Localization of abasic sites and single-strand breaks in mitochondrial DNA from brain of aged rat, treated or not with caloric restriction diet.

According to the "mitochondrial theory of aging" the lifelong accumulation of various kinds of damage to mitochondrial DNA (mtDNA) has been related to the age-dependent mitochondrial bioenergetic dysfunction. Caloric restriction (CR) diet is able to prevent or delay the onset of several age-related damages to mtDNA. The effects of aging and CR on the presence of abasic sites and single-strand breaks of the sugar-phosphate backbone in mtDNA have been analyzed by applying Ligation Mediated-PCR to a H strand region of brain mtDNA from young and old ad libitum-fed and old CR-treated rats. The region, encompassing the Direct Repeat 1 of the 4,834 bp-long deletion, is highly damaged in the old ad libitum-fed animals with respect to the young ones, whereas in the CR rats it shows a much lower extent of damage. The data confirm, at single nucleotide resolution, the protective effect of CR on the age-related mtDNA damage.

Mycobacterium porcinum strains isolated from bovine bulk milk: implications for Mycobacterium avium subsp. paratuberculosis detection by PCR and culture.

In this study, the isolation of 52 mycobactin-independent fast growing mycobacteria from 631 bulk milk samples (8.2%), is reported. These strains, isolated during a bulk milk survey for Mycobacterium avium subsp. paratuberculosis (Map), strongly affected Map detection both by PCR and by culture, as they gave a positive IS900 PCR signal and resulted to totally inhibit the growth of Map when spotted on HEYM slants already inoculated with 200 microl of 10-fold dilutions containing from 5 x 10 to 5 x 10(3)Map cells/ml. 16S rRNA gene sequencing, using the MicroSeq 500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems), was performed on a subset of six strains, identifying Mycobacterium porcinum with 100% homology in all six cases. The 52 strains were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene, which confirmed the identification of M. porcinum for all the isolates. Using specific primers designed on the Map-IS900 sequence and on the M. porcinum sequence determined in this study, a 1385bp sequence from the M. porcinum genome was characterized. This IS900-like sequence showed 82% homology with Map IS900. From our findings the following results emerged: (a) any culture showing one or more M. porcinum colonies represents a potential "false negative" result and should therefore be considered as contaminated; (b) IS900-like elements could be more widespread than was previously thought; (c) IS900 PCR positive results should be interpreted cautiously, as confirmed by the evidence that the primer pair used in this study resulted not to be specific.

Application of universal primers for identification of Foot-and-mouth disease virus and Swine vesicular disease virus by PCR and PCR-ELISA.

Two approaches for simultaneous identification of both Foot-and-mouth disease virus (FMDV) and Swine vesicular disease virus (SVDV) are described: (1) a single-step reverse transcription-PCR with three primers and (2) a PCR-ELISA assay with two universal primers for genome amplification and two virus-specific probes for identification. These methods are based on the use of 3D gene universal PCR primers, the structure of which was optimized and refined due to the close relationship between the two viruses belonging to different genera of the Picornaviridae family. In procedure (1), a three-primer PCR containing one universal antisense primer and two virus-specific primers was shown to differentiate between FMDV and SVDV in one reaction, due to the different length of the amplified DNA fragments (600 and 340 base pairs, respectively). In procedure (2), the two viruses were identified by PCR-ELISA, i.e. PCR for the 3D gene followed by two parallel hybridizations with FMDV and SVDV-specific probes in microplate wells and ELISA detection. The application of universal primers could halve the number of PCR experiments in both cases, as compared to the usual virus-specific PCR procedures. Also, we investigated the 3D gene structure of several SVDV strains isolated at different times. No essential changes were detected in the regions coding for conserved motifs of the RNA-dependent RNA polymerase recognized by our universal primers. The multi-primer PCR was successfully tested on 38 FMDV and 15 SVDV strains, and the PCR-ELISA on 32 FMDV and 16 SVDV strains including clinical material from disease cases.

HIV p17 enhances lymphocyte proliferation and HIV-1 replication after binding to a human serum factor.

