M. L. Villa

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.

Cellular immune factors associated with mother-to-infant transmission of HIV

ObjectiveTo study a possible correlate of protection in mother-to-infant transmission of HIV infection. In particular, to determine whether lack of HIV-specific T-helper (TH) function as indicated by HIV and non-HIV antigen-stimulated interleukin (IL)-2 production of mother and/or newborn peripheral blood leukocytes (PBL) is associated with mother-to-infant transmission of HIV. MethodsPBL from 21 HIV-seropositive pregnant women and 23 cord blood leukocytes (CBL) from their offspring were studied for in vitro TH function by IL-2 production in response to HIV and non-HIV antigens. Polymerase chain reaction (PCR) and viral culture assays were performed to determine HIV infection of the infants. ResultsPBL from 10 out of 21 (48%) mothers and from eight out of 23 (35%) CBL samples responded to two or more out of five synthetic gp160 envelope (env) peptides. Three of the 23 (13%) offspring were shown to be HIV-infected by PCR and/or viral culture on follow-up. All three infected infants were from a subset whose CBL did not exhibit env-specific TH immunity. ConclusionOur results demonstrate that fetal T cells can be primed to HIV env determinants in utero, suggest that HIV-specific TH immunity may be protective in newborns, and provide a possible means for identifying newborns who are at risk for HIV infection.

Immunological activation markers in the serum of African and European HIV-seropositive and seronegative individuals.

Objective: The concentration of type 1 and type 2 cytokines and fibroblast‐associated apoptosis‐1 soluble receptor (sAPO‐1/Fas) was analysed in the sera of Ugandan and Italian HIV‐1‐seropositive and seronegative individualls. The data were compared to determine whether the immunological status of these groups was different. Methods: Sixty‐seven Ugandan and 30 Italian HIV‐positive patients were analysed and stratified according to CD4 counts (group 1, > 500×106/l; group 2, 200–500×106/l; group 3, < 200×106/l). Sera from 15 Ugandan and 11 Italian HIV‐negative blood donors were also analysed. Serum concentration of type 1 cytokines [interleu‐kin (IL)‐2, IL‐12, and interferon (IFN)‐&ggr;] and type 2 cytokines (IL‐4 and IL‐10), and sAPO‐1/Fas were measured by enzyme‐linked immunosorbent assay. Results: Serum levels of IL‐2, IFN‐&ggr; and IL‐10, but not of IL‐4 and IL‐12, were elevated in HIV‐positive group 1 and 2 Africans compared with HIV‐positive Italian individuals. IL‐4 was mildly augmented in HIV‐positive group 3 African patients. Serum concentration of sAPO‐1/Fas was reduced in HIV‐positive Africans compared with HIV‐positive Italian individuals. Finally, serum levels of IL‐2 and IL‐10 were increased and sAPO‐1/Fas reduced when sera of HIV‐negative African healthy controls were compared with their Italian counterparts. The ratio of type 1/type 2 cytokines was roughly 1.0 in HIV‐negative African controls, and much greater than 1.0 in HIV‐negative Italian controls. Conclusions: These preliminary findings indicate that immune activation is present in African HIV infection. Furthermore, these data raise the possibility that abnormal immune activation and increased susceptibility to antigen‐induced cell death is present even in HIV‐negative African controls.

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.

Reduced natural killer cell activity and IL-2 production in malnourished cancer patients

Natural killer (NK) cell activity was measured in the peripheral blood mononuclear cells (PBMC) from malnourished (MN) and well-nourished (WN) cancer patients and in healthy controls. A marked depression of NK activity was observed in MN cancer patients with moderate protein-calorie malnutrition (PCM), but not in WN cancer patients nor in the healthy controls. The depression of NK activity did not correlate with the localisation of the tumour, patients age or body weight reduction. The defective NK activity of PBMC from MN cancer patients was restored to normal by rIL-2, but not by alfa-rIFN. Parenteral nutrition of MN patients with the proper amount of proteins and calories quickly corrected the depressed NK activity, indicating a central role of malnutrition in the genesis of their immune disfunction. PBMC from MN cancer patients produced lower amounts of IL-2, as compared with healthy controls, when stimulated in vitro; the most frequently affected were the responses to recall antigens such as influenza virus vaccine (FLU), while those to allogeneic PBMC (ALLO) and phytohaemagglutinin (PHA) were less affected. However, for each patient the ability to produce IL-2 in vitro did not correlate with NK activity, thus showing how the impairment of NK activity is not subsequent to a decreased production of endogenous IL-2. In summary, it can be concluded that malnutrition, rather than malignancy, plays a major role in the immune dysfunction of cancer patients.

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.

Restorative effect of total parenteral nutrition on natural killer cell activity in malnourished cancer patients

Decreased natural killer cell activity (NKCA) is associated with malnutrition in both cancer and non-cancer patients. We have studied the effect of total parenteral nutrition (TPN) on NKCA in 9 malnourished cancer patients, candidates for surgery. TPN was administered for a median of 10 days (range 7-11), providing 1.5-fold the estimated resting energy expenditure, with 30% as fat. Calorie:nitrogen ratio was 150:1. Basal human recombinant interferon-alpha 2a (rIFN-alpha 2a) and human recombinant IL-2 rIL-2) activated NKCA were measured, as were the main nutritional parameters, prior to and after TPN. NKCA increased in all patients and reached the normal range in 5, 3 and 4 subjects, respectively, for basal, rIFN-alpha 2a and rIL-2 activated NKCA. As regards nutritional assessment, body weight and IgM levels significantly increased from 47.7 to 50.1 kg and from 174 to 237 mg/dl, respectively. This study demonstrates that a 10-day TPN course increases and sometimes restores normal NKCA. Such effect was constant and preceded nutritional changes.

The breakdown of the cytokine network subsequent to human immunodeficiency virus infection.

The acquired immunodeflciency syndrome (AIDS) is a clinically multifaceted disease induced by infection with the human immunodeficiency virus (HIV). HIV infection results in a complex pattern of immunologic alterations that leads to the development of AIDS in the majority of HIV seropositive (HIV+) individuals. The reduction in CD4 T lymphocyte counts is the hallmark of HIV infection; nevertheless, long before the reduction in CD4 counts reaches critical levels, a series of profound and complex defects that impair the function of CD4 T lymphocytes can be detected. Thus, HIV infection is characterized by quantitative and qualitative defects affecting CD4 T lymphocytes. It was suggested recently that programmed cell death (PCD) is an important mechanism leading to CD4 depletion in HIV infection, and that susceptibility of peripheral lymphocytes to PCD is differentially regulated by diverse cytokines. Thus, type 1 cytokines would protect CD4 lymphocytes against PCD, whereas type 2 cytokines would not protect against, and could augment, PCD. We suggest that the qualitative alterations of the immune response provoke the CD4 depletion characteristic of HIV disease via type 2 cytokinemediated augmentation of PCD, and are therefore ultimately responsible for the progression of HIV infection. Finally, we summarize recent data showing that three correlates of disease progression: emergence of HIV strains with syncitium-inducing ability (SI), type 1-to-type 2 cytokine shift, and CD4 depletion, are significantly associated, suggesting a complex interconnected virologic-immunologic pathogenesis of HIV infection.