Biomedical subjects
Publications and source records attributed to G Weber.
In this prospective study, a significant incidence of fever (47%), true bacteremia (15%), and sepsis (12%), were found in 60 cardiac patients treated with an intra-aortic balloon counterpulsation pump. The benefit of antibiotic prophylaxis in this setting should therefore be evaluated.
Multiple endocrine neoplasia type 1 is an autosomal dominant familial cancer syndrome. The responsible gene, MEN1, has been isolated and inactivating mutations have been found in the majority of MEN1 families. The underlying genetic defects in the remaining families may be located in yet unidentified regions of the MEN1 gene. Here, we present novel transcripts of MEN1 which vary in the content of their 5'-untranslated region. All transcript variants display upstream exons correctly spliced to MEN1 exon 2. The most commonly seen splice isoform occurred in a region previously published as human intron 1, a region which shows a high conservation between human and rodent MEN1. This splice variant uses an analogous transcription initiation site and identical splice donor/acceptor sites as a major transcript seen in rodent Men1. The newly identified MEN1 isoforms may represent biologically important transcripts and should thus be studied for mutations in the regions enclosed therein.
Bis-(8-anilinonaphthalene-1-sulfonate) (bis-ANS) causes inactivation of vesicular stomatitis virus (VSV) at micromolar concentrations while butyl-ANS and ANS are effective at concentrations one and two orders of magnitude higher, respectively. VSV fully inactivated by the combined effects of 10 microM bis-ANS and 2.5 kbar hydrostatic pressure elicited a high titer of neutralizing antibodies. Incubation of VSV with >/=2 M urea at atmospheric pressure caused very little virus inactivation, whereas at a pressure of 2.5 kbar, 1 M urea caused inactivation that exceeded by more than two orders of magnitude the sum of the inactivating effects produced by urea and pressure separately. Measurements of bis-ANS fluorescence showed that increasing the urea concentration reduces the pressure required to disrupt the structure. We conclude that anilinonaphthalene sulfonate compounds inactivate VSV by a mechanism similar to that produced by pressure. The most effective antiviral compound was bis-ANS which can be used for the preparation of safe viral vaccines or as an antiviral drug eventually.
We have investigated the endogenous expression of menin, a protein encoded by the gene mutated in multiple endocrine neoplasia type 1 (MEN1). Western blot analysis showed strong expression of menin as a 68 kDa protein in all of 7 human and primate cell lines tested. In a panel of 12 fetal human tissue extracts, 68 kDa menin was readily detected in brain cortex, kidney, pituitary, testis and thymus and weakly detected in thyroid. Reproducible bands other than 68 kDa were observed in adrenal and heart, whereas menin was undetectable in liver, lung, pancreas and skin. Analysis of synchronized HeLa cells revealed no variation in the amount or size of menin throughout the cell cycle. Protein expression was compared between lymphoblastoid cell lines from healthy controls and MEN1 patients carrying nonsense mutations on 1 allele. No truncated protein was detected in either cytoplasmic or nuclear fractions in mutation-carrying cells. The expression level and cellular location of full-length menin did not differ between cell lines derived from MEN1 patients and healthy donors. This suggests that the wild-type allele has been up-regulated in mutation-carrying cells to compensate for the loss of 1 functional allele.
