Clinico-radiological features, molecular spectrum, and identification of prognostic factors in developmental and epileptic encephalopathy due to inosine triphosphate pyrophosphatase (ITPase) deficiency.

Developmental and epileptic encephalopathy 35 (DEE 35) is a severe neurological condition caused by biallelic variants in ITPA, encoding inosine triphosphate pyrophosphatase, an essential enzyme in purine metabolism. We delineate the genotypic and phenotypic spectrum of DEE 35, analyzing possible predictors for adverse clinical outcomes. We investigated a cohort of 28 new patients and reviewed previously described cases, providing a comprehensive characterization of 40 subjects. Exome sequencing was performed to identify underlying ITPA pathogenic variants. Brain MRI (magnetic resonance imaging) scans were systematically analyzed to delineate the neuroradiological spectrum. Survival curves according to the Kaplan-Meier method and log-rank test were used to investigate outcome predictors in different subgroups of patients. We identified 18 distinct ITPA pathogenic variants, including 14 novel variants, and two deletions. All subjects showed profound developmental delay, microcephaly, and refractory epilepsy followed by neurodevelopmental regression. Brain MRI revision revealed a recurrent pattern of delayed myelination and restricted diffusion of early myelinating structures. Congenital microcephaly and cardiac involvement were statistically significant novel clinical predictors of adverse outcomes. We refined the molecular, clinical, and neuroradiological characterization of ITPase deficiency, and identified new clinical predictors which may have a potentially important impact on diagnosis, counseling, and follow-up of affected individuals.

[Epilepsy in Angelman syndrome and the most common electroencephalographic findings]. / Epilepsia en el síndrome de Angelman y hallazgos electroencefalográficos más frecuentes.

INTRODUCTION: Angelman syndrome is a genetic disorder characterised by severe mental retardation, subtle dysmorphic facial features, a characteristic behavioural phenotype, seizures and abnormalities in video electroencephalograms (video EEG). Angelman syndrome may be associated with genetic mechanisms involving the region of chromosome 15q11-13. Up to 90% of cases have epileptic seizures, usually in the early years of life. Videoelectroencephalography patterns with some typical characteristics associated with Angelman syndrome have been reported, although these are not specific to it, and as such it is also useful for early diagnosis, especially in the first months or years of life. OBJECTIVE: To characterise the videoelectroencephalography findings of 17 patients diagnosed with Angelman syndrome, and compare them with previously published studies. PATIENTS AND METHODS: We conducted a retrospective observational study of 34 video EEGs performed on 17 patients diagnosed with Angelman syndrome at the clinical neurophysiology service of the Puerta de Hierro University Hospital in Madrid between 2019 and 2022. The primary objective was to characterise the videoelectroencephalographic findings and compare them with previously published studies. As secondary objectives, we analysed the patterns proposed by Dan and Boyd, and other demographic, genetic and clinical data. RESULTS: Video EEG supported the clinical suspicion in our study, as baseline brain activity was altered in all the patients. We identified a pattern similar to those defined by Dan and Boyd in 88% of the cases, and the type III pattern was the most common in our series. CONCLUSIONS: These findings confirm that video EEG is highly sensitive for the diagnosis of Angelman syndrome, and very useful as a diagnostic biomarker in the early stages of life.

