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The synthesis of active pharmaceutical ingredients (APIs) is commonly perceived as more efficient when performed using continuous-flow methods, whereas batch processes are often seen as less favorable due to their limitations in yield, heat and mass transfer, and safety. This perception largely stems from existing studies that focus on green metrics such as the E-factor and yield. However, a comprehensive comparison of batch and flow processes through full techno-economic analyses (TEA) and life-cycle assessments (LCA) remains underexplored, leaving key aspects of their environmental and economic impacts inadequately assessed. This work addresses this gap by presenting a detailed comparison of batch and flow syntheses of seven industrially relevant APIs, including amitriptyline hydrochloride, tamoxifen, zolpidem, rufinamide, artesunate, ibuprofen, and phenibut. Eleven environmental impact categories within the framework of nine planetary boundaries were assessed, and the study also included an evaluation of capital and operating costs for both production methods. The results demonstrated that, on average, continuous-flow processes are significantly more sustainable with improvements in energy efficiency, water consumption, and waste reduction. Flow processes also show a marked reduction in carbon emissions and up to a 97% reduction in energy consumption, highlighting their potential for greener API manufacturing. Despite these advantages, the study identified areas where the continuous-flow technology requires further development. Specifically, manufacturing certain APIs in flow show lower-than-average improvements in operating expenditure and land system changes, the latter being directly correlated with the consumption of organic solvents, that can be comparable to or even higher than in batch. These challenges highlight the need for further optimization of flow processes to fully realize their potential in API production., Competing Interests: The authors declare no competing financial interest., (© 2025 The Authors. Published by American Chemical Society.)

The bacterial envelope is one of the oldest and most essential cellular components and has been traditionally divided into Gram-positive (monoderm) and Gram-negative (diderm). Recent landmark studies have challenged a major paradigm in microbiology by inferring that the last bacterial common ancestor had a diderm envelope and that the outer membrane (OM) was lost repeatedly in evolution to give rise to monoderms. Intriguingly, OM losses appear to have occurred exclusively in the Terrabacteria, one of the two major clades of bacteria. In this Review, we present current knowledge about the Terrabacteria. We describe their diversity and phylogeny and then highlight the vast phenotypic diversity of the Terrabacteria cell envelopes, which display large deviations from the textbook examples of diderms and monoderms, challenging the classical Gram-positive-Gram-negative divide. We highlight the striking differences in the systems involved in OM biogenesis in Terrabacteria with respect to the classical diderm experimental models and how they provide novel insights into the diversity and biogenesis of the bacterial cell envelope. We also discuss the potential evolutionary steps that might have led to the multiple losses of the OM and speculate on how the very first OM might have emerged before the last bacterial common ancestor., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. Springer Nature Limited.)

This study presents the first model-based optimal shutdown procedure of a multistage continuous crystallization process which aims at the maximization of on-spec production and minimization of the shutdown time. The cooling antisolvent crystallization of Aspirin (acetylsalicylic acid) in a three-stage continuous crystallizer was used as a case study. To address the optimal shutdown problem, several single optimization scenarios were considered to assess the impact of the degrees of freedom, discretization schemes, and optimization settings such as the constraints. The proposed optimal shutdown procedures showed that significant amounts of on-spec crystals can be produced both at fixed and variable shutdown times. Most importantly, the optimal shutdown procedures can match the steady-state productivity, based on the shutdown to steady-state productivity ratio (STSPR) which can easily reach 100%. Moreover, the residual shutdown material, considered as waste, can be dramatically reduced by >80% compared to the current standard shutdown procedures. Given the conflicting nature of the maximization of on-spec production and minimization of the shutdown time, multiobjective optimization of the shutdown operation was also addressed to identify the set of Pareto optimal solutions. Finally, a multicriteria decision-aiding method, based on multiattribute utility theory, was proposed to rank the Pareto optimal solutions to support the decision-making and help identify a suitable and feasible single optimal shutdown solution., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

A simple model is developed for membrane fouling, taking into account two main fouling phenomena: cake formation, due to attached solids on the membrane surface, and pore clogging, due to retained compounds inside the pores. The model is coupled with a simple anaerobic digestion model for describing the dynamics of an anaerobic membrane bioreactor (AnMBR). In simulations, we investigate its qualitative behavior: it is shown that the model exhibits satisfying properties in terms of a flux decrease due to membrane fouling. Comparing simulation and experimental data, the model is shown to predict quite well the dynamics of an AnMBR. The simulated flux best fits the experimental flux with a correlation coefficient r2=0.968 for the calibration data set and r2=0.938 for the validation data set. General discussions are given on possible control strategies to limit fouling and optimize the flux production. We show in simulations that these strategies allow one to increase the mean production flux to 33 L/(h·m 2 ),whereas without control, it was 18 L/(h·m 2 ).

