Characteristics of respiratory viruses' circulation through a six-year period (2016-2022) in a pediatric population in Normandy, France, and the impact of COVID-19 pandemic.

IMPORTANCE: The report highlights an epidemiological change in the circulation of respiratory viruses in pediatric populations due to strategies adopted against COVID-19 pandemic. COVID-19 has resulted in a significant increase in requests for multiplex respiratory research to identify the virus responsible for the symptoms. The diagnostic needs have increased, and the number of samples analyzed in 2021-2022 is equal to the samples analyzed over the four epidemic periods preceding the pandemic. The report suggests the importance of active surveillance of respiratory viruses' circulation and new recommendations for respiratory virus detection in pediatric patients.

In vivo parameters influencing 2-Cys Prx oligomerization: The role of enzyme sulfinylation.

2-Cys Prxs are H2O2-specific antioxidants that become inactivated by enzyme hyperoxidation at elevated H2O2 levels. Although hyperoxidation restricts the antioxidant physiological role of these enzymes, it also allows the enzyme to become an efficient chaperone holdase. The critical molecular event allowing the peroxidase to chaperone switch is thought to be the enzyme assembly into high molecular weight (HMW) structures brought about by enzyme hyperoxidation. How hyperoxidation promotes HMW assembly is not well understood and Prx mutants allowing disentangling its peroxidase and chaperone functions are lacking. To begin addressing the link between enzyme hyperoxidation and HMW structures formation, we have evaluated the in vivo 2-Cys Prxs quaternary structure changes induced by H2O2 by size exclusion chromatography (SEC) on crude lysates, using wild type (Wt) untagged and Myc-tagged S. cerevisiae 2-Cys Prx Tsa1 and derivative Tsa1 mutants or genetic conditions known to inactivate peroxidase or chaperone activity or altering the enzyme sensitivity to hyperoxidation. Our data confirm the strict causative link between H2O2-induced hyperoxidation and HMW formation/stabilization, also raising the question of whether CP hyperoxidation triggers the assembly of HMW structures by the stacking of decamers, which is the prevalent view of the literature, or rather, the stabilization of preassembled stacked decamers.

Biotherapies in stroke.

Stroke is the second leading cause of death worldwide and the most common cause of severe disability. Neuroprotection and repair mechanisms supporting endogenous brain plasticity are often insufficient to allow complete recovery. While numerous neuroprotective drugs trials have failed to demonstrate benefits for patients, they have provided interesting translational research lessons related to neurorestorative therapy mechanisms in stroke. Stroke damage is not limited to neurons but involve all brain cell type including the extracellular matrix in a "glio-neurovascular niche". Targeting a range of host brain cells, biotherapies such as growth factors and therapeutic cells, currently hold great promise as a regenerative medical strategy for stroke. These techniques can promote both neuroprotection and delayed neural repair through neuro-synaptogenesis, angiogenesis, oligodendrogliogenesis, axonal sprouting and immunomodulatory effects. Their complex mechanisms of action are interdependent and vary according to the particular growth factor or grafted cell type. For example, while "peripheral" stem or stromal cells can provide paracrine trophic support, neural stem/progenitor cells (NSC) or mature neurons can act as more direct neural replacements. With a wide therapeutic time window after stroke, biotherapies could be used to treat many patients. However, guidelines for selecting the optimal time window, and the best delivery routes and doses are still debated and the answers may depend on the chosen product and its expected mechanism including early neuroprotection, delayed neural repair, trophic systemic transient effects or graft survival and integration. Currently, the great variety of growth factors, cell sources and cell therapy products form a therapeutic arsenal that is available for stroke treatment. Their effective clinical use will require prior careful considerations regarding safety (e.g. tumorgenicity, immunogenicity), potential efficacy, cell characterization, delivery route and in vivo biodistribution. Bone marrow-derived cell populations such as mesenchymal stromal/stem cells (MSC) or mononuclear cells (MNC), umbilical cord stem cells and NSC are most investigated notably in clinical trials. Finally, we discuss perspectives concerning potential novel biotherapies such as combinatorial approaches (growth factor combined with cell therapy, in vitro optimization of cell products, or co-transplantation) and the development of biomaterials, which could be used as injectable hydrogel scaffold matrices that could protect a cell graft or selectively deliver drugs and growth factors into the post-stroke cavity at chronic stages. Considering the remaining questions about the best procedure and the safety cautions, we can hope that future translational research about biotherapies will bring more efficient treatments that will decrease post-stroke disability for many patients.