TITLE: Epilepsia en el síndrome de Angelman y hallazgos electroencefalográficos más frecuentes.Introducción. El síndrome de Angelman es un trastorno genético caracterizado por retraso mental grave, rasgos faciales dismórficos sutiles, fenotipo conductual característico, crisis epilépticas y anomalías en el videoelectroencefalograma (video-EEG). El síndrome de Angelman puede estar asociado a mecanismos genéticos que involucran a la región del cromosoma 15q11-13. Hasta el 90% de los casos tiene crisis epilépticas, más frecuentemente en los primeros años de vida. Se han descrito patrones videoelectroencefalográficos con algunas características típicas asociadas a síndrome de Angelman, aunque no específicas, por lo que también es útil para el diagnóstico temprano, sobre todo en los primeros meses o años de vida. Objetivo. Caracterizar los hallazgos videoelectroencefalográficos de 17 pacientes diagnosticados de síndrome de Angelman y compararlos con estudios publicados previamente. Pacientes y métodos. Hemos realizado un estudio observacional retrospectivo de 34 video-EEG, realizados entre 2019 y 2022, de 17 pacientes con diagnóstico de síndrome de Angelman, llevados a cabo en el servicio de neurofisiología clínica del Hospital Universitario Puerta de Hierro. El objetivo principal fue caracterizar los hallazgos videoelectroencefalográficos y compararlos con estudios publicados previamente. Como objetivos secundarios, hemos analizado los patrones propuestos por Dan y Boyd, y otros datos demográficos, genéticos y clínicos. Resultados. El video-EEG apoyó la sospecha clínica en nuestro estudio, dado que la actividad cerebral de base se encontraba alterada en todos los pacientes. En el 88% de los casos fue posible identificar un patrón semejante a los definidos por Dan y Boyd, y, en nuestra serie, el patrón de tipo III fue el más frecuente. Conclusiones. Estos hallazgos confirman la alta sensibilidad del video-EEG para el diagnóstico de síndrome de Angelman y su gran utilidad como biomarcador diagnóstico en la primera etapa de la vida.

Fabrication of hierarchical micro-nanotopographies for cell attachment studies.

We report on the development of micro/nanofabrication processes to create hierarchical surface topographies that expand from 50 nm to microns in size on different materials. Three different approaches (named FIB1, FIB2, and EBL) that combine a variety of techniques such as photolithography, reactive ion etching, focused ion beam lithography, electron beam lithography, and soft lithography were developed, each one providing different advantages and disadvantages. The EBL approach was employed to fabricate substrates comprising channels with features between 200 nm and 10 µm in size on polymethylmethacrylate (PMMA), which were then used to investigate the independent or competitive effects of micro- and nanotopographies on cell adhesion and morphology. Rat mesenchymal stem cells (rMSCs) were cultured on four different substrates including 10 µm wide and 500 nm deep channels separated by 10 µm distances (MICRO), 200 nm wide and 100 nm deep nanochannels separated by 200 nm distances (NANO), their combination in parallel (PARAL), and in a perpendicular direction (PERP). Rat MSCs behaved differently on all tested substrates with a high degree of alignment (as measured by both number of aligned cells and average angle) on both NANO and MICRO. Furthermore, cells exhibited the highest level of alignment on PARAL, suggesting a synergetic effect of the two scales of topographies. On the other hand, cells on PERP exhibited the lowest alignment and a consistent change in morphology over time that seemed to be the result of interactions with both micro- and nanochannels positioned in the perpendicular direction, also suggesting a competitive effect of the topographies.

[Thromboembolic disease: analysis of 239 cases]. / Enfermedad tromboembólica venosa: análisis de 239 casos.

AIM: To know the number of patients that are admitted in the hospital with TED or those who have developed it during their stay, analyzing how to manage this disease and make a basis for a prospective study of this disease. METHODS: It is a descriptive and retrospective study of TED diagnosed patients during their stay at the Hospital Clínico San Carlos of Madrid for a 6 month period. Data related with epidemiologic records, diagnosis, treatment and complications of patients with Deep-Vein Thrombosis (DVT), Pulmonary Thromboembolism (PTE) or both (DVT+PTE) are collected. RESULTS: From October 1st of 2003 to March 31st of 2004, 239 patients were diagnosed with TED (64 DVT, 125 PTE y 51 DVT + PTE) when they were discharged from our hospital, with an average age of 73.2 years (standard desviation 13.64). We classify as risky factors with significative statistical differences chemotherapy, acute myocardium infarction and obesity. It has not been found any relation between the treatment used and the development of hemorrhage. Patients with previous episodes of TED had more frequent hemorrhagies than those without such records. Hypokinesia in the right ventricle shown on the echocardiogram supposed a gloomy prognosis of the death for TED as well as the development of DVT + PTE. CONCLUSION: In more than a 50% of patients, TED was PTE and more than a 60% were women. It is important to obtain information about these patients because 2/3 of them are admitted to internal medicine. Chemotherapy, AIM and obesity were factors significatively associated to DVT, PTE and DVT + PTE. Hypokinesia in the right ventricle shown on the echocardiogram supposed a gloomy prognosis for TED as well as the development of DVT + PTE.