E. coli and most other diderm bacteria (those with two membranes) have an inner membrane enriched in glycerophospholipids (GPLs) and an asymmetric outer membrane (OM) containing GPLs in its inner leaflet and primarily lipopolysaccharides in its outer leaflet. In E. coli, this lipid asymmetry is maintained by the Mla system which consists of six proteins: the OM lipoprotein MlaA extracts GPLs from the outer leaflet, and the periplasmic chaperone MlaC transfers them across the periplasm to the inner membrane complex MlaBDEF. However, GPL trafficking still remains poorly understood, and has only been studied in a handful of model species. Here, we investigate GPL trafficking in Veillonella parvula, a diderm Firmicute with an Mla system that lacks MlaA and MlaC, but contains an elongated MlaD. V. parvula mla mutants display phenotypes characteristic of disrupted lipid asymmetry which can be suppressed by mutations in tamB, supporting that these two systems have opposite GPL trafficking functions across diverse bacterial lineages. Structural modelling and subcellular localisation assays suggest that V. parvula MlaD forms a transenvelope bridge, comprising a typical inner membrane-localised MCE domain and, in addition, an outer membrane ß-barrel. Phylogenomic analyses indicate that this elongated MlaD type is widely distributed across diderm bacteria and likely forms part of the ancestral functional core of the Mla system, which would be composed of MlaEFD only., (© 2023. The Author(s).)

Skeletal class III malocclusion is characterized by an evident disharmony and discrepancy between the maxilla and mandible, affecting facial appearance and masticatory function. Usually, the association of transversal and vertical anomalies along with the anteroposterior problem makes the orthodontist's job perceived as a challenge. The ortho-surgical approach can be considered as an efficient alternative for the treatment of skeletal class III malocclusion, and it requires an interdisciplinary relationship between the orthodontist and a maxillofacial surgeon. This case report presents a 25-year-old female patient who consulted with a chief complaint of unsatisfactory facial esthetics. The clinical examination revealed a skeletal class III mainly due to maxilla hypoplasia accompanied with mandibular laterognathism, causing a unilateral posterior crossbite. The patient was treated with the ortho-surgical approach. The treatment objectives were achieved, providing a stable class I occlusion with an acceptable overjet and overbite, coordinated midlines, and a satisfactory facial profile with an excellent esthetic balance between the hard and soft tissue., Competing Interests: The authors have no conflict of interests to declare., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

The objective of the research was to improve the process design of a combined antisolvent-cooling crystallization to reduce the degree of agglomeration of a real active pharmaceutical ingredient product, which was manufactured using a crystallization stage employing a methanol/water solvent system. Knowledge was gained from the use of process analytical technology (PAT) tools to monitor the process variables, allowing particle size, degree of agglomeration, solute concentration, and supersaturation to be tracked throughout the process. Based on knowledge of the solubility behavior and interpretation of the PAT histories, changes were made to the sequences of antisolvent addition and cooling within the crystallization process to reduce agglomeration in the final product. Different seed loadings and seeding addition points were also investigated to maintain operation within lower supersaturation regions of the phase diagram to limit agglomeration and avoid an undesired polymorphic transformation to an unstable form. The improved sequences of operations and seeding conditions did not provide sufficient improvement in the product quality and so were augmented by applying wet milling for further deagglomeration followed by temperature cycling to remove fine particles generated during milling. Open-loop heating and cooling cycles produced some limited improvements, whereas closed-loop direct nucleation control methods using FBRM as a feedback sensor for particle counts per second were much more successful at producing high-quality crystals of the desired polymorphic form. The work shows that understanding the trajectory of the process through the phase diagram to follow appropriate supersaturation profiles gives improved control of the various kinetic mechanisms and can be used to improve the quality of the final product., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

This paper deals with a mathematical analysis of two-steps model of anaerobic digestion process, including dynamics of soluble microbial products (SMP). We propose to investigate effects of the new variable SMP on qualitative properties of the process in different generic cases. Equilibria of the model are graphically established considering qualitative properties of the kinetics and, their stability are proved theoretically and/or verified by numerical simulations. It will shown that the model has a rich qualitative behavior as equilibria bifurcation and multi-stability according to the considered bifurcation parameter.

Novel cost effective, versatile, reconfigurable, reusable and easy to assemble glass capillary microfluidic devices were developed and used to generate micro/nano-materials with controlled size and morphology. The devices are composed of coaxial assemblies of glass capillaries held between two interchangeable plastic blocks fabricated from chemically inert polyoxymethylene copolymer using computer numerical control (CNC) machining. Three different blocks were combined and locked together using Lego® inspired stud-and-hole coupling system to achieve different flow configurations. The device allows a truly axisymmetric round capillary inside a round capillary geometry and self-alignment of capillaries. The synthesis of polyvinylpyrrolidone capped gold nanoparticles and liposomes of controlled size was demonstrated in the co-flow device by mixing the contents of two parallel laminar streams. The flow focusing device was used to generate piroxicam monohydrate crystals of controlled size (10-29 μm) by antisolvent crystallisation. Silver nanoparticles with tailored size (40-90 nm) were prepared in the three-phase device by merging silver nitrate and tannic acid/citrate streams inside droplets. The same device was used to prepare fluorescently labelled double emulsion droplets with controlled number of inner droplets. The droplet morphology was modified and tuned during operation by adjusting the distance between the inner capillaries. Water-in-oil emulsions consisted of Eudragit S100 solution at pH > 7 dispersed in Miglyol® 840 were prepared and gellified in situ over 6 h without fouling. The setup time of the novel devices was reduced from ∼30 min in manually made capillary devices to just several minutes., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

