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Background Darunavir is an anti-HIV protease inhibitor repurposed for SARS-CoV-2 treatment. Objective The aim of this study was to assess the population pharmacokinetics of darunavir in SARS-CoV-2 patients compared with HIV patients. Methods Two separate models were created by means of a nonlinear mixed-effect approach. The influence of clinical covariates on each basic model was tested and the association of significant covariates with darunavir parameters was assessed at multivariate regression and classification and regression tree (CART) analyses. Monte Carlo simulation assessed the influence of covariates on the darunavir concentration versus time profile. Results A one-compartment model well-described darunavir concentrations in both groups. In SARS-CoV-2 patients (n = 30), interleukin (IL)-6 and body surface area were covariates associated with darunavir oral clearance (CL/F) and volume of distribution (Vd), respectively; no covariates were identified in HIV patients (n = 25). Darunavir CL/F was significantly lower in SARS-CoV-2 patients compared with HIV patients (4.1 vs. 10.3 L/h; p

(1) Background: Niemann–Pick type C disease (NPCD) is an autosomal recessive lysosomal storage disorder caused by mutations in the NPC1 or NPC2 genes. The clinical presentation is characterized by visceral and neurological involvement. Apart from a small group of patients presenting a severe perinatal form, all patients develop progressive and fatal neurological disease with an extremely variable age of onset. Different biomarkers have been identified; however, they poorly correlate with neurological disease. In this study we assessed the possible role of plasma NfL as a neurological disease-associated biomarker in NPCD. (2) Methods: Plasma NfL levels were measured in 75 healthy controls and 26 patients affected by NPCD (24 NPC1 and 2 NPC2; 39 samples). (3) Results: Plasma NfL levels in healthy controls correlated with age and were significantly lower in pediatric patients as compared to adult subjects (p = 0.0017). In both pediatric and adult NPCD patients, the plasma levels of NfL were significantly higher than in age-matched controls (p < 0.0001). Most importantly, plasma NfL levels in NPCD patients with neurological involvement were significantly higher than the levels found in patients free of neurological signs at the time of sampling, both in the pediatric and the adult group (p = 0.0076; p = 0.0032, respectively). Furthermore, in adults the NfL levels in non-neurological patients were comparable with those found in age-matched controls. No correlations between plasma NfL levels and NPCD patient age at sampling or plasma levels of cholestan 3β-5α-6β-triol were found. (4) Conclusions: These data suggest a promising role of plasma NfL as a possible neurological disease-associated biomarker in NPCD.

The NLRP3 inflammasome is cytosolic multi-protein complex that induces inflammation and pyroptotic cell death in response to both pathogen (PAMPs) and endogenous activators (DAMPs). Recognition of PAMPs or DAMPs leads to formation of the inflammasome complex, which results in activation of caspase-1, followed by cleavage and release of pro-inflammatory cytokines. Excessive activation of NLRP3 inflammasome can contribute to development of inflammatory diseases and cancer. Autophagy is vital intracellular process for recycling and removal of damaged proteins and organelles, as well as destruction of intracellular pathogens. Cytosolic components are sequestered in a double-membrane vesicle-autophagosome, which then fuses with lysosome resulting in degradation of the cargo. The autophagy dysfunction can lead to diseases with hyperinflammation and excessive activation of NLRP3 inflammasome and thus acts as a major regulator of inflammasomes. Autophagic removal of NLRP3 inflammasome activators, such as intracellular DAMPs, NLRP3 inflammasome components, and cytokines can reduce inflammasome activation and inflammatory response. Likewise, inflammasome signaling pathways can regulate autophagic process necessary for balance between required host defense inflammatory response and prevention of excessive and detrimental inflammation. Autophagy has a protective role in some inflammatory diseases associated with NLRP3 inflammasome, including gouty arthritis, familial Mediterranean fever (FMF), and sepsis. Understanding the interregulation between these two essential biological processes is necessary to comprehend the biological mechanisms and designing possible treatments for multiple inflammatory diseases.

A clinical study has been conducted to test the efficacy of Ecocid® S, a biocidal agent. The active substance is potassium peroxysulphate and is used in clinical practice after the mechanical cleaning of various surfaces that act as potential sources of infection transmission. We determined 29 swabbing points, from which 87 samples were collected with cotton swabs. Swabs were submitted for microbiological testing to evaluate microbial contamination before cleaning, and before and after disinfection with Ecocid® S. We submitted 63 swabs from 21 swabbing points for further statistical analysis. Five swabs were excluded because the presence of bacteria in the swabs before disinfection had not been determined. The clinical study on the efficacy of Ecocid® S disinfectant showed that it is effective with an average reduction in contamination of 95.75%. The disinfectant was also active with a significantly reduced time of action: it was removed with dry paper towels from all sampling points, except the floor scales, only 5 to 10 minutes after application. The time required for the proper preparation of examination tables and other equipment in clinical practice is of vital importance for a smooth workflow.Key words: animals; disinfection; potassium peroxysulphate; Ecocid® S ZMANJŠANJE ŠTEVILA BAKTERIJ PO UPORABI RAZKUŽILA ECOCID®S (RAZKUŽILO NA OSNOVI KALIJEVEGA PEROKSISULFATA) V PROSTORIH KLINIKE ZA MALE ŽIVALIPovzetek: S klinično študijo smo želeli ugotoviti učinkovitosti biocidnega razkužila ECOCID®S. Aktivna snov v razkužilu je kalijev peroksisulfat, ki se na klinikah uporablja za razkuževanje različnih površin, ki predstavljajo ključna mesta za prenos okužb, po njihovem mehaničnem čiščenju. Določili smo 29 vzorčnih mest, na katerih smo z uporabo bombažnih brisov odvzeli 87 vzorcev. Z mikrobiološkim testiranjem smo ugotavljali stopnjo kontaminacije pred čiščenjem, ter pred in po razkuževanju z ECOCID®S. Za statistično obdelavo smo uporabili 63 rezultatov z 21 vzorčnih mest. Pet rezultatov je bilo izločenih iz obdelave, ker je bila že pred razkuževanjem stopnja kontaminacije pod mejo detekcije uporabljene metode. S klinično študijo učinkovitosti razkužila Ecocid®S smo ugotovili povprečno 95,75 % zmanjšanje števila kontaminantov po uporabi razkužila. Razkužilo je bilo učinkovito tudi ob skrajšanem času delovanja le 5 do 10 minut po nanosu. Zaradi zagotavljanja tekočega dela na kliniki je izredno pomemben čas, ki je potreben za ustrezno pripravo površin in druge opreme za pregled živali, zato je razkužilo Ecocid® S primerno za uporabo na veterinarskih klinikah, saj hitro in učinkovito zmanjša bakterijsko kontaminacijo.Ključne besede: živali; dezinfekcija; kalijev peroksisulfat; Ecocid® S

