Thomas Stallmach

Loss of nephrocystin-3 function can cause embryonic lethality, Meckel-Gruber-like syndrome, situs inversus, and renal-hepatic-pancreatic dysplasia.

Many genetic diseases have been linked to the dysfunction of primary cilia, which occur nearly ubiquitously in the body and act as solitary cellular mechanosensory organelles. The list of clinical manifestations and affected tissues in cilia-related disorders (ciliopathies) such as nephronophthisis is broad and has been attributed to the wide expression pattern of ciliary proteins. However, little is known about the molecular mechanisms leading to this dramatic diversity of phenotypes. We recently reported hypomorphic NPHP3 mutations in children and young adults with isolated nephronophthisis and associated hepatic fibrosis or tapetoretinal degeneration. Here, we chose a combinatorial approach in mice and humans to define the phenotypic spectrum of NPHP3/Nphp3 mutations and the role of the nephrocystin-3 protein. We demonstrate that the pcy mutation generates a hypomorphic Nphp3 allele that is responsible for the cystic kidney disease phenotype, whereas complete loss of Nphp3 function results in situs inversus, congenital heart defects, and embryonic lethality in mice. In humans, we show that NPHP3 mutations can cause a broad clinical spectrum of early embryonic patterning defects comprising situs inversus, polydactyly, central nervous system malformations, structural heart defects, preauricular fistulas, and a wide range of congenital anomalies of the kidney and urinary tract (CAKUT). On the functional level, we show that nephrocystin-3 directly interacts with inversin and can inhibit like inversin canonical Wnt signaling, whereas nephrocystin-3 deficiency leads in Xenopus laevis to typical planar cell polarity defects, suggesting a role in the control of canonical and noncanonical (planar cell polarity) Wnt signaling.

Increased Cerebral Infarct Volumes in Polyglobulic Mice Overexpressing Erythropoietin

There is increasing evidence that erythropoietin (Epo) has a protective function in cerebral ischemia. When used for treatment, high Epo plasma levels associated with increases in blood viscosity, however, may counteract beneficial effects of Epo in brain ischemia. The authors generated two transgenic mouse lines that overexpress human Epo preferentially, but not exclusively, in neuronal cells. In mouse line tg21, a fourfold increase of Epo protein level was found in brain only, whereas line tg6 showed a dramatic increase of cerebral and systemic transgene expression resulting in hematocrit levels of 80%. Cerebral blood flow (CBF), as determined by bolus tracking magnetic resonance imaging, was not altered in the tg6 line. The time-to-peak interval for the tracer, however, increased approximately threefold in polyglobulic tg6 mice. Immunohistochemical analysis revealed an increase in dilated vessels in tg6 mice, providing an explanation for unaltered CBF in polyglobulic animals. Permanent occlusion of the middle cerebral artery (pMCAO) led to similar perfusion deficits in wild-type, tg6, and tg21 mice. Compared with wild-type controls, infarct volumes were not significantly smaller (22%) in tg21 animals 24 hours after pMCAO, but were 49% enlarged (P < 0.05) in polyglobulic tg6 mice. In the latter animals, elevated numbers of Mac-1 immunoreactive cells in infarcted tissue suggested that leukocyte infiltration contributed to enlarged infarct volume. The current results indicate that moderately increased brain levels of Epo in tg21 transgenic mice were not sufficient to provide significant tissue protection after pMCAO. The results with tg6 mice indicate that systemic chronic treatment with Epo associated with elevated hematocrit might deteriorate outcome after stroke either because of the elevated hematocrit or other chronic effects.