L-Asparaginase (ASNase) is a vital component of the first line treatment of acute lymphoblastic leukemia (ALL), an aggressive type of blood cancer expected to afflict over 53,000 people worldwide by 2020. More recently, ASNase has also been shown to have potential for preventing metastasis from solid tumors. The ASNase treatment is, however, characterized by a plethora of potential side effects, ranging from immune reactions to severe toxicity. Consequently, in accordance with Quality-by-Design (QbD) principles, ingenious new products tailored to minimize adverse reactions while increasing patient survival have been devised. In the following pages, the reader is invited for a brief discussion on the most recent developments in this field. Firstly, the review presents an outline of the recent improvements on the manufacturing and formulation processes, which can severely influence important aspects of the product quality profile, such as contamination, aggregation and enzymatic activity. Following, the most recent advances in protein engineering applied to the development of biobetter ASNases (i.e., with reduced glutaminase activity, proteolysis resistant and less immunogenic) using techniques such as site-directed mutagenesis, molecular dynamics, PEGylation, PASylation and bioconjugation are discussed. Afterwards, the attention is shifted toward nanomedicine including technologies such as encapsulation and immobilization, which aim at improving ASNase pharmacokinetics. Besides discussing the results of the most innovative and representative academic research, the review provides an overview of the products already available on the market or in the latest stages of development. With this, the review is intended to provide a solid background for the current product development and underpin the discussions on the target quality profile of future ASNase-based pharmaceuticals.

Objectives: Older cognitively impaired adults present a higher risk of hospitalisation and mortality following a visit to the emergency department (ED). Better understanding of avoidable incidents is needed to prevent them and the associated ED presentations in community-dwelling adults. This study aimed to synthetise the actual knowledge concerning these incidents leading this population to ED presentation, as well as possible preventive measures to reduce them., Design: A scoping review was performed according to the Arksey and O'Malley framework., Methods: Scientific and grey literature published between 1996 and 2017 were examined in databases (Medline, Cumulative Index of Nursing and Allied Health, Ageline, Scopus, ProQuest Dissertations/theses, Evidence-based medecine (EBM) Reviews, Healthstar), online library catalogues, governmental websites and published statistics. Sources discussing avoidable incidents leading to ED presentations were included and then extended to those discussing hospitalisation and mortality due to a lack of sources. Data (type, frequency, severity and circumstances of incidents, preventive measures) was extracted using a thematic chart, then analysed with content analysis., Results: 67 sources were included in this scoping review. Five types of avoidable incidents (falls, burns, transport accidents, harm due to self-negligence and due to wandering) emerged, and all but transport accidents were more frequent in cognitively impaired seniors. Differences regarding circumstances were only reported for burns, as scalding was the most prevalent mechanism of injury for this population compared with flames for the general senior population. Multifactorial interventions and implications of other professionals (eg, pharmacist, firefighters) were reported as potential interventions to reduce avoidable incidents. However, few preventive measures were specifically tested in this population., Conclusions: Primary research that screens for cognitive impairment and involves actors (eg, paramedics) to improve our understanding of avoidable incidents leading to ED visits is greatly needed. This knowledge is essential to develop preventive measures tailored to the needs of older cognitively impaired adults., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

An Anaerobic Membrane BioReactors (AnMBR) model is presented in this paper based on the combination of a simple fouling model and the Anaerobic Model 2b (AM2b) to describe biological and membrane dynamic responses in an AnMBR. In order to enhance the model calibration and validation, Trans-Membrane Pressure (TMP), Total Suspended Solid (TSS), COD, Volatile Fatty Acid (VFA) and methane production were measured. The model shows a satisfactory description of the experimental data with R 2 ≈0.9 for TMP data and R 2 ≈0.99 for biological parameters. This new model is also proposed as a numerical tool to predict the deposit mass composition of suspended solid and Soluble Microbial Products (SMP) on the membrane surface. The effect of SMP deposit on the TMP jump phenomenon is highlighted. This new approach offers interesting perspectives for fouling prediction and the on-line control of an AnMBR process., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Current studies show the relevance of geriatric prevention and rehabilitation programs to slow down the development of disability in community-dwelling older adults who are becoming frail. This evidence reveals the importance of improving knowledge on how individual components of frailty and specific disability in basic and instrumental activities of daily living (ADL) are related, to offer early, targeted, and tailored interventions. The objective was to examine the association between each of the five frailty phenotype components (weakness, slowness, exhaustion, low physical activity, weight loss) and disability in specific ADL pertaining to physical aspects (bathing, dressing, cutting toe nails, transportation, shopping, housekeeping, food purchasing, food preparation) and cognitive aspects (finances, telephone, medication). A cross-sectional design involving 1643 community-dwelling older adults (65+) from the longitudinal multi-center FRéLE study was used. Disability was defined as needing help or being unable to perform specific ADL. Multiple logistic regressions were adjusted for socio-demographic characteristics, clinical variables, and for 4 other frailty components. Results showed that low physical activity and slowness were significantly linked to disability in all physical and cognitive aspects of ADL (OR: 1.71-9.42; p<0.05), except using the telephone. Notably, all frailty components except weight loss were associated with disability in the physical aspects of instrumental ADL (transportation, shopping, housekeeping, food purchasing, food preparation) (OR: 1.73-9.42; p<0.05). This study helped identify the relevant frailty components as targets in community-based prevention and rehabilitation programs. Easily imbedded interventions in daily routines should be promoted earlier in the frailty process to delay or reduce disability., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A series of tubes: The continuous manufacture of a finished drug product starting from chemical intermediates is reported. The continuous pilot-scale plant used a novel route that incorporated many advantages of continuous-flow processes to produce active pharmaceutical ingredients and the drug product in one integrated system., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

