Cultural Adaptation and Validation of the Modified Checklist for Autism in Toddlers, Revised with Follow-up in Moroccan Arabic dialect.

BACKGROUND: The prevalence of autism spectrum disorder is increasing worldwide, making screening and early intervention necessary. Several screening instruments have been developed in recent years. The Modified Checklist for Autism in Toddlers Revised with Follow-up (M-CHAT-R/F) is considered to be one of the specific measures designed to identify toddlers at risk for autistic spectrum disorder. OBJECTIVE: The aim of the study was to translate and adapt the original version of M-CHAT-R/F from the English to the Moroccan Arabic language. STUDY DESIGN: Specialized translators and clinicians ensured forward and backward translation of the scale into Moroccan Arabic. Then, a two-stage screening of the M-CHAT-R/F-T was applied to a study sample comprised of 56 toddlers with autistic spectrum disorder (category I) and 96 toddlers with normal development (category II). "Kappa test", "Cronbach's alpha" test, the intra class correlation coefficient, and the area under the curve were determined. RESULT: The average score results of M-CHAT-R/F were 13.12 for category I, while it was 2.24 for category II. The Cronbach's alpha coefficient of the checklist was 0.929. The kappa values ranged from k=0.78 to k=0.97 with a confidence interval of 95% indicating good convergence. The intra-class correlation coefficient ranged from 0.97 to 0.99, which is excellent. The area under the curve in our study was 0.988, an excellent result. CONCLUSION: Efficiency of the Moroccan Arabic version of the MCHAT was demonstrated for screening in the general population.

Stigmatizing attitudes and social perception towards mental illness among Moroccan medical students.

OBJECTIVES: This study aimed to explore medical students' attitudes, social perception, and knowledge towards mental illness and identify the associated factors. METHODS: A cross-sectional study was conducted in the Medical Faculty of Fez. A representative sample of 420 Moroccan students from the first to the seventh years was selected randomly during the academic year (2018/2019). They completed the Mental Illness Clinicians Attitudes scale (MICA) and a self-questionnaire exploring sociodemographic data, the university course, social perception and knowledge towards mental illness, and the potential consideration of psychiatry as a career. RESULTS: The average age of participants was 21.73 years (SD ±2.60), and 72.4% (n=304) of respondents were female (M/F gender ratio=0.39). The participants held stigmatizing attitudes and negative social perceptions towards mental illness, as indicated by the high score of the MICA scale (mean=57.24; SD ±9.95). Females (p=0.02) and married students (p=0.02) showed significantly more tolerant attitudes. The attitudes of students (p=0.37) who completed the psychiatry clerkship were slightly more favorable (p=0.15). There was no significant difference in attitudes according to the level of study (p=0.06). Students with a lower socioeconomic level tended to be less stigmatizing (p=0.08). The assessment of knowledge about mental illness among students objectified major gaps. A total of 17.9% (n=75) considered psychiatry as a career. CONCLUSION: The attitudes of medical students in this study were stigmatizing, and this should without delay motivate Moroccan research, educational and health authorities to investigate further scientific research in this area to address these attitudes and remedies.

Clinical profile of schizophrenia comorbid with obsessive-compulsive symptoms: A comparative study.