There is no recommended protocol for detecting and isolating Clostridium difficile present in food samples. Here, we have evaluated the recovery of C. difficile in meat samples after incubating them in various enrichment broths. The media were as follows: cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, d-cycloserine, cefoxitin, and lysozyme; cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme; and cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, C. difficile moxalactam norfloxacin selective supplement, and lysozyme. Samples were inoculated with various strains and quantities of C. difficile and then enriched in the different broths for 1, 4, and 7 days. C. difficile was isolated on agar plates and detected with quantitative real-time PCR (qPCR). The procedure using enrichment in cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, d-cycloserine, cefoxitin, and lysozyme and incubation for 4 days for qPCR detection and 7 days for isolation (plating on C. difficile agar base with added C. difficile selective supplement and 7% [v/v] defibrinated horse blood after alcoholic shock and centrifugation) was validated. Samples of different kinds of meat and meat preparation were contaminated and used for validation of the chosen protocol. The sensitivity of detection with qPCR was 100%, and the sensitivity of the isolation method was 96%.

Cystatin C is a potent cysteine protease inhibitor that plays an important role in various biological processes including cancer, cardiovascular diseases and neurodegenerative diseases. However, the role of CstC in inflammation is still unclear. In this study we demonstrated that cystatin C-deficient mice were significantly more sensitive to the lethal LPS-induced sepsis. We further showed increased caspase-11 gene expression and enhanced processing of pro-inflammatory cytokines IL-1β and IL-18 in CstC KO bone marrow-derived macrophages (BMDM) upon LPS and ATP stimulation. Pre-treatment of BMDMs with the cysteine cathepsin inhibitor E-64d did not reverse the effect of CstC deficiency on IL-1β processing and secretion, suggesting that the increased cysteine cathepsin activity determined in CstC KO BMDMs is not essential for NLRP3 inflammasome activation. The CstC deficiency had no effect on (mitochondrial) reactive oxygen species (ROS) generation, the MAPK signaling pathway or the secretion of anti-inflammatory cytokine IL-10. However, CstC-deficient BMDMs showed dysfunctional autophagy, as autophagy induction via mTOR and AMPK signaling pathways was suppressed and accumulation of SQSTM1/p62 indicated a reduced autophagic flux. Collectively, our study demonstrates that the excessive inflammatory response to the LPS-induced sepsis in CstC KO mice is dependent on increased caspase-11 expression and impaired autophagy, but is not associated with increased cysteine cathepsin activity.

Purpose The purpose of this paper is to study the microbiological quality of raw milk delivered by 17 vending machines (VM) owned by different Slovenian milk producers. Design/methodology/approach For the determination of hygiene-technical conditions of VM, an observation list that included criteria for estimation of hygiene-technical suitability was made. A total of 51 milk samples were collected in three different seasons. The swabs and the cleaning liquid (eluates) of dispensing nozzles and chambers were also sampled. The main groups of microorganisms were determined by colony count technique according to international standards in all collected samples. Findings The aerobic colony count was higher than 100,000 CFU/mL in 20 (39.2 per cent) of milk samples. Its mean value was 4.8 log10 CFU/mL. The mean values of Enterobacteriaceae, psychrotrophic microorganisms, lipolytes, proteolytes, yeasts and moulds together, coagulase-positive staphylococci and somatic cell count were 3.3 log10 CFU/mL, 4.1 log10 CFU/mL, 3.2 log10 CFU/mL, 3.9 log10 CFU/mL, 2.2 log10 CFU/mL, 2.8 log10 CFU/mL and 5.3 log10 cells/mL, respectively. E. coli was found in 33.3 per cent of milk samples, while Listeria monocytogenes and antibiotics were not detected. The inner surface contamination of the dispensing nozzles and chambers was estimated in the range from 1.8 log10 CFU to 6.0 log10 CFU/cm2. The presence of detergents and disinfectants in supply valve eluates was determined in more than one-third of the samples. The hygienic-technical conditions of observed VM show some deviations from specified hygienic-technical requirements which could influence the safety of raw milk. Research limitations/implications The data about construction and the cleaning practice of VM, included in the experiment, were not available during the inspection facility. Originality/value In the paper the pathogenic and also the spoilage microorganisms in milk in the combination with hygienic conditions of inside surfaces of VM were studied.

