Distribution and phenotype of murine rotavirus-specific B cells induced by intranasal immunization with 2/6 virus-like particles

Virus-like particles containing the rotavirus (RV) internal proteins VP2 and VP6 (2/6-VLP) have been shown to induce serum and fecal antibodies as well as protection in mice after intranasal administration with a mutant of E. coli toxin, LT-R192G. To better understand the origin of fecal IgA induced by this protocol, we studied the RV-specific B cell response in systemic and mucosal lymphoid tissues using a flow cytometry assay that allows quantification and phenotypic characterization of RV-specific B lymphocytes. We also assessed the RV-specific antibody-secreting cells in the spleen and intestinal lamina propria (ILP). A remarkably high frequency of RV-specific B cells was found in the respiratory lymphoid tissues and spleen, of which only a minority expressed the alpha4beta7 integrin (intestinal homing receptor). In contrast, but in accordance with alpha4beta7 expression at the induction site, a very low response was observed in intestinal lymphoid tissues (mesenteric lymph nodes and ILP), which did not increase after a second immunization. Thus, intranasal immunization with a nonreplicating antigen does not induce an important number of RV-specific B cells with an intestinal homing profile.     

Comparative usage of herpesvirus entry mediator A and nectin-1 by laboratory strains and clinical isolates of herpes simplex virus

The herpesvirus entry mediator A (HVEM/HveA) and nectin-1 (HveC/CD111) are two major receptors for herpes simplex virus (HSV). Although structurally unrelated, both receptors can independently mediate entry of wild-type (wt) HSV-1 and HSV-2 by interacting with the viral envelope glycoprotein D (gD). Laboratory strains with defined mutations in gD (e.g. rid1) do not use HVEM but use nectin-2 (HveB/CD112) for entry. The relative usage of HVEM and nectin-1 during HSV infection in vivo is not known. In the absence of a defined in vivo model, we used in vitro approaches to address this question. First, we screened HSV clinical isolates from various origins for receptor tropism and found that all used both HVEM and nectin-1. Second, we determined the numbers of surface receptors on various susceptible and resistant cell lines as well as on primary fibroblasts derived from an individual with cleft lip/palate ectodermal dysplasia (CLPED1). Although CLPED1 cells can only express a defective form of nectin-1, they allowed entry of wild type and mutant HSV strains by usage of either HVEM or nectin-2. Finally, we compared the ability of HVEM and nectin-1 to mediate entry when expressed at varying cell surface densities. Both receptors showed a direct relationship between the number of receptors and HSV susceptibility. Direct comparison of receptors suggests that nectin-1 is more efficient at promoting entry than HVEM. Overall, our data suggest that both receptors play a role during HSV infection in vivo and that both are highly efficient even at low levels of expression.     

Molecular cytogenetic characterization of proximal-type epithelioid sarcoma

Proximal-type epithelioid sarcoma is a recently described soft-tissue tumor that is distinguished from conventional-type epithelioid sarcoma by a far more aggressive clinical course, frequent location in the proximal anatomic regions, and variable rhabdoid morphology. Because of their rarity and peculiar morphology, proximal-type epithelioid sarcomas frequently pose serious diagnostic dilemmas, being easily misdiagnosed as a variety of other malignant neoplasms. To date, the information available on the genetic alterations associated with this tumor entity has been confined to single conventional cytogenetic reports. In this article, we present the results of a conventional and molecular cytogenetic analysis of six proximal-type epithelioid sarcomas. Spectral karyotyping analysis of these cases deciphered the characteristics of several marker chromosomes and complex translocations, leading to the recognition of recurrent rearrangements. The most frequently involved chromosome arm was 22q, and the identification of two cases with a similar translocation, t(10;22), suggests a role for one or more genes on chromosome 22 in the pathogenesis of this tumor and provides an opportunity for finely mapping the translocation-associated breakpoints. Chromosome arm 8q gain was also a frequent event and correlated with gain of MYC gene copy number, as demonstrated by fluorescence in situ hybridization. A review of both cases reported in the literature and those presented in this study reinforced the involvement of chromosomes 8 and 22 and also indicated frequent rearrangements of chromosomes 7, 14, 18, and 20.     