OBJECTIVE: Describe and compare the clinical profile of schizophrenic patients with and without obsessive-compulsive symptoms and obsessive-compulsive disorder (OCD) patients. METHODS: A descriptive and analytical cross-sectional study was carried out at the psychiatry department of Hassan II University Hospital in Fez over 12 months to compare three groups of patients: "schizo-obsessive" (n=32), "schizophrenia" (n=34), and "OCD" (n=46). All participants (n=112) were assessed using the mini-international neuropsychiatric interview (MINI), the Yale-Brown obsessive-compulsive scale (Y-BOCS), the Brown assessment of beliefs scale (BABS), the Hamilton anxiety rating scale (HAM-A), the Beck's depression inventory (BDI-II), the positive and negative syndrome scale (PANSS), and the clinical global impressions-severity scale (CGI-S). RESULTS: The "schizo-obsessive" group differed from the "schizophrenia" group in: more severe psychotic symptoms (mean=64.16±17.049, P<0.001), higher anxiety (mean=8.87±5,655, P<0.001) and depression (mean=7.50±5.989, P<0.001) scores, more prevalent suicide attempts (46.9%), higher illness severity score (mean=5.13±1.157, P=0.02), and more professional disinsertion (78.1%). The "schizo-obsessive" group (mean= 14.47±3.388) had significantly poor insight (P<0.001) compared to the "OCD" group (mean= 8.35±4.542). There were similarities in the obsessive and compulsive themes between the "schizo-obsessive" and the "OCD" groups, with no significant difference of severity (P=0.26). CONCLUSION: A careful assessment of obsessive symptomatology is essential in schizophrenia for better patient management and prognosis.

[Clinical presentation, co-occurrence and suicidality in psychiatric emergency patients with psychogenic non-epileptic seizures]. / Expression clinique, comorbidités psychiatriques et suicidalité chez des patients avec crises psychogènes non épileptiques.

INTRODUCTION: Non-epileptic psychogenic seizures (PNES) are a common condition but little studied in psychiatry. Our study is the first Moroccan of this topic. Our aim was to describe their clinical presentation, comorbidities and suicidality. METHOD: It was a one-year recruitment study conducted in the Department of Psychiatry at the University Hospital of Fez. We collected socio-demographic data. Psychometric evaluation used the MINI criteria for different psychiatric disorders, personality disorders and suicidality, the Hamilton anxiety scale, the Beck depression scale and the Childhood Trauma Questionnaire. RESULTS: Thirty-two patients were included, the majority of whom were female with a low economic level (89 %). The average age of onset average was 23.97 years. The generalized form represented 71.9 % of cases with a triggering factor present in 46 %. The duration of the seizures average was 23.47minutes, and 37.5 % of the patients had at least one attack per day. Among patients, 68.8 % had comorbidity: major depressive disorder MDD (46 %) and post-traumatic stress disorder PTSD (18.6 %). Suicide risk was rated as moderate or severe in 12.5 % of patients, and 40.62 % had a previous suicide attempt. A personality disorder was present in 18.8 % of cases. Finally, a history of child abuse was found in 59.4 % of patients and was correlated to PNES age of onset and co-occurrence depression. CONCLUSION: We found several similarities with previous studies in terms of clinical description and co-occurrences. However, our sample is characterized by more prolonged and more frequent seizures and a higher rate of suicidality.

[The role of the general practitioner in management of psychiatric disorders]. / Le rôle du médecin généraliste dans la prise en charge des pathologies psychiatriques.