Cathepsin L2, a novel human cysteine proteinase produced by breast and colorectal carcinomas.

We have identified and cloned a new member of the papain family of cysteine proteinases from a human brain cDNA library. The isolated cDNA codes for a polypeptide of 334 amino acids that exhibits all of the structural features characteristic of cysteine proteinases, including the active site cysteine residue essential for peptide hydrolysis. Pairwise comparisons of this amino acid sequence with the remaining human cysteine proteinases identified to date showed a high percentage of identity (78%) with cathepsin L; the percentage of identity with all other members of the family was much lower (<40%). On the basis of these structural characteristics, we have tentatively called this novel protein cathepsin L2. The cDNA encoding the mature cathepsin L2 was expressed in Escherichia coli, and after purification, the recombinant protein was able to degrade the synthetic peptide benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, which is commonly used as a substrate for cysteine proteinases. Cathepsin L2 proteolytic activity on this substrate was abolished by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cysteine proteinases, thus providing additional evidence that the isolated cDNA encodes a functional cysteine proteinase. Northern blot analysis of polyadenylated RNAs isolated from a variety of human tissues demonstrated that cathepsin L2 is predominantly expressed in the thymus and testis. This finding is in marked contrast with the wide tissue distribution of most cysteine proteinases characterized to date, including cathepsin L, and suggests that cathepsin L2 may play a specialized role in the thymus and testis. Expression analysis of cathepsin L2 in human tumors revealed a widespread expression in colorectal and breast carcinomas but not in normal colon or mammary gland or in peritumoral tissues. Cathepsin L2 was also expressed by colorectal and breast cancer cell lines as well as by some tumors of diverse origin, including ovarian and renal carcinomas. These results open the possibility that this novel enzyme may be involved in tumor processes, as already reported for other cysteine proteinases, including cathepsin L.

cdc25 cell cycle-activating phosphatases and c-myc expression in human non-Hodgkin's lymphomas.

cdc25A, cdc25B, and cdc25C are a family of human phosphatases that activate the cyclin-dependent kinases at different points of the cell cycle. cdc25A and cdc25B have been shown to have oncogenic potential, and they have been identified as transcriptional targets of c-myc. To determine the role of cdc25 genes in the pathogenesis of human lymphomas and their possible correlation with c-myc deregulation, we have analyzed the expression of cdc25A, cdc25B, and cdc25C and c-myc genes in a series of 63 non-Hodgkin's lymphomas and 8 nonneoplastic lymphoid tissues. The mRNA levels of the three phosphatases in the nonneoplastic tissues were negative or negligible. cdc25B overexpression was detected in 35 tumors (56%). This overexpression was more frequently found in aggressive (81%) than in indolent lymphomas (36%; P < 0.01). cdc25B overexpression was also significantly associated with a higher proliferative activity of the tumors. No cdc25B gene amplification or rearrangements were detected by Southern blot analysis. A biallelic EcoRI polymorphism of cdc25B gene was identified with a similar distribution in patients with lymphoma and in a normal population. cdc25A was overexpressed in three aggressive lymphomas. No detectable cdc25C mRNA levels were seen in any of the tumors. c-myc was overexpressed in 43% of tumors, and it correlated significantly with the presence of cdc25B up-regulation. Twenty-six of 35 (74%) lymphomas with high levels of cdc25B mRNA also showed c-myc overexpression, whereas 27 of 28 (96%) tumors without detectable or with very low cdc25B expression also had undetectable c-myc levels (P < 0.0001). In addition, a significant linear correlation was found between the cdc25B and c-myc mRNA levels (r = 0.575, P < 0.001). These findings suggest that cdc25B overexpression in non-Hodkin's lymphoma may participate in the pathogenesis of aggressive variants, and it may cooperate with c-myc oncogene in the development of these tumors.