[Autologous stem cell transplantation for autoimmune diseases: recommendations from the SFGM-TC]. / Autogreffe des cellules souches hématopoïétiques dans les maladies auto-immunes : recommandations de la SFGM-TC.

Autologous hematopoietic stem cell transplantation is a valid alternative to immunosuppressive treatment in patients with auto-immune disease; however, the role of this approach remains subject to debate. In the attempt to harmonize clinical practices between different French transplantation centers, the French Society of Bone Marrow Transplantation and Cell Therapies (SFGM-TC) set up its fourth annual series of workshops which brought together practitioners from all of its member centers. These workshops took place in September 2013 in Lille. In this article we give an overview regarding the indications of autologous stem cell transplantation in auto-immune diseases as well as recommendations regarding post-transplant follow-up of patients.

An in vitro study to compare 99mTc-stannous colloids and 99mTc-HMPAO for labelling human leukocytes.

AIM: Aim of the present study was to compare in vitro the labelling efficiency (LE) and cell viability (TBE) of autologous leukocytes labelled with (99m)Tc-SnF(2) and (99m)Tc-HMPAO, and to evaluate the quantity and quality of spontaneously released (99m)Tc (SR) from labelled cells at several time points after labelling. METHODS: A total of 14 patients with different diseases and 18 normal subjects were included in this study. A blood sample was collected from each patient; purified autologous leukocytes were divided into 2 samples and labelled with (99m)Tc-SnF(2) and (99m)Tc-HMPAO. LE was evaluated at the end of labelling and TBE and SR were evaluated at 10 min and 1 h, 2 h and 4 h after labelling. RESULTS: LE of (99m)Tc-SnF(2)-WBC was higher than (99m)Tc-HMPAO-WBC (61.2+/-18.7% and 43.3+/-11.3; p<0.0001) and we found an inverse correlation between blood glucose and labelling efficiency for both methods (p=0.02). Minimal differences were also observed between 2 methods after 10 min and 1 h, as far as the cell viability is concerned. The percentage of radioactivity spontaneously released from (99m)Tc-SnF(2)-WBC was significantly higher compared to (99m)Tc-HMPAO-WBC at each time point. Radioactivity released from labelled cells was predominantly (99m)Tc-SnF(2) and (99m)Tc-HMPAO with few free (99m)Tc (<20%). CONCLUSION: Both radiopharmaceuticals are not toxic for WBC. Labelling with (99m)Tc-SnF(2) give a higher LE than with (99m)Tc-HMPAO; however, radiolabelled colloids are more released from labelled cells over a period of 4 h. While (99m)Tc-HMPAO is physiological excreted into gastrointestinal tract, (99m)Tc-SnF(2) can be re-uptaken in vivo by reticulo-endothelial cells of liver and spleen. These findings suggest that (99m)Tc-SnF(2)-WBC might be better than (99m)Tc-HMPAO-WBC for studying inflammatory bowel diseases.

Development and biodistribution of 188Re-SSS lipiodol following injection into the hepatic artery of healthy pigs.

Although intra-arterial radiotherapy with (131)I-labelled lipiodol is a useful therapeutic approach in the treatment of hepatocellular carcinomas, various disadvantages limit its use. Here we describe the development of (188)Re-SSS lipiodol, as well as its biodistribution in the healthy pig after injection into the hepatic artery. The (188)Re-SSS lipiodol was obtained after dissolving a chelating agent, previously labelled with (188)Re, in cold lipiodol. The radiochemical purity (RCP) of the labelling was checked immediately and at 24 and 48 h. The (188)Re-SSS lipiodol was injected into the hepatic artery of six healthy pigs. They were killed 1, 24 and 48 h post injection, for ex vivo counting. An autoradiographic study was performed in three cases. (188)Re-SSS lipiodol was obtained with a yield of 87%+/-9.1%. The immediate RCP was 93%+/-3.4%. This radiolabelling was reproducible and stable at 48 h in plasma: 90.6%+/-1.5% of the activity remained in the lipiodol with an RCP of 91%+/-4%. Ex vivo counting confirmed the predominantly hepatic uptake and revealed weak lung and intestinal uptake. There was very weak urinary elimination (2.3%+/-0.5% at 48 h) and a slightly higher level of intestinal elimination (4.8%+/-1.9% at 48 h). The autoradiographic studies showed (188)Re-SSS lipiodol to be located mainly in sinusoids, like (131)I-lipiodol. By using the method described here, (188)Re-SSS lipiodol can be obtained with a very high yield and a satisfactory RCP. Its biodistribution in the healthy pig is in agreement with data published elsewhere concerning other types of radiolabelling used for lipiodol, except for the very weak urinary and intestinal elimination, which probably indicates better stability of (188)Re-SSS labelling.