Niemann‐Pick disease type C (NPC) is an uncommon lysosomal storage disorder, which is characterized neuropathologically by cholinergic dysfunction and presents clinically with a broad series of neurological signs and symptoms. NPC is inherited as an autosomal recessive trait, caused by mutations in the NPC1 or NPC2 genes. However, recent reports have raised concerns on heterozygous NPC1 gene mutation carriers, which historically have been considered as clinically unaffected, occasionally presenting with clinical parkinsonian syndromes or dementia. In the present study, we aimed at comprehensively assessing clinical, biochemical, and neurophysiological features in heterozygous NPC1 gene mutation carriers. We assessed cholinergic intracortical circuits with transcranial magnetic stimulation, executive functions and plasma oxysterol levels in two families comprising two monozygotic twins with a homozygous NPC1 p.P888S mutation, four patients with a compound heterozygous p.E451K and p.G992W mutation, 10 heterozygous NPC1 p.P888S carriers, 1 heterozygous NPC1 p.E451K carrier, and 11 noncarrier family members. We observed a significant impairment in cholinergic circuits, evaluated with short‐latency afferent inhibition (SAI), and executive abilities in homozygous/compound heterozygous patients and heterozygous asymptomatic NPC1 carriers, compared to noncarriers. Moreover, we reported a significant correlation between executive functions performances and both plasma oxysterol levels and neurophysiological parameters. These data suggest that heterozygous NPC1 carriers show subclinical deficits in cognition, possibly mediated by an impairment of cholinergic circuits, which in turn may mediate the onset of neurological disorders in a subset of patients.

Introduction It is known that HIV infected patients with syphilis are more prone to invasion of T. pallidum into central nervous system. Moreover, the diagnosis of neurosyphilis in HIV infected patients can be particularly difficult and challenging since serological tests in the cerebrospinal fluid (CSF) can often be false negative. Case report A previously healthy 40- year- old male was admitted to neurology department due to acute left side paresthesia, ataxia and diplopia. Since his head CT and CT-angiography scans were without pathological findings and his symptoms started recovering, he was treated conservatively. His symptoms worsened the next day and MR scan revealed right posterior pontine infarction. As part of routine screening for stroke in young patients, he was tested for HIV and syphilis. Serologic tests for syphilis were positive in serum as were screening and confirmatory tests for HIV infection. CD4+ cell count was 282/mm³ and HIV RNA was 9480 copies/ml. CSF analysis showed elevated protein level (0,70 g/L) and lymphocytic pleocytosis (lymphocytes 30/mm³). CSF- RPR and CSF-TPHA were not reactive. However, because of strong clinical suspicion of meningovascular syphilis, additional serological tests for syphilis in CSF, i.e. CSF-IgG-FTA-ABS and CSF-LIA (Line Immuno Assay), were performed: both were positive. After the confirmation of suspected meningovascular syphilis, treatment with intravenous benzylpenicillin was given for 21 days. Neurological symptoms subsided and patient was discharged with minimal neurological sequelae. Conclusion The correct diagnosis of neurosyphilis in HIV infected patients presents a challenge since serologic tests can be false negative. Therefore, different serologic tests with high specifity and sensitivity should be used, newer tests such as LIA and CIA being particularly helpful. Clinicians should be aware of the characteristics of syphilis and HIV coinfection to establish the correct diagnosis and provide adequate treatment, which will minimise neurological impairments among these patients.

A telerehabilitation system for physiotherapy of upper extremities has been developed for patients with Parkinson disease. The gamification system (with Kinect™ sensor) has been applied in a daily clinical practice; 28 patients with Parkinson's disease were included in the pilot study, 26 patients finished the “Fruit picking” game. The preliminary patients' feedback (objective measurements, games score) and the results of the clinical tests (MDS-UPDRS part III (27 vs 29), Jebsen hand test, 9 Hole Peg Test, Box & Blocks Test − 48 vs 52) were encouraging (faster daily activities for up to 10%).

Campylobacteriosis is the most frequently reported bacterial food-borne illness in the European Union and contaminated broiler meat is considered the most important source of infection in humans. The aim of the present study was to evaluate real-time PCR (qPCR) and digital PCR (dPCR) for quantification of Campylobacter jejuni in 75 broiler neck-skin samples collected from a poultry slaughterhouse, and to compare them with the ISO 10272-2 standard plate count method. For qPCR standard curve, C. jejuni-negative neck-skin samples were spiked with C. jejuni suspension with a known number of bacterial cells. The observed CFU/g values by qPCR correlated greatly with the expected values and qPCR showed good performance with the reliable limit of detection (rLOD) and limit of quantification (LOQ) of three and 31 target copies per reaction, respectively. However, both rLOD (1219 CFU/g) and LOQ (12,523 CFU/g) were beyond the EFSA-proposed critical limit of 500–1,000 CFU/g of neck skin. Although C. jejuni cell counts were ≤1,000 CFU/g in only 7/75 samples by plate counting, they were ≤LOQ in 60/75 and ≤rLOD in 26/75 (≤1,000 CFU/g in 24/75) samples by qPCR. A strong and statistically significant correlation was observed between qPCR and dPCR. Both PCR-based methods correlated significantly with the plate count method; however, the correlation was moderate. Using the Bland–Altman analysis, an average agreement was noted between all three methods, although with a large standard deviation. A significant bias toward overestimation in dPCR was observed, probably due to the relatively high number of false positive calls. The linear dynamic range was comparable in both PCR-based methods; however, qPCR proved to be more suitable for routine use. In the future, the establishment of a reliable molecular quantification of C. jejuni in poultry samples showing a wide range of contamination levels down to the proposed critical limit is needed to enable time- and cost-effective surveillance throughout all stages in the food production chain. As both rLOD and LOQ were beyond this limit, a modification of the procedure is suggested to include less sample dilution prior to DNA extraction to enable PCR-based quantification of C. jejuni at the proposed microbiological criteria.

