Mucosal delivery of ACNPV baculovirus driving expression of the Gal-lectin LC3 fragment confers protection against amoebic liver abscess in hamster.

Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA) in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9%) or partial development (21%) of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine.

Cobalt sorption in silica-pillared clays.

Silicon pillared samples were prepared following conventional and microwave irradiation methods. The samples were characterized and tested in cobalt sorption. Ethylenediammine was added before cobalt addition to improve the amount of cobalt retained. The amount of cobalt introduced in the original clay in the presence of ethylenediammine was the highest. In calcined pillared clays the cobalt retention with ethylenediammine was lower (ca. 40%). In all cases the presence of ethylenediammine increased twice the amount of cobalt sorption measured for aqueous solutions.

Analysis of the local kinetics and localization of interleukin-1 alpha, tumour necrosis factor-alpha and transforming growth factor-beta, during the course of experimental pulmonary tuberculosis.

A mouse model of pulmonary tuberculosis induced by the intratracheal instillation of live and virulent mycobacteria strain H37-Rv was used to examine the relationship of the histopathological findings with the local kinetics production and cellular distribution of tumour necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha) and transforming growth factor-beta (TGF-beta). The histopathological and immunological studies showed two phases of the disease: acute or early and chronic or advanced. The acute phase was characterized by inflammatory infiltrate in the alveolar-capillary interstitium, blood vessels and bronchial wall with formation of granulomas. During this acute phase, which lasted from 1 to 28 days, high percentages of TNF-alpha and IL-1 alpha immunostained activated macrophages were observed principally in the interstium-intralveolar inflammatory infiltrate and in granulomas. Electron microscopy studies of these cells, showed extensive rough endoplasmic reticulum, numerous lysosomes and occasional mycobacteria. Double labelling with colloid gold showed that TNF-alpha and IL-1 alpha were present in the same cells, but were confined to separate vacuoles near the Golgi area, and mixed in larger vacuoles near to cell membrane. The concentration of TNF-alpha and IL-1 alpha as well as their respective mRNAs were elevated in the early phase, particularly at day 3 when the bacillary count decreased. A second peak was seen at days 14 and 21-28 when granulomas appeared and evolved to full maturation. In contrast, TGF-beta production and numbers of immunoreactive cells were low in comparison with the advanced phase of the disease. The chronic phase was characterized by histopathological changes indicative of more severity (i.e. pneumonia, focal necrosis and extensive interstitial fibrosis) with a decrease in the TNF-alpha and IL-1 alpha production that coincided with the highest level of TGF-beta. The bacillary counts were highest as the macrophages became large, vacuolated foamy cells, and containing numerous bacilli with immunoreactivity to mycobacterial lipids and lipoarabinomannan (LAM). These macrophages displayed poor and scarce TNF-alpha and IL-1 alpha immunostaining but still strong immunoreactivity to TGF-beta. These cytokine production kinetics and the spatial relationship between immunostained cells and lung lesions corroborate the important role of TNF-alpha and IL-1 alpha in the constitution of granulomas and immune protection during the early phase of the infection, and also suggest an important if not primary role for TGF-beta in the immunopathogenesis of the advanced forms of pulmonary tuberculosis.

Correlation between the kinetics of Th1, Th2 cells and pathology in a murine model of experimental pulmonary tuberculosis.

T-helper 1 (Th1) Th2 kinetics were studied by immunohistochemistry and molecular biology techniques (reverse transcriptase polymerase chain reaction. RT PCR, Southern-blot) during the course of pulmonary tuberculosis induced in BALB/c mice by the intratracheal instillation of the live and virulent strain H-37Rv. The histopathological study clearly showed two phases of the disease. The first one was an acute phase which was characterized by inflammatory infiltrate in the alveolar capillary interstitium, blood vessel and bronchial wall with formation of granulomas. In this acute phase which lasted from 1 to 28 days, a clear predominance of Th1 cells was observed, manifested by a high percentage of interleukin-2 (IL-2) positive cells in the inflammatory infiltrate and granulomas demonstrated by immunohistology, as well as a gradual increment of interferon-gamma (INF-gamma) m-RNA. This was followed by a chronic or advanced phase characterized by pneumonia, focal necrosis and fibrosis, with a Th0 balance due to an equivalent proportion of IL-2 and IL-4 positive cells in the lung lesions, that coincided with the highest level of INF-gamma and IL-4 mRNA. The cytofluorometric analysis of bronchial lavage cells, showed a predominance of CD4 T cells during the acute phase and CD8 T lymphocytes in the chronic phase, gamma-delta T lymphocytes showed two peaks, at the beginning (3 days) and at the end (4 months) of the infection. These results suggest that T-lymphocyte subset kinetics and the pattern of cytokines produced in the lung during tuberculosis infection changed over time and correlate with the type and magnitude of tissue injury.