Dose-response of RAG2-/-/gammac-/- mice to busulfan in preparation for spermatogonial transplantation.

Practical applications of spermatogonial transplantation require good rates of colonization by the donor cells. Recipient testes are usually depleted of competing endogenous spermatogonia by administration of 32-44 mg busulfan kg(-1) body weight before transplantation. However, it is not clear that this is the optimum dose, especially for immunodeficient mice. In the present study, the response of adult RAG2(-/-)/gamma(c)(-/-) (RAG2) male mice to treatment with 10-50 mg busulfan kg(-1) body weight was determined in terms of mortality rates, testicular masses and histology, and colonization of seminiferous tubules by transplanted spermatogonia. Mortality increased from 0 to 50% at doses between 20 mg busulfan kg(-1) and 40 mg busulfan kg(-1), whereas the maximum effects on testicular mass and histology were observed at 20 mg busulfan kg(-1). Colonization of testes by genetically marked spermatogonia after treatment of mice with 20 mg busulfan kg(-1) was equivalent to rates previously reported in recipients treated with 32-44 mg busulfan kg(-1). Thus, 20 mg busulfan kg(-1) appears to be the optimum dose for preparing RAG2 mice for spermatogonial transplantation. However, because the steepness of the dose-response curves indicates that direct administration of busulfan is not ideal for this purpose, 15 mg busulfan kg(-1) was administered to pregnant females at various times between day 10.5 and day 16.5 of gestation to determine whether this would deplete the number of germ cells in male offspring. Although there were large variations in testicular mass and histology, no mortality was observed and administration of busulfan at day 10.5 or 12.5 after mating delayed initiation of spermatogenesis, indicating that prenatal administration of busulfan combined with neonatal transplantation might be an effective method for further increasing rates of colonization by donor spermatogonia.

Genome sequence of the plant pathogen Ralstonia solanacearum.

Ralstonia solanacearum is a devastating, soil-borne plant pathogen with a global distribution and an unusually wide host range. It is a model system for the dissection of molecular determinants governing pathogenicity. We present here the complete genome sequence and its analysis of strain GMI1000. The 5.8-megabase (Mb) genome is organized into two replicons: a 3.7-Mb chromosome and a 2.1-Mb megaplasmid. Both replicons have a mosaic structure providing evidence for the acquisition of genes through horizontal gene transfer. Regions containing genetically mobile elements associated with the percentage of G+C bias may have an important function in genome evolution. The genome encodes many proteins potentially associated with a role in pathogenicity. In particular, many putative attachment factors were identified. The complete repertoire of type III secreted effector proteins can be studied. Over 40 candidates were identified. Comparison with other genomes suggests that bacterial plant pathogens and animal pathogens harbour distinct arrays of specialized type III-dependent effectors.

Diagnosis of osteomyelitis in the diabetic foot with a 99mTc-HMPAO leucocyte scintigraphy combined with a 99mTc-MDP bone scintigraphy.

