GFPT1-Associated Congenital Myasthenic Syndrome Mimicking a Glycogen Storage Disease - Diagnostic Pitfalls in Myopathology Solved by Next-Generation-Sequencing.

GFPT1-related congenital myasthenic syndrome (CMS) is characterized by progressive limb girdle weakness, and less prominent involvement of facial, bulbar, or respiratory muscles. While tubular aggregates in muscle biopsy are considered highly indicative in GFPT1-associated CMS, excessive glycogen storage has not been described. Here, we report on three affected siblings with limb-girdle myasthenia due to biallelic pathogenic variants in GFPT1: the previously reported missense variant c.41G > A (p.Arg14Gln) and the novel truncating variant c.1265_1268del (p.Phe422TrpfsTer26). Patients showed progressive proximal atrophic muscular weakness with respiratory involvement, and a lethal disease course in adulthood. In the diagnostic workup at that time, muscle biopsy suggested a glycogen storage disease. Initially, Pompe disease was suspected. However, enzymatic activity of acid alpha-glucosidase was normal, and gene panel analysis including 38 genes associated with limb-girdle weakness (GAA included) remained unevocative. Hence, a non-specified glycogen storage myopathy was diagnosed. A decade later, the diagnosis of GFPT1-related CMS was established by genome sequencing. Myopathological reexamination showed pronounced glycogen accumulations, that were exclusively found in denervated muscle fibers. Only single fibers showed very small tubular aggregates, identified in evaluation of serial sections. This family demonstrates how diagnostic pitfalls can be addressed by an integrative approach including broad genetic analysis and re-evaluation of clinical as well as myopathological findings.

Concurrent axon and myelin destruction differentiates X-linked adrenoleukodystrophy from multiple sclerosis.

Cerebral disease manifestation occurs in about two thirds of males with X-linked adrenoleukodystrophy (CALD) and is fatally progressive if left untreated. Early histopathologic studies categorized CALD as an inflammatory demyelinating disease, which led to repeated comparisons to multiple sclerosis (MS). The aim of this study was to revisit the relationship between axonal damage and myelin loss in CALD. We applied novel immunohistochemical tools to investigate axonal damage, myelin loss and myelin repair in autopsy brain tissue of eight CALD and 25 MS patients. We found extensive and severe acute axonal damage in CALD already in prelesional areas defined by microglia loss and relative myelin preservation. In contrast to MS, we did not observe selective phagocytosis of myelin, but a concomitant decay of the entire axon-myelin unit in all CALD lesion stages. Using a novel marker protein for actively remyelinating oligodendrocytes, breast carcinoma-amplified sequence (BCAS) 1, we show that repair pathways are activated in oligodendrocytes in CALD. Regenerating cells, however, were affected by the ongoing disease process. We provide evidence that-in contrast to MS-selective myelin phagocytosis is not characteristic of CALD. On the contrary, our data indicate that acute axonal injury and permanent axonal loss are thus far underestimated features of the disease that must come into focus in our search for biomarkers and novel therapeutic approaches.

Microglia damage precedes major myelin breakdown in X-linked adrenoleukodystrophy and metachromatic leukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) and metachromatic leukodystrophy (MLD) are two relatively common examples of hereditary demyelinating diseases caused by a dysfunction of peroxisomal or lysosomal lipid degradation. In both conditions, accumulation of nondegraded lipids leads to the destruction of cerebral white matter. Because of their high lipid content, oligodendrocytes are considered key to the pathophysiology of these leukodystrophies. However, the response to allogeneic stem cell transplantation points to the relevance of cells related to the hematopoietic lineage. In the present study, we aimed to better characterize the pathogenetic role of microglia in the above-mentioned diseases. Applying recently established microglia markers to human autopsy cases of X-ALD and MLD we were able to delineate distinct lesion stages in evolving demyelinating lesions. The immune-phenotype of microglia was altered already early in lesion evolution, and microglia loss preceded full-blown myelin degeneration both in X-ALD and MLD. DNA fragmentation indicating phagocyte death was observed in areas showing microglia loss. The morphology and dynamics of phagocyte decay differed between the diseases and between lesion stages, hinting at distinct pathways of programmed cell death. In summary, the present study shows an early and severe damage to microglia in the pathogenesis of X-ALD and MLD. This hints at a central pathophysiologic role of these cells in the diseases and provides evidence for an ongoing transfer of toxic substrates primarily enriched in myelinating cells to microglia.

