Randomized study of phentolamine mesylate for reversal of local anesthesia.

Local anesthetic solutions frequently contain vasoconstrictors to increase the depth and/or duration of anesthesia. Generally, the duration of soft-tissue anesthesia exceeds that of pulpal anesthesia. Negative consequences of soft-tissue anesthesia include accidental lip and tongue biting as well as difficulty in eating, drinking, speaking, and smiling. A double-blind, randomized, multicenter, Phase 2 study tested the hypothesis that local injection of the vasodilator phentolamine mesylate would shorten the duration of soft-tissue anesthesia following routine dental procedures. Participants (122) received one or two cartridges of local anesthetic/vasoconstrictor prior to dental treatment. Immediately after treatment, 1.8 mL of study drug (containing 0.4 mg phentolamine mesylate or placebo) was injected per cartridge of local anesthetic used. The phentolamine was well-tolerated and reduced the median duration of soft-tissue anesthesia in the lip from 155 to 70 min (p < 0.0001).

Effects of feline immunodeficiency virus on feline monocyte-derived dendritic cells infected by spinoculation.

During type 1 human immunodeficiency virus infection, not only can dendritic cells (DCs) prime T cells against the virus, but they can also infect them in trans. Feline AIDS is caused by feline immunodeficiency virus (FIV) and is considered a model for the human illness because the two diseases have many features in common. Little is known about the interaction of feline DCs with FIV; therefore, this study attempts to tackle such an issue. Infection of feline monocyte-derived DCs (MDDCs) was attempted by spinoculation with FIV strains Petaluma (FIV-Pet) and M2. FIV-Pet was released rapidly in the supernatants of both infected MDDCs and activated T cells after spinoculation. It is shown that FIV-Pet was produced by MDDCs by monitoring viral content in the supernatants of infected MDDCs, by intracellular staining for p25 and by showing its cytopathic effect. Although activated T cells were better substrates for FIV replication, leading to prolonged viral shedding, both immature MDDCs and MDDCs matured with lipopolysaccharide supported virus production, mostly during the first 2 days after infection. At later times, FIV induced syncytium formation by MDDCs. Concerning the FIV receptors, MDDCs were shown to be CD134-negative and CXCR4-positive, a phenotype compatible with permissiveness to FIV-Pet. These results also suggest that maturation is not hampered by FIV infection and that virus exposure itself does not induce MDDC maturation. It is also shown that infected MDDCs can infect activated PBMCs efficiently in trans. It is concluded that MDDCs can be infected by FIV, although infection does not appear to influence their functionality.

Development of feline immunodeficiency virus ORF-A (tat) mutants: in vitro and in vivo characterization.

A functional ORF-A is essential for efficient feline immunodeficiency virus replication in lymphocytes. We have characterized a series of mutants of the Petaluma strain, derived from p34TF10 and having different combinations of stop codons and increasingly long deletions in ORF-A. Six clones proved fully replicative in fibroblastoid Crandell feline kidney cells and monocyte-derived macrophage cultures but failed to replicate in T cell lines and primary lymphoblasts. Cats inoculated with three selected mutants had considerably milder infections than controls given intact ORF-A virus. In vivo, the mutants maintained growth properties similar to those in vitro for at least 7 months, except that replication in lymphoid cells was strongly reduced but not ablated. One mutant underwent extensive ORF-A changes without, however, reverting to wild-type. Antiviral immune responses were feeble in all cats, suggesting that viral loads were too low to represent a sufficiently powerful antigenic stimulus.

