Oxidative DNA damage in reconstituting T cells is associated with relapse and inferior survival after allo-SCT.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) is the only curative treatment option for a number of hematologic malignancies. Its therapeutic potential relies on the potency of donor T cells to eliminate residual malignant cells, the so-called graft-versus-leukemia (GVL) effect. Disease relapse remains the most frequent treatment failure and is associated with poor outcome. Therefore, it is inevitable to decipher mechanisms that weaken GVL. In recent years, studies of tumor biology have revealed that metabolic remodeling of the micromilieu can critically regulate immune responses. Accumulation of reactive oxygen species leads to a metabolic condition known as oxidative stress, which can severely hamper T cells. Currently, only a few studies, mainly using preclinical models, have demonstrated the occurrence of oxidative stress after allo-SCTs. Therefore, we sought to investigate oxidative stress in a well-characterized group of patients who underwent allo-SCT and its impact on reconstituting T cells. We identified high concentrations of serum 8-hydroxydeoxyguanosine (8-OHdG) as an established biomarker for oxidative stress. 8-OHdG is one of the major products of DNA oxidation, which is normally rapidly removed. After allo-SCT, T cells accumulated oxidative DNA damage. High cellular 8-OHdG content (8-OHdGhi) was associated not only with signs of enhanced T-cell activation but also premature exhaustion. The inability of 8-OHdGhi T cells to efficiently target malignant cells or produce cytotoxic granzyme B and interferon gamma was associated with a significantly increased relapse risk and a shorter overall survival. Taken together, our novel findings could give reason to focus on bolstering DNA repair in reconstituting T cells as a means to improve GVL efficacy.

[TILGen study-immunological targets in patients with breast cancer : Influence of tumor-infiltrating lymphocytes]. / TILGen-Studie ­ Immunologische Angriffspunkte bei Patientinnen mit Brustkrebs : Einfluss der tumorinfiltrierenden Lymphozyten.

BACKGROUND: The interaction of our immune system with breast cancer (BC) cells prompted the investigation of tumor-infiltrating lymphocytes (TILs) and targeted, tumor antigen-specific immunotherapy. OBJECTIVES: Correlation between TILs and pathological complete response (pCR) after neoadjuvant systemic therapy (NACT). Tumor-specific antigens (TSAs) in HER2+ and triple negative BC and establishment of TSA-specific therapies within the interdisciplinary TILGen study. METHODS: Illustration of the TILGen study design. Assessment of TILs and correlation with pCR within this BC study. RESULTS: pCR was achieved in 38.4% (56/146) and associated with estrogen receptor/progesterone receptor negative (ER-/PR-) and HER2+ tumors. Lymphocytic predominant BC (LPBC) was found in 16.4% (24/146), particularly in ER-/PR- (ER-: 27.3% vs. ER+: 9.9%, PR-: 22.3% vs. PR+: 8.2%), large, and poorly differentiated BC. TILs were significantly correlated with pCR in multivariate analysis. In LPBC, pCR was achieved in 66.7%, whereas it was 32.8% in non-LPBC. CONCLUSIONS: First results confirm the influence of the human immune system on the response to NACT in HER2+ and triple negative BC. TSA-specific immunotherapy might improve the outcome in BC patients but there is an urgent need for comprehensive studies to further investigate this issue.

[Recent development of hypertension and acute renal failure in a 59-year-old woman]. / Neu aufgetretene Hypertonie und akutes Nierenversagen bei einer 59-jährigen Patientin.

We report on a 59-year-old woman who presented with nausea, fatigue, arterial hypertension, and acute renal failure. Clinical examination, laboratory findings of blood and urine and abdominal sonography were inconclusive. Renal biopsy revealed infiltration by a diffuse large B­cell lymphoma. In fluorodeoxyglucose positron emission tomography/computed tomography tracer enrichment was demonstrated in both kidneys, skeletal system and retroperitoneal lymph nodes. Renal function improved already after the first cycle of R­CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). After chemotherapy, a complete remission, normalization of blood pressure and renal function were achieved.