JAK2(V617F) is the predominant mutation in myeloproliferative neoplasms (MPN). Modeling MPN in a human context might be helpful for the screening of molecules targeting JAK2 and its intracellular signaling. We describe here the derivation of induced pluripotent stem (iPS) cell lines from 2 polycythemia vera patients carrying a heterozygous and a homozygous mutated JAK2(V617F), respectively. In the patient with homozygous JAK2(V617F), additional ASXL1 mutation and chromosome 20 allowed partial delineation of the clonal architecture and assignation of the cellular origin of the derived iPS cell lines. The marked difference in the response to erythropoietin (EPO) between homozygous and heterozygous cell lines correlated with the constitutive activation level of signaling pathways. Strikingly, heterozygous iPS cells showed thrombopoietin (TPO)-independent formation of megakaryocytic colonies, but not EPO-independent erythroid colony formation. JAK2, PI3K and HSP90 inhibitors were able to block spontaneous and EPO-induced growth of erythroid colonies from GPA(+)CD41(+) cells derived from iPS cells. Altogether, this study brings the proof of concept that iPS can be used for studying MPN pathogenesis, clonal architecture, and drug efficacy.

Background: Maternally derived duplications of the 15q11-q13 region are the most frequently reported chromosomal aberrations in autism spectrum disorders (ASD). Prader-Willi and Angelman syndromes, caused by 15q11-q13 deletions or abnormal methylation of imprinted genes, are also associated with ASD. However, the prevalence of these disorders in ASD is unknown. The aim of this study was to assess the frequency of 15q11-q13 rearrangements in a large sample of patients ascertained for ASD., Methods: A total of 522 patients belonging to 430 families were screened for deletions, duplications, and methylation abnormalities involving 15q11-q13 with multiplex ligation-dependent probe amplification (MLPA)., Results: We identified four patients with 15q11-q13 abnormalities: a supernumerary chromosome 15, a paternal interstitial duplication, and two subjects with Angelman syndrome, one with a maternal deletion and the other with a paternal uniparental disomy., Conclusions: Our results show that abnormalities of the 15q11-q13 region are a significant cause of ASD, accounting for approximately 1% of cases. Maternal interstitial 15q11-q13 duplications, previously reported to be present in 1% of patients with ASD, were not detected in our sample. Although paternal duplications of chromosome 15 remain phenotypically silent in the majority of patients, they can give rise to developmental delay and ASD in some subjects, suggesting that paternally expressed genes in this region can contribute to ASD, albeit with reduced penetrance compared with maternal duplications. These findings indicate that patients with ASD should be routinely screened for 15q genomic imbalances and methylation abnormalities and that MLPA is a reliable, rapid, and cost-effective method to perform this screening.

Objectives: We undertook this study to determine the prevalence of hypovitaminosis D, its determinants, and its relationships to physical performance, serum parathyroid hormone (PTH) concentration, bone mineral density, and biochemical markers of bone turnover in healthy, ambulatory, pre- and postmenopausal women., Methods: The group studied included 415 women aged 24 to 77 years. Between July and September, we assessed calcium intake and measured serum calcium, phosphorus, albumin, alkaline phosphate, 25-hydroxyvitamin D (25(OH)D), PTH, osteocalcin, and C-terminal cross-linking telopeptide of Type I collagen. We also measured bone mineral density (BMD) by dual-energy radiograph absorptiometry in the spine and total femur. Three tests were used to assess physical performance: timed get-up-and-go test, 5-times-sit-to-stand test, and 2.4 m speed walk., Results: The prevalence of vitamin D insufficiency (<30 ng/mL) was 91%. In multiple logistic regression, the main determinants of hypovitaminosis D were age >55 years (OR 2.14 [95% IC, 1.1-4.1; P = 0.026)], wearing a veil [OR 2 (95% IC, 1.1-4; P = 0.04)], time spent outdoors less than 30 min/d [OR 2.8 (95% IC: 1.4-5.7; P = 0.003)], and daily calcium intake less than 700 mg [OR 2.39 (95% IC, 1.2-4.7; P < 0.01)]. A significant inverse correlation between 25 OH and osteocalcin (r = -0,18, P < 0001), 25 OH, and Type I collagen (r = -0,15, P = 0003) were observed. By Locally Weighted Regression and Scatterplot Smoothing technique, there was an increase in PTH level when S-25(OH)D was below 30 ng/mL. After adjustment for age, both spine BMD and total femoral BMD failed to show any significant correlation with serum 25(OH)D and PTH. There was no correlation between any physical performance tests and 25(OH)D levels., Conclusions: Our study showed that during the summer season, vitamin D insufficiency is very common in healthy adult Moroccan women. Lack of sun exposure and veiled clothing style were the most important factors that influenced hypovitaminosis D. Patients with hypovitaminosis D had a high bone turnover, whereas there was no effect on BMD and physical performance. Further research is needed to evaluate the clinical impact of the above findings.

