A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4

An acceptable live-attenuated dengue virus vaccine candidate should have low potential for transmission by mosquitoes. We have identified and characterized a mutation in dengue virus type 4 (DEN4) that decreases the ability of the virus to infect mosquitoes. A panel of 1248 mutagenized virus clones generated previously by chemical mutagenesis was screened for decreased replication in mosquito C6/36 cells but efficient replication in simian Vero cells. One virus met these criteria and contained a single coding mutation: a C-to-U mutation at nucleotide 7129 resulting in a Pro-to-Leu change in amino acid 101 of the nonstructural 4B gene (NS4B P101L). This mutation results in decreased replication in C6/36 cells relative to wild-type DEN4, decreased infectivity for mosquitoes, enhanced replication in Vero and human HuH-7 cells, and enhanced replication in SCID mice implanted with HuH-7 cells (SCID-HuH-7 mice). A recombinant DEN4 virus (rDEN4) bearing this mutation exhibited the same set of phenotypes. Addition of the NS4B P101L mutation to rDEN4 bearing a 30 nucleotide deletion (Delta30) decreased the ability of the double-mutant virus to infect mosquitoes but increased its ability to replicate in SCID-HuH-7 mice. Although the NS4B P101L mutation decreases infectivity of DEN4 for mosquitoes, its ability to enhance replication in SCID-HuH-7 mice suggests that it might not be advantageous to include this specific mutation in an rDEN4 vaccine. The opposing effects of the NS4B P101L mutation in mosquito and vertebrate systems suggest that the NS4B protein is involved in maintaining the balance between efficient replication in the mosquito vector and the human host.     

Substance use and perceived symptom improvement among patients with bipolar disorder and substance dependence

BACKGROUND: Bipolar disorder (BPD) is the Axis I disorder with the highest risk for coexisting substance use disorder. One explanation for this phenomenon is the 'self-medication hypothesis', which states that some patients experience improvement in psychiatric symptoms as a result of substance use. We thus investigated reasons for substance use and perceived substance-induced improvement in BPD symptoms among patients with current BPD and substance dependence. METHODS: A total of 45 patients received six monthly assessments; 21 also received integrated group therapy (IGT), focusing simultaneously on BPD and substance dependence, while 24 did not receive IGT. Patients reported at intake their current reasons for initiating substance use (including BPD symptoms) and the effects of substance use on those symptoms. RESULTS: Nearly all patients initiated substance use because of at least one BPD symptom, especially depression (77.8%) and racing thoughts (57.8%); most (66.7%) reported improvement in at least one BPD symptom as a result of substance use. Among patients reporting substance-induced improvement in BPD symptoms, those receiving IGT reported fewer days of drug use over the 6-month study period than those not receiving IGT; this difference was not significant among patients without substance-induced improvement in BPD symptoms. LIMITATIONS: The study is limited by its small sample size and by the potential inaccuracy of self-reports regarding the effects of substance use on mood. CONCLUSIONS: Substance dependent patients who report that substance use improves their BPD symptoms may benefit from treatment that focuses simultaneously on both disorders.     

Phenotype delineation of ZNF462 related syndrome

Zinc finger protein 462 (ZNF462) is a relatively newly discovered vertebrate specific protein with known critical roles in embryonic development in animal models. Two case reports and a case series study have described the phenotype of 10 individuals with ZNF462 loss of function variants. Herein, we present 14 new individuals with loss of function variants to the previous studies to delineate the syndrome of loss of function in ZNF462. Collectively, these 24 individuals present with recurring phenotypes that define a multiple congenital anomaly syndrome. Most have some form of developmental delay (79%) and a minority has autism spectrum disorder (33%). Characteristic facial features include ptosis (83%), down slanting palpebral fissures (58%), exaggerated Cupid's bow/wide philtrum (54%), and arched eyebrows (50%). Metopic ridging or craniosynostosis was found in a third of study participants and feeding problems in half. Other phenotype characteristics include dysgenesis of the corpus callosum in 25% of individuals, hypotonia in half, and structural heart defects in 21%. Using facial analysis technology, a computer algorithm applying deep learning was able to accurately differentiate individuals with ZNF462 loss of function variants from individuals with Noonan syndrome and healthy controls. In summary, we describe a multiple congenital anomaly syndrome associated with haploinsufficiency of ZNF462 that has distinct clinical characteristics and facial features.     