Successful Generation of CD19 Chimeric Antigen Receptor T Cells from Patients with Advanced Systemic Lupus Erythematosus.

Although it has been shown that the production of functional chimeric antigen receptor T cells is feasible in patients with B-cell malignancies, it is currently unclear whether sufficient amounts of functional autologous CAR T cells can be generated from patients with autoimmune diseases. Intrinsic T-cell abnormalities and T-cell-targeted immune suppression in patients with autoimmunity may hamper the retrieval of sufficient T cells and their transduction and expansion into CAR T cells. Patients with active systemic lupus erythematosus (SLE) underwent leukapheresis after tapering glucocorticoids and stopping T-cell-suppressive drugs. This material was used as source for manufacturing anti-CD19 CAR T-cell products (CAR) in clinical scale. Cells were transduced with a lentiviral anti-CD19 CAR vector and expanded under good manufacturing practice (GMP) conditions using a closed, semi-automatic system. Functionality of these CAR T cells derived from autoimmune patient cells was tested in vitro. Six SLE patients were analyzed. Leukapheresis could be successfully performed in all patients yielding sufficient T-cell numbers for clinical scale CAR T-cell production. In addition, CAR T cells showed high expansion rates and viability, leading to CAR T cells in sufficient doses and quality for clinical use. CAR T cells from all patients showed specific cytotoxicity against CD19+ cell lines in vitro. GMP grade generation of CD19 CAR T-cell products suitable for clinical use is feasible in patients with autoimmune disease.

Reduced cytotoxicity by mutation of lysine 590 of Pseudomonas exotoxin can be restored in an optimized, lysine-free immunotoxin.

Immunotoxins, which are fusion proteins of an antibody fragment and a fragment of a bacterial or a plant toxin, induce apoptosis in target cells by inhibition of protein synthesis. ADP-ribosylating toxins often have few lysine residues in their catalytic domain. As they are the target for ubiquitination, the low number of lysines possibly prevents ubiquitin-dependent degradation of the toxin in the cytosol. To reduce this potential degradation, we aimed to generate a lysine-free (noK), Pseudomonas exotoxin (PE)-based immunotoxin. The new generation 24 kDa PE, which lacks all but the furin-cleavage site of domain II, was mutated at lysine 590 (K590) and at K606 in a CD22-targeting immunotoxin and activity was determined against various B cell malignancies in vitro and in vivo. On average, K590 mutated to arginine (R) reduced cytotoxicity by 1.3-fold and K606R enhanced cytotoxicity by 1.3-fold compared to wild type (wt). Mutating K590 to histidine or deleting K590 did not prevent this loss in cytotoxicity. Neither stability nor internalization rate of K590R could explain reduced cytotoxicity. These results highlight the relevance of lysine 590 for PE intoxication. In line with in vitro results, the K606R mutant was more than 1.8-fold more active than the other variants in vivo suggesting that this single mutation may be beneficial when targeting CD22-positive malignancies. Finally, reduced cytotoxicity by K590R was compensated for by K606R and the resulting lysine-free variant achieved wt-like activity in vitro and in vivo. Thus, PE24-noK may represent a promising candidate for down-stream applications that would interfere with lysines.

CD137 (4-1BB) stimulation leads to metabolic and functional reprogramming of human monocytes/macrophages enhancing their tumoricidal activity.