BACKGROUND: The aim of this prospective study was to assess the role of 99mTc-HMPAO leucocyte scintigraphy combined with a 99mTc-MDP bone scintigraphy in the diagnosis of the diabetic foot infection (HMPAO-Leu/MDP). METHODS: 75 diabetic patients with suspected osteomyelitis were included. The HMPAO-Leu/MDP scan was considered to be consistent with osteomyelitis when the HMPAO-Leu uptake was concordant in all the incidences with an MDP bone uptake. A HMPAO-Leu uptake without concordant bone MDP activity was considered as a soft-tissue infection. The results of the HMPAO-Leu/MDP scan were compared to the following diagnostic criteria: bone infection was confirmed by radiological follow-up or bone biopsy; the absence of bone infection was confirmed by clinical (healing of the ulcer without antibiotherapy) and radiological follow up. RESULTS: According to these criteria, among the 83 ulcers, bone infection was observed in 41 (49.4%): the HMPAO-Leu/MDP scan was positive in 38 cases, including 14 ulcers with normal or doubtful radiographs at inclusion. In the group of 42 ulcers without proven bone infection, the HMPAO-Leu/MDP scan was negative in 41 cases, including 17 lesions with a soft-tissue infection. CONCLUSION: With a sensitivity of 92.6%, a specificity of 97.6%, the HMPAO-Leu/MDP scan is a reliable tool for the diagnosis of osteomyelitis in the diabetic foot. Neuroarthropathy did not affect the performances of the HMPAO-Leu/MDP scan. Owing to a high spatial resolution this test is very helpful to differentiate bone infection from soft-tissue infection especially in case of neuroarthropathy.

Scintimammography: better detection of small-sized lesions with tomoscintigraphic than planar images, a phantom study.

Planar (99)Tc(m)-methoxyisobutylisonitrile ((99)Tc(m)-MIBI) scintimammography has been used for several years to detect breast cancer tumours, but with low sensitivity for small lesions. Results of tomoscintimammography studies have not been conclusive. We conducted a phantom study to compare the detection of small-sized tumours with planar versus tomoscintigraphic images. We used a data spectrum anthropomorphic fillable breast phantom with two 9.8 mm and 12.4 mm spheres superficially or deep in the breast compartment with sphere/breast activity ratios varying from 3 to 6. We acquired planar and 180 degrees tomoscintigraphic images in each configuration using a double head standard gamma camera. In certain cases we varied different parameters (64x64 matrix or 360 degrees rotation) in a second series of tomoscintigraphic acquisitions. We simultaneously used filtered back-projection reconstruction (FBP) and iterative reconstruction (IR). Planar images were shown by the sphere in 10 out of 25 cases. Tomoscintigraphic images were shown by the sphere in nine out of 25 cases with FBP and in 18 out of 25 with IR. There was a significant difference between IR and FBP (P<0.01) and between planar and IR images (P<0.01), but no significant difference between planar and IR images. The noise/signal ratio was lower with planar images than with the two types of reconstruction (P<0.05) but was not significantly different between the two types of reconstruction. Contrast was lower on planar images than on the two types of reconstruction (P<0.05) and was also better on IR than on FBP images (P<0.05). Granularity was lower for planar images than for reconstruction images (P<0.01) and also lower for IR than for FBP (P<0.01). The tomoscintigraphic reconstructions acquired with a 64x64 matrix were only positive in four out of 10 cases, while they were positive in nine out of 10 with a 128x128 matrix. We concluded that, in this phantom study, tomoscintimammography with IR provides a significant improvement in the detection of small-sized breast tumours compared with planar images. In addition, for tomoscintigraphic images, a 128x128 matrix is preferable to a 64x64 matrix. Those results have, of course, to be confirmed in vivo in a large population of patients with small-sized breast lesions.

Biodistribution of 111indium-labeled macrophages infused intravenously in patients with renal carcinoma.

PURPOSE: We have previously reported a clinical trial on the intravenous injection of autologous activated macrophages (AAM) in 15 patients with renal carcinoma. The present paper concerns scintigraphic investigations performed in 11 of these patients after injection of 111indium oxinate-radiolabeled AAM. METHODS: AAM were prepared from mononuclear cells (MNC) collected by apheresis from patients treated simultaneously with granulocyte-macrophage colony-stimulating factor (GM-CSF). MNC were cultured for 6 days in the presence of GM-CSF and exposed for 18 h to gamma-interferon, the AAM were then separated by elutriation and injected. RESULTS: After intravenous infusion, radiolabeled AAM were transiently retained in the lungs, where they predominated in the first hour. Later on, radioactivity accumulated in liver and spleen and then decreased from the first and second day, respectively. In one patient, two foci of radioactivity were detected in the lungs 1 h after injection, and persisted thereafter. Their association with tumor lesions was uncertain. This observation possibly resulted from the presence of granulocytes in the radiolabeled AAM populations of this patient. It seems that MNC collected from GM-CSF-treated patients and cultured in the presence of GM-CSF enables the differentiation of granulocytes. CONCLUSIONS: A series of 11 investigations confirms the previously reported distribution pattern of intravenously injected AAM. It is possible that in patients treated with hematopoietic cell-mobilizing agents, granulocytes develop in cultures designed to produce monocyte-derived antigen-presenting cells.