Sequence and functional analysis of the Streptomyces phaeochromogenes plasmid pJV1 reveals a modular organization of Streptomyces plasmids that replicate by rolling circle.

pJV1 is an 11 kb, high-copy-number conjugative Streptomyces phaeochromogenes plasmid that replicates by the rolling circle mechanism (RCR). Sequencing combined with functional analysis of deletion, insertion and frameshift mutations was used to characterize the genes involved in plasmid transfer and chromosome mobilization (Cma), the single-strand origin for RCR and an associated strong incompatibility (Sti) determinant. pJV1 contains two essential transfer genes whose expression is regulated by an adjacent repressor gene with similarity to the GntR family of regulators. A consensus sequence specific for the helix-turn-helix motifs of repressor proteins of Streptomyces plasmids is proposed. Unregulated expression of the transfer genes by inactivation of the repressor is lethal. Three additional genes increase intramycelial plasmid spread resulting in pock formation but, unlike the essential transfer genes, are not required for Cma. The pJV1 transfer genes and their regulatory region, but not the minimal replication region encoding the double-strand replication origin and replication protein, are similar in their sequence and arrangement to those of the Streptomyces nigrifaciens plasmid pSN22, revealing a modular organization of Streptomyces RCR plasmids.

Presentation of synthetic peptide antigen encoded by the MAGE-1 gene by granulocyte/macrophage-colony-stimulating-factor-cultured macrophages from HLA-A1 melanoma patients.

The recent identification of the sequences of the peptides derived from a number of human melanoma-associated antigens has presented opportunities for developing a specific-peptide-based vaccine in this form of cancer. Since antigen-presenting cells (APC) play a crucial role in the induction of the T-cell-mediated immune response, we examined whether or not ex vivo cultured APC, bearing the appropriate MHC restricting elements, when pulsed with a relevant melanoma-specific cytotoxic-T-lymphocyte (CTL)-determined peptide, can present the peptide to the CTL. Here we show that a population of cells, derived from the monocyte/macrophage lineage from peripheral blood and grown in granulocyte/macrophage-colony-stimulating factor, exhibit many essential characteristics of "professional" APC (dendritic-type morphology with a proportion of the population, the B7 molecule, and high levels of MHC class I and class II molecules, CD11b and CD54 molecules) and are capable of efficiently presenting the nonapeptide, EADPTGHSY, encoded by the melanoma antigen MAGE-1 gene, to the MAGE-1-specific CTL clone, 82/30. These results suggest that this type of autologous ex vivo cultured population of professional APC, when pulsed with the relevant-CTL-determined peptide, can serve as a novel type of candidate vaccine for active specific immunization against HLA-A1-positive patients with melanoma expressing the MAGE-1 antigen.

Recombinant outer surface protein C ELISA for the diagnosis of early Lyme disease.

To compare the sensitivity of a new ELISA for IgM antibodies to Borrelia burgdorferi that uses a recombinant outer surface protein C (rOspC) with those of a whole cell (WC) ELISA and an immunoblot assay for the diagnosis of early Lyme disease, serum specimens from 82 consecutive patients with physician-documented erythema migrans were analyzed. To compare the specificities of the three assays, serum specimens from 50 patients without a history of Lyme disease and from an area in which B. burgdorferi is not endemic were analyzed. The sensitivities of the WC ELISA, immunoblot assay, and IgM rOspC ELISA were 28%, 29%, and 46%, respectively, while the specificities were 100%, 100%, and 98%, respectively. The IgM rOspC ELISA is a convenient, readily automated, easily standardized serologic test that is significantly more sensitive for the diagnosis of early Lyme disease than either WC ELISA or immunoblot assay.