Immunotherapies have heralded a new era in the cancer treatment. In addition to checkpoint inhibitors, agonistic antibodies against co-stimulatory immune receptors hold the potential to invoke efficient antitumor immunity. Targeting CD137 has gained momentum based on its ability to drive NK- and T-cell-based responses. CD137-engaging mAbs have already entered clinical trials for different types of tumors showing promising results. Despite the efforts to translate CD137-mediated immunotherapy into clinical practice, little remains known regarding the role of CD137 in human monocytes/macrophages.We found CD137 being expressed on monocytes of healthy controls and at even higher levels in patients with multiple myeloma or CLL. CD137HI(GH) monocytes displayed a distinct phenotypic, transcriptomic, and metabolic profile. They possessed an increased phagocytic capacity enabling superior antibody-dependent phagocytosis (ADPC) of multiple myeloma and lymphoma cells that were treated with anti-CD38 or anti-CD20 mAbs. Triggering CD137 promoted both metabolic and tumoricidal activity in an extracellular signal-regulated kinase (ERK)-dependent fashion. In addition, we observed a phenotypic, transcriptomic, and functional skewing towards a M1-like phenotype.Overall, we introduce CD137 as a positive immune checkpoint on human monocytes/macrophages, which can have therapeutic implications especially in view of synergistic effects when combining CD137 agonists with tumor-targeting antibodies.

Inflammation-induced glycolytic switch controls suppressivity of mesenchymal stem cells via STAT1 glycosylation.

Mesenchymal stem cells (MSCs) represent key contributors to tissue homeostasis and promising therapeutics for hyperinflammatory conditions including graft-versus-host disease. Their immunomodulatory effects are controlled by microenvironmental signals. The MSCs' functional response towards inflammatory cues is known as MSC-"licensing" and includes indoleamine 2,3-dioxygenase (IDO) upregulation. MSCs use tryptophan-depleting IDO to suppress T-cells. Increasing evidence suggests that several functions are (co-)determined by the cells' metabolic commitment. MSCs are capable of both, high levels of glycolysis and of oxidative phosphorylation. Although several studies have addressed alterations of the immune regulatory phenotype elicited by inflammatory priming metabolic mechanisms controlling this process remain unknown. We demonstrate that inflammatory MSC-licensing causes metabolic shifts including enhanced glycolysis and increased fatty acid oxidation. Yet, only interfering with glycolysis impacts IDO upregulation and impedes T-cell-suppressivity. We identified the Janus kinase (JAK)/signal transducer and activator of transcription (STAT)1 pathway as a regulator of both glycolysis and IDO, and show that enhanced glucose turnover is linked to abundant STAT1 glycosylation. Inhibiting the responsible O-acetylglucosamine (O-GlcNAc) transferase abolishes STAT1 activity together with IDO upregulation. Our data suggest that STAT1-O-GlcNAcylation increases its stability towards degradation thus sustaining downstream effects. This pathway could represent a target for interventions aiming to enhance the MSCs' immunoregulatory potency.

Direct evidence to support the immunosurveillance concept in a human regressive melanoma.

The concept of immunosurveillance against cancer has been an extensively debated question over the last decades. Multiple indirect arguments have supported the view that the immune system may control, at least in certain cases, malignant cell growth while direct demonstration is still lacking in the human. In an attempt to address this issue, we have selected a study model, namely spontaneously regressive melanoma. In previous series of experiments, the variability of T cell receptors (TCRs) in the lymphocytes infiltrating a regressive tumor lesion was investigated. Results demonstrated that clonal T cell populations, precisely defined through their V-D-J junctional sequences, were amplified in situ. One clone was predominant, expressing the V beta 16 variable gene segment. A specific anti-V beta 16 TCR mAb was generated here to purify and functionally characterize the corresponding cells. A tumor-infiltrating lymphocyte-derived V beta 16+ T cell line was developed using this reagent. These in vitro cultured cells were found to express the in vivo predominant TCR sequence exclusively and to display an HLA-B14-restricted cytotoxic activity against the autologous tumor cells. Immunohistochemical experiments, performed with the anti-V beta 16 mAb, showed that the corresponding CTLs are present in the tumor area, some of them being closely opposed to the melanoma cells. Together, these studies demonstrate the existence of a local adaptive immune response clinically associated to tumor regression, thus strongly supporting the validity of the immunosurveillance concept in certain human tumors.

Analysis of T cell receptor variability in tumor-infiltrating lymphocytes from a human regressive melanoma. Evidence for in situ T cell clonal expansion.