Purification, biochemical characterization and immunogenicity of SA5K, a secretion antigen of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) secretory proteins are generally considered important antigens for immune protection against tuberculosis (TB). An 8.3-kDa secretory antigen of MTB and Mycobacterium bovis bacillus Calmette-Guérin (BCG), called SA5K, was recently identified and cloned in our laboratory. In this report, recombinant SA5K containing a histidine hexamer was expressed in Escherichia coli and purified to investigate its biochemical structure and to establish whether it was immunogenic for healthy sensitized and nonsensitized human donors and for patients infected with MTB. The protein nucleotide sequence was shown to be identical in BCG and in MTB. SA5K revealed an abnormal electrophoretic mobility in SDS-PAGE that made it look lighter than it is in Western blotting. While recombinant SA5K was poorly recognized by T lymphocytes from patients with pulmonary TB, it elicited proliferation of CD4+ T lymphocytes in the vast majority of healthy individuals sensitized to mycobacterial antigens by BCG vaccination. At a serum dilution of 1 : 80, antibodies reacting against recombinant SA5K were found in 67% of sera from TB patients and in 73% of sera from healthy subjects. The percentage of positive subjects dropped at higher serum dilutions, but no significant difference in the recognition rate was observed between TB patients and healthy donors and between healthy vaccinated and nonvaccinated subjects. Owing to the high percentage of sera from healthy subjects who recognized SA5K in Western blotting, the antigen seems to exhibit, at least in the present form, a poor specificity for an employment for a serodiagnosis of TB.

TT virus levels in the plasma of infected individuals with different hepatic and extrahepatic pathology.

TT virus (TTV) infection is extremely widespread in the general population. A sensitive real-time PCR assay was developed that quantitated accurately the most prevalent TTV genotypes in Italy. When used to test 217 individuals for TTV viraemia, the overall prevalence was 94%. Viraemia levels varied widely amongst individual subjects, with no major differences related to gender or age. The highest TTV titres were in haemophiliacs and in patients with non-A-E hepatitis, but they did not differ from the group with miscellaneous diseases. HIV- and HCV-infected subjects and patients with primary liver diseases had TTV loads similar to those of healthy individuals.

Molecular properties, biology, and clinical implications of TT virus, a recently identified widespread infectious agent of humans.

TT virus (TTV) was first described in 1997 by representational difference analysis of sera from non-A to non-G posttransfusion hepatitis patients and hence intensively investigated as a possible addition to the list of hepatitis-inducing viruses. The TTV genome is a covalently closed single-stranded DNA of approximately 3.8 kb with a number of characteristics typical of animal circoviruses, especially the chicken anemia virus. TTV is genetically highly heterogeneous, which has led investigators to group isolates into numerous genotypes and subtypes and has limited the sensitivity of many PCR assays used for virus detection. The most remarkable feature of TTV is the extraordinarily high prevalence of chronic viremia in apparently healthy people, up to nearly 100% in some countries. The original hypothesis that it might be an important cause of cryptogenic hepatitis has not been borne out, although the possibility that it may produce liver damage under specific circumstances has not been excluded. The virus has not yet been etiologically linked to any other human disease. Thus, TTV should be considered an orphan virus.

Qualitative and quantitative requirements for CD4+ T cell-mediated antiviral protection.

CD4+ Th cells deliver the cognate and cytokine signals that promote the production of protective virus-neutralizing IgG by specific B cells and are also able to mediate direct antiviral effector functions. To quantitatively and qualitatively analyze the antiviral functions of CD4+ Th cells, we generated transgenic mice (tg7) expressing an MHC class II (I-Ab)-restricted TCR specific for a peptide derived from the glycoprotein (G) of vesicular stomatitis virus (VSV). The elevated precursor frequency of naive VSV-specific Th cells in tg7 mice led to a markedly accelerated and enhanced class switching to virus-neutralizing IgG after immunization with inactivated VSV. Furthermore, in contrast to nontransgenic controls, tg7 mice rapidly cleared a recombinant vaccinia virus expressing the VSV-G (Vacc-IND-G) from peripheral organs. By adoptive transfer of naive tg7 CD4+ T cells into T cell-deficient recipients, we found that 105 transferred CD4+ T cells were sufficient to induce isotype switching after challenge with a suboptimal dose of inactivated VSV. In contrast, naive transgenic CD4+ T cells were unable to adoptively confer protection against peripheral infection with Vacc-IND-G. However, tg7 CD4+ T cells that had been primed in vitro with VSV-G peptide were able to adoptively transfer protection against Vacc-IND-G. These results demonstrate that the antiviral properties of CD4+ T cells are governed by the differentiation status of the CD4+ T cell and by the type of effector response required for virus elimination.