Increased collagen synthesis in skin fibroblasts from patients with primary hypertrophic osteoarthropathy. Evidence for trans-activational regulation of collagen transcription.

OBJECTIVE: To investigate collagen synthesis in skin fibroblasts from patients with primary hypertrophic osteoarthropathy (HOA), a disorder characterized clinically by skin thickening. METHODS: Collagenase-digestible protein, messenger RNA (mRNA) levels, and transcriptional activity of the alpha 1(I) procollagen gene were assessed in skin-derived fibroblast lines. RESULTS: Compared with fibroblasts from uninvolved skin, fibroblasts from involved skin had elevated levels of collagen synthesis and alpha 1(I) procollagen mRNA, and increased transcriptional activity of the alpha 1(I) procollagen promoter. CONCLUSION: Abnormalities of collagen synthesis in fibroblasts from patients with primary HOA can be accounted for, at least in part, by a trans-activated up-regulation of collagen transcription.

Use of recombinant OspC from Borrelia burgdorferi for serodiagnosis of early Lyme disease.

Infection with Borrelia burgdorferi, the etiologic agent of Lyme disease, is associated with an early and dominant humoral response to the spirochete's 23-kDa outer surface protein C (OspC). We have cloned and expressed OspC as a fusion protein in Escherichia coli and have shown that patient serum samples react with it in an enzyme-linked immunosorbent assay (ELISA) (S. J. Padula, A. Sampieri, F. Dias, A. Szczepanski, and R. W. Ryan, Infect. Immun. 61:5097-5105, 1993). Now we have compared the detection of B. burgdorferi-specific immunoglobulin M antibodies in 74 individuals with culture-positive erythema migrans by a whole-cell ELISA, immunoblot, and the recombinant OspC (rOspC) ELISA. Seventy-six negative controls were also studied. With all of the tests, there was a statistically significant association between the duration of disease and the frequency of a positive result. With the rOspC ELISA, the predictive value of a positive test was 100% and the predictive value of a negative test was 74%. Similar results were obtained with the whole-cell ELISA and with the immunoblot using as the source of test antigen a strain of B. burgdorferi which expresses abundant levels of OspC. We conclude that the use of rOspC in an ELISA is a convenient, readily automated, and easily standardized test for the serodiagnosis of early Lyme disease.

Molecular characterization and expression of p23 (OspC) from a North American strain of Borrelia burgdorferi.

We have found that sera from patients with early stages of Lyme disease contain predominant immunoglobulin M reactivity to a major 23-kDa protein (p23) from Borrelia burgdorferi 2591 isolated in Connecticut. To characterize this immunodominant antigen, we cloned and sequenced p23 and found it to be 83% identical by nucleotide sequence and 75% identical by amino acid sequenced to pC (recently renamed OspC), an abundantly expressed protein on the outer surface of PKo, a European strain of B. burgdorferi (B. Wilske, V. Preac-Mursic, S. Jauris, A. Hofmann, I. Pradel, E. Soutschek, E.Schwab, G. Will, and G. Wanner, Infect. Immun. 61:2182-2191, 1993). In addition, immunoelectron microscopy localized p23 to the outer membrane, confirming that p23 is the strain 2591 homolog of OspC. The North American strain B31, commonly used in serologic assays for Lyme disease, does not express OspC. Northern (RNA) blot analysis detected low levels of ospC mRNA in B31, and DNA sequencing of the ospC gene from B31 revealed a 54-bp deletion in the upstream regulatory region, possibly accounting for the low transcriptional activity of ospC. The ospC coding region from B31 was cloned and antibody-reactive OspC was expressed in Escherichia coli. An immunoglobulin M enzyme-linked immunosorbent assay using recombinant OspC as the target antigen shows promise for the serodiagnosis of early stages of Lyme disease.