Structure Prediction for Peptides Capable of Inducing Antibody Formation in Mice

A simple method for the sequence prediction of peptides capable of thein vivo stimulation of antibody production in mice without conjugation with protein carriers was proposed on the basis of literature data on the structure of T-helper epitopes active in vivo. According to this approach, a potentially active peptide should contain a nine-membered sequence with a hydrophobic amino acid residue in the first position and a positively charged residue in the ninth position. The efficiency of this approach was confirmed by the presence of such sequences in the previously described synthetic peptides with immune activities, by the application of this approach to the choice of immunogenic fragments within the sequences of various proteins that exhibited further the specific activity, and by the construction of immunogenic peptides on the basis of inactive natural sequences.     

Infection of Mice,Ferrets, and Rhesus Macaques with a Clinical Mumps Virus Isolate

In recent years, many mumps outbreaks have occurred in vaccinated populations worldwide. The reasons for these outbreaks are not clear. Animal models are needed to investigate the causes of outbreaks and to understand the pathogenesis of mumps virus (MuV). In this study, we have examined the infection of three animal models with an isolate of mumps virus from a recent outbreak (MuV-IA). We have found that while both ferrets and mice generated humoral and cellular immune responses to MuV-IA infection, no obvious signs of illness were observed in these animals; rhesus macaques were the most susceptible to MuV-IA infection. Infection of rhesus macaques via both intranasal and intratracheal routes with MuV-IA led to the typical clinical signs of mumps 2 weeks to 4 weeks postinfection. However, none of the infected macaques showed any fever or neurologic signs during the experimental period. Mumps viral antigen was detected in parotid glands by immunohistochemistry (IHC). Rhesus macaques represent the best animal model for the study of mumps virus pathogenesis.     

Expressive Aphasia as a Presentation of Encephalitis with Bartonella henselae Infection in an Immunocompetent Adult

Objective: To show the first clinically reported case of Cat Scratch Disease (CSD) presenting as a focal neurologic deficit in an immunocompetent adult.Patient: 59-year-old male with a history of a previous stroke.Results: Examination showed an expressive aphasia, word substitution errors, and impaired repetition. A head CT and MRI showed no acute changes. The EEG findings were non-focal and did not show any epileptiform activity. The patient had a history of contact with stray kittens and previous axillary lymphadenopathy. Bartonella henselae serology titers were IgG positive 1:1024 (< 64) and IgM positive 1:20 (< 16). After antibiotic administration, the patient’s symptoms and aphasia resolved.Conclusions: Focal presentations concerning for stroke or partial seizure activity may have underlying infectious etiology. We recommend consideration of CSD in the differential diagnosis of any adult with a history of lymphadenopathy, fever, and recent contact with a cat who presents with neurologic complications.     

含体内激活启动子的重组减毒鼠伤寒沙门菌诱导转基因鼠细胞免疫应答

以庚型肝炎病毒 (hepatitisGvirus ,HGV)转基因小鼠为模型 ,探讨逆转免疫耐受的方法及与HGV致病性的关系。首先采用鼠伤寒沙门菌pagC基因的启动子 (PpagC)为转录调控元件 ,构建宿主体内表达HGVNS3抗原的重组减毒鼠伤寒沙门菌 ,并口服接种免疫HGV转基因小鼠。结果证明对诱导转基因小鼠血清HGVNS3抗体无明显影响 ,但对小鼠血清HGV抗原含量、肝组织HGV抗原及HGVmRNA表达量有明显抑制作用。体外培养脾细胞表现出针对HGVNS3抗原的细胞免疫反应 ,并检测到Th1 型细胞因子IFN γ。过继转移实验证明T细胞可能是通过IFN γ介导的机制抑制转基因鼠体内HGV的表达及复制。组织病理学检查显示 ,转基因小鼠肝组织见淋巴细胞浸润等轻度炎性变。T细胞免疫耐受消除的结果提示 ,这种宿主体内激活目的抗原表达的口服疫苗有望成为治疗病毒性肝炎的新方法。     