Immunomodulatory placental-expanded, mesenchymal stromal cells improve muscle function following hip arthroplasty.

BACKGROUND: No regenerative approach has thus far been shown to be effective in skeletal muscle injuries, despite their high frequency and associated functional deficits. We sought to address surgical trauma-related muscle injuries using local intraoperative application of allogeneic placenta-derived, mesenchymal-like adherent cells (PLX-PAD), using hip arthroplasty as a standardized injury model, because of the high regenerative and immunomodulatory potency of this cell type. METHODS: Our pilot phase I/IIa study was prospective, randomized, double blind, and placebo-controlled. Twenty patients undergoing hip arthroplasty via a direct lateral approach received an injection of 3.0 × 108 (300 M, n = 6) or 1.5 × 108 (150 M, n = 7) PLX-PAD or a placebo (n = 7) into the injured gluteus medius muscles. RESULTS: We did not observe any relevant PLX-PAD-related adverse events at the 2-year follow-up. Improved gluteus medius strength was noted as early as Week 6 in the treatment-groups. Surprisingly, until Week 26, the low-dose group outperformed the high-dose group and reached significantly improved strength compared with placebo [150 M vs. placebo: P = 0.007 (baseline adjusted; 95% confidence interval 7.6, 43.9); preoperative baseline values mean ± SE: placebo: 24.4 ± 6.7 Nm, 150 M: 27.3 ± 5.6 Nm], mirrored by an increase in muscle volume [150 M vs. placebo: P = 0.004 (baseline adjusted; 95% confidence interval 6.0, 30.0); preoperative baseline values GM volume: placebo: 211.9 ± 15.3 cm3 , 150 M: 237.4 ± 27.2 cm3 ]. Histology indicated accelerated healing after cell therapy. Biomarker studies revealed that low-dose treatment reduced the surgery-related immunological stress reaction more than high-dose treatment (exemplarily: CD16+ NK cells: Day 1 P = 0.06 vs. placebo, P = 0.07 vs. 150 M; CD4+ T-cells: Day 1 P = 0.04 vs. placebo, P = 0.08 vs. 150 M). Signs of late-onset immune reactivity after high-dose treatment corresponded to reduced functional improvement. CONCLUSIONS: Allogeneic PLX-PAD therapy improved strength and volume of injured skeletal muscle with a reasonable safety profile. Outcomes could be positively correlated with the modulation of early postoperative stress-related immunological reactions.

The congenital clubfoot - immunohistological analysis of the extracellular matrix.

PURPOSE: Congenital clubfoot is one of the most common limb disorders in humans and its etiology is still unclear. In order to better understand the pathogenesis of patients with primary clubfoot, we examined whether there are quantitative changes in the extracellular matrix (ECM; based on common interstitial collagens [C] like CI and CIII, microfilamentous collagens like CVI, noncollagenous proteins like undulin, and enzymes like matrixmetalloproteinase [MMP]-2 and tissue inhibitor of matrixmetalloproteinase [TIMP]-2 that are known to play a role in fibrogenesis and fibrolysis) of muscles involved in the foot deformity of patients with primary clubfoot corresponding to fibrosis. PATIENTS AND METHODS: Thirty patients (age ranging from 4 months to 5 years and 7 months) with primary clubfoot were examined (23 male and 7 female patients), among whom 18 patients were affected on one side and 12 affected on both sides. Twenty-five biopsies were taken during the first operative foot correction (Crawford-McKay) and 5 in the context of relapses. Muscle biopsies were taken from the muscles involved in the defect (Musculus [M.] gastrocnemius and M. tibialis anterior) and from the M. vastus lateralis of the M. quadriceps femoris, which were treated as healthy comparison muscles. Quantitative analysis of the components of the ECM was performed using a computer-assisted fibrosis measurement of the immunohistochemically processed tissue samples. RESULTS: We found higher values for M. gastrocnemius for CI, CIII, CVI and undulin in comparison with M. vastus lateralis. However, values for TIMP-2 were reduced. We found no significant differences for the components of M. tibialis anterior and M. vastus lateralis. There were no quantitative differences between male and female or between patients affected on one side and both sides. In patients who underwent relapse surgery, CI, CIII, CVI, and undulin of the gastrocnemius were significantly higher, while TIMP-2 was significantly lower. CONCLUSION: In the present study, we found manifest fibrosis in gastrocnemius due to quantitative changes in the ECM. In contrast to other studies, we found increasing fibrosis not just in contracted tissues but also in the muscle itself. Further studies are needed to clarify whether these changes are primarily responsible for the malfunction or whether they occur secondarily in the consequence of the dysfunction.