p16MTS1/CDK4I mutations and concomitant loss of heterozygosity at 9p21-23 are frequent events in squamous cell carcinoma of the larynx.

We have examined the presence of p16MTS1/CDK4I gene deletions, mutations and methylation status, and 9p21-23 deletions in a series of 46 squamous cell carcinomas of the larynx and paired normal mucosa previously characterized for cyclin D1 gene amplification and overexpression. pRb expression was also examined by immunohistochemistry. p16MTS1/CDK4I mutations were found in 10/46 (22%) carcinomas and hypermethylation in 2/31 (7%). Loss of heterozygosity at 9p21-23 was found in 24 out of 42 (57%) carcinomas examined. All p16MTS1/CDK4I mutated cases and the two hypermethylated carcinomas showed 9p21-23 loss of heterozygosity. The loss of heterozygosity correlated with advanced local invasion (P=0.0045), lymph node metastases (P=0.0326), stage IV of the tumors (P=0.0058), and existence of cyclin D1 amplification/overexpression (P < 0.03). Only one out of 37 carcinomas was negative for pRb expression. No alterations in p16 gene or 9p21-23 loss of heterozygosity were detected in this case. These findings indicate that p16MTS1/CDK4I is frequently inactivated by gene mutation, hypermethylation, and allelic deletions in a significant subset of squamous cell carcinomas of larynx. Since 9p21-23 loss of heterozygosity was more frequently detected than p16MTS1/CDK4I mutations, and mutated carcinomas invariably had loss of heterozygosity, allelic losses probably precede the p16MTS1/CDK4I mutations. Their association with cyclin D1 deregulation in advanced carcinomas could indicate a possible cooperative effect in the progression of these neoplasms.

Basilar artery thrombosis in a child heterozygous for factor V Leiden mutation.

Activated protein C resistance, usually because of factor V Leiden mutation, is considered to be the most common hereditary prothrombotic condition. A 9-year-old male with a basilar artery stroke and activated protein C resistance is described. The patient, found to be heterozygous for factor V Leiden mutation, is one of several recent reports that suggest that activated protein C resistance is an important risk factor for spontaneous arterial thrombosis in infancy and childhood.

NOTCH1 mutations identify a genetic subgroup of chronic lymphocytic leukemia patients with high risk of transformation and poor outcome.

NOTCH1 has been found recurrently mutated in a subset of patients with chronic lymphocytic leukemia (CLL). To analyze biological features and clinical impact of NOTCH1 mutations in CLL, we sequenced this gene in 565 patients. NOTCH1 mutations, found in 63 patients (11%), were associated with unmutated IGHV, high expression of CD38 and ZAP-70, trisomy 12, advanced stage and elevated lactate dehydrogenase. Sequential analysis in 200 patients demonstrated acquisition of mutation in one case (0.5%) and disappearance after treatment in two. Binet A and B patients with NOTCH1-mutated had a shorter time to treatment. NOTCH1-mutated patients were more frequently refractory to therapy and showed shorter progression-free and overall survival after complete remission. Overall survival was shorter in NOTCH1-mutated patients, although not independently from IGHV. NOTCH1 mutation increased the risk of transformation to diffuse large B-cell lymphoma independently from IGHV, with this being validated in resampling tests of replicability. In summary, NOTCH1 mutational status, that was rarely acquired during the course of the disease, identify a genetic subgroup with high risk of transformation and poor outcome. This recently identified genetic subgroup of CLL patients deserves prospective studies to define their best management.