Sepsis is defined as the systemic inflammatory response to infection. Phospholipase A(2) (PLA(2)) plays an important role in inflammation processes by initiating the production of inflammatory mediators. The role of cytosolic PLA (cPLA(2)) has not yet been identified in inflammatory and infectious disease clinical settings. The aim of the present research was to determine whether cPLA(2) activity has a role during sepsis. Since neutrophil activation has been documented during sepsis, these cells were chosen as a model to evaluate the function of cPLA(2) in this clinical setting. cPLA(2 )was studied at 3 levels: activity, protein expression, and messenger RNA (mRNA). Neutrophils from 32 septic patients with and without bacteremia were examined. cPLA(2) activity was measured using labeled phosphatidyl choline vesicles as a substrate, and total PLA(2) was determined by the release of labeled arachidonic acid from prelabeled cells. A significant increase in cPLA(2) activity, protein expression, and total PLA(2) activity in neutrophils was detected during sepsis. mRNA levels, detected by reverse transcriptase-polymerase chain reaction, were significantly higher during sepsis, indicating that the increase in the amount of cPLA(2) is regulated on the mRNA level. The significant elevation of cPLA(2) activity and expression in neutrophils during sepsis suggests that this enzyme plays a major role in neutrophil function in this clinical setting. (Blood. 2000;95:660-665)
The applicability of free flow electrophoresis (FFE) was expanded towards processing of sensitive cells. The chamber medium was adjusted to a physiologic pH of 7.35 by a mixture of N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid) (EPPS) and 2,2-bis(hydroxymethyl)-2,2'2"-nitrilotriethanol (BISTRIS). These substances proved to be nontoxic to sensitive cells such as human smooth muscle or thyroid cells. They enhanced the electrical conductivity of the medium only slightly so that a new cell electrophoresis separation medium could be prepared, which contained 30 mM NaCl together with or without 1 mM CaCl2 but did not generate problems of overheating the fluid. Suspended in this medium, human smooth muscle cells as well as human thyroid carcinoma cells remained viable single cells for at least 120 min. After this period they could be recultured to form monolayers. If electrophoresed in the Octopus preparative FFE device, they migrated as single cells and did not clot; therefore, their electrophoretic behavior could be determined exactly.
The present study focuses on the establishment and characterization of a new follicular thyroid carcinoma cell line. The human cell line ML-1 was derived from a dedifferentiated follicular thyroid carcinoma relapse, which progressed despite preceding surgery followed by two radioiodine therapies. More than 90% of the cells of this line express thyroglobulin, chondroitin sulfate, and vimentin antigens, but only about 70% show cytokeratin filaments and a negative surface charge density such as human erythrocytes. More importantly, cells of this line are able to take up iodine and/or glucose both in vitro and in vivo and to secrete thyroglobulin, chondroitin sulfate, and fibronectin into the interstitial space. In addition, triiodothyronine is released constitutively into culture supernatants. Moreover, it is also suitable for xenotransplantation studies because it is tumorigenic in NMRI nude mice in vivo. The cell line forms tumors with follicular structures when transplanted to nude mice. Due to these unique features the ML-1 cell line can be considered as a very suitable test model for pharmacological and cell biological studies. Since chemicals may interfere with the production of thyroid hormones, this cell line represents also a tool for toxicological investigations.
FIA with pulsed amperometric detection (PAD) is used for the determination of metal-binding carbohydrates in plant extracts. Results of direct FIA measurements agree very well with those of corresponding HPLC-PAD analyses. The proposed method is used to determine total carbohydrates in isolated low-molecular-weight fractions of plant roots, which have been obtained after extraction at different pH values. The results are compared with those obtained by corresponding metal determinations (Ca, Mg, Zn, Mn) by AAS and also with constant potential amperometric detection at a copper working electrode. The latter detection mode is more sensitive, but less selective for carbohydrates.
In this study, we assessed cognitive function and neurophysiological development in congenitally hypothyroid (CH) children. We performed a cross-sectional study at the outpatient Pediatric Clinic and Department of Neurophysiology at San Raffaele Hospital, Milan. The study enrolled 25 CH patients (6.00-10.83 years of age) detected by neonatal screening, and 34 healthy control children (4-11 years of age). Patients and controls had comparable scores at neuropsychological tests (WISC-R), and at auditory P300 tests. In contrast, we found significantly longer LLSEP latencies in CH patients (p < 0.03). CH patients treated 30 days after birth showed lower scores at neuropsychological tests, but not at neurophysiological tests, compared to patients who started the replacement therapy earlier. Patients with more severe fetal hypothyroidism (T4 levels at diagnosis < or = 2 micrograms/dl) had lower neuropsychological scores, and similar neurophysiological results, compared with patients with moderate fetal hypothyroidism. The severity of fetal hypothyroidism and early treatment influence the mental outcome of CH patients. Neurophysiological results show that central nervous system damage occurs in some patients despite early treatment.