The nutcracker syndrome refers to compression of the left renal vein between the aorta and the superior mesenteric artery. After a review of the literature, the authors emphasize the uncommon incidence of this pathology, and the interest of imaging in the survey of this affection.

Dravet syndrome (DS) is a genetically determined epileptic encephalopathy mainly caused by de novo mutations in the SCN1A gene. Since 2003, we have performed molecular analyses in a large series of patients with DS, 27% of whom were negative for mutations or rearrangements in SCN1A. In order to identify new genes responsible for the disorder in the SCN1A-negative patients, 41 probands were screened for micro-rearrangements with Illumina high-density SNP microarrays. A hemizygous deletion on chromosome Xq22.1, encompassing the PCDH19 gene, was found in one male patient. To confirm that PCDH19 is responsible for a Dravet-like syndrome, we sequenced its coding region in 73 additional SCN1A-negative patients. Nine different point mutations (four missense and five truncating mutations) were identified in 11 unrelated female patients. In addition, we demonstrated that the fibroblasts of our male patient were mosaic for the PCDH19 deletion. Patients with PCDH19 and SCN1A mutations had very similar clinical features including the association of early febrile and afebrile seizures, seizures occurring in clusters, developmental and language delays, behavioural disturbances, and cognitive regression. There were, however, slight but constant differences in the evolution of the patients, including fewer polymorphic seizures (in particular rare myoclonic jerks and atypical absences) in those with PCDH19 mutations. These results suggest that PCDH19 plays a major role in epileptic encephalopathies, with a clinical spectrum overlapping that of DS. This disorder mainly affects females. The identification of an affected mosaic male strongly supports the hypothesis that cellular interference is the pathogenic mechanism., Competing Interests: The authors have declared that no competing interests exist.

Chromosomal rearrangements are found in a subset of patients with autism. Duplications involving loci associated with behavioural disturbances constitute an especially good candidate mechanism. The Williams-Beuren critical region (WBCR), located at 7q11.23, is commonly deleted in Williams-Beuren microdeletion syndrome (WBS). However, only four patients with a duplication of the WBCR have been reported to date. Here, 206 patients with autism spectrum disorders were screened for the WBCR duplication by quantitative microsatellite analysis and multiple ligation-dependent probe amplification. One male patient with a de novo interstitial duplication of the entire WBCR of paternal origin was identified. The patient had autistic disorder, severe language delay and mental retardation, with mild dysmorphism. The present report concerns the first patient with autistic disorder and a WBCR duplication. This observation indicates that the 7q11.23 duplication could be involved in complex clinical phenotypes, ranging from developmental or language delay to mental retardation and autism.

Posterior-reversible encephalopathy syndrome (PRES) is a recently clinicoradiologic entity caused by numerous medical conditions and characterized by acute-neurologic disorders, such as headaches, confusion, seizures associated with arterial hypertension. MRI characteristics are typical. The rapid diagnosis is of capital importance due to a potential reversibility.

Unlabelled: Factor VII deficiency (or hypoproconvertinemia) is a rare inherited bleeding disorder that can cause hemarthrosis similar to that seen in hemophilia. We report a case., Case Report: A 28-year-old woman experienced recurrent spontaneous hemarthrosis in both elbows and one knee starting at 2 years of age. She sought advice for an episode of bleeding in the left knee. The prothrombin level was decreased to 15% and the activated partial thromboplastin time was normal. Radiographs disclosed advanced joint destruction in the right shoulder, both elbows, the left knee, and both ankles. She described similar joint symptoms in two cousins. Plasma factor VII was less than 10%. A diagnosis of arthropathy due to an inherited bleeding disorder was given. Triamcinolone hexacetonide was injected into the joint; fresh frozen plasma was given concomitantly as an intravenous infusion., Discussion: Factor VII deficiency is an extremely uncommon bleeding disorder with an estimated prevalence of 1/300,000 to 1/500,000. Bleeding occurs only in homozygotes whose factor VII level is less than 20%. Hemarthrosis is less common than hemophilia, although the characteristics of joint destruction are similar in the two conditions.