Pharmacological characterization of six trkB antibodies reveals a novel class of functional agents for the study of the BDNF receptor.

BACKGROUND AND PURPOSE: By interacting with trkB receptors, brain-derived neurotrophic factor (BDNF) triggers various signalling pathways responsible for neurone survival, differentiation and modulation of synaptic transmission. Numerous reports have implicated BDNF and trkB in the pathogenesis of various central nervous system affections and in cancer, thus representing trkB as a promising therapeutic target. In this study, we used an antibody-based approach to search for trkB-selective functional reagents. EXPERIMENTAL APPROACH: Six commercially available polyclonal and monoclonal antibodies were tested on recombinant and native, human and rodent trkB receptors. Functional and pharmacological characterization was performed using a modified version of the KIRA-elisa method and radioligand binding studies. Western blot analyses and neurite outgrowth assays were carried out to determine the specificity and selectivity of antibody effects. The survival properties of one antibody were further assessed on cultured neurones in a serum-deprived paradigm. KEY RESULTS: The functional trkB-selective antibodies showed distinct pharmacological profiles, ranging from partial agonists to antagonists, acting on trkB receptors through allosteric modulations. The same diversity of effects was observed on the mitogen-activated protein kinase signalling pathway downstream of trkB and on the subsequent neurite outgrowth. One antibody with partial agonist activity demonstrated cell survival properties by activating the Akt pathway. Finally, these antibodies were functionally validated as true trkB-selective ligands because they failed activating trkA or trkC, and contrary to BDNF, none of them bind to p75(NTR). CONCLUSIONS AND IMPLICATIONS: These trkB-selective antibodies represent a novel class of pharmacological tools to explore the pathophysiological roles of trkB and its potential therapeutic relevance for the treatment of various disorders.

Epstein-Barr virus related opsoclonus-myoclonus-ataxia does not rule out the presence of occult neuroblastic tumors.

Opsoclonus-myoclonus-ataxia (OMA) secondary to Epstein-Barr virus (EBV) infection has only been described in three pediatric patients. Previous reports suggested that evidence for a recent EBV infection in the absence of an occult neoplasm would predict a favorable prognosis for OMA as well as no tumor development. We present the case of a 20-month-old child with OMA associated with a microbiologically documented acute EBV infection and an occult thoracic ganglioneuroblastoma diagnosed 5 months later.

Dual effect of nitric oxide donors on cyclooxygenase-2 expression in human mesangial cells.

Nitric oxide (NO) is emerging as a key regulator of gene expression, capable of playing either positive or negative roles. The results of this study indicate that NO exerts a dual effect on cyclooxygenase-2 (COX-2) expression in human mesangial cells (HMC). Treatment of HMC with NO synthase inhibitors attenuated interleukin-1beta (IL-1beta/tumor necrosis factor-alpha (TNF-alpha)-elicited COX-2 protein and mRNA expression, suggesting a positive role of endogenous NO on COX-2 induction. However, NO donors (sodium nitroprusside [SNP] and S-nitroso-N-acetylpenicillamine [SNAP]) amplified cytokine-elicited COX-2 expression at early time points of treatment (up to 8 h for mRNA and up to 24 h for protein expression), but were inhibitory at later times. Oligonucleotide decoy experiments confirmed the importance of nuclear factor kappaB (NF-kappaB) activation for COX-2 induction by IL-1beta/TNF-alpha. Treatment with N(G)-nitro-L-arginine methyl ester (L-NAME) did not affect initial activation of NF-kappaB by IL-1beta/TNF-alpha, but unveiled an inhibitory effect of NO generation on NF-kappaB activity after 4 h. In HMC supplemented with SNP, cytokine-induced NF-kappaB activation was potentiated at early times of induction (5 to 15 min), but inhibited at later times (1 to 4 h), suggesting a dual effect of NO donors on NF-kappaB activation. Interestingly, IkappaBalpha protein levels followed a reciprocal pattern of expression: IkappaBalpha levels were lower at early times of induction in NO donor-supplemented cells; however, after 1 h of treatment, IkappaBalpha levels became higher than in cells treated only with cytokines. In the presence of SNP, cytokine-elicited IkappaBalpha mRNA induction was initially delayed, but was amplified at later times. These changes in IkappaBalpha expression could contribute to the dual effects of NO donors on NF-kappaB activation and COX-2 expression in HMC.

