We report on 11 "high-titer patients" who entered our ABOi kidney transplant program with initial titers of 1:256 or above. Seven of 11 patients (64%) could successfully be transplanted with our modified ABO-apheresis protocol. Four of 11 high-titer patients did not reach target isoagglutinine titers of 1:4 or less and therefore did not undergo transplantation. We conclude that intensified preoperative IA renders a majority of high-titer patients suitable candidates for ABOi kidney transplantation.
Subject(s)
ABO Blood-Group System , Blood Component Removal/methods , Blood Group Incompatibility , Kidney Transplantation/methods , Adult , Aged , Cohort Studies , Female , Humans , Immunosorbent Techniques , Immunosuppressive Agents/therapeutic use , Kidney Diseases/therapy , Male , Middle Aged , Treatment OutcomeABSTRACT
BACKGROUND: Atherosclerosis is a chronic inflammatory response of the arterial wall to injury. Macrophage migration inhibitory factor (MIF), a cytokine with potent inflammatory functions, was thus considered to be important in atherosclerotic lesion evolution. METHODS AND RESULTS: We studied the presence and distribution of MIF immunoreactivity (MIF-IR) and MIF mRNA in internal mammary arteries with a normal histology and arteries with plaques in different stages of human atherosclerosis. To address a potential role for the coactivator Jab1 as a cellular mediator of MIF effects in vascular tissue, we correlated the expression of MIF to that of Jab1 by using immunohistochemistry and coimmunoprecipitation. We further sought to determine a potential functional role for endothelium-derived MIF in early atherogenesis by studying the effects of oxidized LDL on MIF expression in cultured human umbilical vascular endothelial cells. The results showed that MIF-IR and Jab1-IR are found in all cell types present in atherosclerotic lesions, that MIF-IR is upregulated during progression of atherosclerosis, that MIF is produced locally in the arterial wall, and that all MIF(+) cells are simultaneously Jab1(+). Coimmunoprecipitation experiments demonstrated in vivo complex formation between MIF and Jab1 in plaques. MIF expression in human umbilical vascular endothelial cells and a macrophage line was upregulated after stimulation with oxidized LDL. CONCLUSIONS: MIF is produced abundantly by various cells in all types of human atherosclerotic lesions and thus may play an important role in early plaque development and advanced complicated lesions. MIF-Jab1 complexes could serve critical regulatory functions in atherosclerotic lesion evolution.
Subject(s)
Arteriosclerosis/metabolism , Arteriosclerosis/pathology , Macrophage Migration-Inhibitory Factors/biosynthesis , Aortic Diseases/metabolism , Aortic Diseases/pathology , Arteriosclerosis/genetics , COP9 Signalosome Complex , Carotid Artery Diseases/metabolism , Carotid Artery Diseases/pathology , Cells, Cultured , Coronary Artery Disease/metabolism , Coronary Artery Disease/pathology , DNA-Binding Proteins/analysis , DNA-Binding Proteins/immunology , Disease Progression , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Fibrosis , Humans , Immunohistochemistry , Inflammation/metabolism , Inflammation/pathology , Intracellular Signaling Peptides and Proteins , Lipoproteins, LDL/pharmacology , Macrophage Migration-Inhibitory Factors/genetics , Macrophage Migration-Inhibitory Factors/immunology , Macrophages/drug effects , Macrophages/metabolism , Mammary Arteries/cytology , Mammary Arteries/metabolism , Peptide Hydrolases , Transcription Factors/analysis , Transcription Factors/immunology , Transcription, Genetic , Up-RegulationABSTRACT
Post-transplantation lymphoproliferative disorder is an infrequent complication after hematopoietic stem cell transplantation. It is hypothesized that lack of T-cell surveillance following transplantation permits reactivation of latent EBV leading to polyclonal B-cell expansion and finally outgrowth of a predominant clone. Most cases are of donor origin. Here, we describe an 8-year old boy with early onset post-transplantation lymphoproliferative disorder following matched-unrelated stem cell transplantation for high-risk T-cell leukemia whose disease was unusual for two reasons. First, his B-cell clone was of host origin and, in contrast to the few PTLD of host origin described so far, not associated with autologous reconstitution. Secondly, using clonal analysis, we could retrospectively show that the B-cell clone emerged during consolidation chemotherapy for T-cell leukemia, 3 months before stem cell transplantation.