AIM: Psychiatric disorders represent a huge burden of disease worldwide. Significant gaps in their diagnosis and treatment remain. In Morocco, an observed shortage of psychiatrists would logically call for their collaboration with family physicians. Our objectives were to analyse perceptions of general practitioners' capacity to manage psychiatric disorders, to study the difficulties encountered in their management of these pathologies, and to assess their degree of collaboration with existing mental health structures. METHODS: A cross-sectional nationwide survey of family physicians practicing in urban and rural areas in the private and public sectors in the Kingdom of Morocco was organised. Carried out over a period of 10 months (April 2016 to January 2017), the study was based on an anonymised self-administered questionnaire comprising 23 items. Were excluded from the sample: physicians who had tested the questionnaire prior to its distribution, medical students, family physicians employed in administrative services, resident doctors in university hospitals, and questionnaires with incomplete answers. RESULTS: All in all, five hundred and four questionnaires were distributed, with a participation rate of 79.8%, while 57.9% of the respondents were men. Their seniority as doctors ranged from 1 year to 60 years with average duration of 19.5±11 years. An overwhelming majority of the doctors had obtained their doctorates in Morocco, while 8.4% had been trained abroad. The majority of the doctors surveyed worked in private practice in urban areas (53.1%), and most of them (81.9%) agreed that the management of psychiatric disorders is a public health priority. The participating physicians stated that they received an average of five patients per day suffering from mental disorders, representing 17.3% of their medical consultations. According to their perceptions, the most frequent psychiatric pathologies were depression, somatoform disorders and sleep disturbances, which were cited by 92.9%, 78.8% and 78.5% of them respectively. Regarding the diagnosis of psychiatric pathologies, 60.2% of the doctors considered bipolar disorder as a difficult pathology to diagnose, followed by schizophrenia, autism and dementia, while the pathologies reported as the most difficult to treat were addictive behaviours, schizophrenia, bipolar disorder, autism and dementia. Physicians felt a need for training, primarily pertaining to anxiety disorders, schizophrenia and bipolar disorders (49.5%, 35% and 33.7%, respectively). Nearly a quarter of the respondents (22.4%) mentioned a need for training in management of all psychiatric illnesses. Regarding the reasons for their referral of patients to mental health care structures, 65.4% of the physicians justified their doing so by a need for hospitalisation, while in 43.7% of cases, the transfer was carried out at the request of the patient or his family. The difficulties mentioned by the respondents were firstly a lack of adapted means of care (77.5%), followed by their insufficient training in mental healthcare (52.4%) and absence of collaboration with mental health professionals (52.4%). CONCLUSION: For the majority of the physicians surveyed, psychiatry represented a public health priority, and their prioritising provides a probable explanation for their positive perception of their role in the management of mental disorders. However, we went on to observe that a number of difficulties constituted obstacles to their role in management of mental disorders and induced them to refer their patients to psychiatrists. Our analysis highlighted the impact of continuing education on physicians' attitudes and the interest of collaboration between practitioners. Efforts in university teaching and continuous education should be encouraged in view of enabling family physicians to more effectively cope with the demands of daily practice and meet the expectations of the overall population.

[Reversible catatonia after the abrupt discontinuation of clozapine: Case report]. / Une catatonie réversible suite à l'arrêt brutal de la clozapine. À propos d'un cas.

In this paper, we report the case of a patient, aged 26, with schizophrenia who was admitted to psychiatric emergencies for catatonia, one week after abrupt discontinuation of clozapine. An improvement was seen after only two days of the reintroduction of clozapine alone. This catatonia is reversible and it responds magnificently to the reintroduction of clozapine. And we conclude that patients and their caregivers need to be educated about the effects of abrupt cessation of clozapine administration.

Psychoactive substances use and associated factors among middle and high school students in the North Center of Morocco: a cross-sectional questionnaire survey.

BACKGROUND: Data on psychoactive substance (PAS) consumption among adolescents in the North Center of Morocco are not at all available. Therefore, the current study aimed at investigating the prevalence and the determinants of psychoactive substances use among middle and high school students in this region. METHODS: A cross-sectional study was conducted from April 2012 to November 2013 in public middle and high schools in the North Central Region of Morocco. An anonymous self-administered questionnaire was used to assess psychoactive substances use among a representative sample of school students from the 7th to the 12th grade, aged 11-23 years, selected by stratified cluster random sampling. Factors associated with psychoactive substance use were identified using multivariate stepwise logistic regression analyses. RESULTS: A total of 3020 school students completed the questionnaires, 53.0 % of which were males. The overall lifetime smoking prevalence was 16.1 %. The lifetime, annual and past month rates of any psychoactive substance use among the study subjects were 9.3, 7.5, and 6.3 % respectively. Cannabis recorded the highest lifetime prevalence of 8.1 %, followed by alcohol 4.3 %, inhalants 1.7 %, psychotropic substances without medical prescription 1.0, cocaine 0.7, heroine 0.3, and amphetamine with only 0.2 %. Psychoactive substance use was associated with males more than females. The risk factors identified by multivariate stepwise logistic regression analyses were being male, studying in secondary school level, smoking tobacco, living with a family member who uses tobacco, and feeling insecure within the family. CONCLUSIONS: The prevalence among all school students reported by the current study was comparable to the national prevalence. Efforts to initiate psychoactive substance prevention programs among school students should be made by designing such programs based on the significant factors associated with psychoactive substance use identified in this study.