Antibiotics have always appeared miraculous, saving innumerable lives. However, the unwise use of antimicrobial drugs has led to the appearance of resistant bacteria. The purpose of this study was to evaluate antimicrobial resistance in Escherichia coli (n =160) isolated from food of animal origin. The focus was on E. coli -producing extended-spectrum β-lactamases. E. coli was chosen because it is a part of the normal microbiota in mammals and can enter the food chain during slaughtering and food manipulation. Subsequently, its resistance genes can be transferred to pathogenic bacteria and human microbiota. Phenotypic and genotypic analyses of selected antimicrobial resistances were carried out together with a molecular analysis of virulence genes. E. coli isolates from food of animal origin were compared with clinical E. coli strains isolated from the human intestinal tract. Extended-spectrum β-lactamase-producing E. coli isolates were found in 9.4% of food isolates and in 1.8% of intestinal isolates. Phylogenetically, the majority of food (86.3%) and intestinal E. coli (58.1%) isolates were found to belong to the commensal phylogenetic groups A and B1. The distribution of 4 of 14 analyzed virulence factors was similar in the food and intestinal isolates. Strains isolated from food in Slovenia harbored resistance genes and virulence factors, which can constitute a problem for food safety if not handled properly.

In antibiotic residue analyses the first step of screening is just as important as the following steps. Screening methods need to be quick and inexpensive, but above all sensitive enough to detect the antibiotic residue at or below the maximum residue limit (MRL). In the case of a positive result, the next step is conducted and further methods are used for confirmation. MRLs stated in European Union Regulation 37/2010 for tetracyclines in raw milk are: 100 µg kg(-1) for tetracycline, 100 µg kg(-1) for oxytetracycline, 100 µg kg(-1) for chlortetracycline and no limit for doxycycline because it is prohibited for use in animals from which milk is produced for human consumption. The current five-plate microbiological screening method for the detection of antibiotic residues in raw milk was found to be simple and inexpensive, but not specific, sensitive and reliable enough to detect tetracycline at MRL in routine raw milk screening procedures. Spiking samples with tetracycline at the MRL level and applying them on Bacillus cereus ATCC 11778 microbiological plates often gave false-negative results, indicating that tetracyclines may have to be inactivated or masked. Tetracyclines seem to bind to a certain component in milk. Consequently, when applying samples to the B. cereus microbiological plate the antibiotic cannot inhibit the growth of B. cereus which disables the formation of inhibition zones on the test plate. After adding the appropriate amount of citric acid into the milk samples, we solved the problem of false-negative results. During the validation 79 samples of milk were spiked with tetracyclines at different concentrations: 100 µg kg(-1) for tetracycline, 100 µg kg(-1) for oxytetracycline, 80 µg kg(-1) for chlortetracycline and 30 µg kg(-1) for doxycycline. Concentrations used in the validation matched the requirements for MRLs (they were either at or below the MRLs) stated in European Union Regulation 37/2010. The sensitivity of the validation was 100%.

International audience; The high incidence of lung-damaging life-threatening respiratory infections in infants may be related to the immaturity of their immune systems. To determine whether lung immune features differ in early life compared with those in adulthood, whole lung as well as lung T lymphocyte and DC responses were investigated in BALB/c neonates versus adults. Higher expression of GATA-3 and rapid and sustained production of type 2 cytokines by lung explants after in vitro exposure to anti-CD3 was the hallmark of the neonatal period, suggestive of a Th2 bias. Neonatal lung GATA-3-producing cells were identified as CD31, CD4 and CD8 double-negative T lymphocytes, a subset found at a higher frequency in neonatal than adult lung. The neonatal lungs contained fewer conventional DCs, with a lower ratio of CD103 to CD11b DCs, and a much lower number of plasmacytoid DCs in comparison with adult lungs. Yet, when stimulated in vivo by BCG, neonatal lung DCs matured and primed adult naıve CD4 T cells toward Th1 as efficiently as adult BCG-primed lung DCs. Conversely,both adult and neonatal BCG-primed lung DCs induced a Th2 cytokine response from neonatal naıve lymph node Tcells, suggestive of anintrinsic feature of neonatal T lymphocytes.

To study the adipokines concentration and glucose homoeostasis in the early-second trimester of women who will develop gestational diabetes mellitus (GDM).Maternal plasma and fetal amniotic fluid samples were prospectively collected between 2006 and 2007 at the time of mid-trimester amniocentesis. Eight patients found to be affected by GDM were compared with 10 control patients with a normal pregnancy course. Adipokines leptin and adiponectin, as well as insulin and glucose concentration both in amniotic fluid and maternal plasma were compared between cases and controls. HOMA-IR (homeostatic model assessment for insulin resistance) was also calculated both for amniotic fluid and maternal serum.The amniotic fluid adiponectin concentration was higher in women who would develop GDM than in controls (29.9 ng/ml, 95% CI 26.7-49.8 ng/ml, versus 14.9 ng/ml, 95% CI 13.5-18.8 ng/ml), p 0.05). No difference was shown for leptin both in amniotic fluid and maternal serum. Insulin concentrations in the amniotic fluid were found to be lower in GDM than in controls, while HOMA-IR-index resulted lower in amniotic fluid and higher maternal serum (p 0.05).Our data suggests that an earlier alteration in the fetal glucose metabolism will precede the glucose dysmetabolism in pregnancies later complicated by GDM.

Retinol (ROH) is an essential micronutrient required for normal fetal development and an essential molecule for antioxidant processes. To investigate the putative role of ROH as a marker of preeclampsia in early second trimester amniotic fluid (AF). Case–control study comparing the concentration of ROH and other antioxidants such as uric acid, vitamin E and malondialdehyde (MDA) in second trimester AF in patients that later developed preeclampsia with normal pregnancies. The concentration of ROH in amniotic fluids of women that later developed preeclampsia was significantly higher than those of uncomplicated pregnancies (66.72 µg/l (49.00–70.56) vs. 44.4 µg/l (31.9–51.17), p

We report the case of an 18-month-old girl who presented a complete III right cranial nerve paralysis which disappeared spontaneously in approximately three weeks without any medical therapy. Magnetic Resonance (MR) investigation performed three days after paralysis onset showed a focal area of contrast enhancement at the third cranial nerve emergence. MR performed three months later, with an absence of symptoms, showed a less evident but persistent contrast enhancement at the cranial nerve emergence. We briefly discuss the causes of inflammatory nerve involvement since a parainfective cause of nerve paralysis was implicated.