Malignant melanomas are often infiltrated by T lymphocytes. It is postulated that the presence of tumor-infiltrating lymphocytes (TIL) reflects ongoing immune responses against transformed cells. Such "responses" appear generally inefficient with the potential exception of infrequent clinical situations characterized by spontaneous tumor regression. We have characterized here the molecular structure of the T cell receptor beta chain expressed by TILs in a case of regressive melanoma. Advantage was taken of the PCR technology to study T lymphocytes directly without cell culture. Experimentally validated V beta subfamily specific primers were used to evaluate the V beta usage in TILs and control samples. Our results reveal that clonal T cell populations, precisely defined by their V-D-J junctional sequences, are amplified at the tumor site. The existence of such local antigen-driven selections support the hypothesis that antitumor responses may indeed take place in regressive melanoma.

Immunostimulatory cytokines in somatic cells and gene therapy of cancer.

The use of immunostimulatory cytokines has become an increasingly promising approach in cancer immunotherapy. The major goal is the activation of tumour-specific T lymphocytes capable of rejecting tumour cells from patients with low tumour burden or to protect patients from a recurrence of the disease. Strategies that provide high levels of immunostimulatory cytokines locally at the site of antigen have demonstrated pre-clinical and occasional clinical efficacy. Animal models using poorly immunogenic tumours revealed that tumour cells genetically engineered to produce cytokines like IL-2, IL-4, IL-7, IL-12, IFNs, GM-CSF or TNF-alpha were found to be effective in eradicating disseminated tumours. Experimental data obtained from these different animal models are reviewed here to provide an overview of this rapidly evolving field. The data obtained so far from clinical trials involving cytokine gene-modified cells have provided important information regarding the feasibility, safety, immunological effects and occasional clinical responses.

The PD-1/PD-L1 axis contributes to immune metabolic dysfunctions of monocytes in chronic lymphocytic leukemia.

Immune dysfunctions in chronic lymphocytic leukemia (CLL) contribute to tumor immune escape and attenuate immune-based therapies. Monocytes/macrophages represent key components of cancer immune surveillance and effectors for antibody-mediated antitumor effects. Monocytes display an altered subset composition in CLL. Moreover, we find a changed metabolic phenotype: glucose uptake, glucose transporters and expression of glycolytic molecules are reduced. Our data establish a link between glycolytic competence and monocyte-mediated phagocytosis of tumor cells. Furthermore, we report that CLL monocytes express Bruton's tyrosine kinase (BTK). Our observations suggest that using BTK inhibitors in CLL might further aggravate the observed immune metabolic defects in monocytes. Triggering the programmed cell death-1 (PD-1) checkpoint on monocytes hampers glycolysis, phagocytosis and BTK signaling. Conversely, disrupting PD-1/PD-L1 signaling reverses these immune metabolic dysfunctions. Taken together, our findings imply a novel metabolic interplay between CLL cells and monocytes and that blocking PD-1/PD-L1 might restore metabolic together with antitumor activity of CLL monocytes/macrophages.

Phase I trial of intravenously administered endotoxin (Salmonella abortus equi) in cancer patients.

We report a phase I study in cancer patients being treated with i.v. bolus injections of highly purified lipopolysaccharide (LPS) Salmonella abortus equi. Twenty-four patients with disseminated cancer received escalating doses of LPS at 2-week intervals. Dose escalation was performed in six dose levels treating 3-6 patients at each level. Dose levels 1 and 2 consisted of 0.15 and 0.3 ng/kg, respectively. Further dose escalation up to 5.0 ng/kg was enabled by pretreatment with ibuprofen, which attenuated the constitutional side effects of LPS. The maximum tolerated dose was 4.0 ng/kg with dose-limiting toxicity being World Health Organization grade III hepatic toxicity. Hematological changes included transient decreases in WBCs affecting granulocytes, monocytes, and lymphocytes in a marked different pattern. Endogenous cytokine release occurred in an LPS dose-dependent manner as measured by tumor necrosis factor-alpha, interleukin-6, and macrophage colony-stimulating factor serum levels. Moderate antitumor activity in colorectal cancer was observed in the case of 2 patients. Phase II trials of LPS are currently in progress.

