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Background: Recreational use of nitrous oxide (N 2 O) leads to neurological disorders including combined subacute degeneration of spinal cord, psychological disorders, and thrombosis. Serum or urine N 2 O assays could not be routinely performed. Hence, it is necessary to investigate other biological markers such as metabolic markers. We aimed here to challenge the three main biological markers used for the diagnosis of nitrous oxide abuse as total vitamin B12, homocysteine, and methylmalonic acid., Methods: We retrospectively collected clinical and biological data from 52 patients with known, documented chronic N 2 O abuse and associated clinical signs (peripheral neuropathy disability score or thrombosis event). Sera and plasma total vitamin B12, methylmalonic acid (MMA), and homocysteine were performed to identify the most specific marker of chronic N 2 O intoxication and related clinical outcomes., Results: Plasma homocysteine was almost consistently increased in case of N 2 O chronic consumption, whereas MMA increase and total vitamin B12 decrease are not systematically found. Our results showed that none of the markers are correlated with levels of N 2 O consumptions. However, homocysteine and MMA are correlated with clinical severity, but MMA seems to be a better marker of clinical severity., Conclusion: There is no specific marker of nitrous oxide abuse according to levels of consumption, total vitamin B12 decrease could not be used either as consumption or as severity marker. However, we showed that homocysteine is consistently increased and could be used as marker of recent N 2 O consumption. On the other hand, we showed that MMA could be used as a marker of clinical gravity., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

The detection of immunoglobulin G (IgG) oligoclonal bands (OCB) in cerebrospinal fluid (CSF) by isoelectric focusing (IEF) is a valuable tool for the diagnosis of multiple sclerosis. Over the last decade, the results of our clinical research have suggested that tears are a non-invasive alternative to CSF. However, since tear samples have a lower IgG concentration than CSF, a sensitive OCB detection is therefore required. We are developing the first automatic tool for IEF analysis, with a view to speeding up the current visual inspection method, removing user variability, reducing misinterpretation, and facilitating OCB quantification and follow-up studies. The removal of band distortion is a key image enhancement step in increasing the reliability of automatic OCB detection. Here, we describe a novel, fully automatic band-straightening algorithm. The algorithm is based on a correlation directional warping function, estimated using an energy minimization procedure. The approach was optimized via an innovative coupling of a hierarchy of image resolutions to a hierarchy of transformation, in which band misalignment is corrected at successively finer scales. The algorithm's performance was assessed in terms of the bands' standard deviation before and after straightening, using a synthetic dataset and a set of 200 lanes of CSF, tear, serum and control samples on which experts had manually delineated the bands. The number of distorted bands was divided by almost 16 for the synthetic lanes and by 7 for the test dataset of real lanes. This method can be applied effectively to different sample types. It can realign minimal contrast bands and is robust for non-uniform deformations.

Cryptosporidium parvum is a major cause of diarrheal illness and was recently potentially associated with digestive carcinogenesis. Despite its impact on human health, Cryptosporidium pathogenesis remains poorly known, mainly due to the lack of a long-term culture method for this parasite. Thus, the aim of the present study was to develop a three-dimensional (3D) culture model from adult murine colon allowing biological investigations of the host-parasite interactions in an in vivo-like environment and, in particular, the development of parasite-induced neoplasia. Colonic explants were cultured and preserved ex vivo for 35 days and co-culturing was performed with C. parvum. Strikingly, the resulting system allowed the reproduction of neoplastic lesions in vitro at 27 days post-infection (PI), providing new evidence of the role of the parasite in the induction of carcinogenesis. This promising model could facilitate the study of host-pathogen interactions and the investigation of the process involved in Cryptosporidium-induced cell transformation.

