Attenuated poxvirus expressing three immunodominant CMV antigens as a vaccine strategy for CMV infection.

BACKGROUND: Human cytomegalovirus (CMV) infection is an important risk factor in the post-transplant (Tx) recovery phase for both hematopoietic stem cell Tx (HSCT) and solid organ Tx (SOT) recipients. CMV infection may be prevented or controlled by simultaneously inducing both CMV-specific neutralizing antibody (nAb) and cellular immunity. Soluble (s) UL55 (surface glycoprotein), UL83 (tegument protein) and UL123/e4 (nuclear protein) are immunodominant in eliciting both CMV nAb and cellular immunity. An attenuated poxvirus, modified vaccinia Ankara (MVA) was selected to develop this vaccine strategy in Tx recipients, because of its clinical safety record, large foreign gene capacity, and capability to activate strong humoral and cellular immune responses against recombinant antigens. OBJECTIVES: A subunit vaccine that targets multiple CMV antigens will be used to gain maximal coverage and protective function against CMV infection. rMVA simultaneously expressing sUL55, UL83 and UL123/e4 will be generated, and humoral and cellular immunity it elicits will be characterized, after murine immunization and in vitro to amplify clinical recall responses. STUDY DESIGN: rMVA will be constructed in two steps using UL123/e4-pLW22 followed by sUL55-UL83-pLW51 transfer plasmids. Western blots will be used to characterize expression levels of each antigen. Primary immunity will be evaluated in mouse models, while recall responses to the virally expressed CMV antigens will be assessed in human peripheral blood. RESULTS: We generated CMV-MVA via homologous recombination, and demonstrated high expression levels of sUL55, UL83 and UL123/e4 by Western blot. CMV-MVA immunization potently induced both humoral and cellular immunity to sUL55, UL83 and UL123 after murine immunization, and cellular immunity to UL83 and UL123 by in vitro amplification of T cell recall responses in human PBMC. CONCLUSIONS: rMVA promotes high level expression of three immunodominant CMV antigens, which is reflected in results of immunization studies in which high titers of UL55-specific antibodies and CD4+ T-help are detected, as well as high levels of UL83-specific and moderate levels of UL123-specific CD8+ CTL.     

Pathogenic anti-DNA antibodies in SLE: idiotypic families and genetic origins

We have adopted an idiotypic approach to study the double stranded DNA (dsDNA) binding antibodies of systemic lupus erythematosus (SLE). Three anti-idiotypic reagents, 8.12, 3I, and F4, identify cross reactive idiotypes that are each expressed on anti-dsDNA antibodies in the sera of many patients with SLE. These idiotypic antibodies are implicated in the pathogenesis of SLE as they are present in immune complex deposits in the kidneys of patients with SLE glomerulonephritis. The autoantibody associated idiotypes are also expressed on antibodies that do not bind DNA. We are investigating the origin of the pathogenic anti-dsDNA antibodies of SLE by comparing the autoantibodies, the antibodies to foreign antigens, and the myeloma proteins that express each SLE associated idiotype. In conjunction with serological analysis of these idiotypic systems, molecular genetic studies indicate that both the 8.12 and the 3I autoantibody associated idiotypes may be germline encoded, while the F4 idiotype is generated by somatic mutation. The data further suggest that the antigenic specificity of the pathogenic anti-DNA antibodies of SLE is acquired through somatic mutation of germline immunoglobulin genes. By studying the regulation of genes capable of encoding pathogenic autoantibodies, in both SLE patients and non-autoimmune individuals, we may be able to elucidate the pathogenesis of autoimmune disease and begin to design more effective therapeutic interventions.     

Adaptive changes in early and late blind: a FMRI study of verb generation to heard nouns