Suicidal behaviors among Moroccan school students: prevalence and association with socio-demographic characteristics and psychoactive substances use: a cross-sectional study.

BACKGROUND: Suicidal behavior is a major cause of injury and death worldwide, especially among adolescents and young adults. Few studies have tackled this issue in the Arab world. The present study investigated the prevalence and the risk factors of suicidal behaviors among Moroccan school students. METHODS: From April 2012 to November 2013, a cross-sectional study was conducted in the North-Centre region of Morocco among students in public secondary schools selected using stratified cluster random sampling. The data were collected via anonymous self-administered questionnaires. The Mini International Neuropsychiatric Interview was used in its Moroccan Colloquial Arabic version to assess suicidality according to the DSM-IV criteria. RESULTS: A total of 3020 students (53 % boys) aged 11-23 years (average age = 16 ± 2.1 years) were included in the study. The prevalence of suicide ideation, suicide planning and suicide attempts during the last month were 15.7, 6.3, and 6.5 % respectively. Univariate analyses demonstrated that suicidal behaviors followed different epidemiological patterns. According to the multivariate analyses, the risk factors for all suicidal behaviors among Moroccan school students were the female gender, middle school level, urban locations, low family income, parents' divorce, tobacco consumption and psychoactive substances (alcohol and cannabis) use. CONCLUSIONS: The intervention of preventive programs has become an emergency to overcome the issue of suicidality in Morocco. Further researches on adolescents' suicidal behaviors are suggested to update temporal data and assess the effectiveness of potential interventions.

Odocoileus hemionus deer adenovirus is related to the members of Atadenovirus genus.

The Odocoileus hemionus deer adenovirus (OdAdV-1) causes systemic and local vasculitis and proves extremely lethal for mule deer. To characterize the virus, part of the genome flanking the fiber gene was cloned and sequenced. The sequence revealed two open-reading frames that mapped to pVIII hexon-associated protein precursor and fiber protein of several other adenoviruses. The highest amino acid homology for pVIII and fiber was found with the members of the proposed Atadenovirus genus: ovine adenovirus isolate 287 (OAdV-287), bovine adenovirus 4 (BAdV-4) and duck adenovirus 1 (DAdV-1). The homology with bovine adenovirus type 3 (BAdV-3) proved low. The E3 region was not found between the gene for pVIII and fiber. These data suggest that OdAdV-1 is a member of the Atadenovirus genus.

Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease.

Since targeting of recombinant adenovirus vectors to defined cell types in vivo is a major challenge in gene therapy and vaccinology, we explored the natural diversity in human adenovirus tissue tropism. Hereto, we constructed a library of Ad5 vectors carrying fibers from other human serotypes. From this library, we identified vectors that efficiently infect human cells that are important for diverse gene therapy approaches and for induction of immunity. For several medical applications (prenatal diagnosis, artificial bone, vaccination, and cardiovascular disease), we demonstrate the applicability of these novel vectors. In addition, screening cell types derived from different species revealed that cellular receptors for human subgroup B adenoviruses are not conserved between rodents and primates. These results provide a rationale for utilizing elements of human adenovirus serotypes to generate chimeric vectors that improve our knowledge concerning adenovirus biology and widen the therapeutic window for vaccination and many different gene transfer applications.

Treatment of malignant gliomas with a replicating adenoviral vector expressing herpes simplex virus-thymidine kinase.