Disregulation of p16MTS1/CDK4I protein and mRNA expression is associated with gene alterations in squamous-cell carcinoma of the larynx.

To determine the relationship between p16MTS1/CDK4I expression, gene inactivation and 9p21 loss of heterozygosity (LOH) in the development of laryngeal carcinomas, we have examined p16MTS1/CDK4I protein and mRNA expression in a series of 7 normal and 36 tumoral tissues, and the presence of gene alterations and 9p21 LOH. Fifteen tumors (42%) showed low levels of pl6MTS1/CDK4I protein expression (similar to normal samples), 7 carcinomas (19%) expressed higher levels, and no protein expression was seen in 14 tumors (39%). No gene alterations were detected in 11 of the 15 tumors (73%) with protein levels similar to normal tissues. Most of the cases with absence of protein expression (86%) had gene alterations. Of the 7 tumors with protein over-expression, 4 showed frameshift or point mutations (2 cases each). mRNA analysis showed pl6MTS1/CDK4I -gene expression in 12 of 17 carcinomas examined. Gene alterations were detected in 9 of the 12 mRNA-positive tumors and in 2 of the 5 negative carcinomas. Concordant expression of p16alpha and p16beta transcripts was observed in all tumors. 9p21 LOH was detected in 23 carcinomas, 18 of which (78%) showed associated p16MTS1/CDK4I -gene alterations. These results indicate that disregulation of p16MTS1/CDK4I protein and mRNA expression is a frequent phenomenon in laryngeal carcinomas commonly associated with gene alterations and 9p21 LOH. The relative number of discrepancies between protein and mRNA expression and the presence of genetic alterations indicate that a comprehensive study of the gene including all these parameters may be necessary to assess the role of this gene in the pathogenesis of such tumors.

Collagenase-3 expression is associated with advanced local invasion in human squamous cell carcinomas of the larynx.

Collagenase-3 (MMP-13) is a matrix metalloproteinase recently identified on the basis of differential expression in normal breast tissues and in breast carcinoma. To date, collagenase-3 expression has been reported only in breast carcinomas and in articular cartilage of arthritic patients; the presence and possible implication of this enzyme in the progression of other malignant tumours are unknown. In this study collagenase-3 mRNA expression has been analysed by northern blot in a series of 35 matched squamous cell carcinomas of the larynx and the corresponding adjacent non-neoplastic tissues. In addition, mRNA expression of membrane type 1-matrix metalloproteinase (MT1-MMP) and gelatinase A, two matrix metalloproteinases which have the ability to activate collagenase-3 in vitro, was also examined in the same cases. No collagenase-3 expression was detected in any of the 35 normal mucosae, but collagenase-3 mRNA was observed in 20 of the 35 carcinomas (57 per cent). Western blot analysis revealed the presence of collagenase-3 protein in those carcinomas with high levels of mRNA expression, whereas no protein was detected in the carcinomas with negative mRNA expression, or in any of the normal tissues. The protein was localized predominantly in tumour epithelial cells. Collagenase-3 expression correlated significantly with better histological differentiation of the tumours (p = 0.026), as well as with advanced local invasion (p = 0.026). Collagenase-3 upregulation was also significantly associated with MT1-MMP and gelatinase A overexpression. These findings suggest that collagenase-3 expression may contribute to the progression of a significant subset of squamous cell carcinomas of the larynx and that its coordinate overexpression with MT1-MMP and gelatinase A may have a cooperative effect in the progression of the tumours.