Expression in Escherichia coli of a dominant immunogen of Trypanosoma cruzi recognized by human chagasic sera.

A genomic clone expressing a Trypanosoma cruzi antigen in Escherichia coli was identified using human chagasic sera. Chagasic antibodies affinity purified on extracts of this clone recognized a high-molecular-weight protein expressed in all developmental stages of the parasite life cycle, as well as in various T. cruzi strains. The antigen is associated with the cytoskeleton of the parasite and localizes along the attachment region between the flagellum and the cell body. Antibodies to the recombinant antigen were detected in the sera of 115 chagasic patients from different endemic regions, but not in sera of patients with leishmaniasis, T. rangeli infection, or other parasitic diseases. Our data suggest that the presence of antibodies to this antigen may be specifically associated with Chagas' disease.     

Accuracy of a commercial enzyme-linked immunosorbent assay for CagA in patients from Brazil with and without gastric carcinoma

We validated a commercial enzyme-linked immunosorbent assay for the detection of anti-CagA antibodies in Brazilian patients with Helicobacter pylori infection. The test presented high sensitivity (97.4%) and specificity (88.9%) when employed in patients without gastric carcinoma. However, in gastric carcinoma patients, the test was neither sensitive nor specific enough to detect cagA-positive H. pylori infection.     

Mapping of B-Cell Epitopes in a Trypanosoma cruzi Immunodominant Antigen Expressed in Natural Infections

Tc40 is an immunodominant antigen present in natural Trypanosoma cruzi infections. This immunogen was thoroughly mapped by using overlapping amino acid sequences identified by gene cloning and chemical peptide synthesis. To map continuous epitopes of the Tc40 antigen, an epitope expression library was constructed and screened with sera from human chagasic patients. A major, linear B-cell epitope spanning residues 403 to 426 (PAKAAAPPAA) was identified in the central domain of Tc40. A synthetic peptide spanning this region reacted strongly with 89.8% of the serum samples from T. cruzi-infected individuals. This indicates that the main antigenic site is defined by the linear sequence of the peptide rather than a conformation-dependent structure. The major B-cell epitope of Tc40 shares a high degree of sequence identity with T. cruzi ribosomal and RNA binding proteins, suggesting the existence of cross-reactivity among these molecules.     

Longitudinal study of daily variation of rats' behavior in the elevated plus-maze

We conducted a longitudinal study about daily variation of Wistar male rats' behavior in the elevated plus-maze (EPM) evaluated in the 1st, 2nd, 3rd, 6th, 12th, and 18th months of life. Animals were submitted to the plus-maze in 12 sessions at 2-h intervals (n=72, 6 per time point). Spontaneous rest-activity rhythm of four animals was assessed by observation of 24-h videotape records. Time series were analyzed by Cosinor method. Behavioral rates on the six occasions and in light and dark phases were compared by means of two-way ANOVA with repeated measures. Exploratory behavior in EPM was smaller in the light phase and in older animals. Higher values of open and closed arms exploration were observed in the first and third months of the dark phase, and in the first month of the light phase. Adjustment to the 24-h period was significant at all stages for rest-activity data, number of entries in closed arms, and time on center, and for three to five stages for open-arm exploration. In general, 24 h variability was more pronounced in younger animals compared with older ones. The present study showed that: (1). a significant amount of total variability of the behavioral indexes analyzed could be attributed to 24 h variation, (2). light/dark phases differences in EPM exploration were present at all developmental stages, (3). older Wistar rats explored less the EPM and were less active in their home cage compared with younger ones, and (4). behavioral indexes (EPM) decrease was phase related and partially related to a reorganization of rest-activity rhythm.     