Background: IgG concentrations in cerebrospinal fluid generally range from 20 to 45 mg/L. In multiple sclerosis immune reactions lead to intrathecal synthesis of specific IgGs that can be detected in biological fluid samples both quantitatively and qualitatively by isoelectric focusing of supplementary oligoclonal IgG bands., Method: A simple tool, using the MATLAB application, to facilitate and improve isoelectric focusing profile analysis is presented and evaluated in terms of its sensitivity, repeatability and reproducibility. A comparison between human readers and semi-automatic method has also been performed., Results: Results from the semi-automatic method were found to be equivalent or superior to generally employed laboratory methods. Repeatability analysis for semi-automatic processing yielded coefficients of variation (CVs) in the 3-7% range, and using a sample with an estimated IgG concentration of 200 mg/L, four bands were still visible after dilution to 5 mg/L, corresponding to band concentrations of 1.1-1.6 mg/L. Discordances between visual inspection and automatic analysis only appear at threshold levels for interpretation (the gray zone)., Conclusion: The semi-automatic method has acceptable performance for routine implementation.

Premature rupture of membranes (PRM) affects 5 to 15% of pregnancies, leading to prematurity and neonatal infection. PRM can be identified by through various amniotic fluid proteins in vaginal secretions. The aim of this study is to compare two immunochromatographic tests based on the detection of insulin-like growth factor binding protein (IGFBP-1) and alpha-foeto protein (AFP) for one of the two tests in cervico-vaginal secretions. Two tests, Actim(®) Prom and Amnioquick(®) Duo were performed on 80 pregnant women with suspected PRM. Amnioquick(®) Duo allows the simultaneous detection of IGFBP-1 and AFP with an automated incubation and reading. The number of positive results is similar (Khi-deux = 0.173, p = 0.6773) for IGFBP-1 between the two tests and there is a good agreement (K = 0.621), with a proportion of negative results of 86%. The number of positive results for AFP is more important in comparison to IGFBP-1. Results positive/positive (Actim(®) Prom/Amnioquick(®)) for IGFBP-1 seems to be related to the time when tests have been performed, that is to say in the last weeks of pregnancy. In conclusion, both tests have similar performance, but there is a risk of false positive results with AFP, this can be explained by the presence of non-visible blood in samples. An automated incubation and reading allows a good reproducibility. Moreover, the computer data storage improve the post-analytical quality.

Regional pneumococcal observatories in region Centre, created in 1997, participate with the others pneumococcal observatories alongside the National Reference Center for Pneumococci and the Institut de Veille Sanitaire at the monitoring of the evolution of resistance of pneumococci to antibiotics in France. Between 1997 and 2007, 2427 strains of Streptococcus pneumoniae were isolated in part from cerebrospinal fluids, blood and middle ear fluid, from children and adults. The prevalence of pneumococci with a decreased susceptibility to penicillin (PDSP) decreased strongly in region Centre: 56.8 % in 2001, 39.6 % en 2007. These data are similar to the French national data over the same period., (Copyright (c) 2009 Elsevier Masson SAS. All rights reserved.)

There are several interferon-alpha (IFN-alpha) subtypes. Mechanism of disparity in biological effects among members of IFN-alpha subtypes remains unexplained. Biological activity of IFN-alpha is mediated in part by induction of intracellular antiviral proteins. We studied whether differences in biologic effects of IFN-alpha subtypes may rely on their antiviral protein inducing effect. Intracellular induction of MxA protein and anti-virus-induced cytopathic effect (CPE) activity of 11 IFN-alpha subtypes in human amnion WISH cells have been studied. MxA protein quantitation in cell lysates was performed by immunochemiluminescence assay and anti-virus-induced CPE activity was assessed by protection against vesicular stomatitis virus (VSV)-induced CPE. Range of MxA values was high when cells were treated with 10 and 100 IU/ml of each IFN-alpha subtype. Levels of MxA correlated with anti-VSV-induced CPE obtained with 10 IU/ml IFN-alpha subtype. Together our data show a disparity in MxA-inducing activity of IFN-alpha subtypes and suggest that differences in anti-VSV-induced CPE of IFN-alpha subtypes in WISH cells can be related to their different ability to induce MxA.

