Gino Doria

A microcomputer program for probit analysis of interleukin-2 (IL-2) titration data

IL-2 activity is commonly estimated in cell culture supernatants by an IL-2-dependent cell proliferation assay. This method is both reproducible and sensitive. However, it often appears from the literature that statistical analysis of the titration data either is disregarded or, when performed, is based on statistically incorrect assumptions. The proposed method is based on the principle of biological assay by parallel lines as applied to probit analysis of quantitative responses. The procedure has been embodied in a simple and interactive computer program which automatically estimates the IL-2 concentration in the biological sample, in terms of U/ml, and provides its standard error and confidence limits. This program is also suitable for quantitative determination of other biologically active substances that show a sigmoid dose/response relationship.

Regulation of cytokine production in aging: use of recombinant cytokines to upregulate mitogen-stimulated spleen cells

We investigated the production of IL-2 and IFN-gamma (Th1 type) and IL-4 (Th2 type) cytokines by mitogen-activated spleen cells from young, adult and old mice. Cytokine production was evaluated in culture supernatants by CTLL proliferation (IL-2), ELISA (IFN-gamma), CT4.S proliferation (IL-4) and in mRNA extracted from activated CD4+ cells by RT-PCR (IL-2, IFN-gamma and IL-4). Results show that the production of IL-2, as protein and mRNA, is profoundly depressed by aging, whereas that of IFN-gamma, as protein and mRNA, firstly declines and then increases with age. The production of IL-4, as protein, monotonically declines with aging whereas, as mRNA, firstly decreases and then increases above the level in young mice. Spleen cells in culture were also incubated with mitogens and with a recombinant cytokine (IL-1 beta, IL-2, IL-3, IL-4, IL-12 or IFN-gamma) at various concentrations. It was found that recombinant cytokines by and large enhance cytokine production when the level induced by mitogens only is low. This conclusion applies to IL-2 and IFN-gamma production as protein and mRNA. The addition of recombinant cytokines also increases the production of IL-4 at the protein level in spleen cells from old mice but, at the mRNA level, only in spleen cells from young mice. This finding suggests age-related changes in IL-4-specific mRNA transcription rate and post-transcriptional half-life as well as translation kinetics.

Melatonin Restores Immunodepression in Aged and Cyclophosphamide‐Treated Mice

Melatonin, the main hormone of the pineal gland, when chronically injected into young mice or mice immunodepressed by aging or by cyclophosphamide treatment, was able to enhance the antibody response to a T-dependent antigen. The enhancement of antibody response was associated with increased induction of T helper cell activity and IL-2 production as evidenced in mice immunodepressed by aging or by cyclophosphamide treatment. These observations suggest that melatonin may be successfully used in the therapy of immunodepressive conditions.

Hormone replacement therapy affects various immune cell subsets and natural cytotoxicity

The effects of hormone replacement therapy (HRT) on lymphocytes and granulocytes have never been determined in detail. Ten healthy menopausal women (age 49-51 years; menopause less than 2 years) were treated for 6 months by administering transdermal estradiol (100 micrograms/day for 21 consecutive days) and oral medroxyprogesterone acetate (10 mg/day from day 10 to day 21). Days 22-28 were therapy-free. All subjects were examined during the first and the last month of treatment: evaluations were carried out on days 0, 8, 21 and 28. CD4+CD45RO+ cells were found to be significantly reduced on day 8. CD56+ cells and CD8+CD11b+ cells were decreased on day 21 and recovered basal level on day 28. Natural killer cell function was transiently increased on day 8 and greatly reduced on day 21. During the first month of therapy, the expression of Leu8 and CD11b antigens on granulocyte membranes was significantly affected by HRT. Taken together, the results indicate that HRT selectively affects various immune cell subsets.

Differential effects of gonadectomy on thymic stromal cells in promoting T cell differentiation in mice

Twenty-six week-old BDF1 mice were gonadectomized and grafted with thymus from irradiated (8.5 Gy) newborn, 6-week-old, or 26-week-old mice. One month later, grafted thymuses were recovered and examined in terms of thymocyte numbers, subpopulations and proliferative responses to Concananavlin A (Con A). The growth of the irradiated thymus was significantly higher in gonadectomized (Gx) than in sham-operated (Sham) mice and the magnitude of thymic growth was apparently age-dependent, as it was greater for newborns than for older mice. Con A response of thymocytes was also significantly higher in Gx mice than in Sham mice, and the magnitude of the response declined with advancing age of the thymus donors. Flow cytometric analysis revealed that a significant increase in the percentage of CD4+CD8- was observed in thymus grafts showing high Con A responses. However, this effect of Gx on the thymus graft was dependent on age of the thymus donor. Namely, newborn thymus grafts could grow equally well in both Gx and Sham recipients, whereas thymus grafts from 6- and 26-week-old mice could grow well only in Gx, but not in Sham recipients. The number of thymocytes was comparable in thymus grafts from 6- and 26-week-old mice, but the proliferative response to Con A was higher in the former than in the latter graft. Collectively, Gx appeared to promote immigration of thymocyte precursors into the thymus and to enhance proliferation and differentiation of thymocytes towards CD4+CD8- T cells, in an age-related manner.

Genetic control of immune responsiveness, aging and tumor incidence.