About half of the patients with paraneoplastic diseases develop an immune response against neuronal antigens expressed by both the tumor and the nervous system. In 31 patients with anti-Hu antibodies and 19 patients with anti-Yo antibodies, we searched for the presence of additional non-neuronal auto-antibodies and further studied whether the presence of such auto-antibodies was correlated with a specific oncological or neurological presentation. Positive antinuclear antibodies (ANA) were found at a titer of 1:40 or above in 48% patients with anti-Hu antibodies, and in 37% patients with anti-Yo antibodies. Anti-cytoplasmic antibodies were also detected in 42% patients with anti-Yo antibodies. No specific correlation between the presence of non-neuronal auto-antibodies and clinical characteristics of the patients could be identified. In particular, neither the type of underlying cancer, the overall survival, the tumor response to treatment, nor the neurological presentation were related to the serological status.

The precise immune mechanisms of neuronal death in anti-Hu-associated paraneoplastic encephalomyelitis (PEM) are unclear. We performed an immunohistochemical study on postmortem brain tissue from 11 patients with anti-Hu-associated PEM to further characterize the immune reaction and to ascertain possible mechanisms of neuronal death. To analyze inflammatory infiltrates, antibodies against lymphocyte subpopulations (CD3, CD20, CD4, CD8), macrophage and activated microglia (CD68), major histocompatibility complex (MHC) classes I and II (HLA-ABC and HLA-DR), and the intercellular adhesion molecules (ICAM) -1 and -3 were used. Cell death mechanisms were defined using antibodies against the cytotoxic protein TIA-1, the C9neo component of complement, the Fas receptor (CD95) and its ligand, the apoptosis effector activated caspase-3, and the apoptosis inhibitor Bcl-2. A great number of T cells expressing the cytotoxic protein TIA-1 was observed, mainly in clusters around neurons. ICAM-1 immunoreactivity was increased in the neuropil and reactive astrocytes in areas of inflammation within the central nervous system and in satellite cells of pathological dorsal root ganglia surrounding apparently normal sensory neurons. By contrast, Fas, FasL, C9neo, and activated caspase-3 immunoreactivities were negative in pathological areas. Bcl-2 immunoreactivity was found in satellite cells, but not in sensory neurons of normal and pathological dorsal root ganglia. Our data point out to an induction of a cytotoxic, non-apoptotic, neuronal death in anti-Hu-associated PEM. The increased ICAM-1 immunoreactivity may favor the infiltration of lymphocytes in the pathological areas.

We reviewed 200 patients with paraneoplastic encephalomyelitis (PEM) and anti-Hu antibodies to show possible clinical differences with respect to previous series, and to identify patient, tumour and treatment-related characteristics associated with neurological disability and survival. The median age of the 200 patients was 63 years (range 28-82 years) and 75% were men. The predominant neurological syndromes were sensory neuropathy (54%), cerebellar ataxia (10%), limbic encephalitis (9%) and multifocal involvement (11%). Sensorimotor neuropathies with predominant motor involvement were observed in only 4% of the patients. Pathological or X-ray evidence of a tumour was obtained in 167 patients (83%) and was a small-cell lung cancer (SCLC) in 74% of those with histological diagnosis. Coexistence of extrathoracic tumours with SCLC was rare (0.5%). Positive Hu immunoreactivity was observed in the extrathoracic tumours of six out of seven patients in whom autopsy or long-term follow-up ruled out a coexisting SCLC. PEM preceded the diagnosis of the tumour in 71% of patients (mean delay +/- SD 6.5 +/- 7.0 months; range 0.1-47 months). In the 24 patients in whom the tumour diagnosis was the initial event, PEM predicted the progression or relapse of the tumour in 87% of them. No tumour was found in 33 patients, including four who had a post-mortem study and four with >5 years of follow-up. In a logistic regression analysis, treatment of the tumour, associated or not with immunotherapy, was an independent predictor of improvement/stabilization of PEM [odds ratio 4.56; 95% confidence interval (CI) 1.62-12.86]. Cox multivariate analysis indicated that the variables independently associated with mortality were: age >60 years [relative risk (RR) 1.49; 95% CI 1.05-2.12], Rankin score at diagnosis >3 (RR 1.60; 95% CI 1.12-2.28), more than one area of the nervous system affected (RR 1.61; 95% CI 1.08-2.40), and absence of treatment (RR 2.56; 95% CI 1.76-3.71). We conclude that, unlike previous series, the majority of our patients were male, and there was a low occurrence of predominantly motor neuropathies and extrathoracic tumours coexisting with SCLC. When the diagnosed extrathoracic tumour expresses Hu antigens, further tests to rule out a coexisting SCLC are probably unnecessary. Finally, the predictors of mortality and PEM evolution found in the study may be important in the design of future therapeutic protocols, and emphasize the importance of early diagnosis and treatment of the underlying tumour.