OBJECTIVE: To analyse the role of recombinant HIV-1 protein p17 in the modulation of cell activity. METHODS: Peripheral blood mononuclear cells (PBMC) obtained from healthy donors were cultured in the presence or absence of p17 with mitogens such as phytohaemagglutinin or interleukin-2 and their response assayed by cell proliferation. Cross-linking experiments were employed to investigate the presence of a binding between p17 and factor(s) present in human serum. An immunoenzymatic assay for p24 antigen detection was used to analyse the effect of the addition of exogenous p17 to cultures of PBMC infected with HIV-1 in vitro. RESULTS: Purified recombinant p17 protein at a concentration of 0.25 microg/ml significantly increased the proliferation of preactivated PBMC obtained from healthy donors. This effect was obtained by binding p17 to factor(s) present in human serum and observed on both CD4+ and CD8+ T cells. Recombinant p17 also induced an increased rate of HIV-1 replication, probably due to enhanced T-cell proliferation. The activity of p17 protein was inhibited by anti-p17 antibodies generated by injecting recombinant p17 in rabbits, but not by human antibodies generated during the natural course of HIV infection. CONCLUSION: Characterization of the human factor(s) and identification of the interacting p17 epitope(s) will improve our understanding of the mechanisms used by HIV to efficiently replicate in our organisms.

Lack of polarized type 1 or type 2 cytokine profile in asymptomatic HIV-1-infected patients during a two-year bimonthly follow-up.

The production of type 1 (interferon or IFN-gamma) and type 2 (interleukin or IL-4 and IL-10) cytokines by mitogen-stimulated peripheral blood mononuclear cells (PBMCs) obtained from asymptomatic human immunodeficiency virus-seropositive (HIV+) patients untreated with any antiviral, antibacterial or antimycotic drugs, and from healthy individuals, was evaluated by quantitative ELISA. Patients who were HIV+ were characterized by the absence of abnormal cytokine production. The level of each cytokine differed among individuals in the same group with intersubject variations greater for HIV+ patients than for healthy individuals. The longitudinal evaluation of IFN-gamma, IL-4 and IL-10 production showed intrasubject variations which were particularly marked in HIV+ patients. Accordingly, HIV+ patients and, to a lesser extent, healthy individuals were characterized by a wide spectrum of possible profiles, which were confined to type 0 phenotype. In HIV+ patients no correlation was found between each cytokine level and the number of CD4+ T cells, not even in those with a falling CD4+ T-cell count and clinical symptoms.

A further step in the evolution of rabbit hemorrhagic disease virus: the appearance of the first consistent antigenic variant.

Rabbit hemorrhagic disease virus (RHDV) is a noncultivable calicivirus that infects rabbits (Oryctolagus cuniculus) and causes epidemics of an acute fatal hepatitis. In 1997 we identified two RHDV isolates from geographically distant Italian regions, which differed antigenically from the reference strain RHDV.Bs89. In fact, they were not reactive with mAb 1H8, that is able to protect rabbits from RHD and showed a low reactivity with the rabbit convalescent serum raised against RHDV.Bs89. Experimental infection of rabbits with either RHDV isolates confirmed their high pathogenicity and their peculiar antigenic profile; nevertheless, rabbits vaccinated with the current vaccine were protected against challenge infection with these isolates. Sequence comparison definitely demonstrated that the two isolates originated from the same RHDV variant and that the similarity of their structural protein (VP60) sequences with the RHDV.Bs89 is equal to 93%. This variant was named RHDVa since shows consistent genetic and antigenic differences from the wild-type RHDV. In particular, 44% of amino acid substitutions in RHDVa VP60 were located between amino acids 344 and 370, where the similarity with RHDV.Bs89 drops to 70%, suggesting that this region probably contains the epitope recognized by mAb 1H8. In addition, this paper presents preliminary data concerning the amino acids of VP60 involved in the hemagglutination site of the virus.

T cells from individuals in advanced stages of HIV-1 infection do not proliferate but express activation antigens in response to HIV-1-specific antigens.