Analysis of the Mycobacterium bovis hsp60 promoter activity in recombinant Mycobacterium avium.

A clinical isolate of Mycobacterium avium was transformed with a new shuttle plasmid containing the Escherichia coli beta-galactosidase reporter gene under the control of the Mycobacterium bovis bacillus Calmette-Guérin (BCG) hsp60 promoter. beta-Galactosidase activity was assayed spectrophotometrically in bacterial homogenates of the recombinant strain (M. avium::lacZ) and used for quantification of the hsp60 promoter strength in different conditions of extra- and intracellular growth. Very low levels of beta-galactosidase were recorded during the exponential phase of in vitro growth, while they increased progressively during the late exponential and stationary phases. A significant increase in enzyme activity was also induced in exponentially growing cells by shifting the incubation temperature from 37 to 45 degrees C, but not from 37 to 42 degrees C nor from 30 to 42 degrees C. No induction of the promoter was observed by adding hydrogen peroxide to the cultures. Finally, beta-galactosidase levels were quantified during growth of M. avium::lacZ in murine macrophages. Soon after phagocytosis and, to a lesser extent at 1, 5 and 7 days after infection, increased levels of bacterial beta-galactosidase were observed indicating an increment in transcriptional activity of hsp60 promoter both at early phases of infection and during the course of intracellular growth.

Identification and molecular cloning of a novel secretion antigen from Mycobacterium tuberculosis and Mycobacterium bovis BCG.

A novel protein called SA-5K was identified in Mycobacterium bovis BCG (BCG) short-term culture filtrates (CFs) by means of a recently described monoclonal antibody (mAb), L8D8. This protein had an apparent molecular mass (MM) of 5 kDa, as judged by Western blotting after sodium dodecyl sulphate-polyacrylamide gel electrophoresis in reducing conditions, and did not seem to contain any sugar or lipid substituents. In the present work, SA-5K was purified from BCG CFs by affinity chromatography. A protein that could be detected in Western blot but not by standard protein staining techniques was obtained. When SA-5K was subjected to aminoterminal sequencing, the 10 amino acids (aa) found matched the first 10-aa sequence deduced from an open reading frame (ORF) of M. tuberculosis. The ORF encodes a polypeptide, likely to include a signal for secretion, with an estimated MM of 8.3 kDa after signal peptide cleavage. The secretory nature of SA-5K was confirmed by the fact that it could only be detected in CFs, but not in other BCG subcellular fractions. After size exclusion chromatography, reactivity with mAb L8D8 was found to peak in the 45-50- and 14-16-kDa fractions. The latter MM was close to that estimated from the ORF of M. tuberculosis, implying that the 5-kDa antigen detected initially by Western blot in reducing conditions was a portion of SA-5K released after reduction of a disulphide bridge. The presence of the gene for SA-5K in BCG and its identity were confirmed by PCR (polymerase chain reaction) with specific primers and restriction analysis: the PCR product was slightly shorter in BCG than in M. tuberculosis. The gene coding for SA-5K was cloned by PCR from BCG and M. tuberculosis DNA and was expressed in Escherichia coli.

Characterization of antigens recognized by new monoclonal antibodies raised against culture filtrate proteins of Mycobacterium bovis bacillus Calmette-Guérin.