A 57 year-old woman developed acute limbic encephalitis and brainstem dysfunction. Anti-HU antibodies were repeatedly detected in serum and CSF. Postmortem examination showed necrotic and hemorrhagic lesions in the temporal lobes characteristic of herpes simplex virus encephalitis, which was confirmed by immunocytochemistry, and Purkinje cell loss with proliferation of Bergman glia and myelin loss in the external aspect of the dentate nuclei characteristic of paraneoplastic encephalitis. PCR-assay performed on temporal tissue extracts was positive for HSV-1. There was no identifiable neoplasm. This unusual association raises the possibility of a link between the two diseases.

Paraneoplastic neurological diseases are a group of neurological disorders associated with neoplastic tumors but not due to tumoral extension, metabolic, infectious, vascular or toxic complications of these tumors or their treatment. In the majority of paraneoplastic neurological disorders, circulating autoantibodies directed against neurons have been found in the serum and/or the CSF suggesting, and in some cases implicating, autoimmunity in the pathophysiology of these diseases. The finding of autoimmune phenomena during the course of paraneoplastic neurological disorders is of importance: from a practical point of view, since the detection of anti-neuronal autoantibodies is of great diagnostic help and should lead to the thorough search of the associated tumor often at an early stage of its development; from a theoretical point of view, these disorders represent a peculiar type of molecular mimicry. Tumoral neontigens having structural homology or identity with neuronal autoantigens elicit autoreactivity. The immunological effector mechanisms involved in the pathophysiology of paraneoplastic syndromes appear to differ according to the disease: autoantibodies are pathogenic in Lambert-Eaton syndrome whereas, in paraneoplastic cerebellar degeneration and in the Hu syndrome, the cellular immune response might play a greater role.

A 67-year-old woman was admitted for intestinal pseudoobstruction associated with peripheral sensitive neuropathy. Jejunal biopsies performed during laparotomy, showed axonal degeneration and lympho-plasmocytic infiltration in myenteric plexus. High titer of seric anti-Hu antibodies suggested a paraneoplastic syndrome. Thoracic CT scan showed mediastinal lymph nodes. Their histological examination confirmed the diagnosis of metastatic small-cell lung carcinoma. Her condition gradually deteriorated despite supportive parenteral nutrition, chemotherapy, steroids and intravenous immunoglobulins. She died 12 months after the onset of symptoms.

Vascular thrombosis remains severe complication in patients with nephrotic syndrome. Both venous and arterial thrombosis are observed. We report three new cases of arterial thrombosis in patients with nephrotic syndrome. The role of acquired hemostasis disorders, inducing hypercoagulability, is predominant. Extramembranous glomerulonephritis remains the most frequent cause of nephrotic syndrome, complicated by vascular thrombosis. Treatment is based on anticoagulation and corticosteroid therapy. Search for proteinuria should be part of the etiology work-up in all patients with vascular thrombosis of undetermined origin.

We report a case of a 23-year-old patient admitted for a right femur fracture resulting from a traffic accident. An intra-alveolar haemorrhage occurred 48 hours later, with asphyxia anaemia, haematic bronchial aspirations, and bilateral alveolar opacities at chest X-ray. This symptomatology was associated with fever, sub-conjunctival petechiae, major hypocholesterolemia, deterioration of renal function, and cholestasis. All these features suggested a fat embolism. Other possible aetiologies were discarded because of normal cardiovascular and immunologic systems and absence of infection. The outcome under symptomatic treatment was satisfactory within 15 days. The occurrence of intra-alveolar haemorrhage in post-traumatic fat embolism is a rare event caused by pulmonary capillary obstruction by fat emboli.

Vascular involvement, usually venous thrombosis, is common in Behçet's disease. Arterial manifestations, usually aneurysms or more rarely occlusion, are less common. We analyzed 13 cases of Behçet's disease with arterial complications. This fourth series in the literature was collected over 11 years. There were 12 men and 1 woman, mean age 41 +/- 7 years. Mean delay to arterial complications was 5.8 years (maximum 20 years) after the first sign of the disease. In the 13 patients in our series, there were a total of 18 arterial lesions, including one aneurysm and one stenosis of the internal carotid artery. There were 2 cases (10%) with lesions of the aorta (aneurysms). Approximately half of the arterial lesions (7/18) involved the femoral artery. There were 11 localizations on the femoropopliteal axis (11 aneurysms). One aneurysm of the humeral artery was the only localization in the upper limb. Vascular involvement was limited to arterial lesions in 9/13 patients with multiple lesions at different localizations in 2 patients. In two others, a second arterial localization occurred secondarily. All patients except one were operated. Mid-term complications are: 1 death and 4 thrombi including 3 with good results after reoperation. In these patients with Behçet's disease, risk of severe complications is important, requiring long-term surveillance.