Comparison of Tc-99m-labelled antileukocyte fragment Fab' and Tc-99m-HMPAO leukocyte scintigraphy in the diagnosis of bone and joint infections: a prospective study.

Between January and July 1998, we conducted a prospective study to compare Tc-99m-labelled antigranulocyte monoclonal antibody fragment Fab' (LEUKOSCAN) scintigraphy versus Tc-99m-hexamethylpropyleneamine oxime (Tc-99m-HMPAO)-labelled leukocyte scintigraphy (HMPAO-LS) for the diagnosis of unselected patients with bone and joint infection. Twenty-three patients (16 men and 7 women; mean age, 67 years) with suspected bone infection were explored successively with bone scintigraphy, HMPAO-LS and LEUKOSCAN scintigraphy. Thirty-two foci were studied (diabetic foot = 11, prosthetic material = 8, joint disease = 4, others = diagnosed in 18 cases, eight on the basis of bacteriological and histological examination of surgical or puncture specimens, with or without radiographic signs, and 10 on the basis of clinical course and radiographic findings. Overall sensitivity, specificity and accuracy were 86%, 72% and 78%, respectively, for LEUKOSCAN scintigraphy (12 true positives (TP), 13 true negatives (TN), 5 false positives (FP), 2 false negatives (FN)), 93%, 100% and 96%, respectively, for HMPAO-LS (13TP, 18TN, 0FP, 1FN), and 100%, 17% and 53.3%, respectively, for bone scintigraphy. In this small series, LEUKOSCAN scintigraphy was found to be less specific for the diagnosis of osteomyelitis than HMPAO-LS. In addition, the interpretation of LEUKOSCAN scintigraphy is more difficult than HMPAO-LS for the diagnosis of bone infection in the diabetic foot, and would appear to be less discriminating for differentiating soft tissue infection from osteitis in the case of plantar perforating ulcers.

A novel A10E homozygous mutation in the HSD3B2 gene causing severe salt-wasting 3beta-hydroxysteroid dehydrogenase deficiency in 46,XX and 46,XY French-Canadians: evaluation of gonadal function after puberty.

Severe 3beta-hydroxysteroid dehydrogenase (3betaHSD) deficiency is a rare form of congenital adrenal hyperplasia resulting from mutations in the HSD3B2 gene that impair steroidogenesis in both the adrenals and gonads and cause salt-wasting in both sexes and incomplete masculinization of the external genitalia in genetic males. About two thirds of the reported patients are 46,XY. We describe two French-Canadian patients from two families without a known relationship who presented with severe salt-wasting 3betaHSD deficiency in infancy. Although the diagnosis was considered clinically, plasma steroid profiles were confusing. We have thus directly sequenced DNA fragments generated by PCR amplification of the four exons, exon-intron boundaries, and the 5'-flanking regions of the HSD3B2 gene. Sequencing of exon II revealed the presence of a C to A transversion in both alleles of these two cases, thus converting codon 10 (GCA), which codes for Ala, into GAA, encoding Glu. This Ala is highly conserved in the vertebrate 3betaHSD gene family and is located in the putative NAD-binding domain of the enzyme. The mutant type II 3betaHSD enzyme carrying an A10E substitution exhibited no detectable activity in intact transfected Ad293 cells. Both homozygous patients share the same haplotype, spanning approximately 3.3 centimorgans surrounding the HSD3B2 locus, which is consistent with a founder effect for this missense mutation. The 46,XY patient presented with ambiguous genitalia at birth and underwent normal masculinization at puberty, but was azoospermic at 18.5 yr of age. The 46,XX patient presented progressive breast development, menarche, and evidence of progesterone secretion. The only previously reported cases with pubertal follow-up revealed paternity in one male and hypogonadism in one female. These findings demonstrate the complex relationships between the genotype and the gonadal phenotype in severe 3betaHSD deficiency and the difficulty in predicting fertility.