Explanted cryopreserved allografts: a morphological and immunohistochemical comparison between arterial allografts and allograft heart valves from infants and adults

OBJECTIVE Life expectancy of cryopreserved allografts implanted in infants is different from those implanted in adults. A morphological study of explanted allograft heart valves was performed to determine the mechanism of deterioration and to compare cryopreserved arterial and heart valve allografts from adult patients with those explanted from infants. METHOD Between 1987 and 1996, 209 cryopreserved allografts were implanted: 125 valved conduits or monocusps to reconstruct the right ventricular outflow tract in congenital heart disease, 50 allograft heart valves to treat native aortic and prosthetic aortic valve endocarditis and 34 cryopreserved arterial allografts to replace mycotic aortic aneurysms or infected aortic prosthetic grafts. Two months to 8 years after implantation, 23 heart valve allografts, 11 right-sided and 12 left-sided, and four arterial allografts had to be explanted for reasons such as degeneration, recurrent infection, aneurysm formation or rupture. Besides conventional staining, immunohistochemical detection of cell populations was performed as follows: CD45RO, CD3 and CD43 for T lymphocytes, CD20 for B lymphocytes, CD68 for macrophages, protein S100 for Langerhans-cells, vimentin for fibroblasts, alpha-actin for smooth muscle cells and factor VIII for endothelial cells. RESULTS Explanted cryopreserved allografts were all fibrotic, acellular, non-vital and without endothelial cells. The fibrous tissue was preserved. T lymphocytes, indicating rejection, were found in all right-sided allografts from the paediatric population, but only in 9% of left-sided valves explanted from adults and in one of the four of arterial allografts. Macrophages and Langerhans-cells were found only in right-sided allografts from paediatric patients. CONCLUSION Right-sided cryopreserved allografts from a paediatric population showed ongoing cellular rejection. By contrast, there was only a weak T-cell mediated rejection to adult heart valve and arterial allografts. Therefore, similar long-term results can be expected in adult arterial and heart valve allografts, whereas longevity of right-sided heart valve allograft in the paediatric age group seems endangered by cellular rejection.

c-Jun N-Terminal Kinase 2 Deficiency Protects Against Hypercholesterolemia-Induced Endothelial Dysfunction and Oxidative Stress

Background— Hypercholesterolemia-induced endothelial dysfunction due to excessive production of reactive oxygen species is a major trigger of atherogenesis. The c-Jun-N-terminal kinases (JNKs) are activated by oxidative stress and play a key role in atherogenesis and inflammation. We investigated whether JNK2 deletion protects from hypercholesterolemia-induced endothelial dysfunction and oxidative stress. Methods and Results— Male JNK2 knockout (JNK2−/−) and wild-type (WT) mice (8 weeks old) were fed either a high-cholesterol diet (HCD; 1.25% total cholesterol) or a normal diet for 14 weeks. Aortic lysates of WT mice fed a HCD showed an increase in JNK phosphorylation compared with WT mice fed a normal diet (P<0.05). Endothelium-dependent relaxations to acetylcholine were impaired in WT HCD mice (P<0.05 versus WT normal diet). In contrast, JNK2−/− HCD mice did not exhibit endothelial dysfunction (96±5% maximal relaxation in response to acetylcholine; P<0.05 versus WT HCD). Endothelium-independent relaxations were identical in all groups. A hypercholesterolemia-induced decrease in nitric oxide (NO) release of endothelial cells was found in WT but not in JNK2−/− mice. In parallel, endothelial NO synthase expression was upregulated only in JNK2−/− HCD animals, whereas the expression of antioxidant defense systems such as extracellular superoxide dismutase and manganese superoxide dismutase was decreased in WT but not in JNK2−/− HCD mice. In contrast to JNK2−/− mice, WT HCD displayed an increase in O2− and ONOO− concentrations as well as nitrotyrosine staining and peroxidation. Conclusions— JNK2 plays a critical role as a mediator of hypercholesterolemia-induced endothelial dysfunction and oxidative stress. Thus, JNK2 may provide a novel target for prevention of vascular disease and atherosclerosis.

Erythropoietin-induced excessive erythrocytosis activates the tissue endothelin system in mice.