We evaluated the interaction between oncolytic, replication-competent adenoviral vectors and the herpes simplex virus-1 thymidine kinase (HSV1-tk) gene/ganciclovir (GCV) suicide system for the treatment of malignant gliomas. We constructed a panel of replication-competent adenoviral vectors in which the luciferase (IG.Ad5E1(+). E3Luc) or HSV1-tk gene (IG.Ad5E1(+).E3TK) replace the M(r) 19,000 glycoprotein (gp19K) coding sequence in the E3 region. IG.Ad5E1. IG.Ad5.ClipLuc and IG.AdApt.TK are E1-deleted viruses that contain the luciferase or the HSV1-tk gene in the former E1 region driven by the human cytomegalovirus promoter. IG.Ad5. Sarcoma 1800HSA.E3Luc contains an irrelevant gene in the E1 region, whereas the gp19K coding sequence in the E3 region is replaced by the luciferase gene as in the replicating virus IG.Ad5E1(+).E3Luc. For in vitro experiments, we used a panel of human glioma cell lines (U87 MG, T98G, A172, LW5, and U251), a rat gliosarcoma cell line (9 L), and human lung (A549) and prostate carcinoma (P3) cell lines. In vitro, GCV sensitivity (10 microg/ml) was studied in U87 MG cells after infection at a multiplicity of infection of 1 and 10. A s.c. U87 MG glioma xenograft model was established in NIH-bg-nu-xid mice. Tumors of 100-150 mm(3) were treated with a single injection of adenovirus 10(9) IU suspended in 100 microl of PBS, and GCV 100 mg/kg was administered i.p. twice daily for 7 days. The cytopathic effect of all three replication-competent adenoviral vectors was similar to the cytopathic effect of wild-type adenovirus 5 on all human cell lines tested, indicating that deletion of the E3 gp19K sequences did not affect the oncolytic effect of the vectors. In vitro, luciferase expression was the same for both E1-deleted vectors (IG.Ad5.ClipLuc and IG.Ad5. Sarcoma 1800HSA.E3Luc), demonstrating the strength of the internal E3 promoter even in the absence of E1A. However, in vitro expression levels obtained with replication-competent IG.Ad5E1(+). E3Luc were 3 log higher (allowing infection with a 2-3-log lower multiplicity of infection) in the human cell lines. In U87 MG glioma cells, the oncolytic effect of replication-competent IG.Ad5E1(+).E3TK was significantly enhanced by the addition of GCV and greatly exceeded the cytotoxicity of replication-incompetent IG.AdApt.TK combined with GCV. In established s.c. U87 MG glioma xenografts, a single injection of IG.Ad5E1(+).E3TK resulted in a significant slowing of tumor growth and prolonged survival compared with injection of IG.AdApt.TK. Addition of GCV slowed tumor growth, further adding to survival. In conclusion, the oncolytic effect of replicating adenoviral vectors and HSV1-tk/GCV have potent antitumor effects in gliomas. When combined, these two approaches are complementary, resulting in a significantly improved treatment outcome. In addition, replication-competent adenoviral vectors missing the E3 gp19K coding sequences, have oncolytic efficacy comparable with wild type. In combination with high expression levels obtained with the natural E3 promoter, such vectors are promising new anticancer agents.

Highly efficient transduction of human monocyte-derived dendritic cells with subgroup B fiber-modified adenovirus vectors enhances transgene-encoded antigen presentation to cytotoxic T cells.

The efficiency of dendritic cells (DC) as immunotherapeutic vaccines critically depends on optimal delivery of target Ags. Although DC modified by subgroup C type 5 recombinant adenoviruses (rAd5) provide encouraging results, their clinical application is hampered by the need for high viral titers to achieve sufficient gene transfer, due to the lack of the Ad5 fiber receptor. We now demonstrate that rAd5 carrying subgroup B Ad fibers are up to 100-fold more potent than classical rAd5 for gene transfer and expression in human DC, rAd5 with a type 35 fiber (rAd5F35) being the most efficient vector. This improvement relates to a greater and faster virus entry and to an increased transgene expression especially following DC maturation. Furthermore, these new vectors possess enhanced synergistic effects with other activation signals to trigger DC maturation. Consequently, rAd5F35-infected DC engineered to express the gp100 melanoma-associated Ag largely exceed rAd5-infected DC in activating gp100-specific CTL. Finally, the DC infection pattern of rAd5F35 is fully conserved when DC are in the vicinity of primary skin-derived fibroblasts, suggesting this vector as a candidate for in vivo targeting of DC. Thus, subgroup B fiber-modified rAd5 constitute a major breakthrough in the exploitation of ex vivo rAd-targeted DC as clinically relevant vaccines and may also be suitable for in vivo genetic modification of DC.