Objective: To look for serum factors detectable early after head injury and predictive of heterotopic bone formation., Patients and Methods: In this prospective study of a homogeneous population of 31 men with severe brain injury, blood samples were obtained 3 months after the accident, and levels of serum factors influenced by bone metabolism were compared between patients with and without heterotopic bone formation. As extensive fractures can influence serum factors, the patients without heterotopic bone formation were divided into two groups based on whether they had major fractures. Radionuclide bone scanning was used to validate patient classification., Results: The group with heterotopic bone formation had significantly higher serum alkaline phosphatase levels (P < 0.01) and significantly lower serum leptin levels (P < 0.01), as compared to the other two groups. Body mass index and serum creatinine were comparable in the three groups., Conclusion: Leptin may be associated with the development of heterotopic bone formation. The antiosteogenic effect of leptin mediated by hypothalamic neurons may be impaired by hypothalamic damage related to severe brain injury.

Interferon alpha (IFN-alpha) is synthesized in response to viral infections. MxA protein, induced specifically by IFN-alpha and beta, expressed in peripheral blood cells, is detected more consistently than circulating IFN-alpha in serum of patients with viral infections. Thus, activation of the IFN-alpha/MxA system can be used as additional marker of the presence of a virus in patients. Therefore MxA protein and IFN-alpha levels were measured in patients with multiple sclerosis (MS), a chronic neurological disease of unknown etiology, in order to investigate the possible role of viruses in the expression of this disease. The means of MxA values obtained by using an immunochemiluminescent assay were significantly higher in blood of patients with remitting (n = 197) or relapsing (n = 39) multiple sclerosis (MS) patients and in patients with viral infections than in blood from healthy controls (n = 25) and from patients with bacterial infections (n = 12). Intra-individual variance in MxA levels in seven clinically stable remitting patients with MS was observed in the course of a follow-up, and high MxA levels were detected in three of them in blood samples collected consecutively over several months. By using an ultra sensitive assay, a higher MxA-inducer activity was obtained with sera from MS patients (n = 39) than with those from healthy controls (n = 12). Experiments with neutralizing antibodies proved that this activity in serum from patients was due to IFN-alpha, whereas IFN-alpha could not be detected by other methods. Altogether these results demonstrate that there is an activation of the IFN-alpha/MxA system in MS patients, which is consistent with the hypothesis that a viral infection may be associated with MS., (Copyright 2001 Wiley-Liss, Inc.)

Coxsackieviruses B (CVB) (B1-B6), positive-strand RNA viruses, cause a variety of diseases. CVB4 may have a causal role in insulin-dependent diabetes mellitus. IFN-alpha inhibits CVB replication; however, the mechanism is not well known. The interferon-alpha-inducible human MxA protein exerts an antiviral activity against negative-strand RNA viruses and against Semliki Forest virus, a positive-strand RNA virus. To test the antiviral spectrum of MxA against CVB4, we took advantage of stably transfected Vero cells expressing MxA (Vero/MxA) in 98% of cells. Compared with control cells, in Vero/MxA cells, CVB4 yields were dramatically reduced and expression of the VP1 CVB protein analyzed by immunofluorescence was highly restricted. Furthermore, the accumulation of positive- and negative-strand CVB4 RNA was prevented as shown by in situ hybridization and RT-PCR. These results indicate that the antiviral activity of MxA extends to CVB4 and that its replication cycle is inhibited at an early step in Vero/MxA cells., (Copyright 2001 Academic Press.)

A Herpes Consensus allows the simultaneous detection of 6 human herpesviruses: herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), human cytomegalovirus (HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), and human herpes virus 6 (HHV-6). This technique was used first to examine retrospectively 100 DNA extracts from 95 CSF and 5 aqueous fluids, prepared by treatment by saturated NaCl followed by ethanol precipitation (n = 63) or by simple boiling (n = 37) and stored at -80 degrees C, and secondly to test prospectively 38 CSF samples for which two DNA extracts were prepared with commercially available DNA extraction kits. In all cases, the results were compared with those of an "in-house" PCR. Concordant results between both PCR and the Herpes Consensus techniques were obtained in 61 of 63 DNA extracts prepared by treatment by saturated NaCl (97%) and in only 31 of 37 boiled samples (84%). Both commercially available methods of DNA extraction examined appear to be suitable for Herpes Consensus PCR, although they cannot remove completely PCR inhibitors that must be sought in case of negative results. This preliminary study shows that the Herpes Consensus method should be of value for rapid diagnosis of herpesvirus infections on condition that it is performed on purified DNA extracts.