Intratumoral consumption of indium-111-labeled platelets in a child with splenic hemangioma and thrombocytopenia.

The authors report Kasabach-Merritt syndrome (KMS) in a patient with thrombocytopenia and splenic hemangioma. A 13-month-old boy with a history of anemia, thrombocytopenia, and abdominal mass was admitted to the hospital. The scintigraphic studies showed that a large mass contiguous to the spleen was responsible for the platelet uptake. After partial splenectomy, the platelet count returned to normal. This report of KMS in a child with splenic hemangioma suggests that the scintigraphic studies are mandatory to confirm diagnosis. Indium-111-labeled platelets are useful in identifying hemangiomatous sequestration of platelets in patients with thrombocytopenia.

Contribution of 99Tcm-DMSA scintigraphy to aetiological diagnosis in renal transplant recipients with impaired renal function.

Routine 99Tcm-dimercaptosuccinic acid (DMSA) scintigraphy was performed in a series of 24 kidney transplant recipients with impaired renal function. Diagnostic findings on planar and tomoscintigraphic acquisitions obtained 3 and 4 h after the injection of 130-140 MBq 99Tcm-DMSA were compared with the diagnosis established by fine-needle biopsy in 13 patients and by clinical course and other examinations (ultrasonography, bacteriology) in 11 patients. Renal scintigraphy demonstrated segmental defects in patients with rejection (n = 2/6), immunosuppressor nephrotoxicity (n = 2/6), acute pyelonephritis (n = 3/3), renal artery stenosis (n = 1/1) and obstructive lymphocele (n = 1/1). Diffuse lack of uptake was observed in one patient with severe renal failure. The scintigram was normal in 14 patients, including three with lesions histologically compatible with graft rejection or immunosuppressor nephrotoxicity. 99Tcm-DMSA was thus found to contribute little to the differential diagnosis between graft rejection and immunosuppressor nephrotoxicity. However, it may be useful for identifying specific disease states, particularly acute pyelonephritis, seen as well-delimited systematized defects. 99Tcm-DMSA scintigraphy could also be used in late follow-up after pyelonephritis in renal transplant recipients.

Treatment of metastatic renal cell carcinoma with activated autologous macrophages and granulocyte--macrophage colony-stimulating factor.

Fifteen patients with progressive metastatic renal cell carcinoma were treated with granulocyte-macrophage colony-stimulating factor and intravenous infusions of activated autologous macrophages (AAMs). The latter were prepared from leukapheresis-separated mononuclear cells cultured in the presence of granulocyte-macrophage colony-stimulating factor, exposed to gamma interferon, and submitted to elutriation to separate AAMs. Three intravenous injections of AAMs were performed within a 2-week interval. This treatment cycle was repeated once or twice, in cases of tumor response or stabilization. Ninety-seven preparations containing a mean 3 x 10(9) AAMs were administered and usually well tolerated. One partial response, eight stabilizations and six progressions were observed. The median time to progression and median overall survival time after inclusion were 7 and 9 months, respectively. The cells injected did not accumulate substantially in tumor lesions, as shown by scintigraphic imaging of indium-111-labeled AAMs. Thus, combined granulocyte-macrophage colony-stimulating factor and AAM treatment was well tolerated and resulted in transitory stabilization (n = 8) or partial regression (n = 1) in 9 of 15 patients.

A new insight into the molecular basis of 3beta-hydroxysteroid dehydrogenase deficiency.

Classical 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD) deficiency is a rare form of congenital adrenal hyperplasia that impairs steroidogenesis in both the adrenals and gonads resulting from mutations in the HSD3B2 gene, causing varying degrees of salt-loss in both sexes and incomplete masculinization of the external genitalia in genetic males. To date a total of 34 mutations (including 5 frameshift, 4 nonsense, 1 in-frame deletion, 1 splicing and 23 missense mutations) have been identified in the HSD3B2 gene. Results from functional charaterization studies of the mutant proteins agrees with the prediction that no functional type II 3beta-HSD isoenzyme is expressed in the adrenals and gonads of the patients with the severe salt-losing form, whereas the nonsalt-losing form causes an incomplete loss in enzymatic activity, thereby leaving sufficient enzymatic activity to prevent salt loss. Recent studies have highlighted the fact that various mutations appear to have a drastic effect upon the stability of the protein, therefore providing molecular evidence of a new mechanism involved in classical 3beta-HSD deficiency. Finally, the functional characterization of the missense mutations known to be involved in this autosomal recessive disorder provides valuable information concerning the structure-function relationships of the 3beta-HSD enzyme superfamily.