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The aim of the present paper is to describe and compare services for adults with intellectual disability (ID) and mental health needs in five European countries: Austria, England, Greece, Ireland and Spain. A framework and structure for collecting information about service provision was designed. This information was collected through a mixture of interviews with service providers, questionnaires and a review of the research literature within each country. Information was collected on historical context, policy, legislation, assessment, treatment and the structure of services for people with ID and mental health problems. Overall, the needs of those with additional mental health needs have not been specifically addressed at a national level with perhaps the exception of England and Ireland, although there are still gaps in services in these nations. Normalization has been adopted in each of the five countries, and there are moves toward deinstitutionalization, integration and inclusion. Families and self-advocacy groups have grown. The pace of this change varies between and even within countries. The main findings of the study include: unclear policy, trends for legislative changes, increased prevalence of mental health problems, inadequate generic service provision, a need for specialist mental health services, a need for improved interconnections of services, and a need for training developments. Policy and legislation in the five European countries under consideration tend to separate the disability aspects of people with ID from their mental health needs. Consequently, the service needs of this group remain largely invisible. This might be a direct reflection of policy clarity and legislation, or could be the result of a failure to implement existing guidelines. This has a detrimental effect on the lives of people with ID, and their families and carers.
An outbreak of Streptococcus pneumoniae serotype 1 occurred in a closed community that was characterized by poverty and crowding. Vaccine was administered to individuals aged >2 years; no new cases occurred among vaccine recipients. Six weeks after vaccination, carriage of serotype 1, but not of other serotypes, decreased 8.8-fold. This suggests that the reduction in serotype 1 carriage reflects the natural course of the outbreak rather than a vaccine effect. Polysaccharide vaccine may be helpful in terminating pneumococcal outbreaks but may not affect pneumococcal carriage.
The aim of this preliminary study was to evaluate the acoustic patterns of the cries of hypothyroid newborns at the time of diagnosis and after the beginning of therapy. Cries were recorded at the nursery of the San Raffaele Hospital, Milan, Italy from 12 full-term subjects (three boys and nine girls) affected by congenital hypothyroidism. Results show that untreated hypothyroid infants at first recording had fewer voiceless and partially voiced cries than normal controls. The percent distribution of this pattern did not change at the second recording after the onset of substitutive therapy. Also, untreated hypothyroid infants had many more cry units showing a vibrato contour than did controls, and this pattern did not change after the onset of treatment. Starting, maximum, minimum, and end frequencies measured on the fundamental were significantly lower in the hypothyroid sample. Four hypothyroid subjects recorded before therapy and within 4 weeks after therapy onset significantly augmented their fundamental frequency parameters; however, in 25% of the sample, sound parameters remained unaltered after 3 or more weeks of treatment. To our knowledge the present preliminary study is the first one performed on follow-up of hypothyroid newborns and indicates that both central and peripheral damage might influence the pattern of crying in untreated hypothyroid infants.
We report here the characterization of an alternative splice variant of prointerleukin-1alpha (proIL-1alpha), constitutively expressed by the normal adult rat testis. In addition to the classical 32K proIL-1alpha (32proIL-1alpha) messenger RNA, the testis produced a shorter variant encoding a putative protein of 24K (24proIL-1alpha). In situ hybridization demonstrated constitutive expression of the splice transcript in the seminiferous tubules. This alternative complementary DNA lacked the fifth exon, harboring the calpain cleavage site essential for generation of mature 17K IL-1alpha. This was verified by calpain treatment, producing the expected cleavage products of recombinant 32proIL-1alpha, but not of 24proIL-1alpha. Similarly, expression in COS-7 cells demonstrated processing of 32proIL-1alpha to the mature 17K form and secretion, whereas 24proIL-1alpha remained unprocessed. Both 32proIL-1alpha and 24proIL-1alpha showed a dose-dependent stimulatory effect in a thymocyte proliferation assay, although at lower potency than mature 17K IL-1alpha. In contrast, when tested on hCG-stimulated Leydig cells in vitro, a dose-dependent inhibition of testosterone production was obtained with mature 17K IL-1alpha and at a lower potency with 32proIL-1alpha, whereas 24proIL-1alpha was inactive. In conclusion, the three IL-1 bioactive proteins described here contribute to IL-1 protein heterogeneity and may serve as constitutive paracrine mediators in the testis.