Synthetic niche to modulate regenerative potential of MSCs and enhance skeletal muscle regeneration.

Severe injury to the skeletal muscle often results in the formation of scar tissue, leading to a decline in functional performance. Traditionally, tissue engineering strategies for muscle repair have focused on substrates that promote myogenic differentiation of transplanted cells. In the current study, the reported data indicates that mesenchymal stromal cells (MSCs) transplanted via porous alginate cryogels promote muscle regeneration by secreting bioactive factors that profoundly influence the function of muscle progenitor cells. These cellular functions, which include heightened resistance of muscle progenitor cells to apoptosis, migration to site of injury, and prevention of premature differentiation are highly desirable in the healing cascade after acute muscle trauma. Furthermore, stimulation of MSCs with recombinant growth factors IGF-1 and VEGF165 was found to significantly enhance their paracrine effects on muscle progenitor cells. Multifunctional alginate cryogels were then utilized as synthetic niches that facilitate local stimulation of seeded MSCs by providing a sustained release of growth factors. In a clinically relevant injury model, the modulation of MSC paracrine signaling via engineered niches significantly improved muscle function by remodeling scar tissue and promoting the formation of new myofibers, outperforming standalone cell or growth factor delivery.

Experimental investigation and histopathological identification of acute thermal damage in skeletal porcine muscle in relation to whole-body SAR, maximum temperature, and CEM43 °C due to RF irradiation in an MR body coil of birdcage type at 123 MHz.

PURPOSE: This study is an investigation of the relationship between several characteristic parameters and acute thermal damage in porcine skeletal muscle. MATERIAL AND METHODS: Fourteen pigs under injection anaesthesia were placed into a magnetic resonance body coil and exposed for different time durations to different specific energy absorption rate (SAR) levels at 123 MHz. Local temperatures were measured using four temperature sensors. Sensors 1-3 were placed in skeletal muscle and one sensor was placed in the rectum. Sensors 1 and 2 were placed in hot-spot areas and sensor 3 was placed at the periphery of the animals. The pigs were exposed to whole-body SAR (SAR-wb) between 2.5 W/kg and 5.2 W/kg for 30 or 60 min. Three animals received no SAR. After each experiment, muscle samples adjacent to the positions of sensors 1-3 were taken for frozen section analysis. Three characteristic parameters were chosen for investigation: SAR-wb, maximum sensor temperature (T-max), and cumulative equivalent minutes at 43 °C (CEM43 °C). RESULTS: Histopathological criteria were established to detect acute thermal tissue damage in frozen sections such as widening of intercellular space between the muscle fibres and loss of glycogen. Clear tissue damage thresholds were found for T-max and CEM43 °C, though not for SAR-wb. For all animals with high thermal exposure, damage was also found for muscle samples adjacent to the peripheral sensor 3. CONCLUSIONS: Both T-max and CEM43, are able to predict thermal damage in porcine muscle. However, CEM43 is the less ambiguous parameter. The reasons for the occurrence of the aforementioned damage at low local temperatures at the animals' periphery remain unclear and further investigations are needed.

Eosinophils in hereditary and inflammatory myopathies.