The endothelium controls blood flow and pressure by releasing several vasoactive factors, among them the vasodilator nitric oxide (NO) and the potent vasoconstrictor endothelin‐1 (ET‐1). Although increased NO levels have been found in excessive erythrocytosis, little is known concerning ET‐1 expression in this condition. Thus, we examined the endothelin system in transgenic mice that due to constitutive overexpression of erythropoietin (Epo) reached hematocrit levels of ~80%. Surprisingly, despite generalized vasodilatation, polycythemic mice exhibited a two‐ to fivefold elevation in ET‐1 mRNA levels in aorta, liver, heart, and kidney. In line with this, increased expression of ET‐1 protein was detected in the pulmonary artery by immunohistochemical analysis. Compared with their wild‐type littermates, aortic rings of Epo transgenic animals exhibited a marked reduction in vascular reactivity to ET‐1 and big ET‐1, but this effect was abrogated upon preincubation with the NO synthase inhibitor N‐nitro‐l‐arginine methyl ester (L‐NAME). Pretreatment of polycythemic mice with the ETA receptor antagonist darusentan for 3 wk significantly prolonged their survival upon acute exposure to L‐NAME. Taken together, these results demonstrate for the first time that excessive erythrocytosis induces a marked activation of the tissue endothelin system that results in increased mortality upon blockade of NO‐mediated vasodilatation. Because ETA antagonism prolonged survival after acute blockade of NO synthesis, endothelin may be regarded as a contributor to the adverse cardiovascular effects of erythrocytosis and may thus represent a new target in the treatment of cardiovascular disease associated with erythrocytosis.

Metal-responsive transcription factor-1 (MTF-1) is essential for embryonic liver development and heavy metal detoxification in the adult liver

Metal‐responsive transcription factor‐1 (MTF‐1) activates the transcription of metallothionein genes and other target genes in response to heavy metal load and other stresses such as hypoxia and oxidative stress. It also has an essential function during embryo‐genesis: targeted disruption of Mtf1 in the mouse results in lethal liver degeneration on day 14 of gestation. Here we studied Mtf1 knockout mice at embryonic and adult stages, the latter by means of conditional knockout. Hepatocytes from Mtf1 null mutant and wild‐type embryos were taken into culture on day 12.5 of gestation. Both initially appeared normal, but mutant cells were lost within a few days. Furthermore, Mtf1 null hepatocytes were poorly, if at all, rescued by cocultivation with wild‐type rat embryo hepatocytes, indicating a cell‐autonomous defect. When the Mtf1 gene was excised by Cre recombinase after birth in liver and bone marrow and to a lesser extent in other organs, mice were viable under non‐stress conditions but highly susceptible to cadmium toxicity, in support of a role of MTF‐1 in coping with heavy metal stress. An additional MTF‐1 function was revealed upon analysis of the hematopoietic system in conditional knockout mice where leukocytes, especially lymphocytes, were found to be severely underrepresented. Together, these findings point to a critical role of MTF‐1 in embryonic liver formation, heavy metal toxicity, and hematopoiesis.— Wang, Y., Wimmer, U., Lichtlen, P., Inderbitzin, D., Stieger, B., Meier, P. J., Hunziker, L., Stallmach, T., Forrer, T., Rülicke, T., Georgiev, O., Schaffner, W. Metal‐responsive transcription factor‐1 (MTF‐1) is essential for embryonic liver development and heavy metal detoxification in the adult liver. FASEB J. 18, 1071–1079 (2004)

Aberrant positioning of trophoblast and lymphocytes in the feto-maternal interface with pre-eclampsia