Human rotavirus specific T cells: quantification by ELISPOT and expression of homing receptors on CD4+ T cells

Using an intracellular cytokine assay, we recently showed that the frequencies of rotavirus (RV)-specific CD4(+) and CD8(+) T cells secreting INFgamma, circulating in RV infected and healthy adults, are very low compared to the frequencies of circulating cytomegalovirus (CMV) reactive T cells in comparable individuals. In children with acute RV infection, these T cells were barely or not detectable. In the present study, an ELISPOT assay enabled detection of circulating RV-specific INFgamma-secreting cells in children with RV diarrhea but not in children with non-RV diarrhea without evidence of a previous RV infection. Using microbead-enriched CD4(+) and CD8(+) T cell subsets, IFNgamma-secreting RV-specific CD8(+) but not CD4(+) T cells were detected in recently infected children. Using the same approach, both CD4(+) and CD8(+) RV-specific T cells were detected in healthy adults. Furthermore, stimulation of purified subsets of PBMC that express lymphocyte homing receptors demonstrated that RV-specific INFgamma-secreting CD4(+) T cells from adult volunteers preferentially express the intestinal homing receptor alpha4beta7, but not the peripheral lymph node homing receptor L-selectin. In contrast, CMV-specific INFgamma-secreting CD4(+) T cells preferentially express L-selectin but not alpha4beta7. These results suggest that the expression of homing receptors on virus-specific T cells depends on the organ where these cells were originally stimulated and that their capacity to secrete INFgamma is independent of the expression of these homing receptors.     

ERP correlates of transposed-letter similarity effects: Are consonants processed differently from vowels?

Recent research has shown that pseudowords created by transposing letters are very effective for activating the lexical representation of their base words (e.g., relovution activates REVOLUTION). Furthermore, pseudoword transpositions of consonants are more similar to their corresponding base words than the transposition of vowels. We report one experiment using pseudowords created by the transposition of two consonants, two vowels, and their corresponding control conditions (i.e., the replacement of two consonants or two vowels) in a lexical decision task while Event Related Potentials (ERPs) were recorded. The results showed a modulation of the amplitude of the N400 component as a function of the type of pseudoword (transposed-letter versus replacement letter pseudowords), and this modulation was different for transposed consonants and vowels. These results suggest that consonants and vowels play a different role during word processing.     

Immunity in HIV-1-Infected Adults with a Previous State of Moderate-Severe Immune-Suppression and More Than 500 CD4+ T Cell After Highly Active Antiretroviral Therapy

We evaluated phenotypic and functional parameters of immune restoration of 27 HIV-infected patients on highly active antiretroviral therapy (HAART) (HIV-cases) with HIV-RNA levels below detectable limits at least during 18 months, and CD4+ cell per microliter higher than 500 at the moment of the study and lower than 300 anytime before. These patients were compared with 11 HIV-controls that never had less than 500 CD4+ cell per microliter and 20 healthy-controls (HIV seronegative subjects) in a cross-sectional study. HIV-cases had lower counts of naïve CD4+ than HIV-controls and healthy-controls. HIV-patients (both HIV-cases and HIV-controls) showed higher values of naïve and memory CD8+ counts than healthy-controls. TREC-bearing cell levels were significantly lower in HIV-cases than in healthy-controls. Peripheral blood mononuclear cells (PBMC) cultures, HIV-cases had lower values in proliferation to streptokinase (SK) and tetanus toxin (TT) than in healthy-controls. HIV-cases had lower IFN-γ and higher IL-5 production with pokeweed than healthy-controls (P < 0.01). However, IL-5 production of HIV-cases after TT stimulation was lower than in HIV-controls and healthy-controls. Total IgG and IgG1 levels were significantly higher in HIV-cases than in HIV-controls and healthy-controls. Also, IgM levels were significantly higher in HIV-cases than in healthy-controls. Nevertheless, IgG2 levels were significantly lower in HIV-cases and HIV-controls than in healthy-controls. The levels of specific Igs antipneumococcal capsular polysaccharide and TT were significantly lower in HIV-cases than in healthy-controls. HIV-patients with a previous state of severe-moderate immunosuppression normalizing their CD4+ counts have a incomplete immune reconstitution after HAART. Long-term consequences of this subclinical immune deficiency remain to be determined.     