Capillary blood of febrile children was lysed by using a lysis buffer containing ascorbic acid. MxA quantitation was performed by an immunochemiluminescent assay. The MxA values were significantly higher in capillary blood of infants with viral infections due to adenovirus (n = 5), rotavirus (n = 15), or respiratory syncytial virus (n = 28), than in capillary whole blood from infants with bacterial infections (n = 6) and healthy control patients (n = 20). A strong correlation was found between the MxA values in capillary whole blood and peripheral whole blood (r' = 0.86, P < 0.0001, n = 48). The MxA values found at these two sites were compared with the levels of IFN-alpha obtained by a dissociation enhanced lanthanide fluoroimmunoassay. A correlation between these two values was found. The results show that the combination of collection of blood by finger prick and specific immunochemiluminescent assay for MxA protein measurement may be of value for the diagnosis of viral infections in children., (Copyright 1999 Wiley-Liss, Inc.)

Type I interferon system is an important part of host's innate defense mechanisms against viral infections. The type I interferons mediate in part their antiviral effect via induction of various proteins. Among them the most widely known are 2'-5' oligoadenylate synthetase (2'-5' OAS) and a protein kinase (PKR). MxA, an other antiviral protein, is specifically induced by the type I interferons. The MxA protein contains the dynamin signature, which is implicated in transport processes. The MxA protein appears to block the replication of certain viruses at poorly defined steps. There are substantial differences in the antiviral activity of MxA between virus types. Indeed, the replication of vesicular stomatitis virus and influenza virus is inhibited by MxA, but not the one of type I herpes simplex virus. Measurements of interferon alpha and MxA levels may be of high value in clinical practice. Interferon alpha can be detected by using a bioassay based on the interferon alpha ability to protect cultured cells from the cytopathic effect caused by a selected challenged virus, or by using immunological techniques. The current bioassays are the most sensitive methods but they are cumbersome and lengthy, even though simplifications have been proposed. Immunological techniques are easier, however they do not explore the biological activity of the circulating interferon. The presence of type I interferon in biological samples (serum, plasma, cerebro-spinal fluid, cultured cell supernatants) can be indirectly assessed by capability of interferon alpha to induce in vitro the synthesis of MxA in a dose dependent manner in cultured cells. Following to the lysis of the cells, the induced MxA can be quantitated and hence the type I-interferon concentration can be determinated in samples. The quantitation of MxA protein in peripheral blood lysates can be useful as a specific marker of acute viral infections. A minute amount of whole blood (15 mul) is sufficient which facilitates its use in pediatrics. The specifically type-I-interferons inducible MxA protein is also a potential useful marker in the management of interferon alpha-treatment. Moreover, the detection of interferon alpha and antiviral proteins constitute an indirect approach for investigating the hypothesis of the role of viruses in chronic diseases with suspected infectious aetiology.