This article reports the results of investigations into the process of cell death induced in the Chinese hamster ovary cell K1 subclone (CHO K1) by okadaic acid (OA), a hydrophobic polyether produced by marine dinoflagellates. The IC50 was about 13 nM OA after 24 h of treatment, as determined using neutral red. With the MTT assay, the IC50 was 25 nM, although in this case 25% of the initial staining was still observed at 100 nM. Hoechst staining showed that mitotic figures accumulated at 12 nM OA after a 24- or 48-h treatment. In experiments limited to a 3-day treatment without changing the medium, CHO K1 cells were engaged in the death process at 50 nM OA after about 20 h and at 10 nM OA after 48 h. In many cells nuclear fragmentation that resulted in the apparent appearance of vesicles correlated with increasing cellular volume. But additional cell fragmentation was not observed with any treatment, and the chromatin material seemed to progressively disappear inside the cells. DNA fragmentation was analyzed by electrophoresis and with the TUNEL technique. With both techniques, the DNA was fragmented by 48 h in both 25 and 50 nM OA. Electrophoresis showed that both adherent and nonadherent cells were affected. Annexin-positive/ propidium iodide (PI)–negative cells were rarely observed after OA treatment. Some were seen under the scanning cytometer after 20 h at 50 nM OA or after 48 h at 10 nM OA, but they were never detected by flow cytometry. Most of the time scanning cytometry showed either unstained cells or PI-positive (annexin-positive or -negative) cells (48 h, 50 nM, or 72 h, 10 nM). Flow cytometry cytograms showed two cell subpopulations: one composed of a majority of smaller cells, the other of larger cells. The larger cells markedly decreased with time and OA treatment (50 and 100 nM). Stained-cell counting showed that all cells that stained were both annexin- and PI positive and that most PI-positive cells were smaller. Ki67 antigen labeling showed the proliferative activity of CHO K1 cultures but also demonstrated the loss of this activity in smaller cells treated with 50 nM OA for 48 h. We concluded that in our culture conditions the main OA target within CHO K1 cultures was dividing cells. Our results suggest that cells with disturbed metaphase–anaphase enter apoptosis, leading to necrotic daughter cells. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 383–394, 2003

Six slaughter batches deriving from six typical industrial broiler flocks were examined for the presence, quantity and genetic characteristics of contaminating Campylobacter jejuni (C. jejuni) during various stages of slaughtering and carcass processing. To assess the contamination dynamics of the carcasses, the analyses were always conducted on neck-skin samples from the same pre-selected and carefully marked carcasses in each batch. The skin samples were taken sequentially at three successive slaughter-line locations in the evisceration room, after three-day refrigeration and after three-day freezing procedure. Caecal samples from the same animals were also tested, as well as samples from the slaughterhouse environment before and after slaughtering. The samples were analysed by the ISO10272 isolation method; campylobacters from neck-skin samples were also quantified. Isolates were species-identified and genotyped by pulsed-field gel electrophoresis (PFGE). On average, the highest C. jejuni skin contamination was detected at the first sampling point (post-plucking), suggesting that the majority of Campylobacter contamination actually occurs before the entrance to the eviscerating room, probably during the preceding plucking stage. In two out of five positive batches, an additional increase in contamination was recorded after the evisceration step. An evident trend of increasing contamination level was detected when successive batches were compared at each of two initial sampling sites in the evisceration room, indicating an accumulation of contaminating C. jejuni at some point before the evisceration room. Three-day refrigeration and three-day freezing caused a 4.5- and 142-fold drop in mean C. jejuni CFU counts, respectively. All pre-slaughtering samples from the slaughterhouse environment were negative and all post-slaughtering samples, except water from the scalding tank, were positive. Pulsotypes were limited: altogether five different types were detected, typically one type per batch. The PFGE results from the slaughterhouse environment isolates indicate that cross-contamination is possible (multiple pulsotypes detected in e.g. eviscerating machine). Nevertheless, this was not confirmed in carcasses: analyses of neck-skin isolates suggest that carcasses are contaminated by their own caecal/farm/flock pulsotype.

Scopo del lavoro è stato quello di confrontare l'angiografia digitale arteriosa (ADA) con l'angiografia a risonanza magnetica (ARM) nei controlli nel tempo di pazienti con aneurismi intracranici sottoposti ad embolizzazione con Guglielmi Detachable Coils (GDC). Sono stati effettuati 49 studi comparativi (6 entro 48 ore dal trattamento, 12 a tre mesi, 12 a sei mesi, 14 a un anno e 5 a due anni dall'embolizzazione) in 28 pazienti con aneurismi cerebrali embolizzati con GDC, utilizzando 3D Time-of-Flight (TOF) angio-RM e angiografia digitale arteriosa. Nei casi di embolizzazione parziale abbiamo valutato la presenza e la morfologia dell'eventuale residuo. In tutti i casi è stata valutata la pervietà dei vasi vicini l'aneurisma embolizzato. Nei casi di occlusione completa (36/49, 73,4%) è stata riscontrata una buona correlazione fra le immagini ottenute con angio-RM e quelle angiografia digitale arteriosa. In caso di occlusione parziale (13/49, 26,5%) solo in 7 casi la ARM è stata in grado di visualizzare flusso all'interno dell'aneurisma. In 24 casi su 49 (48,9%) non è stata ottenuta con ARM una adeguata visualizzazione dei vasi vicini alla lesione, sia per le piccole dimensioni di tali vasi che per perdita di segnale da flusso turbolento dovuto probabilmente alla presenza delle spirali. In conclusione l'angio-RM non può sostituire la angiografia digitale arteriosa nei controlli nel tempo di aneurismi embolizzati con GDC. Infatti la scarsa risoluzione della tecnica non sempre consente di visualizzare adeguatamente un eventuale residuo pervio. La ARM va limitata in casi in cui esistano importanti controindicazioni alla esecuzione di ADA e se esiste una buona correlazione con le immagini angiografiche.