Characterization of the sleep-wake patterns in mice lacking fatty acid amide hydrolase

STUDY OBJECTIVES: Oleamide and anandamide are fatty acid amides implicated in the regulatory mechanisms of sleep processes. However, due to their prompt catabolism by fatty acid amide hydrolase (FAAH), their pharmacologic and behavioral effects, in vivo, disappear rapidly. To determine if, in the absence of FAAH, the hypnogenic fatty acid amides induce an increase of sleep, we characterized the sleep-wake patters in FAAH-knockout mice [FAAH (-/-)] before and after sleep deprivation. DESIGN: FAAH (-/-), FAAH (+/-), and FAAH (+/+) mice were implanted chronically for sleep, body temperature (Tb), and locomotor activity (LMA) recordings. Sleep-wake states were recorded during a 24-hour baseline session followed by 8 hours of sleep deprivation. Recovery recordings were done during the 16 hours following sleep deprivation. Total amount of wake, slow-wave sleep, and rapid eye movement sleep were calculated and compared between genotypes. The electroencephalographic spectral analysis was performed by fast Fourier transform analysis. Telemetry recordings of Tb and LMA were carried out continuously during 4 days under baseline conditions. SETTING: N/A. PATIENTS OR PARTICIPANTS: FAAH (-/-) mice and their heterozygote (+/-) and control (+/+) littermates were used. INTERVENTIONS: Sleep deprivation. MEASUREMENTS AND RESULTS: FAAH (-/-) mice possess higher values of slow-wave sleep and more intense episodes of slow-wave sleep than do control littermates under baseline conditions that are not related to differences in Tb and LMA. A rebound of slow-wave sleep and rapid eye movement sleep as well an increase in the levels of slow-wave activity were observed after sleep deprivation in all genotypes. CONCLUSION: These findings support the role of fatty acid amides as possible modulators of sleep and indicate that the homeostatic mechanisms of sleep in FAAH (-/-) mice are not disrupted.     

Lymphoepithelioma-like carcinoma of the bladder: three cases with clinicopathological and p53 protein expression study

Lymphoepithelioma-like carcinoma of the bladder is an uncommon neoplasm, of which 49 cases have been described in the English literature, none of which has been studied for p53 protein expression. We studied three muscle-infiltrating cases of this tumor using immunohistochemical, in situ hybridization and polymerase chain reaction (PCR) methods. The three cases were positive for epithelial markers and negative for lymphoid antigens in the tumoral syncytial areas. The intensive infiltrate of small cells was negative for epithelial and positive for lymphoid markers. This population was mainly made up of cytotoxic T-lymphocytes, positive for TIA-1. p53 protein was intensely positive in more than 90% of the epithelial component nuclei, being negative in the lymphoid cells. PCR study did not show mutations on p53. Both lymphocytes and epithelium were negative for Epstein–Barr virus markers, such as the latent membrane protein and EBER (Epstein–Barr-encoded RNA). The prognosis was very good after radiotherapy and chemotherapy treatment, preserving the bladder despite the muscle infiltration. The presence of an intense cytotoxic T-lymphocyte population may be related to this good prognosis. Both aspects, p53 protein status and T-lymphoid population, had never been studied before in bladder lymphoepithelioma-like carcinoma.     

Retinoids and spermatogenesis: lessons from mutant mice lacking the plasma retinol binding protein.

Using Rbp4-null mice as models, we have established for the first time the kinetics of the spermatogenetic alterations during vitamin A deficiency (VAD). Our data demonstrate that the VAD-induced testicular degeneration arises through the normal maturation of germ cells in a context of spermatogonia differentiation arrest. They indicate that retinoic acid (RA) appears dispensable for the transition of premeiotic to meiotic spermatocytes, meiosis, and spermiogenesis. They confirm that RA plays critical roles in controlling spermatogonia differentiation, spermatid adhesion to Sertoli cells, and spermiation, and suggest that the VAD-induced arrest of spermatogonia differentiation results from simultaneous blocks in RA-dependent events mediated by RA receptor gamma (RARgamma) in spermatogonia and by RARalpha in Sertoli cells. They also provide evidence that expression of major RA-metabolizing enzymes is increased in mouse Sertoli cells upon VAD and that vitamin A-deficient A spermatogonia differ from their RA-sufficient counterparts by the expression of the Stra8 gene.     

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection by duplex PCR assay based on telomeric sequences

We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of Trypanosoma cruzi and Trypanosoma rangeli in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of T. cruzi and 1/4th of T. rangeli cell equivalents without complementary hybridization. In addition, the PCR-based assay was robust enough for direct application to difficult biological samples such as Reduviid feces or guts and was capable of recognizing all T. cruzi and T. rangeli strains and lineages. Because the assay primers amplify entirely different target sequences, no reaction interference was observed, facilitating future adaptation of this assay to an automated format.