We studied the in vitro HIV-1 antigen-stimulated production of IFN-gamma and IL-4 in HIV-1-infected patients and its relationship with viral replication as assessed through the plasma level of HIV-1 RNA. The levels of IFN-gamma and IL-4 were higher in supernatants of stimulated whole blood cultures than in stimulated peripheral blood mononuclear cell cultures, therefore whole blood cultures were used in the rest of the study. Specific IFN-gamma and IL-4 responses to HIV-1 p24 antigen were observed in HIV-1-infected patients but not in healthy controls (n = 23). A lower proportion of individuals with a positive IFN-gamma response to HIV-1 p24 was observed in patients at a declining clinical stage: 62% in asymptomatic patients (CDC group A, n = 16) versus 19% in symptomatic patients (CDC groups B and C, n = 21; P = 0.007, chi2 testing), whereas the proportion of individuals with a positive IL-4 response to HIV-1 p24 was almost similar in both groups of patients (25% versus 23.8%). Increased IL-4 production by HIV-1 p24-activated immunocompetent cells of patients and a predominant IL-4 response to HIV-1 p24 (with IL-4/IFN-gamma > 1) were positively correlated with an increased viral load. In contrast, there was no correlation between the mitogen-stimulated production of IL-4 and IFN-gamma and the viral load in plasma. The CD8 T cells from whole blood of patients, but not from controls played a significant role in the HIV-1 p24-activated production of IFN-gamma and IL-4. In conclusion, HIV-1-antigen-stimulated whole blood appears to be a valuable tool to study the production of IL-4 in HIV-1-infected patients. The cytokine profile pattern in response to epitopes of HIV-1 gag p24 may play an important role in the host immune response to HIV-1.

We investigated the biological properties of interferon (IFN)-alpha produced by Sendai virus (SV)-activated whole blood cultures in 20 patients infected with human immunodeficiency virus (HIV)-1 and 24 healthy controls. Supernatants of cultures were assayed for IFN-alpha by using an immunological method (DELFIA), biological methods and an in-vitro MxA induction assay. The levels of intracellular MxA protein were detected by an immunochemiluminescence assay. The levels of IFN-alpha in patients measured by DELFIA were significantly lower than those in healthy controls (P < 0.0001), but the antiviral activity of IFN-alpha in patients infected with HIV-1 was lower than predicted from DELFIA. The IFN-alpha produced by cells of patients infected with HIV-1 was able to induce MxA protein in human amnions WISH cells but was unable to protect these cells against Vesicular Stomatitis Virus (VSV)-induced cytopathic effects. A relative increased capability to induce the production of MxA protein in vitro was observed with the IFN-alpha contained in culture supernatant of virus-activated whole blood of HIV-1-infected patients with increased levels of MxA in their peripheral blood. These data suggest that biological properties of IFN-alpha produced in the course of HIV-1 infection are different from those observed with IFN-alpha of healthy subjects.

The immune response against viral pathogens include specific and non specific mechanisms. Cytokines are peptides which can play a role in the non specific immunity. Type I interferons (IFNalpha/beta) are the most effective antiviral cytokines. Interferons alpha are represented by a large familly of structurally related genes while the IFNbeta is encoded by a single gene. Type I interferon genes are located on the chromosome 9, and are segregated in a "modern" and an "ancestral" group with distinctive effects onto the cells. Type I interferons consist of 5 alpha helices, 4 of which are closely held together. Type I interferons receptor is a member of the class II cytokine receptor familly. It is a heterodimer composed of polypeptidic chains naimed IFNAR-1 and IFNAR-2. The type I interferons inducers are viruses, inactivated viruses and certain synthetic molecules. The molecular mechanisms of the induction of IFNbeta have been extensively described whereas the factors that play a role in the regulation of IFNalpha are not so well characterized. Two regulating domains located on the IFNbeta gene promotor are involved. The activation of these regulating domains leads to adverse effects by interacting with two intracellular factors (IRF1 and IRF2). IRF1 enhances the synthesis of IFNbeta but other pathways may be involved as well. The different IFNalpha sub-types have not only synergistic but also antagonistic effects. The synthesis of the different subtypes depends on cell lines and IFNalpha inducers. The N terminal part of IFNalpha is a major determinant in the diversity of the biological effects of IFNalpha sub-types which probably involve changes of the conformation of the type I receptor resulting from interactions between the receptor and its ligand. The type I interferons confer resistance to viruses, especially by enhancing the synthesis of intracellular antiviral proteins.