It is not known whether eosinophilic myositis is a specific histopathological feature of limb girdle muscular dystrophy 2A (LGMD2A). Number and location of eosinophils in skeletal muscle biopsies (n=100) was analysed by Giemsa and modified hematoxylin/eosin staining in patients with genetically confirmed myopathies (LGMD2A, LGMD2B, LGMD2L, facioscapulohumeral muscular dystrophy, dystrophinopathy), histologically confirmed idiopathic inflammatory myopathies (sporadic inclusion body myositis (sIBM), dermatomyositis (DM), polymyositis), amyotrophic lateral sclerosis (neurogenic control), and normal controls. The number of eosinophils/mm² was significantly higher in LGMD2A, PM, DM, and sIBM compared to controls but not significantly higher than other myopathies. A large overlap in the number of eosinophils/mm2 between all groups was seen. In all disease groups eosinophils were mainly found endomysially (46- 88%) and intra- and perivascularly (4-37%). There was no correlation between the numbers of eosinophils/mm² and (i) age at biopsy and (ii) the duration of the disease. The extent of myopathic, fibrotic, and inflammatory changes did not differ in samples with high and low eosinophil count. Eosinophils seem to represent an unspecific histological finding in hereditary and inflammatory myopathies, but also amyotrophic lateral sclerosis.

Cerebral expression of neuroglobin and cytoglobin after deep hypothermic circulatory arrest in neonatal piglets.

INTRODUCTION: Deep hypothermic circulatory arrest (DHCA) is used in corrective cardiac surgery for complex congenital heart disease. Endogenous protective mechanisms may be responsible for the prevention of brain damage after hypothermic ischemia. Neuroglobin and cytoglobin are expressed in brain cells and appear to modulate hypoxic-ischemic brain injury. However, their neuroprotective potency is still not understood. Thus the aim of this study was to detect the influence exerted by DHCA on their expression. METHODS: The effects of DHCA were analyzed in a neonatal piglet model with cardiopulmonary bypass, DHCA of 60 and 120 min and subsequent reperfusion of 6h. Complete histological analysis and changes in the mRNA expression of neuroglobin and cytoglobin were measured in the brain. RESULTS: In comparison to animals without DHCA, neuroglobin expression was stable after 60 min DHCA and neuronal cell necrosis in the cortex was mild (< 10 %). Neuroglobin expression was significantly reduced after 120 min DHCA, which was accompanied by substantial neuronal cell necrosis (> 50 %). Cytoglobin expression did not differ significantly between animals with neuronal necrosis vs. sham. CONCLUSION: Constitutive expression levels of neuroglobin may explain the mild neuronal injury after 60 min DHCA. Significant neuronal cell death correlates with reduced neuroglobin expression and might reflect a limited capacity to compensate for ischemic injury. Both respiratory cell proteins may constitute attractive targets for therapeutic modulation of gene regulation, but further studies are necessary.

In vivo visualization of locally transplanted mesenchymal stem cells in the severely injured muscle in rats.

Autologous mesenchymal stem cells (MSCs) have been shown to improve the functional outcome after skeletal muscle trauma. The mechanisms behind this improvement have to be answered prior to a future clinical application. We investigated for the first time the in vivo distribution and behavior of MSCs after local transplantation into a severely injured muscle with magnetic resonance imaging (MRI). Autologous rat MSCs were labeled with very small iron oxide nanoparticles (VSOPs) and transplanted into the soleus muscle 1 week after an open crush injury. Distribution and migration of the cells were evaluated in vivo over time by the repeated performance of high-resolution MRI at 7 T. Three and 6 weeks after transplantation, the muscles were histologically analyzed. The labeled MSCs could be visualized inside the traumatized muscles 24 h after transplantation showing characteristic signal reductions in T2*-weighted sequences. The hypointense signal could be followed over 6 weeks and could be easily discriminated from the structures of the injured muscle. The cell pools did not migrate inside the muscle and showed a decrease in volume over time. Prussian blue-stained histologic sections showed a topographical correlation of the respective MRI signal and nanoparticle-labeled cells. Fusion events of marked cells with regenerating myofibers could be observed. The presented study demonstrates for the first time the feasibility of an in vivo tracking of MSCs with MRI after a severe skeletal muscle injury. The investigated method can be a powerful tool both in experimental setups and in possible clinical applications of stem cell-supported skeletal muscle regeneration.