Effective protection against Mycobacterium tuberculosis may be achieved in experimental animals by immunization with proteins secreted by tuberculous bacilli in the extracellular milieu during growth. In this study, monoclonal antibodies were raised against Mycobacterium bovis bacillus Calmette-Guérin (BCG) culture filtrate proteins or live BCG, in an attempt to identify novel mycobacterial secretion antigens: the localization of the antigens recognized by the monoclonal antibodies within the mycobacterial cell was studied and interspecies reactivity was also investigated. The monoclonal antibodies obtained recognized proteins of molecular mass ranging from 5 to 82 kDa, with a prevailing frequency in the 30 kDa region. Three of the monoclonal antibodies recognized proteins present only in culture filtrates, one reacted with a cytoplasmic antigen, while the remaining antibodies recognized components which were mainly associated with the cell wall and the cytoplasmic membrane. The chemical nature and possible identity of the antigens was checked. Three monoclonal antibodies are likely to react with novel mycobacterial antigens of 5, 42 and 82 kDa, respectively.

Comparative analysis of subcellular distribution of protein antigens in Mycobacterium bovis bacillus Calmette-Guérin.

The distribution of protein antigens in purified subcellular fractions of Mycobacterium bovis bacillus Calmette-Guérin (BCG) was comparatively analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with specific monoclonal antibodies and polyclonal sera. The 19- and 38-kDa lipoproteins were mainly detected in the cell wall and cell membrane enriched fractions, and they were extracted from the former by Triton X-114 and Nonidet P-40. The 65-kDa heat-shock protein (hsp) was present in the cytoplasmic fraction and only trace amounts were found in the crude cell wall preparation. In contrast, the 14-kDa hsp was highly represented in the cell wall fraction, besides being present in cytoplasmic fraction. Both superoxide dismutase (SOD) and antigen 85 complex (Ag 85) were abundantly released in culture medium, and to a lower extent, they were present in the cell wall fraction; SOD was present in comparable amounts also in the cytoplasmic fraction, while Ag 85 was far less represented in the same. Sera from mice immunized with culture filtrate (CF) proteins of BCG recognized several antigens in CFs, which were not detectable in cell wall, cell membrane, and cytoplasmic fractions, indicating that CF proteins include secreted antigens which have not yet been identified.

The role of antibody concentration and avidity in antiviral protection.

Neutralizing antibodies are necessary and sufficient for protection against infection with vesicular stomatitis virus (VSV). The in vitro neutralization capacities and in vivo protective capacities of a panel of immunoglobulin G monoclonal antibodies to the glycoprotein of VSV were evaluated. In vitro, neutralizing activity correlated with avidity and with neutralization rate constant, a measure of on-rate. However, in vivo, protection was independent of immunoglobulin subclass, avidity, neutralization rate constant, and in vitro neutralizing activity; above a minimal avidity threshold, protection depended simply on a minimum serum concentration. These two biologically defined thresholds of antibody specificity offer hope for the development of adoptive therapy with neutralizing antibodies.

Correlation of tolerogenicity of a viral antigen with its immunogenicity.

Induction of B cell tolerance or activation was analyzed with vesicular stomatitis virus (VSV) glycoprotein (G) expressed as a neo-self Ag. A membrane form of VSV-G expressed in all tissues, including the bone marrow, induced unresponsiveness at both the Th and B cell level, whereas a soluble form of VSV-G expressed peripherally in liver and kidney did not tolerize B cells and only reversibly anergized Th cells. Interestingly, a similar correlation was found for activation of mature lymphocytes. When mature normal spleen cells were transferred into the two transgenic mouse lines, the membrane form of VSV-G was strongly immunogenic for both Th and B cells, and high VSV-G-specific IgG Ab titers were induced in these transgenic mice. In contrast, spleen cells transferred into mice expressing the soluble form of VSV-G were not activated, and no VSV-G specific Abs were induced. These results indicate that highly immunogenic Ags are strongly tolerogenic for both immature B and T cells.

Cloning of an unidentified T-cell receptor alpha chain gene from a vesicular stomatitis virus-specific helper T-cell clone.