Like T cells from healthy subjects, those of HIV-1-infected patients are capable of expressing activation antigens on their surface after antigenic or mitogenic stimulation, but their proliferative activity is strongly reduced or even absent, especially in patients with advanced stages of the disease. The characteristic of expressing activation antigens in response to different stimuli in the absence of cell proliferation is shared by CD4+ and CD8+ T-cell subsets from HIV-1-infected patients. The number of T cells capable of expressing CD25 and CD71 in response to HIV-1-related antigens but not of proliferating increased significantly with the progression of the disease, but the number of T cells capable of expressing the two activation antigens in response to the classic tetanus toxoid recall antigen decreased. The higher numbers of T cells capable of responding to HIV-1-related antigens in conjunction with a reduction in the number of T cells responding to recall antigens may explain the occurrence of different infections, including opportunistic microorganisms, during the more advanced stages of HIV-1 infection. Because the increase in the number of HIV-1 antigen-responding T cells (defined by CD25 and CD71 activation antigen expression) is a characteristic of symptomatic HIV-1-infected patients, expression (by flow cytometry) of these activation antigens on T cells in response to HIV-1 antigens could be used as a new marker of disease progression.

Flow cytometric analysis of activation markers on stimulated T cells and their correlation with cell proliferation.

The expression of activation antigens, namely CD25, CD69, CD71, and HLA-DR on T cells from 15 healthy individuals stimulated with different mitogens and specific antigens was evaluated by immunofluorescence assay and flow cytometric analysis and compared with cell proliferation as a function of [3H]thymidine incorporation. CD69 was the earliest expressed antigen on stimulated cells, while HLA-DR was the latest. Regardless of the stimulus used, lymphocytes expressing CD25 and CD71 were always more numerous than cells expressing CD69 and HLA-DR. Variations in the proportion of CD4+ and CD8+ T cells expressing each activation marker were observed with different antigenic stimuli. The expression of each activation marker showed overall agreement with the [3H]thymidine incorporation assay in discriminating between positive and negative immune response. However, no correlation was observed between the percentage of CD25-, CD69-, CD71-, and HLA-DR-positive T cells and the amount of [3H]thymidine incorporation. Moreover, low doses of mitogens and antigens as well as short time of stimulation were sufficient to induce T cells to express activation antigens but not to proliferate. Our data show that results obtained by flow cytometry and [3H]thymidine incorporation may differ qualitatively, at least under certain conditions; this suggests that the 2 assays are complementary, and when combined, may gives a clearer understanding of events leading to efficient cell-mediated immune response.

Heat-killed Bacillus subtilis inhibits T-cell proliferative response to mitogens and recall antigens.

Heat-killed vegetative forms of Bacillus subtilis were found to impair considerably the capacity of human T-lymphocytes to secrete interleukin-2 (IL-2) and to proliferate (in terms of [3H]thymidine incorporation) after phytohaemagglutinin (PHA) stimulation. B. subtilis was also found to interfere with T-cell proliferation induced by concanavalin A (Con A) and the recall antigen tetanus toxoid (TT). The suppressive activity was dependent on bacterial concentration, and was not ascribed to mitogen, medium-nutrient absorption or cell killing. Moreover, B. subtilis did not interfere with mitogen-induced IL-2 receptor expression on the T-cell surface. On the other hand, B. subtilis did not interfere with T-cell proliferation induced by phorbol myristate acetate (PMA) and ionomycin stimulation. All data obtained suggest the binding of B. subtilis subcomponents to- or very close to-the T-cell receptor (TCR). Identification and purification of the basic structure(s) or component(s) of B. subtilis with TCR antagonist activity in vitro will help to exploit different aspects of T-cell activity and development, and possibly, will provide a means of specific control or modification of the immune response.

Detection of Mycoplasma genitalium from urethral swabs of human immunodeficiency virus-infected patients.

Mycoplasma have been suggested as co-factors in the pathogenesis of acquired immune deficiency syndrome (AIDS). The prevalence of urethral infection by Mycoplasma genitalium was determined by polymerase chain reaction (PCR) with urethral swabs from 35 HIV-infected patients at different stages of the disease (all of them were heterosexual men). M genitalium was detected in 2 out of 19 non-AIDS (stage A and B) patients and in a similar proportion (1 out of 14; 7.1%) of samples from healthy individuals. A dramatic increase in the frequency of M. genitalium detection was observed in samples of AIDS (stage C) patients. In fact, 9 out of 16 (56.2%) specimens tested positive by PCR. We found no association in AIDS patients between M. genitalium infection and CD4 count, Human Immunodeficiency Virus (HIV) p24 antigenemia or opportunistic infection.