Differential activation of mononuclear phagocytes in cerebellar malformation associated with Walker-Warburg syndrome.

Walker-Warburg syndrome (WWS) is an autosomal recessive disorder with alterations affecting the CNS that are characteristic of type-II lissencephaly and dysplasia/hypoplasia of the cerebellum. Other than these features, WWS is typically also accompanied by muscular dystrophy and abnormalities affecting the eyes. There is at present little information on the state of microglial and mononuclear phagocytic cell responses within the brain in WWS. In this case report, we present evidence for focal and differential activation of mononuclear phagocytes specifically confined to the dysplastic cerebellum of an infant at 5 months of age, diagnosed with WWS.

Selenoprotein N muscular dystrophy: differential diagnosis for early-onset limited mobility of the spine.

Early spinal rigidity is a nonspecific feature reported in diseases such as neuromuscular and central movement disorders. We present a male patient with rigid spine muscular dystrophy caused by newly identified compound heterozygote mutations of the selenoprotein N gene and discuss this disease as a possible differential diagnosis for early-onset reduced spine mobility. Rigid spine muscular dystrophy is a rare myopathy presenting in childhood with a typical combination of stable or slowly progressive mild to moderate muscle weakness, limitation in flexion of the spine, and progressive restrictive ventilatory disorder. The clinical features of our patient include early-onset rigidity of his spine, scoliosis, mild muscular weakness predominantly of neck and trunk flexors, and restrictive ventilatory disorder. Biopsy of the biceps muscle revealed nonspecific myopathic changes, and molecular analysis confirmed the diagnosis of rigid spine muscular dystrophy. Thus, neuromuscular diseases such as muscular dystrophy must be considered in all patients presenting with early spinal rigidity, and genetic determination is a possible way to determine the diagnosis.

Large-dose pretreatment with methylprednisolone fails to attenuate neuronal injury after deep hypothermic circulatory arrest in a neonatal piglet model.

Conflicting results have been reported with regard to the neuroprotective effects of steroid treatment with cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA). We evaluated the mode and severity of neuronal cell injury in neonatal piglets after prolonged DHCA and the possible neuroprotective effect of systemic pretreatment (>6 h before surgery) with large-dose methylprednisolone (MP). Nineteen neonatal piglets (age, <10 days; weight, 2.1 +/- 0.5 kg) were randomly assigned to 2 groups: 7 animals were pretreated with large-dose systemic MP (30 mg/kg) 24 h before surgery, and 12 animals without pharmacological pretreatment (saline) served as control groups. All animals were connected to full-flow CPB with cooling to 15 degrees C and 120 min of DHCA. After rewarming to 38.5 degrees C with CPB, animals were weaned from CPB and survived 6 h before they were killed, and the brain was prepared for light and electron microscopy, immunohistochemistry, and TUNEL-staining. Quantitative histological studies were performed in hippocampus, cortex, cerebellum, and caudate nucleus. Systemic pretreatment with large-dose MP lead to persistent hyperglycemia but no significant changes of cerebral perfusion. Necrotic and apoptotic neuronal cell death were detected in all analyzed brain regions after 120 min of DHCA. In comparison to the control group, large-dose pretreatment with systemic MP lead to an increase of necrotic neuronal cell death and induced significant neuronal apoptosis in the dentate gyrus of the hippocampus (P = 0.001). In conclusion, systemic pretreatment with large-dose MP fails to attenuate neuronal cell injury after prolonged DHCA and induces regional neuronal apoptosis in the dentate gyrus.

Megacolon in adulthood after surgical treatment of Hirschsprung's disease in early childhood.

