Giuseppe Falcone

A specific adenosine phosphorylase, distinct from purine nucleoside phosphorylase.

Several enzymes, catalyzing the reversible phosphorolysis of purine nucleosides have been described in eucaryotic cells and in micro-organisms [l-6] . The best known is the ‘purine nucleoside phosphorylase’ (EC 2.4.2.1) acting on the nucleosides of hypoxanthine and guanine. Adenosine is not used as substrate by this enzyme [6-81. Phosphorolysis of adenosine has been reported in Salmonella typhimurium [9], where a single enzyme protein appears to act on inosine, guanosine and adenosine. Adenine was found to be substrate for purine nucleoside phosphorylase of four mammalian sources, but its unfavourable kinetic parameters with respect to those of hypoxanthine and guanine are against the role of adenine as a physiological substrate [lO,l l] . In Mycoplasma both adenosine and inosine phosphorolysis have been observed, but no attempt has been made to correlate the two activities to different proteins [12]. The separation of adenosine phosphorylase from the purine nucleoside phosphorylase has never been reported so far, even though Miech and Coll. have presented indirect evidence that in Schistosoma Mansoni worms adenosine phosphorylase activity is a separate entity from purine nucleoside phosphorylase: this conclusion is based on differences in the pH-activity curves and studies with product and nucleoside analogues inhibitors [ 131. The data presented in this paper give the first direct evidence that at least in B. subtilis the phosphorolysis of adenosine and that of inosine and guanosine are catalyzed by distinct enzyme proteins, which can be

Comparison of three restriction endonucleases in IS1245-based RFLP typing of Mycobacterium avium

IS1245-based restriction fragment length polymorphism (RFLP) analysis has been proposed recently for molecular typing of Mycobacterium avium isolates. As there is no standardised method with respect to the optimal restriction enzyme, three restriction endonucleases were tested for analysis of 17 human isolates. The restriction endonucleases, selected on the basis of the physical maps of IS1245 and of the highly homologous IS1311, were BsaAI, that cleaves IS1245, PvuII, that cleaves IS1311, and NruI, that cleaves both IS1245 and IS1311. All the restriction endonucleases yielded polymorphic and complex RFLP patterns. However, BsaAI- and NruI-generated bands were more evenly distributed and easier to detect than PvuII-generated bands, most of which clustered in a narrow zone of the fingerprint. In some cases, DNA digestion with BsaAI or NruI yielded probe-specific restriction fragments of molecular size lower than expected. Moreover, digestion with NruI, which was expected to generate the highest numbers of bands in all the isolates, yielded fewer bands than were obtained with BsaAI or PvuII in 14 and 5 isolates, respectively. These findings might suggest the existence of unidentified IS1245-related insertion element(s) in M. avium isolates. Computer analysis of the IS1245-based RFLP patterns of M. avium isolates showed that the restriction endonucleases were capable, although with minor differences, of defining distinct banding patterns and clusters of identical or highly related isolates, thus confirming IS1245-based RFLP analysis as a useful technique for epidemiological studies.

Auto-anti-idiotypic antibodies inhibit T-cell-mediated hypersensitivity in BCG-infected mice

It is shown that serum from mice heavily infected with BCG contains antibodies which block the cell transfer of delayed-type hypersensitivity (DTH) to purified protein derivative (PPD) when BCG-immune cells were preincubated in it. This suppressive activity is antigen specific in that the serum does not block the cell transfer of contact sensitivity to oxazolone. However, the suppressive activity is not antigen directed in that it is absorbed neither by PPD-coupled Sepharose beads nor by PPD-pulsed normal peritoneal exudate cells. On the other hand, the activity can be absorbed to BCG-immune T cells and eluted from a Sepharose column conjugated with affinity-purified mouse anti-PPD antibodies. The possibility that antireceptor antibodies arise during the BCG infection and regulate DTH reaction is discussed.

Epstein-Barr virus-transformed human B lymphocytes produce natural antibodies to histones

To study the mechanism(s) responsible for the appearance of Epstein-Barr virus (EBV)-induced anti-histone autoantibodies, peripheral blood B lymphocytes from healthy donors were infected with EBV and the resulting lymphoblastoid cell lines were tested for secretion of antibodies reacting with histones. It was found that EBV-transformed cells produce IgM antibody reactive with histones and that the frequency of EBV-inducible circulating B lymphocytes that produce antibodies to histones is at least 10(-5). Moreover, in cultures of tonsillar lymphoid cells, the enrichment in CD5+ B lymphocytes increases the percentage of EBV-transformed cultures making anti-histone IgM antibodies. EBV may therefore, also in vivo, induce natural anti-histone antibody by polyclonal B-cell activation without any requirement of antigen to trigger antibody response.