Borrelia persica Infection in Immunocompetent Mice - A New Tool to Study the Infection Kinetics In Vivo

Borrelia persica, a bacterium transmitted by the soft tick Ornithodoros tholozani, causes tick-borne relapsing fever in humans in the Middle East, Central Asia and the Indian peninsula. Immunocompetent C3H/HeOuJ mice were infected intradermally with B. persica at varying doses: 1 x 10⁶, 1 x 10⁴, 1 x 10² and 4 x 10⁰ spirochetes/mouse. Subsequently, blood samples were collected and screened for the presence of B. persica DNA. Spirochetes were detected in all mice infected with 1 x 10⁶, 1 x 10⁴ and 1 x 10² borrelia by real-time PCR targeting the flaB gene of the bacterium. Spirochetemia developed with a one- to two-day delay when 1 x 10⁴ and 1 x 10² borrelia were inoculated. Mice injected with only four organisms were negative in all tests. No clinical signs were observed when infected mice were compared to negative control animals. Organs (heart, spleen, urinary bladder, tarsal joint, skin and brain) were tested for B. persica-specific DNA and cultured for the detection of viable spirochetes. Compiled data show that the target organs of B. persica infections are the brain and the skin. A newly developed serological two-tiered test system (ELISA and western blot) for the detection of murine IgM, IgG and IgA antibody titers against B. persica showed a vigorous antibody response of the mice during infection. In conclusion, the infection model described here for B. persica is a platform for in vivo studies to decipher the so far unexplored survival strategies of this Borrelia species.     

Breed-Specific Hematological Phenotypes in the Dog: A Natural Resource for the Genetic Dissection of Hematological Parameters in a Mammalian Species

Remarkably little has been published on hematological phenotypes of the domestic dog, the most polymorphic species on the planet. Information on the signalment and complete blood cell count of all dogs with normal red and white blood cell parameters judged by existing reference intervals was extracted from a veterinary database. Normal hematological profiles were available for 6046 dogs, 5447 of which also had machine platelet concentrations within the reference interval. Seventy-five pure breeds plus a mixed breed control group were represented by 10 or more dogs. All measured parameters except mean corpuscular hemoglobin concentration (MCHC) varied with age. Concentrations of white blood cells (WBCs), neutrophils, monocytes, lymphocytes, eosinophils and platelets, but not red blood cell parameters, all varied with sex. Neutering status had an impact on hemoglobin concentration, mean corpuscular hemoglobin (MCH), MCHC, and concentrations of WBCs, neutrophils, monocytes, lymphocytes and platelets. Principal component analysis of hematological data revealed 37 pure breeds with distinctive phenotypes. Furthermore, all hematological parameters except MCHC showed significant differences between specific individual breeds and the mixed breed group. Twenty-nine breeds had distinctive phenotypes when assessed in this way, of which 19 had already been identified by principal component analysis. Tentative breed-specific reference intervals were generated for breeds with a distinctive phenotype identified by comparative analysis. This study represents the first large-scale analysis of hematological phenotypes in the dog and underlines the important potential of this species in the elucidation of genetic determinants of hematological traits, triangulating phenotype, breed and genetic predisposition.     

Inter- and Intra-Host Viral Diversity in a Large Seasonal DENV2 Outbreak

Background

High genetic diversity at both inter- and intra-host level are hallmarks of RNA viruses due to the error-prone nature of their genome replication. Several groups have evaluated the extent of viral variability using different RNA virus deep sequencing methods. Although much of this effort has been dedicated to pathogens that cause chronic infections in humans, few studies investigated arthropod-borne, acute viral infections.