BRCA1 can stimulate gene transcription by a unique mechanism.

Most familial breast and ovarian cancers have been linked to mutations in the BRCA1 gene. BRCA1 has been shown to affect gene transcription but how it does so remains elusive. Here we show that BRCA1 can stimulate transcription without the requirement for a DNA-tethering function in mammalian and yeast cells. Furthermore, the BRCA1 C-terminal region can stimulate transcription of the p53-responsive promoter, MDM2. Unlike many enhancer-specific activators, non-tethered BRCA1 does not require a functional TATA element to stimulate transcription. Our results suggest that BRCA1 can enhance transcription by a function additional to recruiting the transcriptional machinery to a targeted gene.

New insight into the molecular basis of 3beta-hydroxysteroid dehydrogenase deficiency: identification of eight mutations in the HSD3B2 gene eleven patients from seven new families and comparison of the functional properties of twenty-five mutant enzymes.

Classical 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3betaHSD) deficiency is a form of congenital adrenal hyperplasia that impairs steroidogenesis in both the adrenals and gonads resulting from mutations in the HSD3B2 gene and causing various degrees of salt-wasting in both sexes and incomplete masculinization of the external genitalia in genetic males. To identify the molecular lesion(s) in the HSD3B2 gene in the 11 patients from the seven new families suffering from classical 3betaHSD deficiency, the complete nucleotide sequence of the whole coding region and exon-intron splicing boundaries of this gene was determined by direct sequencing. Five of these families were referred to Morel's molecular diagnostics laboratory in France, whereas the two other families were investigated by Peter's group in Germany. Functional characterization studies were performed by Simard's group in Canada. Following transient expression in 293 cells of each of the mutant recombinant proteins generated by site-directed mutagenesis, the effect of the 25 mutations on enzyme activity was assessed by incubating intact cells in culture with 10 nM [14C]-DHEA as substrate. The stability of the mutant proteins has been investigated using a combination of Northern and Western blot analyses, as well as an in vitro transcription/translation assay using rabbit reticulocyte lysates. The present report describes the identification of 8 mutations, in seven new families with individuals suffering from classical 3betaHSD deficiency, thus increasing the number of known HSD3B2 mutations involved in this autosomal recessive disorder to 31 (1 splicing, 1 in-frame deletion, 3 nonsense, 4 frameshift and 22 missense mutations). In addition to the mutations reported here in these new families, we have also investigated for the first time the functional significance of previously reported missense mutations and or sequence variants namely, A82T, A167V, L173R, L205P, S213G and K216E, P222H, T259M, and T259R, which have not previously been functionally characterized. Furthermore, their effects have been compared with those of the 10 previously reported mutant enzymes to provide a more consistent and comprehensive study. The present results are in accordance with the prediction that no functional 3betaHSD type 2 isoenzyme is expressed in the adrenals and gonads of the patients suffering from a severe salt-wasting form of CAH due to classical 3betaHSD deficiency. Whereas the nonsalt-losing form also results from missense mutation(s) in the HSD3B2 gene, which cause an incomplete loss in enzyme activity, thus leaving sufficient enzymatic activity to prevent salt wasting. The functional data described in the present study concerning the sequence variants A167V, S213G, K216E and L236S, which were detected with premature pubarche or hyperandrogenic adolescent girls suspected to be affected from nonclassical 3betaHSD deficiency, coupled with the previous studies reporting that no mutations were found in both HSD3B1 and/or HSD3B2 genes in such patients strongly support the conclusion that this disorder does not result from a mutant 3betaHSD isoenzyme. The present study provides biochemical evidence supporting the involvement of a new molecular mechanism in classical 3betaHSD deficiency involving protein instability and further illustrates the complexity of the genotype-phenotype relationships of this disease, in addition to providing further valuable information concerning the structure-function relationships of the 3betaHSD superfamily.