Literacy for blind people requires learning Braille. Along with others, we have shown that reading Braille activates visual cortex. This includes striate cortex (V1), i.e., banks of calcarine sulcus, and several higher visual areas in lingual, fusiform, cuneus, lateral occipital, inferior temporal, and middle temporal gyri. The spatial extent and magnitude of magnetic resonance (MR) signals in visual cortex is greatest for those who became blind early in life. Individuals who lost sight as adults, and subsequently learned Braille, still exhibited activity in some of the same visual cortex regions, especially V1. These findings suggest these visual cortex regions become adapted to processing tactile information and that this cross-modal neural change might support Braille literacy. Here we tested the alternative hypothesis that these regions directly respond to linguistic aspects of a task. Accordingly, language task performance by blind persons should activate the same visual cortex regions regardless of input modality. Specifically, visual cortex activity in blind people ought to arise during a language task involving heard words. Eight early blind, six late blind, and eight sighted subjects were studied using functional magnetic resonance imaging (fMRI) during covert generation of verbs to heard nouns. The control task was passive listening to indecipherable sounds (reverse words) matched to the nouns in sound intensity, duration, and spectral content. Functional responses were analyzed at the level of individual subjects using methods based on the general linear model and at the group level, using voxel based ANOVA and t-test analyses. Blind and sighted subjects showed comparable activation of language areas in left inferior frontal, dorsolateral prefrontal, and left posterior superior temporal gyri. The main distinction was bilateral, left dominant activation of the same visual cortex regions previously noted with Braille reading in all blind subjects. The spatial extent and magnitude of responses was greatest on the left in early blind individuals. Responses in the late blind group mostly were confined to V1 and nearby portions of the lingual and fusiform gyri. These results confirm the presence of adaptations in visual cortex of blind people but argue against the notion that this activity during Braille reading represents somatosensory (haptic) processing. Rather, we suggest that these responses can be most parsimoniously explained in terms of linguistic operations. It remains possible that these responses represent adaptations which initially are for processing either sound or touch, but which are later generalized to the other modality during acquisition of Braille reading skills.     

A critical function for type I interferons in cancer immunoediting

'Cancer immunoediting' is a process wherein the immune system protects hosts against tumor development and facilitates outgrowth of tumors with reduced immunogenicity. Although interferon-gamma (IFN-gamma) is known to be involved in this process, the involvement of type I interferons (IFN-alpha/beta) has not been elucidated. We now show that, like IFN-gamma, endogenously produced IFN-alpha/beta was required for the prevention of the growth of primary carcinogen-induced and transplantable tumors. Although tumor cells are important IFN-gamma targets, they are not functionally relevant sites of the actions of the type I interferons. Instead, host hematopoietic cells are critical IFN-alpha/beta targets during development of protective antitumor responses. Therefore, type I interferons are important components of the cancer immunoediting process and function in a way that does not completely overlap the functions of IFN-gamma.     

Cloning and Sequencing of a Candida albicans Catalase Gene and Effects of Disruption of This Gene

Catalase plays a key role as an antioxidant, protecting aerobic organisms from the toxic effects of hydrogen peroxide, and in some cases has been postulated to be a virulence factor. To help elucidate the function of catalase in Candida albicans, a single C. albicans-derived catalase gene, designated CAT1, was isolated and cloned. Degenerate PCR primers based on highly conserved areas of other fungal catalase genes were used to amplify a 411-bp product from genomic DNA of C. albicans ATCC 10261. By using this product as a probe, catalase clones were isolated from genomic libraries of C. albicans. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 487 amino acid residues. Construction of a CAT1-deficient mutant was achieved by using the Ura-blaster technique for sequential disruption of multiple alleles by integrative transformation using URA3 as a selectable marker. Resulting mutants exhibited normal morphology and comparable growth rates of both yeast and mycelial forms. Enzymatic analysis revealed an abundance of catalase in the wild-type strain but decreasing catalase activity in heterozygous mutants and no detectable catalase in a homozygous null mutant. In vitro assays showed the mutant strains to be more sensitive to damage by both neutrophils and concentrations of exogenous peroxide that were sublethal for the parental strain. Compared to the parental strain, the homozygous null mutant strain was far less virulent for mice in an intravenous infection model of disseminated candidiasis. Definitive linkage of CAT1 with virulence would require restoration of activity by reintroduction of the gene into mutants. However, initial results in mice, taken together with the enhanced susceptibility of catalase-deficient hyphae to damage by human neutrophils, suggest that catalase may enhance the pathogenicity of C. albicans.     

Comparison of assays to detect cytomegalovirus shedding in the semen of HIV-infected men

We sought to determine the optimal assays for cytomegalovirus (CMV) shedding in semen. Over a 2-month period, 149 HIV-1-infected men who have sex with men each provided up to three semen specimens. Specimens were tested for CMV by culture, rapid assay (shell vial) and polymerase chain reaction (PCR). By culture, 30% of seminal plasma and 28% of seminal cell specimens grew CMV. By rapid assay, results were 38 and 33%, respectively. By PCR, 56% of seminal cell specimens demonstrated CMV: 20% in a single semen specimen; 33% in two specimens; and 34% in all three specimens. Overall, 69% of men had CMV detected by PCR in at least one seminal cell specimen. By quantitative PCR, 14% had ten, 14% had 100, 16% had 1000, and 12% had 10 000 copies in 6.25 μl of semen analyzed. Adjusting for initial CD4+ cell count, men with CMV shedding demonstrated by PCR at the first visit were approximately four times as likely to shed CMV at a subsequent visit (RR 4.28, CI: 2.30–7.95). CMV shedding was associated with decreased CD4+ cell counts in peripheral blood (P=0.05). It is concluded that the PCR assay provided the greatest sensitivity among the three detection methods.     