Pseudomonas aeruginosa infection: Activation of B suppressor cells which affect cell cooperation in the induction phase of contact sensitivity to oxazolone in mice

Pseudomonas aeruginosa depresses contact sensitivity to oxazolone in mice by activating B suppressor cells within the lymph nodes that drain the site of sensitization. These suppressor cells affect the induction phase of the immune response by blocking the cooperation between “stimulator” macrophages and “initiator” T cells on one hand and “recruited” T cells on the other.

A human monoclonal autoantibody isolated from a patient with infectious mononucleosis reactive with both self antigens and Epstein-Barr virus nuclear antigen (EBNA)

In order to investigate the mechanism(s) by which Epstein-Barr virus (EBV) induces the outcome of autoantibodies during infectious mononucleosis (IM), a human IgM (k) monoclonal antibody to cytoskeletal filaments of epithelial cells has been prepared by EBV transformation of peripheral blood B lymphocytes obtained from a patient with IM. The antibody was also found to react with smooth muscle of frozen sections of human stomach tissue by immunofluorescence, and with the Epstein-Barr nuclear antigen (EBNA) by an enzyme-linked immunosorbent assay. These findings demonstrate at the clonal level the epitope homology between hosts cell antigens and EBV-encoded nuclear antigen, which might have relevance in EBV-induced autoimmunity.

LPS-Induced Enhancement of Plaque-Forming Cell Response to Bromelain-Treated Syngeneic Erythrocytes in Mouse Peritoneal Cell Cultures

The effect of bacterial lipopolysaccharide (LPS) on the development of plaque-forming cells (PFC) against bromelain-treated syngeneic mouse red blood cells (Br-MRBC) was studied in peritoneal cell (PC) cultures. It was found that LPS enhances the development of PFC to Br-MRBC and increases DNA synthesis in PC cultures. The LPS-induced enhancement of PFC to Br-MRBC, however, does not appear to require cell proliferation, since it also occurred in PC cultures pretreated with mitomycin C. In addition, the LPS-induced B lymphocytes blastogenesis is under the control of macrophages, while cell differentiation of precursor B lymphocytes into cells actively producing antibodies against Br-MRBC is regulated by suppressor T lymphocytes.

Mouse footpad infection byPseudomonas aeruginosa: Evidence for delayed hypersensitivity to specific bacterial antigen

Mouse hind footpad inoculation with 1×107 viablePseudomonas aeruginosa cells produces a long-lasting, self-limiting disease process characterized by a bacterial multiplication that parallels the swelling of the infected footpad. Regional popliteal and inguinal lymph nodes and spleen of infected animals show cellular modifications which are almost entirely due to lymphocyte proliferation, as indicated by spontaneous DNA synthesis experiments in vitro. Furthermore, mice which had been infected in their footpad either 20 or 27 days previously and challenged withP. aeruginosa antigens into the controlateral footpad show, 24 h later, a marked increase in regional popliteal lymph node weight, indicating the development of delayed hypersensitivity toPseudomonas antigens.

PSEUDOMONAS-AERUGINOSA INFECTION DEPRESSES CONTACT SENSITIVITY TO OXAZOLONE BY ENHANCING SUPPRESSOR CELL-ACTIVITY

The depression of contact sensitivity to oxazolone in mice infected withPseudomonas aeruginosa was studied. In oxazolone-sensitized mice,P. aeruginosa infection affects cell proliferation in the lymph nodes draining the site of sensitization. This impaired cell proliferation does not seem to be due to an altered lymphocyte reactivity, since lymph node and spleen cells from infected animals show a normal mitotic responsiveness to both T and B cell mitogens. In addition, the draining lymph nodes and spleens of mice exhibiting a depressed response to oxazolone contain a cell population able actively to suppress the response to the same antigen of syngeneic recipients sensitized immediately before the cell transfer. These suppressor cells require antigenic stimulation and appear to act on the induction phase of contact sensitivity.

Polyclonal B Cell Activators Inhibit Contact Sensitivity to Oxazolone in Mice by Potentiating the Production of Anti-Hapten Antibodies that Induce T Suppressor Lymphocytes Acting through the Release of Soluble Factors

Polyclonal B cell activators (PBAs) such as purified protein derivative, lipopolysaccharide, Staphylococcus aureus strain Cowan I (StaCwI), and Pseudomonas aeruginosa inhibit contact sensitivity to oxazolone in mice when given 24 h before sensitization. This suppression, transferable from immunodepressed animals to oxazolone-sensitized recipients with immune serum, has been shown to be due to the early appearance of anti-hapten antibodies. These antibodies elicit T suppressor cells which release soluble factor(s) capable of inhibiting the passive transfer of contact sensitivity.