Enrico Clerici

Characterization of type 1 and type 2 cytokine production profile in physiologic and pathologic human pregnancy

Antigen‐ and mitogen‐stimulated cytokine production by peripheral blood mononuclear cells (PBMC) of 50 pregnant women and 31 age‐ and sex‐matched non‐pregnant controls were analysed to determine whether changes in cytokine production occur during normal and pathologic human gestation. The pregnant women, consecutively enrolled during a 3‐month period, were undergoing a normal, non‐pathologic pregnancy at the time of entry into the study, and underwent ultrasound examination to ascertain the exact week of pregnancy and the vitality of the fetus. Forty of the 50 pregnancies (80%) terminated physiologically with the birth of normal babies. Spontaneous abortions were observed in 5/50 (10%) women, and five women gave birth to newborns small for gestational age (SGA). A decrease in the production of IL‐2 and interferon‐gamma (IFN‐γ) accompanied by an increase in production of IL‐4 and IL‐10, was observed in normal pregnancy, with the lowest quantities of IL‐2 and IFN‐γ and the highest quantities of IL‐4 and IL‐10 present in the third trimester of pregnancy. Statistically significant increased production of both IL‐2 and IFN‐γ and reduced production of IL‐10 characterized pathologic pregnancies and distinguished them from normal pregnancies. These preliminary data suggest that a type 2 cytokine profile may be associated with normal human pregnancy, whereas the lack of a dominant type 2 cytokine profile may be indicative of a pathologic pregnancy.

Multiple defects of T helper cell function in newly diagnosed patients with Hodgkin's disease

T helper cell (TH) function, as assessed by interleukin-2 (IL-2) production and [3H]thymidine incorporation, was studied in 47 newly diagnosed untreated patients with Hodgkins disease (HD) and 34 healthy controls. Three different stimuli were used to stimulate in vitro peripheral blood mononuclear cells (PBMC): influenza A vaccine (FLU), HLA alloantigens (ALLO) and phytohaemagglutinin (PHA). Four different patterns of TH function were observed in HD patients: (1) IL-2 production in response to all of the stimuli (40%); (2) IL-2 production in response to ALLO and PHA but not to FLU (26%); (3) IL-2 production in response to PHA alone (19%); and (4) failure to respond by IL-2 production to any of the three of the stimuli (15%). Thus, defective in vitro TH function was detected in the majority of these patients (60%). Defective TH function was observed in none of the 34 controls. Severely compromised TH function (patterns 3 and 4) tended to be associated with more advanced clinical presentation and more compromised haematological parameters (P < 0.05). The IL-2 production assay was more sensitive than the proliferative assay as only 30% of the HD patients failed to proliferate in response to FLU, and none failed to proliferate in response to either ALLO or PHA; this assay can detect subtle, multiple patterns of immune dysregulation in untreated HD patients. Our results suggest that HD is associated with a fundamental dysregulation in TH function, illustrate the complexity of such dysregulation, and raise the possibility that HD progression will be associated with a type-1-type-2 switch in immunoregulatory cytokine production.

Retinoids, breast cancer and NK cells.

N-(4-hydroxyphenyl) retinamide (4-HPR) is a synthetic retinoid which reduces the incidence of experimental tumours in animals and has been chosen for its weak toxicity to be tested as a chemopreventive agent in humans. The mechanism of antineoplastic action is still unknown but a possible immunoenhancing effect may be postulated. We investigated the NK activity of PBMC from a group of women treated with 4-HPR as a part of a large scale randomised phase III trial on chemoprevention of contralateral disease in mastectomised women. After 180 days of treatment the NK activity was augmented 1.73 times as compared to that of patients given a placebo. The NK activity of PBMC from 4-HPR treated women is maximised, being higher than the basal and even the rIL-2 or alfa-rIFN stimulated activity of controls. For this reason in the majority of cases it cannot be further augmented by incubation with either rIL-2 or alfa-rIFN in vitro. The increased NK activity of 4-HPR treated women is not due to an enhanced production of endogenous IL-2, because PBMC cultures from patients treated with 4-HPR or placebo, incubated in vitro with a panel of different stimulators (recall antigens, PHA, allogeneic and xenogeneic cells) produce similar amounts of IL-2. The functional activity, but not the number of NK cells is increased in 4-HPR treated women. The mechanism by which 4-HPR stimulates NK activity is not a function of direct action on NK cells. Indeed incubation of PBMC from blood donors with 4-HPR or its major metabolite N-(4-methoxyphenyl) retinamide (4-MPR) does not modify their natural cytotoxicity.

HLA Antigens in Diabetic Children

Ninety insulin-dependent diabetic children were HLA-typed in order to elucidate the role played by HLA complex-linked genes in the pathogenesis of diabetes melHtus of childhood. HLA-Aw30 and HLA-Bw35 were significantly increased and decreased, respectively, in the diabetic group as compared with controls. In relation to age at onset of diabetes, HLA-B8 was significantly increased in the 0-5-year group. By dividing the patients according to the season at onset of the disease, only HLA-Aw30 in the October-January group reached the level of significance (P=0.05).

