Inna Saporoschetz

Inadequate interleukin 2 production. A fundamental immunological deficiency in patients with major burns.

We studied the production of the two major mediators of cellular immune responses, Interleukin 1 (IL-1) and Interleukin 2 (IL-2), by the peripheral blood mononuclear cells of 23 burn patients (16 men, seven women, mean age 48.9 years) compared with 23 matched controls (16 men, seven women, mean age 46.7 years). Serial measurements were made of IL-1 production by adherent mononuclear cells after stimulation with lipopolysaccharide and of IL-2 production by lymphocytes after stimulation with phytohemagglutinin (PHA). Eighty determinations of IL-2 production by lymphocytes from 12 patients with greater than 30% body surface area burn revealed a mean IL-2 production of 0.71 u as compared with a mean of 1.23 u for patients with less than 30% burns (p = 0.04). Patients with greater than 30% body surface area burns had significantly reduced IL-2 production (p ≤ 0.05) until 60 days after injury, whereas those with smaller burns had reduced IL-2 production only at 20–29 and 30–39 days postburn. Nine burn patients with systemic sepsis showed significantly lower IL-2 production (p = 0.03) at 10–29 days postburn than nonseptic patients, and significantly less IL-2 production during septic episodes. Eight patients with greater than 50% suppression of lymphocyte response to PHA produced less IL-2 (0.4 u) than patients with less than 50% suppression, (1.07 u, p = 0.004). IL-1 production was significantly elevated as compared with controls (4.45 u vs. 3.6 u, p = 0.05) early after injury, but was subsequently within the normal range regardless of burn size. The percentage of circulating helper T-lymphocytes, the principal source of IL-2, was also reduced, although this did not always correlate with IL-2 production, which remained depressed after recovery of the helper T-cell population. These results indicate that failure to produce IL-2, a powerful mediator of cellular immune responses, is an important mechanism underlying the defective cell mediated immunity seen in burn patients.

Association of anergy with an immunosuppressive peptide fraction in the serum of patients with cancer.

Abstract To study the relation between circulating immunosuppressive factors and anergy in patients with cancer, we tested 53 patients with cancer for hypersensitivity to skin-test antigens and 2,4-dinitrochlorobenzene. Of 41 patients with negative skin tests, 27 (66 per cent) had immunosuppressive serum (≥70 per cent inhibition of lymphocyte stimulation by phytohemagglutinin). None of 12 with positive skin tests had immunosuppressive serum. Thus, anergy and serum immunosuppressive activity were correlated (p<0.05). After ion-exchange chromatography, most of the immunosuppressive activity in cancer serum was in the first peak, fraction I. The same fraction from subjects without cancer contained no detectable immunosuppressive activity. Mean total immunosuppressive activity of cancer serum was 166.0 ± 97.5 (S.D.) units, and that of non-cancer serum was 30.4 ± 9.2 units (p<0.05). Diafiltration of fraction I. from cancer serum yielded a fraction (< 10,000 daltons) that was highly immunosuppressive. Thus, ane...

Correlation Between Anergy and a Circulating Immunosuppressive Factor Following Major Surgical Trauma

In order to clarify the relationship between anergy and immunosuppressive activity in the serum, we studied 46 previously well patients before and at three, five, seven and 28 days after surgery. Delayed hypersensitivity was measured by skin testing with four common recall antigens, and serum immunosuppressive activity was determined by the ability of the patients serum in 10% concentration to suppress by 50% or more the phytohemagglutinin (PHA) stimulation of normal human lymphocytes as compared to pooled normal serum. Prior to surgery, all patients manifested delayed hypersensitivity to one or more antigens, and no patient had immunosuppressive serum. Fifteen patients underwent minor surgery under general anesthesia and did not develop anergy or immunosuppressive serum. Thirty-one patients underwent major cardiovascular surgery. Thirteen of these patients became anergic by day 3 after operation, and 11 of the 13 developed immunosuppressive serum. Eighteen patients maintained delayed hypersensitivity after major surgery, and only three developed immunosuppressive serum. The correlation between anergy and immunosuppressive serum was highly significant (p < 0.001). There was a significant difference in the degree of suppressive activity in the serum of the anergic and reactive patient groups for each postoperative day studied until day 28, when there was recovery of delayed hypersensitivity and lack of immunosuppressive serum. The occurrence of postoperative anergy and immunosuppressive serum was not related to the patients age, sex, number of perioperative blood transfusions or duration of anesthesia but was associated with an increase in postoperative infectious complications (p < 0.05) and in postoperative days in the hospital (p < 0.01). Pooled immunosuppressive serum from anergic patients was fractionated by ion exchange chromatography, gel filtration and preparative high voltage electrophoresis. The majority of the immunosuppressive activity could be accounted for by an electrophoretically homogenous polypeptide-containing fraction not identified in the serum of patients undergoing minor surgery or in normal individuals. We conclude that anergy occurring after major operative trauma is associated with the appearance of a circulating immunosuppressive molecular species and that these events are in turn associated with increased patient morbidity and increased length of hospitalization.