A vesicular stomatitis virus glycoprotein-specific, class II restricted, CD4+ T-cell clone was obtained and the unidentified T-cell receptor alpha chain cloned in order to establish a T-cell receptor (TCR) alpha chain transgenic mouse line. Polymerase chain reaction (PCR) strategies have been developed to clone TCR chain genes starting from T-cell cDNA. There remain difficulties, however, in cloning the functional TCR alpha chain due to the complexity of the protocols applied if the variable (V) alpha region rearranged is not known. The strategy described here allows the identification and cloning of alpha chains that are not recognized by any of the anti-V alpha monoclonal antibodies available: three 5' consensus V alpha primers designed from all known V alpha gene sequences and a primer specific for the constant (C) alpha region were used and the PCR product sequenced. The T-cell clone displayed two alpha gene rearrangements, only one of which giving rise to a functional protein. The alpha chain used by the T-cell clone contained a V alpha 3.1 and a J alpha region which has been described only at the genomic level, but never in a functional TCR. The complete alpha chain gene was cloned by enriching the alpha chain-encoding cDNA by ligation-anchored PCR and using an alpha specific primer pair. The use of the present method, even if the sequence of the 5' untranslated (UT) region of the alpha chain is not known, is also discussed.

Role of T helper cell precursor frequency on vesicular stomatitis virus neutralizing antibody responses in a T cell receptor beta chain transgenic mouse.

During most immune responses, T cells help antigen-specific B cells to make antibodies against the antigen. One of the contributions of T cells to antibody production is the induction of isotype switching from IgM to IgG, which is the most abundant isotype in blood serum during recall responses. Other features of memory responses are faster kinetics and higher titers of antibody in the serum. What causes a primary immune response to be different from a secondary is not yet very clear and, particularly, the influence of precursor frequencies of T and B cells on memory responses still remains to be answered. To address this issue, a transgenic (tg) mouse line (ADA) was developed; it expresses the beta chain (V beta 2) of a major histocompatibility complex class II-restricted T cell receptor (TcR) specific for the glycoprotein (G) of vesicular stomatitis virus (VSV) serotype Indiana (VSV-IND). These mice exhibit an increased precursor frequency of VSV-specific CD4+ T cells that leads to enhanced neutralizing IgG production against VSV in vivo in unprimed mice. The data indicate that increased frequency of naive specific helper T cells alone may account for features of a memory phenotype such as high titer of antibodies and isotype switching.

Induction of protective cytotoxic T cells with viral proteins.

Induction of CD8+, class I-restricted T cells by non-infectious, exogenous antigens has been documented for model protein antigens such as ovalbumin and for major histocompatibility complex restricted short peptides in viral and tumor systems. However, the protective capacity of cytotoxic T cells induced by conventional proteins has not been tested in vivo so far. We, therefore, evaluated the induction of protective cytotoxic T cells against three different full-length recombinant viral proteins derived from a baculovirus expression system, i.e. the glycoprotein and nucleoprotein of lymphocytic choriomeningitis virus (LCMV) and the nucleoprotein of vesicular stomatitis virus (VSV). These viral proteins induced cytotoxic T cells in a T helper cell-independent fashion which lysed infected target cells in vitro and protected mice from viral replication, immunopathological disease and growth of a tumor expressing the same antigen as a tumor antigen. These results are surprising, since it had been shown earlier for completely inactivated nonreplicating viral vaccines and again here for beta-propiolactone-inactivated VSV or UV-light inactivated LCMV that nonreplicating viral vaccines were incapable of inducing protective cytotoxic T cells. Our data show that immunization of mice with as little as 10 micrograms of non-infectious viral proteins triggered long-lasting CD8+ T cell-mediated antiviral immunity. It was found that the protein alone was only weakly able to induce cytotoxic T cells, and that association with cellular debris functioned as an adjuvant. These findings may be relevant for our understanding of the phenomenon of cross-priming and have obvious implications for vaccine strategies.

Vesicular stomatitis virus Indiana glycoprotein as a T-cell-dependent and -independent antigen.