A rare cerebral tumour is described with a particular enhancement pattern in post-contrast medium administration magnetic resonance images. The tumour was a supratentorial, multifocal haemangioblastoma, whose solid components did not enhance in pulse sequences acquired after contrast medium administration (double dose), but only in later acquisitions (after about 1 hour). This particular behaviour suggests using contrast medium at high doses with late acquisitions when a cerebral haemangioblastoma is suspected.

This paper evaluates the result of endovascular treatment in aneurysms arising in the internal carotid artery, intracranial tract, using Guglielmi detachable coils. We selected one years, from November 1995 to November 1996 in which we used GDC to breat 26 patients with intracranial aneurysm; 14 of them in the carotid siphon. Patients' ages ranged from 29 to 77 years (9 women and 5 men). Following, the Yasargil classification we divided the aneurysmal origin into: ICA lateral site (posterior communicating artery), 7 cases; ICA inferior site, 3; ACI medial distal site, 1; ICA bifurcation site, 3. Regarding size, the aneurysm was: small in 7 patients, medium in 6 and large in 1. The occlusion percentage was total in 9 patients, sub-total in 4, partial in 1. The clinical presentation was with subarachnoid haemorrhage in 11 patients (6 of them were treated within 3 days from the onset of symptoms); in three of them aneurysm detection was occasional (real occasional in 2, occasional in 1). All 14 patients were evaluated on discharge with the Glasgow Outcome Scale (GOS) modified, according to the Guglielmi proposals: 10 out of 14 patients had an excellent GOS with no deaths. All treatment was performed in anaesthesia with heparin infusion 5000 Ul ev in bolus + 1000 Ul each hour); the heparin infusion was suspended 12 hours after the treatment and then paediatric Aspirin or Fraxiheparin was administered for 4–5 days. No perforation of the aneurysmal sac occurred during treatment or cerebral haemorrhage due to the catheter positioning. In one patient with H-H IIIa, with a wide neck aneurysm and siphon dysplasia rebleeding occurred 21 days after treatment requiring prompt surgery; on discharge the GOS was fair. Regarding thrombo-embolic complications, one patient with H-H grade IIIa, had a hypodense ischaemic lesion, in a temporoparietal site ipsilateral to the aneurysm, without associated symptoms. Another patient, with IIIa H-H grade, presented a thromboembolic complication after treatment; the CT scan revealed multiple hypodense lesions in sub- and supratentorial sites; the GOS was poor on discharge with associated permanent neurological deficit. No patient died as a direct of the treatment. The radiation dose to patient and operator should not be underestimated: in our experience we had three cases of transient localised alopecia. The angiographic controls after treatment, at discharge and after 3–6 months, showed persistent occlusion; in one case of sub-total occlusion, total occlusion was achieved by re-do surgery. The GOS remained stable at 3–6 months after treatment in patients with an excellent result; one patient with GOS fair at discharge reached an excellent result; the other two patients with GOS poor and fair have improved during time reaching a fair and good outcome respectively.

The purpose of this study was to investigate radiation doses in cerebral perfusion computed tomography (CT) examination. As a part of routine patient monitoring, data were collected on patients in terms of the skin dose and CT dose index (CTDIvol) and dose-length product (DLP) values. For the estimation of the dose to the lens a phantom study was performed. Dose values for skin and lens were below the threshold for deterministic effects. The results were also compared with already published data. For better comparison, the effective dose was also estimated. The values collected on patients were in the ranges 230-680 mGy for CTDI and 2120-2740 mGy cm for DLP, while the skin dose and estimated effective dose were 340-800 mGy and 4.9-6.3 mSv, respectively. These values measured in the phantom study were similar, while the doses estimated to the lens were 53 and 51 mGy for the right and left lens, respectively.

Attraverso una valutazione retrospettiva di 26 casi di meningiomi intracranici abbiamo voluto correlare l'entità dell'edema perilesionale con determinati aspetti neuroradiologici. La valutazione dei risultati è stata effettuata con metodi statistici ed è emersa una significativa correlazione fra l'entità dell'edema e la presenza di compressione corticale, l'apporto vascolare prevalente da vasi piali e, in misura minore, le dimensioni del tumore. Questi rilievi possono risultare importanti al fine di dimostrare l'ipotesi di un ruolo attivo delle cellule neoplastiche nella genesi della sofferenza del tessuto cerebrale adiacente (edema citotossico).

An atmospheric pressure plasma deposition for P(d,L)LA (PLA) film was used to modify polymer surface properties using 1,2-diaminopropane and acrylic acid as precursors. These two different plasma coatings result in a high density of amino groups (PLA-NH2) and carboxylic groups (PLACOOH) onto PLA surface as demonstrated by Fourier transform infra-red (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). Plasma coatings modified PLA surface wettability and proteins adsorption from fetal bovine serum (FBS), influencing cell adhesion and proliferation of 3T3 mouse fibroblast, MC-3T3 E1mouse pre-osteoblast, and HaCaT cells (human keratinocytes). In particular both coatings increased pre-osteoblast and keratinocyte adhesion while no effect was observed on fibroblast. Moreover, cell proliferation assessed after 48h by Tox-8 assay was significantly higher for osteoblast cells and keratinocyte seeded onto both PLA-NH2 and PLACOOH compared to cells seeded onto normal PLA. On the basis of the obtained data, the atmospheric pressure plasma deposition described might represent an innovative and useful tool for bone and skin tissue engineering.