Adenovirus-mediated interleukin 3 beta gene transfer by isolated limb perfusion inhibits growth of limb sarcoma in rats.

Cytokine gene transfer using (multiple) intratumoral injections can induce tumor regression in several animal models, but this administration technique limits the use for human gene therapy. In the present studies we describe tumor growth inhibition of established limb sarcomas after a single isolated limb perfusion (ILP) with recombinant adenoviral vectors harboring the rat IL-3 beta gene (IG.Ad.CMV.rIL-3 beta). In contrast, a single intratumoral injection or intravenous administration did not affect tumor growth. Dose-finding studies demonstrated a dose-dependent response with a loss of antitumor effect below 1 x 10(9) IU of IG.Ad.CMV.rIL-3 beta. Perfusions with adenoviral vectors bearing a weaker promoter (MLP promoter) driving the rIL-3 beta gene did not result in antitumor responses, suggesting that the rIL-3 beta-mediated antitumor effect depends on the amount of rIL-3 beta protein expressed by the infected cells. Furthermore, it was shown by direct comparison that ILP with IG.Ad.CMV.rIL-3 beta in the ROS-1 osteosarcoma model is at least as efficient as the established therapy with the combination of TNF-alpha and melphalan. Treatment with IG.Ad.CMV.rIL-3 beta induced a transient dose-dependent leukocytosis accompanied by an increase in peripheral blood levels of histamine. Leukocyte infiltrations were also histopathologically demonstrated in tumors after perfusion. These results demonstrate that ILP with recombinant adenoviral vectors carrying the IL-3 beta transgene inhibits tumor growth in rats and suggest that cytokine gene therapy using this administration technique might be beneficial for clinical cancer treatment.

Infection efficiency of type 5 adenoviral vectors in synovial tissue can be enhanced with a type 16 fiber.

OBJECTIVE: To obtain an adenoviral vector with increased infection efficiency in the synovial tissue compared with conventional vectors based on adenovirus serotype 5 (Ad5), without compromising the specificity of infection. METHODS: Coxsackie adenovirus receptor (CAR) expression was assessed in cultured synoviocytes. Chimeric adenoviruses based on Ad5 but carrying the DNA encoding the fiber of adenovirus from subgroup B (Adll, 16, 35) or D (Ad24, 28, 33, 45, or 47) were constructed and produced on PER.C6 cells. The gene transfer efficiency of these chimera was tested on cultured synoviocytes and peripheral blood mononuclear cells (PBMC). RESULTS: No surface expression of CAR protein was observed on synoviocytes. CAR messenger RNA expression of synoviocytes was found to be low. Of all fiber chimeric vectors tested, vectors carrying the fiber of Ad16 (Ad5.fib16) were most potent, yielding approximately150 times increased transgene expression in cultured synoviocytes compared with those of Ad5. Flow cytometry showed that the increase in transgene expression was caused by the transduction of higher percentages of synoviocytes and higher gene expression per synoviocyte. Experiments with 500 virus particles/cell of Ad5.GFP or Ad5.fib16.GFP resulted in an infection efficiency of 0.6% and 1% in PBMC and 43% and 76% in synoviocytes, respectively. CONCLUSION: Synoviocytes hardly express CAR, which hampers Ad5-mediated gene transfer. Ad5.fib16 is superior to Ad5 vectors for transducing synoviocytes, without compromising the specificity of infection. Our data suggest that Ad5.fib16-mediated gene transfer to synovial tissue improves the therapeutic window.

Improved adenovirus vectors for infection of cardiovascular tissues.