The value of soluble receptor for tumor necrosis factor type II (sTNFRII) as a strong and early predictor of HIV disease progression was suggested. Recently it has been reported that sTNFRII may provide an indication of the HIV load. In this work we focused on the relationship between sTNFRII and HIV burden in 95 HIV-1+ patients without AIDS grouped according to the 1993 classification of the CDC as group A, n = 55, and group B, n = 40. Compared with healthy controls, higher values of sTNFRII were obtained in all groups of HIV-1 infected patients (P < 0.001), but we found no inverse correlation between sTNFRII and CD4+ lymphocyte counts in CDC group A and B of the disease, and no correlation with log RNA copy number in patients with CD4 T-cell counts > 499/microl. A correlation was obtained between sTNFRII and the viral load in patients with CD4 T-cell counts ranging from 200 to 499/microl, but only in CDC group B patients (P < 0.01, n = 26). There was no correlation between the variations of sTNFRII and HIV-1 RNA levels in 19 CDC group A and 15 CDC group B clinically stable patients in the course of a short follow up. The plasma level of sTNFRII do not appear as a valuable surrogate marker of the plasma level of HIV-1 RNA in patients. Further investigations are needed to define the mechanism of the raised level of sTNFRII in HIV-1 infected patients.

Interferon-alpha (IFN-alpha) is an important molecule in the antiviral response, but cells from HIV-1-infected individuals show a reduced ability to secrete IFN-alpha. We investigated an association between an imbalance of type 1/type2 cytokines and the production of IFN-alpha in HIV-1 infection. We used whole blood culture to study the cytokine production profile, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), in response to HIV-1 antigens and to study the Sendai Virus and HSV-1-induced-production of IFN-alpha in seven HIV-1-infected patients. An impaired synthesis of IFN-alpha was obtained in patients with a predominant IL-4 production (IL-4 > IFN-gamma), and we found a positive correlation between the ex vivo production of IFN-alpha and the IFN-gamma/IL-4 ratio but not with the HIV RNA copy number in plasma. We investigated the role of T-cell-derived cytokines in the in vitro production of IFN-alpha by PBMC from eight healthy donors, activated with Sendai Virus or HSV-1. Whereas type 2 cytokines (IL-4, IL-13) inhibited virus-induced IFN-alpha synthesis, on the contrary, type 1 cytokines (IL-2, IFN-gamma) enhanced it. A disarray in the T-cell-derived cytokine response may play a role in the defect of IFN-alpha production in HIV-1-infected individuals. Further investigations are needed to explore this hypothesis.

Interferon alpha (IFNalpha), a type I interferon, can be considered as a viral infection marker because this cytokine is induced during many viral infections. However, it is quite difficult to detect IFNalpha in sera. Investigations are interested in various intra-cellular IFNalpha-induced proteins as viral infection markers. However the activity of these enzymes increased not only in response to type I IFNs but also to type II IFN. MxA protein can be detected in the cytoplasm of IFNalpha/beta-treated cells, whereas other cytokines, including IFNgamma, are poor inducers. Using an immunochemiluminescent assay, we studied MxA protein in whole blood of 34 patients with various viral infections. The whole blood was drawn into sterile vacuum tubes containing heparin or EDTA. MxA values were relatively similar in heparin-treated samples and EDTA-treated samples, with differences not exceeding 1 ng/ml. The levels of MxA protein were compared in whole blood obtained by using two different lysis procedures. A correlation was found between the MxA levels obtained by using procedure I and procedure II, but higher amounts of MxA protein were found with procedure II. The second procedure is rapid and more convenient than the other and it is carried out in one step which reduce technical problems. High levels of MxA protein were found in peripheral blood cells of patients with acute viral infections (Rotavirus, Adenovirus, RSV, CMV), but MxA protein was not elevated in bacterial infections. The MxA levels were also studied in peripheral blood of 32 HCV positive patients. MxA protein was not found in most of IFNalpha-untreated patients, even those with high viral load. In contrast, high levels of MxA protein were found in IFNalpha-treated patients. MxA quantitation can be considered as a specific marker of acute viral infections, and could be useful in the management of treatment with IFNalpha.