Deletions and loss of expression of p16INK4a and p21Waf1 genes are associated with aggressive variants of mantle cell lymphomas.

Mantle cell lymphoma (MCL) is molecularly characterized by bcl-1 rearrangement and cyclin D1 gene overexpression. Some aggressive variants of MCL have been described with blastic or large cell morphology, higher proliferative activity, and shorter survival. The cyclin-dependent kinase inhibitors (CDKIs) p21Waf1 and p16INK4a have been suggested as candidates for tumor-suppressor genes. To determine the role of p21Waf1 and p16INK4a gene alterations in MCLs, we examined the expression, deletions, and mutations of these genes in a series of 24 MCLs, 18 typical, and 6 aggressive variants. Loss of expression and/or deletions of p21Waf1 and p16INK4a genes were detected in 4 (67%) aggressive MCLs but in none of the typical variants. Two aggressive MCLs showed a loss of p16INK4a expression. These cases showed homozygous deletions of p16INK4a gene by Southern blot analysis. An additional aggressive MCL in which expression could not be examined showed a hemizygous 9p12 deletion. Loss of p21Waf1 expression at both protein and mRNA levels was detected in an additional aggressive MCL. No p21Waf1 gene deletions or mutations were found in this case. The p21Waf1 expression in MCLs was independent of p53 mutations. The two cases with p53 mutations showed p21Waf1 and p16INK4a expression whereas the 4 aggressive MCLs with p16INK4a and p21Waf1 gene alterations had a wild-type p53. p21Waf1 and p16INK4a were expressed at mRNA and protein levels in all typical MCLs examined. No gene deletions or point mutations were found in typical variants. Two typical MCLs showed an anomalous single-stranded conformation polymorphism corresponding to the known polymorphisms at codon 148 of p16INK4a gene and codon 31 of p21Waf1 gene. These findings indicate that p21Waf1 and p16INK4a alterations are rare in typical MCLs but the loss of p21Waf1 and p16INK4a expression, and deletions of p16INK4a gene are associated with aggressive variants of MCLs, and they occur in a subset of tumors with a wild-type p53 gene.

Regulation of cyclooxygenase-2 expression in human mesangial cells--transcriptional inhibition by IL-13.

Activated mesangial cells may play an important part in glomerulonephritis. Cytokines can modulate the release of prostanoids by human mesangial cells (HMC). We have investigated the effects of pro-inflammatory stimuli on COX-2 expression in HMC and its potential modulation by interleukin (IL)-13. HMC released increased amounts of prostaglandin E2 (PGE2) after treatment with several combinations of IL-1 beta, tumor necrosis factor (TNF)-alpha and/or lipopolysaccharide. Increases in PGE2 correlated with the induction of COX-2 protein expression. The accumulation of PGE2 elicited by a combination of IL-1 beta/TNF-alpha correlated closely with the temporal pattern of COX-2 protein expression, which reflected the induction of COX-2 mRNA. IL-13 inhibited IL-1 beta/TNF-alpha-elicited PGE2 production, as well as COX-2 protein and mRNA expression in a concentration-dependent fashion. With 50 ng.mL-1 IL-13 these parameters were inhibited by 90, 80 and 84%, respectively. In HMC transfected with the 5' regulatory region of the COX-2 gene, IL-13 suppressed cytokine-induced promoter activation. Our results suggest that COX-2 expression is a major target for IL-13-mediated abrogation of prostaglandin release by HMC and support that this process takes place by transcriptional inhibition of the COX-2 gene.

Posttranscriptional regulation of human iNOS by the NO/cGMP pathway.