Methods and Principal Findings

We deep sequenced the complete genome of ten DENV2 isolates from representative classical and severe cases sampled in a large outbreak in Brazil using two different approaches. Analysis of the consensus genomes confirmed the larger extent of the 2010 epidemic in comparison to a previous epidemic caused by the same viruses in another city two years before (genetic distance = 0.002 and 0.0008 respectively). Analysis of viral populations within the host revealed a high level of conservation. After excluding homopolymer regions of 454/Roche generated sequences, we found 10 to 44 variable sites per genome population at a frequency of >1%, resulting in very low intra-host genetic diversity. While up to 60% of all variable sites at intra-host level were non-synonymous changes, only 10% of inter-host variability resulted from non-synonymous mutations, indicative of purifying selection at the population level.

Conclusions and Significance

Despite the error-prone nature of RNA-dependent RNA-polymerase, dengue viruses maintain low levels of intra-host variability.     

Favus of Scrotum Due to Trichophyton rubrum in Immunocompetent Patients: A Clinical,Mycological and Ultrastructural Study

Mycopathologia - To characterize the clinical and mycological features of favus of scrotum due to Trichophyton rubrum. A single-site prospective study was carried out in an outpatient dermatology...     

Pulmonary Abnormalities in Mice with Paracoccidioidomycosis: A Sequential Study Comparing High Resolution Computed Tomography and Pathologic Findings

Background

Human paracoccidioidomycosis (PCM) is an endemic fungal disease of pulmonary origin. Follow-up of pulmonary lesions by image studies in an experimental model of PCM has not been previously attempted. This study focuses on defining patterns, topography and intensity of lung lesions in experimentally infected PCM mice by means of a comparative analysis between High Resolution Computed Tomography (HRCT) and histopathologic parameters.

Methodology

Male BALB/c mice were intranasally inoculated with 3×10⁶ Paracoccidioides brasiliensis (Pb) conidia (n = 50) or PBS (n = 50). HRCT was done every four weeks to determine pulmonary lesions, quantify lung density, reconstruct and quantify lung air structure. Lungs were also analyzed by histopathology and histomorphometry.

Results

Three different patterns of lesions were evidenced by HRCT and histopathology, as follows: nodular-diffuse, confluent and pseudo-tumoral. The lesions were mainly located around the hilus and affected more frequently the left lung. At the 4^th week post-challenge HRCT showed that 80% of the Pb-infected mice had peri-bronchial consolidations associated with a significant increase in upper lung density when compared with controls, (−263±25 vs. −422±10 HU, p<0.001). After the 8^th and 12^th weeks, consolidation had progressed involving also the middle regions. Histopathology revealed that consolidation as assessed by HRCT was equivalent histologically to a confluent granulomatous reaction, while nodules corresponded to individual compact granulomas. At the 16^th week of infection, confluent granulomas formed pseudotumoral masses that obstructed large bronchi. Discrete focal fibrosis was visible gradually around granulomas, but this finding was only evident by histopathology.

Conclusions/Significance

This study demonstrated that conventional HRCT is a useful tool for evaluation and quantification of pulmonary damage occurring in experimental mouse PCM. The experimental design used decreases the need to sacrifice a large number of animals, and serves to monitor treatment efficacy by means of a more rational approach to the study of human lung disease.     

Recovery and Genetic Characterization of a West Nile Virus Isolate from China

West Nile virus(WNV) is an important neurotropic flavivirus that is widely distributed globally. WNV strain XJ11129 was first isolated in Xinjiang, China, and its genetic and biological characteristics remain largely unknown. In this study,phylogenetic and sequence analyses revealed that XJ11129 belongs to lineage 1 a and shares high genetic identity with the highly pathogenic strain NY99. Then, the full-length genomic c DNA of XJ11129 was amplified and assembled using a modified Gibson assembly(GA) method. The virus(named r XJ11129) was successfully rescued in days following this method. Compared with other wild-type WNV isolates, r XJ11129 exhibited virulence indistinguishable from that of the NY99 strain in vivo. In summary, the genomic and virulence phenotypes of r XJ11129 were characterized in vivo and in vitro, and these data will improve the understanding of the spread and pathogenesis of this reemerging virus.     