Molecular diagnosis of infective endocarditis by PCR amplification and direct sequencing of DNA from valve tissue

We used broad-range eubacterial PCR amplification followed by direct sequencing to identify microbial pathogens in heart valve material from 29 patients with histologically confirmed infective endocarditis and 23 patients free of infective endocarditis. Microorganisms cultured by conventional techniques matched those identified by PCR in 21 cases. PCR alone identified the causative agent in three cases (Streptococcus bovis, Staphylococcus cohnii, and Coxiella burnetii), allowing better patient management. PCR corrected the initial bacteriological diagnosis in three cases (Streptococcus bovis, Streptococcus mutans, and Bartonella henselae). Among the 29 cases of histologically confirmed infective endocarditis, PCR findings were positive in 27 cases and were consistent with the bacterial morphology seen at Gram staining (26 cases) or with the results obtained by immunohistologic analysis with an anti-C. burnetii monoclonal antibody (one case). In two other cases of histologically confirmed infective endocarditis, PCR remained negative in a blood culture-negative case for which no bacteria were seen at histological analysis and in one case with visualization of cocci and blood cultures positive for Enterococcus faecalis. Ten clinical diagnoses of possible infective endocarditis were ruled out by histopathological analysis of the valves and subsequently by PCR. PCR was negative in 13 of the 14 patients in whom infective endocarditis was rejected on clinical grounds; the other patient was found to have Coxiella burnetii infective endocarditis on the basis of PCR and histopathological analysis and was subsequently included in the group of 29 definite cases. In total, PCR contributed to the diagnosis and management of infective endocarditis in 6 of 29 (20%) cases.     

Well-being therapy of generalized anxiety disorder

BACKGROUND: There is increasing awareness that the goal of treatment in generalized anxiety disorder (GAD) should not simply be a response, but restoration of normal function. The aim of this study was to apply a novel psychotherapeutic approach for increasing the level of remission in GAD. METHODS: Twenty patients with DSM-IV GAD devoid of comorbid conditions were randomly assigned to 8 sessions of cognitive behavioral therapy (CBT) or the sequential administration of 4 sessions of CBT followed by other 4 sessions of well-being therapy (WBT). Assessment methods included the Anxiety and Depression Scales of Paykel's Clinical Interview for Depression, Ryff's Psychological Well-being Scales and Kellner's Symptom Questionnaire. A one-year follow-up was undertaken. RESULTS: Significant advantages of the CBT-WBT sequential combination over CBT only were observed with both observer and self-rated methods after treatment. Such gains were maintained at follow-up. CONCLUSIONS: These preliminary results suggest the feasibility and clinical advantages of adding WBT to the treatment of GAD. They lend support to a sequential use of treatment components for achieving a more sustained recovery.     

Mycobacterium bovis BCG vaccination as prophylaxis against Mycobacterium ulcerans osteomyelitis in Buruli ulcer disease

Mycobacterium ulcerans disease, or Buruli ulcer (BU), causes significant morbidity in West Africa. Clinically, the disease presents in the skin as either nonulcerative or ulcerative forms and often invades bones either subjacent to the skin lesion (contiguous osteomyelitis) or remote from the skin lesion (metastatic osteomyelitis). Osteomyelitis represents a severe form of the disease that often requires numerous surgical interventions, even amputations. Surgery is accepted as the present definitive treatment for BU. In the absence of an effective drug treatment, the need for the development of preventive and control strategies becomes paramount. No specific vaccine, however, is presently available for BU. Of 372 consecutive patients in Benin presenting with BU (confirmed by microbiological and histopathological analyses) whose Mycobacterium bovis BCG scar statuses were known, 196 children (<15 years old) and 108 adults had neonatal BCG vaccination scars. Of 196 children with BCG scars, 17 (8.7%) had osteomyelitis, while 7 of 28 children without BCG scars (25.0%) had osteomyelitis. Of 108 adults with BCG scars, 17 (15.7%) had osteomyelitis, while 14 of 40 adults without BCG scars (35.0%) had osteomyelitis. Our results show that effective BCG vaccination at birth provides significant protection against the development of M. ulcerans osteomyelitis in children and adults. Therefore, health authorities should give attention to the enhancement of neonatal BCG vaccination coverage in all countries of Africa where BU is endemic. Protection against severe forms of BU and childhood tuberculosis would likewise be improved by this intervention.