To identify improved adenovirus vectors for cardiovascular gene therapy, a library of adenovirus vectors based on adenovirus serotype 5 (Ad5) but carrying fiber molecules of other human serotypes, was generated. This library was tested for efficiency of infection of human primary vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Based on luciferase, LacZ, or green fluorescent protein (GFP) marker gene expression, several fiber chimeric vectors were identified that displayed improved infection of these cell types. One of the viruses that performed particularly well is an Ad5 carrying the fiber of Ad16 (Ad5.Fib16), a subgroup B virus. This virus showed, on average, 8- and 64-fold-increased luciferase activities on umbilical vein ECs and SMCs, respectively, compared to the parent vector. GFP and lacZ markers showed that approximately 3-fold (ECs) and 10-fold (SMCs) more cells were transduced. Experiments performed with both cultured SMCs and organ cultures derived from different vascular origins (saphenous vein, iliac artery, left interior mammary artery, and aorta) and from different species demonstrated that Ad5.Fib16 consistently displays improved infection in primates (humans and rhesus monkeys). SMCs of the same vessels of rodents and pigs were less infectable with Ad5.Fib16 than with Ad5. This suggests that either the receptor for human Ad16 is not conserved between different species or that differences in the expression levels of the putative receptor exist. In conclusion, our results show that an Ad5-based virus carrying the fiber of Ad16 is a potent vector for the transduction of primate cardiovascular cells and tissues.

Simultaneous detection of NOS-3 protein expression and nitric oxide production using a flow cytometer.

Nitric oxide (NO) is involved in the regulation of SMC proliferation during intimal hyperplasia as has been shown by the inhibitory effect on intimal hyperplasia of adenovirus-mediated ceNOS overexpression in injured arteries in pig. Good assays to quantify the NO-producing enzymes, i.e., NO synthases (NOS), are essential to analyze the mechanism of action of NO in this process. We have developed novel flow cytometric assays for the simultaneous detection of NOS-3 protein, using NOS-3 specific antibodies, and NO production using 4,5-diaminofluorescein-diacetate (DAF-2/DA). The presence of NOS-3 protein and NO production is demonstrated on human A549 and HepG2 cells infected with a NOS-3 adenovirus (Ad.NOS-3). A comparative study showed that the flow cytometric assays are equally sensitive as Western blot analysis, the citrulline assay, or the Sievers assay. On human endothelial and SMC, NOS-3 protein and NO production were simultaneously detected with the assays, both under basal conditions and after Ad.NOS-3transduction. Simultaneous analysis of NOS-3 protein and NO production, made possible by the here-described novel flow cytometric assays, is of significant value to those investigating NOS-3 and NO.

The influence of synovial fluid on adenovirus-mediated gene transfer to the synovial tissue.

OBJECTIVE: To determine the effect of synovial fluid (SF) from rheumatoid arthritis (RA) patients on adenovirus type 5 (Ad5)-mediated gene transfer to synoviocytes, and to explore new strategies for vector development based on the neutralization data obtained. METHODS: SF was derived from 63 randomly selected R4 patients. Ten samples were used to study the effect of SF on Ad5-mediated gene transfer in synoviocytes. IgG and <100-kd fractions were purified from these 10 SF, and their effect on gene transfer was determined. Neutralizing activity against wild-type Ad5 (wt-Ad5), wt-Ad26, wt-Ad34, wt-Ad35, and wt-Ad48 was tested in the SF from the remaining 53 patients. RESULTS: Seven of 10 SF samples inhibited Ad5-mediated gene transfer. Purified antibodies exhibited inhibition patterns similar to those seen with unfractionated SF. In 5 of 10 SF samples, low molecular weight fractions inhibited gene transfer at low dilutions. Neutralization of wt-Ad35 by SF from RA patients was less frequent than neutralization of other wt-Ad tested (4% versus 42-72%; n = 53). CONCLUSION: SF from 70% of the RA patients contained neutralizing antibodies that hamper Ad5-mediated gene transfer to synoviocytes. The activity of neutralizing antibodies may be circumvented in the majority of RA patients when vectors based on an Ad35 backbone are used.