Nitric oxide (NO) and cGMP may exert positive or negative effects on inducible NO synthase (iNOS) expression. We have explored the influence of the NO/cGMP pathway on iNOS levels in human mesangial cells. Inhibition of NOS activity during an 8-h stimulation with IL-1beta plus tumor necrosis factor (TNF)-alpha reduced iNOS levels, while NO donors amplified iNOS induction threefold. However, time-course studies revealed a subsequent inhibitory effect of NO donors on iNOS protein and mRNA levels. This suggests that NO may contribute both to iNOS induction and downregulation. Soluble guanylyl cyclase (sGC) activation may be involved in these effects. Inhibition of sGC attenuated IL-1beta/TNF-alpha-elicited iNOS induction and reduced NO-driven amplification. Interestingly, cGMP analogs also modulated iNOS protein and mRNA levels in a biphasic manner. Inhibition of transcription unveiled a negative posttranscriptional modulation of the iNOS transcript by NO and cGMP at late times of induction. Supplementation with 8-bromo-cGMP (8-BrcGMP) reduced iNOS mRNA stability by 50%. These observations evidence a complex feedback regulation of iNOS expression, in which posttranscriptional mechanisms may play an important role.

Tetrahydrobiopterin modulates cyclooxygenase-2 expression in human mesangial cells.

Tetrahydrobiopterin (BH4) biosynthetic pathways are stimulated under inflammatory conditions. The newly synthesized BH4 serves as a cofactor for optimal activity of inducible nitric oxide synthase (NOS2). In human mesangial cells (HMC), BH4 is also a limiting factor for NOS2 expression. In this study we show that BH4 availability can also play a modulatory role in the expression of cyclooxygenase 2 (COX-2) in HMC. Supplementing HMC with the BH4 donor sepiapterin potentiated IL-1beta/TNF-alpha-induced COX-2 expression by approximately 2-fold. This effect was abolished by methotrexate. In contrast, the NOS inhibitor L-NAME and the soluble guanylate cyclase inhibitor ODQ did not block sepiapterin amplification of COX-2 expression. Moreover, sepiapterin was found to modulate the tyrosine phosphorylation of several cellular substrates, an early event which occurred well before the induction of NOS2 could be evidenced. These findings suggest a role for BH4 in the modulation of mesangial cell responses to pro-inflammatory stimuli.

Possible carcinogenic effects of X-rays in a transgenerational study with CBA mice.

A lifetime experiment using 4279 CBA/J mice was carried out to investigate whether the pre-conceptual exposure of sperm cells to X-ray radiation or urethane would result in an increased cancer risk in the untreated progeny, and/or increased susceptibility to cancer following exposure to a promoting agent. The study consisted of four main groups, namely a control group (saline), a urethane group (1 mg/g body wt) and two X-ray radiation groups (1 Gy, 2 Gy). At 1, 3 and 9 weeks after treatment, the males of these four parental groups were mated with untreated virgin females. The offspring of each parental group was divided into two subgroups: one received s.c. urethane (0.1 mg/g body wt once) as a promoter, the other saline, at the age of 6 weeks. All animals were evaluated for the occurrence of tumours. K-ras oncogene and p53 tumour suppressor gene mutations were investigated in frozen lung tumour samples. The female offspring of male parents exposed to X-rays 1 week before their mating showed a trend towards a higher tumour incidence of the haematopoietic system than the F1 controls. In addition, a higher percentage of bronchioloalveolar adenocarcinomas in male offspring born to irradiated paternals mated 1 week after X-ray treatment points to a plausible increased sensitivity of post-meiotic germ cell stages towards transgenerational carcinogenic effects. On the other hand, no increased tumour incidence and malignancy were observed in the offspring born to irradiated paternals mated 3 and 9 weeks after X-ray treatment. Paternal urethane treatment 1, 3 and 9 weeks prior to conception did not result in significantly altered incidence or malignancy of tumours of the lung, liver and haematopoietic tissue in the offspring. K-ras mutations increased during tumour progression from bronchioloalveolar hyperplasia to adenoma. Codon 61 K-ras mutations were more frequent in lung tumours of urethane-promoted progeny from irradiated parents than from control parents. P53 mutations were absent from these lung alterations.