Abstract Pregnancy represents the growth of an allograft where fetal trophoblast cells evade immune rejection and invade maternal tissue. There should be a balance between fetal trophoblast and maternal immune-responsive cells and alterations in the proportion of these cells may relate to pregnancy disorders. To test this, the decidual tissue of placental bed biopsies was examined and trophoblast cells and lymphocytes were quantified morphometrically; spiral arteries were classified as unchanged, transformed or affected by acute atherosis. Normal pregnancy (n=19) was characterized by the transformation of about one half of all spiral arteries within the placental bed. We found that 40% of all lymphocytes were CD56+ uterine NK cells and 60%, CD3+ T-lymphocytes; about 30% of these were CD8+ T cells. Intrauterine growth retardation in the context of preeclampsia (n=15) was accompanied by reduced trophoblast numbers within smaller and more tortuous arteries and an increase in the proportion of CD56+ uterine NK cells and CD8+ T lymphocytes in the decidua (70% of all CD3+ cells). In the case of pre-eclampsia without fetal growth retardation (n=14) no increase in CD56+ uterine NK cells was seen, while CD8+ T lymphocytes were significantly increased compared with the normal level (50% of all CD3+ cells). Fetal growth retardation is associated with poor transformation of spiral arteries and characterized by an increase of uterine NK cells. Symptoms of pre-eclampsia are independently associated with an increase in the cytotoxic T subset of decidual lymphocytes. Pre-eclampsia and related fetal growth retardation are seemingly caused by an enhancement of the maternal cytotoxic defence against the fetal allograft.

Chronic deciduitis in the placental basal plate: Definition and interobserver reliability

This study tested whether concordance could be achieved for abnormal inflammation in the basal decidua of placental specimens among 6 pathologists experienced in placental pathology. Thirty microscope slides were evaluated by the pathologists for chronic deciduitis. They also scored the severity and extent of inflammation and the presence of plasma cells. No definition of chronic deciduitis was provided. Concordance (5/6 or 6/6 agreement) was achieved in 23 cases (76%). Spearmans rank correlation showed that the diagnosis of chronic deciduitis was almost identical to the assessment of the severity of the inflammation. A regression analysis showed that the perception of severity (and hence chronic deciduitis) was influenced by the other 2 variables, extent and plasma cells. The results were shared with the pathologists, and 25 cases (excluding those with previous 6/6 consensus) were reevaluated. Concordance was now achieved in the 83% of those remaining cases. Using a threshold based on the severity and the extent of lymphocytes, and the presence of plasma cells, pathologists are able to diagnose chronic deciduitis with sufficient concordance to be of value in clinical correlation studies.

MR Autopsy in Fetuses

Objective: The purpose of this study was to determine whether postmortem magnetic resonance imaging (MR autopsy) could serve as an alternative to necropsy of fetuses. The value of MR autopsy in the validation of the obstetric management and in risk counseling concerning future pregnancies is discussed. Methods: 10 consecutive, malformed fetuses were examined by postmortem MRI within 24 h of delivery. Prenatal ultrasound (US) was performed in all fetuses. Complete necropsy served as gold standard. Results: MR autopsy confirmed every US diagnosis responsible for termination. All MRI findings were confirmed by necropsy. In two fetuses, necropsy gave additional information relevant for risk counseling. Histologic examination corrected the diagnosis in one case. Conclusions: MR autopsy provides valuable information previously only available from necropsy. In parents who refuse perinatal necropsy, the information obtained by MR autopsy can be used to validate obstetric management and to evaluate the risk for future pregnancies. Necropsy however remains the gold standard.

Dandy–Walker malformation: prenatal diagnosis and outcome

Prenatal ultrasound identified Dandy–Walker malformation (DWM) in ten singleton pregnancies with concurrent central nervous system (CNS) anomalies and extra‐CNS anomalies in eight cases. DWM was confirmed by postnatal magnetic resonance imaging (MRI) or pathological examination in nine cases. Karyotypes were normal in the seven infants tested. Postnatal neurological and developmental testing in the five survivors showed a spectrum of clinical outcome from minor defects to severe handicap. Postnatal investigation also disclosed additional CNS and extra‐CNS findings not detected on ultrasound, as did autopsy in the other five infants. However, ultrasound diagnosis of DWM is accurate and is an indication for exhaustive screening for concurrent anomalies both within and outside the CNS and in chromosome structure and number, as the prognosis is heavily dependent on associated malformations and karyotype. Copyright