Age-related alterations of the immune system affect both antibody and cell-mediated immune responses, T-cell responses being more severely affected than B-cell responses. Within the T-cell population, aging leads to replacement of virgin by memory cells and to accumulation of cells with signal transduction defects. Changes in T-cell subsets and in cytokine production profiles may produce suitable conditions for T-cell-mediated disregulation of antibody responses characterized by the production of low affinity and self-reactive antibodies. Also B-cells exhibit intrinsic defects and natural killer (NK) cell activity a profound loss in old mice. Whether age-related immune disfunctions influence life span and tumor incidence has been examined in mice genetically selected for high or low antibody responsiveness. It has been found that genetic selection of vigorous antibody responses in most cases produces mice with longer life span and lower lymphoma incidence. Moreover, the results of genetic segregation experiments indicate that antibody responsiveness and life span are polygenic traits regulated by a small number of the same or closely linked loci. Mice genetically selected for high or low mitotic responsiveness to PHA exhibit low or high tumor incidence, respectively, but no difference in life span, suggesting that T-cell activity is restricted to immune surveillance of neoplastic transformation. Studies on mice genetically selected for resistance or sensitivity to chemical carcinogenesis have uncovered loci that control both resistance to tumor induction and longevity while have no effects on immunity and disease incidence. Thus, the relative role of the immune system in conditioning the duration and the biological quality of life remains to be determined.

Effect of synthetic thymic humoral factor (THF‐γ2) on T cell activities in immunodeficient ageing mice

Immunodeficient ageing (C57BL/10 × DBA/2)F1 mice were treated by a single injection of synthetic thymic hormones and 4 days later their thymus and spleen cells were assayed in vitro for T cell activities. A few nanograms of THF–γ2 were found to raise the frequency of mitogen‐responsive T cells in thymus and spleen cell populations as well as the frequency of cytokine‐producing splenic T cells, up to the levels observed in young mice. Moreover, injection of THF‐γ2 was found to restore T cell growth factor (TCGF) production by mitogen‐stimulated spleen cells. Also, the helper activity of spleen cells was enhanced by this treatment and increased with increasing theTHF‐γ2 dose over a wide range. Similarly, the effects of thymopentin and thymosin–α1 on T helper cell activity increased with increasing the injected dose, but the efficiencies of THF‐γ2 and thymopentin were, respectively. 400‐fold and eight‐fold greater than that of thymosin–α1.

Aging of the recipients but not of the bone marrow donors enhances autoimmunity in syngeneic radiation chimeras

Young and old mice have been lethally irradiated and injected with syngeneic bone marrow cells from young or old donors to investigate whether self reactivity in old mice results from age-related damage of the radioresistant stromal cells and/or of the bone marrow hematopoietic cells. Thymus and spleen cell repopulations and mitotic responses at 3 months after irradiation are lower in old than in young recipients, suggesting age-related accumulation of stromal cell damage in the thymus as well as in other central and peripheral lymphoid tissues. The same efficiency of bone marrow cells from young and old donors to repopulate the thymus and spleen in recipients of equal age rules out the detrimental effects of aging on stem cells as well as T and B cell precursors. The serum concentration of auto-antibody and glomerular lesions at 3 and 9 months after irradiation were more pronounced in old than in young recipients and displayed no difference in recipients of equal age, regardless of the age of the bone marrow cell donors. These findings support the possibility that age-related damage of stromal cells induces disregulation of the immune system leading to autoimmune phenomena.

Increased levels of NF-κB inhibitors (IκBα and IκBγ) in the intestinal mucosa of Crohn's disease patients during infliximab treatment

The treatment with infliximab is employed successfully in Crohns disease (CD) but predictors of efficacy are lacking. Activation of the transcription factor NF-kB has been demonstrated in CD and its inhibition is one of the mechanisms by which anti-inflammatory agents exert their effects. We evaluated the production of TNFα by peripheral blood mononuclear cells (PBMC) and the levels of NF-κB family molecules in the intestinal mucosa during infliximab therapy in 12 patients. TNFα was assayed on supernatants of PBMC culture stimulated with PHA or LPS. Immunohistochemistry was also done on intestinal biopsies. In six patients, Western blot analysis of the NF-kB subunit Rel-A, and its inhibitors IκBα and IκBγ was performed on intestinal biopsies and PBMC. The TNFα production by LPS stimulated PBMC showed mild changes, while it was increased by PHA–stimulated PBMC after treatment. The number of inflammatory cells in the intestinal mucosa was reduced (p<0.002) by the treatment. In five out of six cases we detected an increase of the IκBα and IκBγ inhibitor levels in intestinal biopsies after treatment. An increase of IκB inhibitors levels could be one of the mechanisms by which infliximab decreases NF-κB activity and exerts its anti-inflammatory effects.

Cell proliferation and ku protein expression in ageing humans

Previous studies on DNA repair in ageing have demonstrated increased frequencies of single and double strand breaks in lymphocytes from elderly subjects and, as a consequence, decreased efficiency in DNA replication. We have investigated the relationship between cell proliferation and the nuclear expression of ku protein in a human population of 43 subjects of different ages. Ku is an heterodimeric protein composed of two subunits of 70 and 80 kDa, which is involved in the early steps of DNA damage recognition. In the present study, PBL from subjects of different ages were PHA-activated to evaluate the stimulation index and the production of Th1- and Th2-type cytokines. Moreover, nuclear extracts were obtained from activated lymphocytes to evaluate by a gel retardation assay the presence and the functional activity of the heterodimer ku 70/80. Our results indicate that ageing affects the mitotic responsiveness and cytokine production to a significant extent, but only marginally the expression of ku 70/80. These findings suggest that the age-related impairment in DNA repair mechanisms are only in part related to the reduced expression of ku protein able to recognize DNA damage.