The neutralizing immunoglobulin M (IgM) response to vesicular stomatitis virus (VSV) has been shown to be largely T-cell independent in several T-cell-deficient models of mice. By using different antigen froms of VSV, VSV antigen doses could be graded in vivo (infectious > > UV inactivated > formalin inactivated). The present study reveals a T-cell-dependent component of the neutralizing IgM response in nude mice given intravenous injections of low doses of noninfectious UV-inactivated VSV serotype Indiana (VSV-IND) only if the mice are transfused with VSV-IND-specific helper T cells. Instead, nude mice immunized with infectious VSV, which leads to greater antigen doses in vivo, were able to mount an IgM response in the absence of T cells. These results indicate that the IgM response to low doses of VSV-IND glycoprotein (G) is T-cell dependent. Nude mice immunized with infectious VSV also made a variable but low VSV-IND-neutralizing IgG response. A VSV-IND matrix (M)-specific helper T-cell line rendered this response more consistent, much higher, and longer lasting. Thus (i) VSV-G induces a mostly T-cell-independent but partially T-cell-dependent IgM (the latter can be visualized best at low doses of antigen) and (ii) the antibody response to VSV in nude mice proceeds through steps, i.e., IgM and IgG, that are dose dependent. The results suggest that the predominant role of helper T cells may be to expand and maintain the individual steps of differentiating B cells.

Impaired immune and acute-phase responses in interleukin-6-deficient mice.

Interleukin-6 (IL-6) is a multifunctional cytokine that regulates various aspects of the immune response, acute-phase reaction and haematopoiesis (for reviews see refs 1, 2). In vitro, leukaemia inhibitory factor, oncostatin M, ciliary neurotrophic factor and interleukin-11 display overlapping activities with IL-6. This functional redundancy may be explained by the interactions of specific binding receptors with a common signal-transducing receptor (gp130) (for reviews see refs 3, 4). To elucidate the unique function of IL-6 in vivo, we have disrupted the IL-6 gene by homologous recombination. IL-6-deficient mice develop normally. They fail to control efficiently vaccinia virus and infection with Listeria monocytogenes, a facultative intracellular bacterium. The T-cell-dependent antibody response against vesicular stomatitis virus is impaired. Further, the inflammatory acute-phase response after tissue damage or infection is severely compromised, whereas it is only moderately affected after challenge with lipopolysaccharide. We conclude that IL-6 production induced by injury or infection is an important in vivo SOS signal which coordinates activities of liver cells, macrophages and lymphocytes.

Characterization of T-helper epitopes of the glycoprotein of vesicular stomatitis virus.

The T-helper (Th) cell epitopes in the glycoprotein (GP) of vesicular stomatitis virus serotype Indiana (VSV-IND) were analyzed with a complete panel of overlapping synthetic peptides. Three Th-cell epitopes in C57BL/6 (H-2b) mice and two epitopes in BALB/c (H-2d) mice were defined by their ability to stimulate in vitro proliferation of virus-primed, CD8+ T-cell-depleted spleen cells in a class II-restricted manner. A series of CD4+, I-Ab-restricted T-cell hybridomas from VSV-primed C57BL/6 mice were characterized by their production of interleukin-2 and interleukin-3 upon stimulation with VSV-IND or purified VSV GP in vitro. Of nine hybridomas derived from three independent fusions, five were specific for amino acids (aa) 415 to 433 (p8) of VSV-IND GP, three recognized aa 52 to 71 (p41), and one reacted against aa 316 to 335 (p17). Fluorocytometric analysis of Th hybridomas or VSV-stimulated T-cell lines with monoclonal antibodies specific for the T-cell receptor V beta chain did not reveal obvious correlations between the T-cell receptor V beta gene segment used and the epitope recognized. All three peptides recognized by H-2b mice and both epitopes recognized by H-2d mice which were characterized in primed T-cell populations were capable of activating specific Th cells in vivo as measured by the induction of antibody class switch from immunoglobulin M (IgM) to IgG. Thus, the epitopes are relevant for VSV GP-specific Th response in vivo and are able to provide functional help for the production of anti-VSV-specific neutralizing IgG antibodies.