Hirschsprung's disease (HD) is a disorder associated with congenital malformation of the enteric nervous system with segmental aganglionosis. Prevailing therapy includes a resection of the affected part of the bowel. However, patients often do not obtain complete functional improvement after surgical treatment. We present the case of a 25-year-old woman who had surgical treatment of HD in early childhood. After that procedure she had clinical features of constipation for years in the end, passing of stool once a week, requiring laxatives and enemas. We diagnosed an incomplete resection of the aganglionic bowel via rectal biopsy and resected the remaining aganglionic segment. Two months after surgery the patient's bowel function improved to a frequency of 1-4 stools per day. We conclude that regular follow-up is required to identify HD patients with persistent alterations of bowel function after surgery. In patients presenting with constipation, recognition of a remaining aganglionic segment or other alterations of the enteric nervous system should be aimed at in an early stage.

The HIV protease inhibitors saquinavir, ritonavir, and nelfinavir induce apoptosis and decrease barrier function in human intestinal epithelial cells.

BACKGROUND AND AIMS: Diarrhoea is a frequent adverse effect of HIV protease inhibitors (PIs) which may be due to intestinal barrier disruption. We investigated whether tight junction dysregulation, apoptosis or necrosis are responsible for this epithelial damage. METHODS: Saquinavir, nelfinavir, and ritonavir were added to the mucosal or serosal side of HT-29/B6 colon cell monolayers. Transepithelial resistance was monitored for 72 h to assess epithelial barrier function. Apoptosis and necrosis were investigated by light and electron microscopy and quantified by nucleosome ELISA and LDH measurement, respectively. Tight junction components were analysed by Western blots of occludin and zonula occludens. Apoptosis induction in normal human intestinal epithelium was examined by measurement of poly(ADP-ribose) polymerase (PARP) cleavage in Western blots of mucosal tissue explants cultured with PIs for 24 h. RESULTS: HIV PIs decreased transepithelial resistance by more than 44% in HT-29/B6 monolayers. Histology revealed massive apoptotic body formation but no evidence for necrosis after PI treatment. Correspondingly, LDH release was lower than 0.2%/h of total LDH, independent of PI treatment, and nucleosomes were increased up to 22-fold after drug treatment versus control. Occludin and zonula occludens-1 expression in the membrane were not diminished. PARP cleavage increased in normal human intestinal tissue treated with PIs. CONCLUSIONS: PI-induced barrier disruption in intestinal epithelial cells is not due to necrosis or tight junction alterations, but to induction of massive apoptosis which may lead to leak-flux diarrhoea in vivo. Our findings suggest that induction of apoptosis by PIs could have potential for antitumour therapy.

Delayed or late-onset type II glycogenosis with globular inclusions.

Three unrelated patients, one girl, one boy, and an adult female, aged 14, 11 and 41 years, respectively, at the time of biopsy, revealed lysosomal glycogen storage, autophagic vacuoles and peculiar globular inclusions of distinct ultrastructure, which were reducing but did not appear like true "reducing bodies" as described in the congenital myopathy "reducing body myopathy". All three patients had residual activity of acid alpha-glucosidase in their muscle biopsy samples. Leukocytes in the girl showed normal acid alpha-glucosidase activity, but in the boy activity was reduced. Molecular genetic analysis of the GAA gene revealed disease-causing mutations in each patient: H568L/R672W, IVS1-13T>G/G615F, and IVS1-13T>G/IVS1-13T>G. Although only one patient with such globular inclusions has been reported up to now, the three patients described here indicate that in the late-onset type of GSD II such inclusions may not be rare.

A 62-year-old man with chronic pyothorax.