A Genetic Analysis of Targeted Growth in Mice

Effects of normal growth regulation on components of phenotypic variance and covariance of body weight were examined in a cross-fostering study of growth between 2 and 10 wk of age in ICR randombred mice. Different early growth rates caused genetic, postnatal maternal and residual environmental variances to increase, but these variances were subsequently reduced by negative autocorrelation between early and later growth. Postnatal maternal variance continued to increase for about 1 wk after weaning but then decreased substantially. Genetic variance caused by preweaning growth followed a pattern of increase and decrease very similar to that of postnatal maternal variance, but this pattern was masked by new genetic variance. Normal growth regulation affects the magnitudes of genetic variances and serial autocorrelations. The timing of these changes suggests that regulation of cell numbers reduces variance near the end of exponential growth, but this may be obscured by subsequent increase in cell size. In contrast with earlier studies, we find that targeted growth reduces both genetically and environmentally determined differences among early growth trajectories. Final size may be determined by an antagonistic balance between early growth rate and age at initiation of puberty.     

Pathogenic and Genotypic Characterization of a Japanese Encephalitis Virus Isolate Associated with Reproductive Failure in an Indian Pig Herd

Background

India is endemic to Japanese encephalitis virus (JEV) and recurrent outbreaks occur mainly in rice growing areas. Pigs are considered to be the amplifying host for JEV and infection in gestating pigs results in reproductive failure. Most studies conducted on JEV infection in Indian pigs have been serological surveys and very little is known about JEV genotypes circulating in pigs. So the potential risk posed by pigs in JEV transmission and the genetic relationship between viruses circulating in pigs, mosquitoes and humans is poorly understood.

Methodology/Principal Findings

This study was conducted in pigs with a history of reproductive failure characterized by stillborn piglets with neuropathological lesions. Japanese encephalitis (JE) suspected brain specimens inoculated intracerebrally into mice and Vero cells resulted in successful isolation of JEV/SW/IVRI/395A/2014. Clinicopathological observations in infected mice, demonstration of JEV antigen in brain, and analysis of the envelope protein identified the swine isolate as being neurovirulent. Phylogenetic analysis based on prM and E gene sequences showed that it belonged to genotype III. This swine isolate was closely related to JEV associated with the 2005 outbreak in India and JaoArS982 from Japan. Phylogenetic analysis of JEV strains collected between 1956 and 2014 in India categorized the GIII viruses into different clades blurring their spatial distribution, which has been discernible in the previous century.

Conclusions/Significance

Isolation of JEV from stillborn piglets and its close genetic relationship with viruses detected at least three decades ago in humans and mosquitoes in Japan suggests that the virus may have been circulating among Indian pigs for several decades. The close similarity between the present swine isolate and those detected in humans affected in the 2005 outbreak in Uttar Pradesh, India, suggests the need for more intensive surveillance of pigs and implementation of suitable strategies to control JE in India.     

Laminin-3B11, a Novel Vascular-type Laminin Capable of Inducing Prominent Lamellipodial Protrusions in Microvascular Endothelial Cells

The basement membrane (BM) proteins laminins, which consist of α, β, and γ chains, support tissue structures and cellular functions. To date only α4 and α5 types of laminins have been identified in the BMs of blood vessels. Our recent study suggested the presence of novel α3B-containing laminins in vascular BMs. Here we identified and characterized the third member of vascular laminins, laminin-3B11 (Lm3B11). RT-PCR analysis showed that microvascular endothelial (MVE) cells and umbilical vein endothelial cells expressed the messages for the α3B, β1, β2, and γ1 chains. In the culture of MVE cells, α3B was associated with β1 and γ1, producing Lm3B11. Recombinant Lm3B11 was overexpressed by introducing the cDNAs of the three chains into HEK-293 cells and purified to homogeneity. Purified Lm3B11 exhibited relatively weak cell adhesion activity through both α3β1 and α6β1 integrins. Most characteristically, Lm3B11 strongly stimulated MVE cells to extend many lamellipodial protrusions. This pseudopodial branching was blocked by an inhibitor for Src or phosphatidylinositol 3-kinase. Consistently, Lm3B11 stimulated the phosphorylation of Src and Akt more strongly than other laminins, suggesting that the integrin-derived signaling is mediated by these factors. The unique activity of Lm3B11 appears to be favorable to the branching of capillaries and venules.     