Antibody-forming foci in soft-agar cultures of human peripheral blood cells

Human peripheral blood lymphocytes (PBL) from healthy blood donors were grown in soft-agar gel with sheep red blood cells (SRBC) and autologous plasma as a source of complement. After 4--6 days incubation, foci of proliferating hemolysin-forming cells, surrounded by a lytic area of 0.2--0.5 mm, were detected on the surface of the plates. The response was antigen specific, since new hemolytic areas were observed on pouring a fresh agar-SRBC mixture over the surface of primary cultures, but not on pouring a mixture containing rat or rabbit erythrocytes. The antibody response was significantly increased by addition to the cultures of polyethylene glycol 6000 (PEG), 8% final concentration. The mean number of foci was 8.4 +/- 2.2 in cultures without PEG and 36.2 +/- 2.3 in PEG+ cultures, both containing 9 X 10(6) lymphocytes. This finding is in agreement with observations on the frequency of precursors of antibody-forming cells among lymphoid populations. The explanation of the mechanism by which PEG 6000 modified the immune reactivity of PBL is not clear. However, we think, that this technique provides a reliable methodology for PBL antigenic stimulation in vitro.

Macrolidic antibiotics: Effects on primary in vitro antibody responses

The effect of two macrolidic antibiotics, josamycin and erythromycin, on the primary immune response in cultures of human peripheral blood mononuclear cells (PBMC), were studied using a soft agar hemolytic plaque assay. Both compounds induced an appreciable reduction in the primary antibody response in total PBMC cultures. The removal of plastic-adherent cells, however, profoundly modified the effect of macrolides on the immune response. Both josamycin and, to a lesser extent, erythromycin enhanced, rather than suppressed, the antibody response. Furthermore, the macrolide-induced immunodepression in cultures of total PBMC was completely reversed by the addition of catalase (8000 U/ml). Taken together, these findings suggest that the macrolide-induced depression of the antibody response depends upon the presence of adherent monocytic cells and is mediated by the production of hydrogen peroxide.

The interference of antibiotics with antigen-specific antibody responses in man.

The effect of a series of antimicrobial drugs on human antigen-specific primary antibody response in vitro was investigated. Of the five agents tested, only streptomycin and gentamicin induced an appreciable reduction in the antibody response; penicillin G, rifampin and amikacin had a poor or irregular effect. The precise mechanisms of action of these substances on mammalian cells remain to be elucidated since an assessment of their capacity to interfere with the immune system is of particular importance in those patients with induced or acquired immunodeficiencies. In this respect, we provide an inexpensive and sensitive method for the preliminary screening of potentially immunosuppressive drugs.

Rat liver regeneration after partial hepatectomy and sympathetic denervation

Abstract A working hypothesis suggesting that the morphologic and metabolic changes seen in the liver after partial hepatectomy may share a common pathogenic pathway with those observed after CCl 4 poisoning (i.e., a massive discharge of the peripheral autonomic nervous system following the stimulation of central sympathetic areas) and that they may be prevented both by surgical and pharmacologic blocking of the sympathetic nervous system, has been tested by studying the behavior of the liver regeneration in rats either subjected or not to the alcoholization of the coeliac and superior mesenteric ganglia followed by partial hepatectomy. In particular, the fat and glycogen content of the regenerating liver, its weight increase, its capacity to incorporate dl -leucine-1-C 14 , and its morphology have been investigated by means of appropriate experimental techniques. According to the statistical evaluation of the results, such parameters of the liver regeneration are not modified in the rats subjected to the sympathetic denervation as compared to controls. The above data, together with other results from the statistical analysis, have been discussed and compared with those reported in the literature. It has been concluded that, while it is clear that the liver regeneration is independent from sympathetic influences, it is not yet possible to rule out that the partial hepatectomy entails a massive discharge of the peripheral autonomic nervous system similar to that observed following CCl 4 poisoning, mainly because such an increased discharge may play only a minor role on the morphologic and metabolic behavior of a partially resected liver, whose circulatory pattern is definitely different from that observed in the liver of animals treated with the steatogenic poison.

Monocyte mediated modulation of the antibody response in vitro.

Human peripheral blood monocytes (PBMC) were isolated by Ficoll-Hypaque density gradient and then fractionated by differential adhesion to plastic surface. Adherent cell-depleted PBMC, non-readherent fraction and firmly adherent fraction so obtained from PBMC, PBMC themselves and a mixture of the above cells, were then sensitized in vitro with sheep erythrocytes (SRBC) so as to produce a primary antigen-dependent, antigen-specific antibody response. It appears that adherent cell-depleted PBMC produce about twice as many haemolytic areas as compared to total PBMC (from 43 to 85). If depleted PBMC are co-cultured with firmly adherent or non-readherent cells, the number of haemolytic areas goes down to 19 or up to 102, respectively. Functional, histochemical, immunochemical and morphological data suggest that the inhibiting firmly adherent fraction is composed of typical phagocytizing cells, while the enhancing cells of the non-readherent fraction are similar to the dendritic cells described in human blood and some lymphoid organs, which do not exhibit active pinocytic activity, but are the principal accessory cells needed to stimulate lymphocyte responses.

Effect of X-irradiation on liver regeneration

Abstract The in vivo incorporation of dl -Leucine-1- 14 C into the total protein and the protein of cell fractions in liver of partially hepatectomized rats, given a whole-body X-irradiation 4 hr after surgery (i.e., within the so-called presynthetic period of liver regeneration) was investigated. The main feature of our results indicates that the whole-body X-irradiation inhibits the incorporation of labeled aminoacid into the soluble, nuclear, and microsomal fractions of regenerating liver as compared with unirradiated partially hepatectomized control rats. On the basis of recent information on the mechanism of protein synthesis, such data are tentatively explained as resulting from disturbance in the synthesis or turnover of nuclear RNAs acting as templates in the production of the new protein molecules necessary to support liver regeneration.