Pseudohypoparathyroidism (PHP) refers to two major variants that generally coexist in the same family, PHP type Ia (PHP Ia), in which both PTH resistance and a constellation of physical features, termed Albright's hereditary osteodystrophy (AHO), are present, and pseudopseudohypoparathyroidism (PPHP), in which AHO occurs without PTH resistance. Most patients with PHP Ia show a partial deficiency (50%) of Gs activity, due to loss of function mutations in Gsalpha gene (GNAS1). The present study reports clinical, biochemical, and molecular data of 8 unrelated families with PHP Ia and PPHP. The 13 exons of GNAS1 were screened for mutations by PCR and direct sequencing of the amplified products. We detected heterozygous mutations in the affected members of the 4 families in which PHP Ia was present. In 2 families 2 previously reported deletions in exons 5 and 7 were found, whereas in the other 2 families, 2 novel frameshift deletions were identified in exons 1 and 11, causing a premature stop codon in the mutant allele. No mutation was detected in the families in which PPHP was the only clinical manifestation. In conclusion, we report the first mutational analysis of Italian patients with PHP Ia and PPHP, and we describe two novel deletions in GNAS1. Furthermore, we confirm that these mutations cannot be detected in families with isolated PPHP, suggesting that these forms of AHO are genetically distinct from PHP Ia.
Pendred's syndrome is a combination of congenital sensorineural hearing loss and iodine organification defect leading to a positive perchlorate test and goiter. Although it is the commonest form of syndromic hearing loss, the variable clinical presentation contributes to the difficulty in securing a diagnosis. The identification of the disease gene (PDS) prompts the need to reevaluate the syndrome to identify possible clues for the diagnosis. To this purpose, in three Italian families presenting with the clinical features of Pendred's syndrome, the molecular analysis was accompanied by full clinical, biochemical, and radiological examination. A correlation between genotype and phenotype was found in the only patient with enlargement of vestibular aqueduct and endolymphatic duct and sac at magnetic resonance imaging. This subject was a compound heterozygote for a deletion in PDS exon 10 (1197delT, FS400) and a novel insertion in exon 19 (2182-2183insG, Y728X). The present study demonstrates for the first time the value of the combination of clinical/radiological and genetic studies in the diagnosis of Pendred's syndrome. The positivity of a perchlorate discharge test and the malformations of membranous labyrinth fit well with the recent achievements on the role of pendrin in thyroid hormonogenesis and the maintenance of endolymph homeostasis.
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Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder with neoplasia of the anterior pituitary, the parathyroid, the endocrine pancreas and other endocrine tissues including the adrenal cortex. The tumor-suppressor gene causing this disease was identified at the gene locus 11q13. We recently reported that adrenocortical carcinomas frequently show loss of heterozygosity (LOH) of 11q13, but do not contain point mutations within the MEN1-coding region. To investigate whether reduced gene expression (for example by mutations within the MEN1 promoter) may contribute to the tumorigenesis of sporadic adrenocortical tumors, 24 adrenocortical specimen were studied by Northern blot analysis. This series included six adrenocortical carcinomas, four cortisol-producing adenomas, six aldosterone-producing adenomas, three endocrine-inactive adenomas and six normal adrenal glands. The presence of LOH of 11q13 was investigated using five polymorphic microsatellite markers (D11S956, PYGM, D11S4939, D11S4946 and D11S987) close to the MEN1 gene. Poly-A mRNA was hybridized with a PCR-generated cDNA probe of the MEN1 gene, a cDNA of the former MEN1 candidate gene phospholipase (PLC) beta3 and a mouse beta-actin cDNA for normalization. LOH of 11q13 was detected in five out of six carcinomas and two inactive adenomas, but in none of the hormone-producing adenomas. Compared with normal adrenals (100+/-6. 5%, mean+/-s.e.m.) MEN1 mRNA in adrenocortical tumors was expressed in similar amounts (carcinomas 109+/-11%, cortisol-producing adenomas 131+/-10%, aldosterone-producing adenomas 113+/-13%, endocrine-inactive adenomas 111+/-2%) with the exception of one adrenocortical carcinoma with low MEN1 mRNA expression (66%). PLCbeta3 mRNA expression showed a variable pattern without reaching significant differences between the groups. We conclude that since mRNA levels were unaltered in the majority of tumors, mutations of the MEN1 gene causing altered gene transcription is unlikely to be a major pathogenic factor of sporadic adrenocortical tumors.