June 2005. Pyothorax-associated lymphoma (PAL) is a rare B-cell non-Hodgkin lymphoma (B-NHL) which develops in the pleural cavity after a 20- to 64-year history of chronic pyothorax. We present here the case of a 62-year-old man who suffered from chronic pyothorax after pneumectomy 44 years ago, and complained of progressive ataxia. A MRI of the head revealed a solitary lesion in the vermis cerebelli, and a biopsy showed a lymphoma displaying immunoblastic features. Immunohistochemistry revealed an aberrant dual B/T phenotype and an Epstein-Barr virus (EBV) type III LMP-1+/EBNA-2+ latency profile. In-situ hybridization disclosed EBV-encoded RNAs in the tumor cells. PCR for the detection of rearranged immunoglobulin heavy chain (IgH) genes followed by GeneScan analysis demonstrated a clonal B-cell population with DNA amplificates of identical size in the brain manifestation, and a large mediastinal tumor analyzed post mortem. Among the largest series of 106 PALs collected through a nationwide survey in Japan, central nervous system (CNS) involvement was detected in 5 (14%) of 36 patients where an autopsy had been performed. To best of our knowledge, this is the first case of a pyothorax-associated lymphoma initially diagnosed on brain biopsy.

Brain-IL-1 beta triggers astrogliosis through induction of IL-6: inhibition by propranolol and IL-10.

BACKGROUND: Gliosis is a characteristic pathology in many central nervous system (CNS) diseases. Cytokines are considered to be effectors of gliosis. It has been shown that pro-inflammatory cytokines such as interleukin (IL)-1 and IL-6 boost glia scar formation. On the other hand, anti-inflammatory cytokines, such as IL-10 and IL-1 receptor antagonist (ra), can act neuroprotectively. Furthermore, various immune mediators and neurotransmitters can modulate the onset of gliosis. MATERIAL/METHODS: We used 100 male Sprague-Dawley rats to investigate the mechanisms of brain-cytokine-induced astrogliosis using an in vivo model of convection-enhanced delivery of cytokines (IL-beta, IL-6, tumor necrosis factor (TNF)-alpha) into the cerebro-ventricular system. The protective effects of the anti-inflammatory cytokine IL-10 and the neurotransmitter propranolol were also investigated. RESULTS: With this paradigm, we could clearly demonstrate that IL-6 is a key cytokine mediating astrogliosis, noticeable in the increased expression of glial fibrillary acidic protein (GFAP). Thus intra-cerebroventricular infusion of IL-6 increased GFAP expression in a dose-dependent manner. Furthermore, GFAP expression was also increased by IL-beta, which correspondingly triggered an IL-6 release into the CSF. Accordingly, TNF-alpha, which did not induce IL-6 release, also did not induce gliosis. On the other hand, substances which decrease IL-beta-induced IL-6 production, such as propranolol and IL-10, also dramatically decreased IL-beta triggered gliosis. CONCLUSIONS: IL-6 infusion, as well as IL-beta-induced IL-6 release into the CSF, increase GFAP expression in the cerebral cortex and hippocampus. Accordingly, blockade of the IL-beta-induced IL-6 release by IL-10 and propranolol decreases GFAP expression.

The endothelin system in Morris hepatoma-7777: an endothelin receptor antagonist inhibits growth in vitro and in vivo.

1. Plasma concentrations of endothelin are increased in patients with hepatocellular cancer as well as in patients with liver metastasis. However, the impact of these findings remains uncertain. 2. We thus analyzed the endothelin system in a rat hepatoma model (Morris hepatoma 7777) in vitro and in vivo. 3. Our study revealed that tissue concentrations of endothelin-1 (ET-1) and big-ET-1, the precursor of ET-1, were significantly elevated in Morris hepatoma 7777 as compared to normal liver. The ETA receptor density was significantly elevated, whereas the density of the ETB receptor was decreased in Morris hepatoma 7777. 4. We could also demonstrate that hepatoma cells secrete ET-1. 5. Exogenously added ET-1 enhances hepatoma cell growth in a dose-dependent manner. Endothelin receptor antagonists (ETA and combined ETA/ETB receptor antagonists) inhibit tumor cell growth in vitro. Since the combined ETA/ETB receptor antagonist was more effective in vitro, we used this compound also for in vivo studies and could demonstrate that a combined ETA/ETB receptor antagonist is able to reduce hepatoma growth in vivo. 6. In conclusion, the endothelin system is activated in Morris hepatoma 7777 and contributes to hepatoma growth. Since endothelin receptor antagonists are well-tolerated upcoming clinically used drugs without major side effects, our data might provide a new pharmacological approach to reduce hepatoma growth in vivo.