International audience; BACKGROUND & AIMS: Mycobacterium bovis Bacillus Calmette-Guérin (BCG), killed by extended freeze-drying (EFD), induces secretion of interleukin-10 and reduces lung inflammation in a mouse model of asthma. We investigated the effects of EFD BCG in mouse models of inflammatory bowel disease. METHODS: EFD BCG was administered subcutaneously to mice with colitis induced by dextran sodium sulfate (DSS), oxazolone, or adoptive transfer of CD4(+)CD45RB(high)Foxp3(-) T cells from C57Bl/6 Foxp3GFP mice to RAG2(-/-) mice. RESULTS: EFD BCG, administered either before induction of DSS and oxazolone colitis or after development of acute or chronic DSS-induced colitis, reduced symptom scores, loss of body weight, and inflammation. Although transfer of CD4(+)CD45RB(high)Foxp3(-) cells induced colitis in RAG2(-/-) mice, administration of EFD BCG at the time of the transfer converted Foxp3(-) T cells to Foxp3(+) T cells and the mice did not develop colitis. EFD BCG protected mice from colitis via a mechanism that required expansion of T regulatory cells and production of interleukin-10 and transforming growth factor β. EFD BCG activated the retinoid X receptor (RXR)-α-peroxisome proliferator-activated receptor (PPAR)-γ heterodimer, blocked translocation of nuclear factor κB to the nucleus, and reduced colonic inflammation; it did not increase the number of colon tumors that formed in mice with chronic DSS-induced colitis. CONCLUSIONS: EFD BCG controls severe colitis in mice by expanding T regulatory cell populations and PPAR-γ and might be developed to treat patients with inflammatory bowel disease.

FcεR1-expressing neutrophils accumulate in the brain of mice infected with Plasmodium berghei (PbANKA) and promote the development of experimental cerebral malaria., The role of the IgE–FcεRI complex in malaria severity in Plasmodium falciparum–hosting patients is unknown. We demonstrate that mice genetically deficient for the high-affinity receptor for IgE (FcεRIα-KO) or for IgE (IgE-KO) are less susceptible to experimental cerebral malaria (ECM) after infection with Plasmodium berghei (PbANKA). Mast cells and basophils, which are the classical IgE-expressing effector cells, are not involved in disease as mast cell–deficient and basophil-depleted mice developed a disease similar to wild-type mice. However, we show the emergence of an FcεRI+ neutrophil population, which is not observed in mice hosting a non–ECM-inducing PbNK65 parasite strain. Depletion of this FcεRI+ neutrophil population prevents ECM, whereas transfer of this population into FcεRIα-KO mice restores ECM susceptibility. FcεRI+ neutrophils preferentially home to the brain and induce elevated levels of proinflammatory cytokines. These data define a new pathogenic mechanism of ECM and implicate an FcεRI-expressing neutrophil subpopulation in malaria disease severity.

Background A combined protocol of voxel-based morphometry (VBM) and diffusion-weighted imaging (DWI) was applied to investigate the neurodevelopment of gray and white matter in autism. Methods Twenty children with autism (mean age = 7 ± 2.75 years old; age range: 4–14; 2 girls) and 22 matched normally developing children (mean age = 7.68 ± 2.03 years old; age range: 4–11; 2 girls) underwent magnetic resonance imaging (MRI). VBM was employed by applying the Template-o-Matic toolbox (TOM), a new approach which constructs the age-matched customized template for tissue segmentation. Also, the apparent diffusion coefficients (ADC) of water molecules were obtained from the analysis of DWI. Regions of interests (ROIs), standardized at 5 pixels, were placed in cortical lobes and corpus callosum on the non-diffusion weighted echo-planar images ( b = 0) and were then automatically transferred to the corresponding maps to obtain the ADC values. Results Compared to normal children, individuals with autism had significantly: (1) increased white matter volumes in the right inferior frontal gyrus, the right fusiform gyrus, the left precentral and supplementary motor area and the left hippocampus, (2) increased gray matter volumes in the inferior temporal gyri bilaterally, the right inferior parietal cortex, the right superior occipital lobe and the left superior parietal lobule, and (3) decreased gray matter volumes in the right inferior frontal gyrus and the left supplementary motor area. Abnormally increased ADC values in the bilateral frontal cortex and in the left side of the genu of the corpus callosum were also reported in autism. Finally, age correlated negatively with lobar and callosal ADC measurements in individuals with autism, but not in children with normal development. Conclusions These findings suggest cerebral dysconnectivity in the early phases of autism coupled with an altered white matter maturation trajectory during childhood potentially taking place in the frontal and parietal lobes, which may represent a neurodevelopmental marker of the disorder, possibly accounting for the cognitive and social deficits.

We showed in a previous study that the intranasal (i.n) delivery of bacille Calmette-Guerin (BCG) to BP2 mice (H-2q) inhibits eosinophilia and bronchial hyperreactivity in a mouse model of asthma. The present work has been performed to characterize the leucocyte lineages recruited to the lungs of mice after i.n. delivery of BCG and potentially involved in the polarization of T lymphocytes. The different antigen-presenting cells (APC) recruited to bronchoalveolar lavage (BAL) and to lung tissue of mice shortly after the delivery of BCG were analysed in parallel as well as their capacity to drive the immune response towards a T helper type 1 cytokine production. Alveolar macrophages (AM) from the BAL were CD11c+, F4/80+ and CD11b-, and in the lung tissue two major populations of potential APC were detected: one CD11c-, F4/80+, CD11b+ and I-Aq- was identified as interstitial macrophages (IM) and a second expressing CD11c+ and I-Aq+ antigens, negative for CD11b and F4/80 markers as leucocytic dendritic cells (DC). Freshly isolated DC up-regulated CD11b and CD40 antigens after overnight culture, but remained negative for CD8alpha antigen, suggesting a myeloid origin. Lung DC which produced high amount of interleukin (IL)-12 were potent inducers of naive CD4+ T lymphocyte priming, as assessed by interferon-gamma (IFN-gamma) production by these naive CD4+ T cells. Lung explants recovered long term after BCG delivery produced sustained levels of IFN-gamma. Our results suggest that AM and particularly DC by secreting IL-12 shortly after BCG delivery induce the long-term persistence of IFN-gamma-secreting T cells percolating in BCG-loaded lung tissue.