Induction of Protective Immunity against Japanese Encephalitis in Mice by Immunization with a Plasmid Encoding Japanese Encephalitis Virus Premembrane and Envelope Genes

A DNA vaccine plasmid containing the Japanese encephalitis (JE) virus premembrane (prM) and envelope (E) genes (designated pcDNA3JEME) was evaluated for immunogenicity and protective efficacy in mice. Two immunizations of 4-week-old female ICR mice with pcDNA3JEME by intramuscular or intradermal injections at a dose of 10 or 100 μg per mouse elicited neutralizing (NEUT) antibodies at titers of 1:10 to 1:20 (90% plaque reduction), and all immunized mice survived a challenge with 10,000 50% lethal doses of the P3 strain of JE virus. A single immunization with 100 μg of pcDNA3JEME did not elicit detectable NEUT antibodies but induced protective immunity. Spleen cells obtained from BALB/c mice immunized once with 10 or 100 μg of pcDNA3JEME contained JE virus-specific memory cytotoxic T lymphocytes (CTLs). BALB/c mice maintained detectable levels of memory B cells and CTLs for at least 6 months after one immunization with pcDNA3JEME at a dose of 100 μg. The CTLs induced in BALB/c mice immunized twice with 100 μg of pcDNA3JEME were CD8 positive and recognized mainly the envelope protein. These results indicate that pcDNA3JEME has the ability to induce a protective immune response which includes JE virus-specific antibodies and CTLs.     

Hepatic Serum Amyloid A1 Aggravates T Cell-mediated Hepatitis by Inducing Chemokines via Toll-like Receptor 2 in Mice

Serum amyloid A is a proinflammatory molecule that induces leukocyte infiltration and promotes neutrophil adhesion to endothelial cells under inflammatory conditions. The aim of this study was to examine whether Saa1 aggravates T cell-mediated hepatitis by inducing chemokines in a liver-specific, Saa1-overexpressing, transgenic (TG) mouse model. We generated TG mice in which Saa1 was overexpressed specifically in liver tissue. The chemokines monocyte chemotactic protein 1 (MCP1), MIP1α, MIP1β, interferon γ-induced protein 10 (IP-10), and eotaxin were induced in Saa1 TG mice. After concanavalin A treatment, Saa1 expression was higher in Saa1 TG mice than in WT mice. More severe liver injury, increased hepatocyte apoptosis, and higher levels of hepatic enzymes were observed in Saa1 TG mice than in WT mice. Liver infiltration of CD4⁺ T cells and macrophages increased after inducing hepatitis. Activation of T cells was higher in Saa1 TG mice than in WT mice, and the populations of Th17 cells and regulatory T cells were altered by overexpressing Saa1 in TG mice. Secretion of various cytokines, such as interferon γ, tumor necrosis factor α, and interleukin 6, increased in Saa1 TG mice. Injecting a Toll-like receptor 2 (TLR2) antagonist in vivo inhibited chemokine expression and IκBα phosphorylation and showed that the induction of chemokines by Saa1 was dependent on TLR2. Hepatic Saa1 accelerated T cell-mediated hepatitis by inducing chemokine production and activating T cells by TLR2. Therefore, Saa1 might be a novel inflammatory factor that acts as a chemokine modulator in hepatitis.