Effects of in vivo endotoxin infusions on in vitro cellular immune responses in humans

Studies of the immune response of patients following major injury have identified significant abnormalities, some of which may be due to the effects of endotoxin. To evaluate the effect of endotoxin on the immune system without conflicting variables, we studied 18 normal, healthy male volunteers each on two occasions. In one study,Escherichia coli endotoxin was administered intravenously at a dose of 4 ng/kg. In the other, saline was given. Blood for immune function studies was obtained at either 0, 4, or 24 hr (seven volunteers), 0, 1, and 4 hr (five volunteers), or 0, 4, and 6 hr (six volunteers) postinfusion. Peripheral blood mononuclear cells (PBMC) were isolated and adjusted to the same concentration. Measurements following endotoxin infusion were compared with those of the same volunteers following saline infusion and with those from normal ambulatory laboratory volunteers. Interleukin 1 (IL-1) production by adherent cells was significantly reduced at 1 hr post endotoxin infusion. Significant decreases in number of mononuclear cells, response to phytohemagglutinin (PHA), and production of IL-2 and IL-1 were observed by 4 hr after endotoxin infusion. No significant changes in percentages of monocytes, lymphocytes, or CD3, CD4, or CD8 lymphocytes were observed at any time. By 24 hr postinfusion all values had returned to normal or, in some cases, supranormal levels. Response to PHA by PBMC from volunteers 4 hr following endotoxin was completely restored byin vitro addition of recombinant human IL-2 but was only marginally improved by IL-1.In vitro addition of indomethacin to PBMC cultures responding to PHA reduced the suppression observed afterin vivo endotoxin but also was not as effective as IL-2. In a fourth study, seven volunteers were treated as above either with two doses (800 mg each) of the cyclooxygenase inhibitor ibuprofen before endotoxin infusion or with ibuprofen alone. Ibuprofen pretreatment completely restored the PBMC response to PHA to normal and caused a significant decrease in the endotoxin-induced suppression of IL-2 production. However, the decrease in circulating PBMC number and adherent cell secretion of IL-1 was not affected by inhibition of the cyclooxygenase pathway. These results suggest that endotoxin has immunomodulatory effects on both adherent mononuclear-cell and T-lymphocyte function and that more than one mechanism is involved.

Circadian rhythm of cytokine secretion following thermal injury in mice: implications for burn and trauma research.

Although there are many reports of circadian variation in hormone secretion, there are only a few reports on the relationship between circadian rhythm and cytokine production. The aim of the present studies was to investigate whether there is a circadian effect on cytokine production of splenic lymphocytes and adherent splenocytes in mice after burn or sham injury. We selected day 7 after injury for our determinations because we have previously shown day 7 is the time of maximal suppression of T cell IL-2 and IFN&ggr; production and maximal increase in adherent cell proinflammatory cytokine secretion in this model. IL-2 and TNF&agr; were chosen as reference cytokines since the former is known to be produced by T cells and the latter by adherent cells of the innate immune system. The results showed that seven days after sham or thermal injury both T cell IL-2 and adherent cell TNF&agr; production were altered by time of injury or time of cell harvest. IL-2 secretion was significantly decreased in burn compared to sham animals when splenocytes were harvested in the morning; the decrease was non-significant when splenocytes were harvested in the afternoon. TNF&agr; secretion was significantly increased in burn vs. sham adherent cells only when injury took place in the morning. The observed circadian variations in cytokine production could have a significant effect on cytokine levels measured in clinical and animal studies of injury and may explain some of the reported discrepancies among these studies.