Glomus tumours are relatively uncommon lesions most frequently found in the extremitis, usually in the digits. They are most often solitary lesions but multiple tumours have been reported. If the triad of pain, tenderness and cold intolerance should raise the clinical suspicion of a digital glomus. The histological exam is necessary in the extra digital glomus. We reported a case of fossa poplitea glomangioma with tibialis nerve compression. The treatment of choice was a surgical exision. But the tumour have been redivided a year later without malign transformation.

Arterial complications of hydatic disease are rarely encountered. We report a false hydatic aneurysm of the thoracoabdominal aorta revealed by ischemic embolism of the lower limbs. Surgical treatment included aorto-aortic prosthesis and albendazol for 6 months. Results at 18 months are excellent with negative hydatic serology. In endemic areas, hydatic disease is a possible cause of false aneurysms. Long-term surveillance is required after curative surgery and medical treatment.

Although the range of applications for antisense oligonucleotides is vast, current research concentrates mainly on virology and oncology. We have conducted in vivo and in vitro investigations of radiolabelling and biodistribution of a 22-mer phosphodiester oligonucleotide injected in athymic mice bearing xenograft of human mammary tumor (coculture: MCF7 and fibroblasts strain AF-11). Tumor/healthy tissue ratio of the 22-mer phosphodiester oligonucleotide fixation is high during the 24 hours after injection instead of fast elimination.

The development of biotechnologies offers considerable potential for new therapeutical approaches, targetting nucleic acids as information bearing molecules responsible for various pathologies. Gene therapy has, up to now, mainly aimed at compensating deficiencies of gene expression, through complex molecular and cellular constructions. Recently, an increasing interest has focused on short nucleotidic sequences (thus relatively easy to synthetize), or oligonucleotides, which could be able to specifically block the expression of mutated or overexpressed genes. When the target of these oligonucleotides is the mRNA of such genes, the strategy is called "antisense". This strategy has already led to a number of successful results in experimental models in vitro. Much more rare are actual effects in animal models and clinical trials are just being sketched. A review on the current state of the art will enlighten the physiological potential of these new molecules but also underline the still numerous issues before controlling therapeutical applications.

Although the disease is well described, the pathogenesis of bone marrow fibrosis in idiopathic myelofibrosis still remains unclear. We previously reported elevated intraplatelet transforming growth factor-beta (TGF-beta) levels in patients with this myeloproliferative disorder, compared with healthy subjects. Here, in a series of 16 patients, we show that TGF-beta expression is also increased in patients' peripheral blood mononuclear cells (PBMC): (i) at the mRNA level analysed by Northern blot hybridization and/or reverse transcription-polymerase chain reaction (RT-PCR); (ii) and/or at the secreted peptide level as evaluated in conditioned media from patients' mononuclear cells by a growth inhibition assay on CC164 cells. By immunostaining with a polyclonal anti-TGF-beta 1 antibody, TGF-beta was localized in morphologically heterogenous cells; these cells were characterized as megakaryocytes by labelling with a gpIIbIIIa monoclonal antibody. Thus we provide evidence that both TGF-beta and megakaryocytes are linked in the pathogenesis of idiopathic myelofibrosis.

Four fluorescent estrogen ligands were investigated as agents for visualization of estrogen receptors in cells: 2-(2,4-dihydroxyphenyl)-6-hydroxy-3-benzofurancarboxylic acid delta-lactone (coumestrol) and 9(11)-dehydro-12-oxoestradiol [12-oxo-1,3,5-(10),9(11)-estratetraene-3, 17 beta-diol] (12-oxoestradiol), which are inherently fluorescent compounds; and tamoxifen [Z)-1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenyl-1-butene) and 4-hydroxytamoxifen [Z)-1-[4-(2-dimethylaminoethoxy) phenyl]-1-(4-hydroxyphenyl)-2-phenyl-1-butene), which become maximally fluorescent only after ultraviolet irradiation. By conventional fluorescence techniques, these agents can be detected down to 10(-8) M in water, but only to 10(-6) to 10(-7) M in protein solutions; however, by photon-counting spectrofluorimetry, coumestrol and 12-oxoestradiol can be detected in protein solutions down to 5 X 10(-10) M. Three of these compounds have good affinity for the estrogen receptor: coumestrol (20%); 12-oxoestradiol (12%); and 4-hydroxytamoxifen (37%), relative to estradiol (100%). Under conditions where autoradiographic controls indicate that most of the estrogen receptor of MCF-7 human breast cancer cells is in the nucleus, we could demonstrate nuclear fluorescence using 10(-9) M concentrations of coumestrol, 12-oxoestradiol, and 4-hydroxytamoxifen. This nuclear fluorescence was abolished by a 200-fold excess of diethystilbestrol and could only be observed through a fluorescence microscope equipped with a microchannel image intensifier and a video camera detector that together provide a sensitivity enhancement of approximately 10(4). These studies indicate that the estrogen receptor in breast cancer cells can be visualized by fluorescence techniques, provided that the visualizing ligands have adequate affinity and specificity for the receptor and appropriate fluorescence characteristics, and provided that the fluorescence instrument has adequate sensitivity to observe fluorescence emission from cells treated with nM concentrations of the fluorescent agents.