Evidence for in situ amplification of cytotoxic T-lymphocytes with antitumor activity in a human regressive melanoma.

We have derived from lymphocytes infiltrating a human regressive melanoma lesion a series of T-cell receptor alpha/beta-dependent, HLA-B14-restricted cytotoxic T-lymphocyte clones reactive against the autologous tumor. Analysis of the T-cell receptor gene expression revealed that all the clones represented a unique cell expressing a V beta 13.1/J beta 1.1 gene segment. T-cell receptor transcripts expressed in the cloned cells were compared to those present in the uncultured tumor tissue. This analysis demonstrated that the specific cytotoxic T-lymphocyte clones characterized in vitro was actually selected and amplified in vivo at the lesion site. These results provide strong evidence that effector T-cells have contributed to tumor regression.

Generation and purification of CD8+ melan-A-specific cytotoxic T lymphocytes for adoptive transfer in tumor immunotherapy.

Tumor antigens that might serve as potential targets for adoptive T-cell therapy have been defined in different tumor entities, especially in malignant melanoma. To generate conditions to induce primary T-cell responses against different HLA-A*0201-restricted melanoma peptides and to allow further expansion of peptide-specific T cells for adoptive transfer, CD8+-purified T cells from healthy donors were stimulated with Melan-A-pulsed autologous dendritic cells. Dendritic cells were generated in vitro from monocytes with granulocyte macrophage colony-stimulating factor, interleukin-4, and transforming growth factor-beta1. After 3-4 weekly stimulation cycles with Melan-A-pulsed DCs, we were able to induce a strong peptide-specific CTL response in vitro. MHC-peptide tetramer staining revealed a frequency of up to 3.5% CD8+/Melan-A+ T cells. Additional antigen-independent expansion with anti-CD3/anti-CD28 monoclonal antibodies together with interleukin-2 gave rise to 600-fold expansion of CD8+ CTLs that maintained Melan-A specificity and were able to efficiently lyse Melan-A-expressing melanoma cells. To enrich antigen-specific T cells in vitro, we used a recently established technology for analysis and sorting of live cells according to secreted cytokines. In the present study, we demonstrated that Melan-A-specific T cells can be purified by magnetic separation according to secreted IFN-gamma. These cells revealed a very potent monospecific CTL response, even at low E:T ratios, against Melan-A-pulsed and Melan-A-expressing target cells. Altogether, our study demonstrated that we have developed an efficient method for generating large numbers of peptide-specific T cells in vitro that may be used for adoptive T-cell transfer in tumor immunotherapy.

NTS influence on transgene expression from mono- and bicistronic plasmid vectors after lipofection in a human fibroblast cell line.

We evaluated the survival, transgene production, and copy numbers of integrated plasmid units per host genome after lipofection with mono- and bicistronic plasmid vectors in different cell lines and under various conditions. The addition of an integration enhancing murine sequence nontranscribed spacer (NTS) to the plasmids increased transfection efficiency, survival, and transgene expression. However, in human fibroblast cells this sequence had only marginal effects on overall plasmid copy number in bulk cultures. Clones producing the highest amounts of the transgene contained only one or two copies of plasmid per genome, independent of cell type and plasmid design.

Induction of tumor-specific cytotoxic T lymphocytes by immunization with autologous tumor cells and interleukin-2 gene transfected fibroblasts.