Health beliefs and attitudes toward the prevention of osteoporosis in older women

OBJECTIVE: A pilot study to determine health belief factors associated with osteoporosis prevention behaviors in peri-and postmenopausal women. DESIGN: We administered a survey to a convenience sample of 60 women aged 40-95 years old in an urban family practice center and an associated retirement community. The self-reported questionnaire addressed demographics, osteoporosis risk factors, current preventive behaviors for osteoporosis, and health beliefs. RESULTS: The majority of women (89%) believed that osteoporosis is a serious condition, but only 29% perceived a personal susceptibility. Women were less concerned about osteoporosis when compared with cancer, cardiovascular disease, and neurologic disorders. Only 40% of women were taking active measures to prevent osteoporosis. There was no significant relationship between active osteoporosis prevention behaviors and five health belief factors (motivation, barrier, active participant in health care, frustration, and benefit) (p >or= 0.43). However, active behaviors to prevent osteoporosis were found to correlate with the single item "I am worried about developing osteoporosis" (p = 0.03). Most women surveyed would be willing to exercise and take calcium and a multivitamin to prevent osteoporosis. CONCLUSION: Few women are taking active measures to prevent osteoporosis despite their belief that it is a serious condition. Our data suggest that most women do not perceive a personal susceptibility to the disease. Only women who reported actively worrying about developing osteoporosis were more likely to be engaged in significant osteoporosis preventive behaviors.     

The effect of membrane fixed charges on diffusion potentials and streaming potentials

1. Electrical potential differences (p.d.''s) have been measured across an in vitro preparation of rabbit gall-bladder.2. When the gall-bladder separates identical bathing solutions, the p.d. is always zero, regardless of the composition of the bathing solution. Hence the gall-bladder is symmetrical: i.e. the mucosal and serosal cell membranes have the same relative permeability coefficients.3. Osmotic water flow causes streaming potentials of up to 20 mV, of a sign indicating greater permeability to cations than to anions.4. At constant osmolarity, streaming potentials increase slightly with NaCl concentration. Streaming potentials decrease considerably with changes in osmolarity resulting from changes in NaCl concentration.5. Diffusion potentials resulting from electrolyte concentration gradients are fitted well by the constant-field equation with the relative permeability coefficients P_Na = 1·00, P_Cl = 0·33, P_K = 2·3. These permeability coefficients are independent of osmolarity and of salt concentration.6. Relative to 0·25 mM-Ca, 5 mM-Ca reduces streaming potentials by 40%, NaCl diffusion potentials by 62%, and potassium diffusion potentials by 43%.7. The aqueous channels through which water and electrolytes traverse the cell membranes of the gall-bladder contain negative fixed charges, which are blocked by Ca. The physiological significance of the charges may be to reduce chloride permeability and thereby to increase the effectiveness of the gall-bladder in concentrating bile.8. The effect of pH, and analogy with surface charges of other cells, suggest that the charges are organic acids of low pK_a.     

Induction of CTL response by a minimal epitope vaccine in HLA A*0201/DR1 transgenic mice: dependence on HLA class II restricted T(H) response

CTL play a pivotal role in the immune response during viral infections. In this study, the HLA class II restricted T(H) requirement for optimal in vivo induction of HLA class I restricted CTL responses has been investigated. Towards this goal, transgenic mice expressing both HLA class I (A*0201 or A2.1) and class II (DRB1*0101 or DR1) molecules have been derived. Immunization of these mice with an HLA A*0201-restricted and CMV-specific CTL epitope (pp65(495-503)), and either of three different tetanus toxin-derived MHC class II-binding T(H) epitopes, resulted in a vigorous CTL response. CTL specific for the pp65(495-503) epitope were dramatically enhanced in mice expressing both the HLA-DR1 and HLA-A*0201 transgenes. Notably, preinjection of three TT peptides (TT(639-652), TT(830-843), and TT(947-967)) increased the capability of HLA A*0201/DR1 Tg mice to respond to subsequent immunization with the T(H) + CTL peptide mixture. These results indicate that the use of HLA A*0201/DR1 Tg mice constitute a versatile model system (in lieu of immunizing humans) for the study of both HLA class I and class II restricted T-cell responses. These studies provide a rational model for the design and assessment of new minimal-epitope vaccines based on their in vivo induction of a pathogen-specific CTL response.