Phytohemagglutinin stimulation of human lymphocytes. Failure to detect an early increase in cyclic AMP concentration.

Abstract Cyclic AMP content and adenylate cyclase activity were determined in human lymphocytes stimulated to proliferate by phytohemagglutinin (PHA). Both commercial (Difco PHA-M) and purified PHA were used at optimal stimulatory concentrations as confirmed by incorporation of [14C]leucine. Cyclic AMP levels were measured by radioimmunoassay. Exposure of lymphocytes to PHA-M alone yielded no significant change in intracellular cAMP levels; however, in the presence of the phosphodiesterase inhibitor, caffeine, PHA-treated cells had lower levels of cAMP than the caffeine controls. That adenylate cyclase remained active in these experiments was shown by marked increases in lymphocyte cAMP levels following exposure to prostaglandin E1 (PGE1). Stimulatory concentrations of PHA-M or purified PHA also failed to induce adenylate cyclase activity in lymphocyte particulate preparations whereas PGE1 or NaF produced significant increases in enzyme activity. Moreover, PGE1, at concentrations which caused a prompt and significant rise in adenylate cyclase activity and in intracellular cAMP levels, inhibited lymphocyte stimulation by PHA.

Long-term immunotherapeutic intervention with pentoxifylline in a mouse model of thermal injury and infection.

Major thermal or traumatic injury often results in abnormalities of immune function, and these abnormalities contribute to the increased susceptibility to infection observed in these patients. Abnormalities of T-cell function, including decreased proliferation and secretion of cytokines are observed following major injury and, conversely, there is markedly increased monokine production. Thus, therapy of this syndrome might logically be aimed at modulating the immune system to upregulate T-cell function and downregulate monocyte hyperactivation. Pentoxifylline (PTX), a methylxanthine derivative, has been shown to be therapeutically effective in several animal models. The purpose of this study was to evaluate PTX and its effect on cytokine production in a mouse model of thermal injury and to study its effect on survival after septic challenge. The results show that PTX therapy after injury can restore T-cell production of IL-2 and downregulate the hyperactive macrophage secretion of proinflammatory cytokines. However, improvement in survival resulting from this therapy following thermal injury and septic challenge depends on timing of dosage.

Stimulatory effects on various immunologic responses by ascites fluids from patients with metastatic malignancy.

Abstract Cancer ascites has been repeatedly shown to suppress a number of immune responses. In this report, we examined a total of 28 unfractionated ascitic fluid samples from patients with various metastatic malignancies and rather unexpectedly found that the majority of these samples were capable of enhancing various immune reactivities. Twelve of 17 samples were shown to increase significantly humoral antibody production in mice when injected 1 day before sheep erythrocytes. Ten of 13 samples significantly enhanced the proliferative response of human peripheral blood mononuclear leukocytes to the T-lymphocyte mitogen, phytohemagglutinin. Twelve ascitic samples were added to mouse mixed lymphocyte cultures and 9 were found to augment significantly the generation of alloreactive cytolytic lymphocytes. Pooled normal human serum was similarly tested as a control and found to have no effect in these systems. Furthermore, we have isolated three active fractions from these cancer ascites fluids by Sephadex G-75 gel filtration. However, each fraction was predominantly responsible for enhancing only one of the three immune responses tested.

Bioassay for antioxidants based on protection of isolated rat liver mitochondria against the photodynamic toxicity of benzo[a]pyrene

Abstract A bioassay for antioxidants, based on the protection of isolated rat-liver mitochondria from the photodynamic toxicity of benzo[ a ]pyrene, is described. The assay has been applied to 92 antioxidants of widely varied structure, including phenols, aliphatic and aromatic amines, heterocyclic nitrogen compounds, aminoazobenzenes, polyenes (β-carotene, xanthophyll and vitamin A), sulphur-containing compounds, ultraviolet absorbers and antioxidant synergists. Data from the assay correlate closely with data from a previously described assay based on the protection of Tetrahymena pyriformis from photodynamic injury.