The rare occurrence of malignant fibrous histiocytoma of the orbit followed combined modality therapy in an adolescent is reported.

Over a period of 4.5 years, 977 discograms were performed on 774 patients, with a complication, septic dis-citis in only one case. Six hundred and eighty-one patients underwent percutaneous treatment of the herniated lumbar disc [chemonucleolysis or nucleoaspiration (Onik procedure)], while in the remaining 93 patients the disco-graphic findings contraindicated percutaneous treatment. Discography plays two important roles, one diagnostic and the other decisional. While confirming the clinical and neuroradiological findings of disc herniation, discography verifies the correct positioning of the needle at the centre of the nucleus pulposus and proves decisive in determining the preferential treatment to be undertaken on the basis of morphological, functional and dynamic data.

The authors report the cases of two patients with CT-documented paramedian mesencephalo-thalamic infarcts, showing language disturbances. The first patient showed a non fluent, transcortical motor-like aphasia, the other had a fluent but severely paraphasic language disorder. The CT study disclosed that it was the dorso-median thalamic nucleus that was mostly involved in both cases. These findings agree with a few previous pathological studies suggesting that the paramedian thalamic nuclei, particularly the dorso-median nucleus may play some role in language disturbances. However the anatomical basis for thalamic aphasia remains speculative, taking into account the importance of cortical connections in the origin of subcortical neuropsychological disturbances.

The aim of the study was to assess the effectiveness and safety of iomeprol (300 mg I/ml) in CT of the brain. Thirty-one patients were enrolled according to strict inclusion and exclusion criteria necessary for a phase II study. The efficacy was judged to be good in 10 patients and excellent in 21. None of the patients showed clinically significant changes in vital parameters, ECG and EEG tracings or laboratory tests. Among the 31 patients there were no subjective reactions and only 1 patient complained of a side-effect. Taking into account the results of this multicentre trial we can conclude that iomeprol is a good contrast medium with excellent local and systemic tolerability.

The development of Computed Tomography and Magnetic Resonance has reduced the diagnostic role of Digital Subtraction Angiography (DSA) in the neuroradiological evaluation of intracranial tumors. DSA is currently an important pre-surgical examination, able to offer important information regarding the type and the entity of neoplastic vascularization. Moreover the development of endo-vascular interventional practices (pre-surgical embolization of meningiomas and endo-arterious chemotherapy of gliomas) has widened the applications of angiography.

We performed 25 control studies in 17 patients with cerebral aneurysms embolized with Guglielmi coils (GDC), both with MR-angiography (MRA) and digital subtraction angiography (DSA). No case studied with MRA showed artifacts due to GDC. In some cases (15/25, 60%) there was a poor visualization of small vessels, distal to the aneurysm (probably caused by turbulent flow due to GDC presence). In cases with complete occlusion (16/25) there was a good correlation between MRA and DSA. At the moment the cases of partial occlusion (9/25) are too few to express a true judgement. In conclusion MRA may become an alternative to DSA, mainly in elderly patients.

Parkinson’s disease (PD) is treated by medication, less with deep brain stimulation and physiotherapy. Different opinions on the clinical meaningfulness of the physiotherapy or recommended intensive physiotherapy were found. Our objectives were to design intensive target-based physiotherapy for upper extremities suitable for telerehabilitation services and examine the clinical meaningfulness of the exergaming at an unchanged medication plan. A telerehabilitation exergaming system using the Kinect sensor was developed; 28 patients with PD participated in the study. The system followed the participants’ movements and adapted the difficulty level of the game in real time. The outcomes of the study showed that seven out of 26 participants could set up the equipment at home alone. Clinical outcomes of Box and Blocks Test (mean: 47 vs. 52, P=0.002, Cohen’s d=0.40), UPDRS III (mean: 27 vs. 29, P=0.001, d=0.22), and daily activity Jebsen’s test; writing a letter (mean: 24.0 vs. 20.6, P=0.003, d=0.23); and moving light objects (mean: 4.4 vs. 3.9, P=0.006, d=0.46) were statistically significant (P

Intravenously administered iodinated contrast media have been demonstrated, since early experience with computed tomography of the brain, to improve clinical value of the procedure for detecting intracranial lesions. There is no universal agreement about the amount and the method of administration of the contrast medium. Many authors maintain that the use of large doses gives better results for the diagnosis of tumors and metastases. The purpose of this paper is to evaluate the tolerance of iopamidol administered by rapid intravenous infusion in a large number of patients undergoing contrast enhanced computed tomography to detect brain metastases. The authors examined 969 consecutive adult patients suffering from lung cancer, brain metastases have been detected in 17% of cases. Adverse reactions to contrast media occurred in 3 patients. Non ionic contrast media are recommended in this diagnostic procedure.

The authors expose their experience in neuroradiological applications of dynamic-CT (D-CT) or angio-CT. The examinations are performed by means of a G.E. 8.800 CT/T. A special software offers automatic analysis of the region of interest. Vascular and tumoral patients are examined trying to evaluate: (a) the blood flow in patients suffering from transient ischaemic attacks and (b) to estimate the possibilities of D-CT in the evaluation of tumoral vascularity and in the identification of characteristic curves for different kinds of tumors.