In a phase I trial designed to study a vaccine composed of autologous tumor cells and interleukin-2 gene transfected fibroblasts we analyzed lymphocytes infiltrating the vaccination site (VIL) in two melanoma patients. Functional studies demonstrated that numbers of MHC class I restricted cytotoxic T cells directed against the autologous tumor had increased at the immunization site in both cases. Analysis of the variability of T cell receptors (TCR) in the VIL of one patient revealed that the cytotoxic T lymphocytes consisted of a predominant population of TCRBV21S3+ T cells. Enrichment of this subpopulation to more than 99% by specific anti-TCRBV21S3 monoclonal antibody linked immunomagnetic beads and sequencing of the TCR-beta chain disclosed exactly the same V-D-J junctional sequence in all eight TCRBV21 transcripts from these VIL. The identical sequence was also detected in all eight TCRBV21 transcripts from the patient's tumor-infiltrating lymphocytes, indicating that the same CTL clone had infiltrated the tumor, circulated in the peripheral blood, and was amplified at the vaccination site. The TCRBV21S3+ T cells were also found to display an MHC class I restricted cytotoxic activity specifically directed against the autologous tumor cells. At the beginning of treatment these cells were undetectable at the vaccination site and delayed-type hypersensitivity testing was negative, contrasting with the positive results after therapy. Thus it is likely that vaccination with autologous tumor cells plus interleukin-2 gene transfected allogeneic fibroblasts had induced not only local accumulation but also an increase in the frequency of circulating tumor specific CTL.

Primary fibroblasts from human adults as target cells for ex vivo transfection and gene therapy.

Diploid fibroblast (dFb) cultures were established from a total of 106 skin and serosa biopsies of human adults. Using an optimized enzymatic dissociation procedure, 10(11) dFb/cm2 skin were obtained from patients younger than 60 years after an average time of 89 +/- 8 days, with a mean population doubling time of 3.87 +/- 1.4 days. Enzymatic dissociation of skin biopsies yielded cultures of significantly higher growth capacity of dFb than those prepared by mechanical dissociation followed by spontaneous outgrowth of cells. The plating efficiency that may be crucial for clonal selection of transfected cells was negligible when dFb were plated without feeder cells at low density, while it was enhanced to 9-24% by the addition of a feeder layer of irradiated human embryonal fibroblasts. DFb secreted various cytokines with spontaneous release of interleukin-6 (IL-6) in high quantities of up to 20 ng/10(6) cells/24 hr. In addition, one-third of the culture secreted substantial amounts of granulocyte-macrophage colony-stimulating factor (GM-CSF), while low amounts of tumor necrosis factor-alpha (TNF-alpha) were detectable in some cases after irradiation of the cells. Comparison of various transfection methods by a transient luciferase expression assay demonstrated that receptor-mediated gene transfer was approximately 10-fold more efficient than cationic lipofection of dFb, while electroporation resulted in substantially less expression of the reporter gene. We conclude that primary dFb can be obtained reproducibly from human adults and represent a suitable target cell population for receptor-mediated gene transfer and cationic lipofection.(ABSTRACT TRUNCATED AT 250 WORDS)

Systematic evaluation of chimeric marker genes on dicistronic transcription units for regulated expression of transgenes in vitro and in vivo.

Plasmid expression vectors combining human cytokine cDNAs and selectable marker genes on dicistronic transcription units were functionally characterized in vitro and in vivo. The internal ribosome entry sequence (IRES) of encephalomyocarditis virus mediated cap-independent translation of the downstream cistron. After cationic lipofection of cells with a dicistronic construct containing the Neor gene downstream of a human interleukin-2 (IL-2) cDNA, all G418-resistant clones secreted high amounts of IL-2. Reversal of the order of the cDNAs was associated with less efficient transgene expression and represented no advantage in comparison to separate expression cassettes. To combine direct in vitro selection of expression with in vivo elimination of cytokine-secreting cells, an improved chimeric cDNA of the Neor and herpes simplex virus (HSV) thymidine kinase (TK) genes was constructed and shown to confer sensitivity to ganciclovir concentrations that can be achieved in human patients. This chimeric marker was coupled on dicistronic constructs with a granulocyte colony-stimulating factor (G-CSF) cDNA as a molecule with easily detectable bioactivity in vivo. Subcutaneous implantation of pCMV.GCSF.ires TK/NEO-transfected CMS-5 cells into syngeneic BALB/c mice resulted in excessive leukocytosis and progressively growing tumors. Treatment with ganciclovir led to normalization of leukocyte counts in all animals, whereas complete regression of tumors was observed in only 3/5 mice. Hypermethylation of the transfected promoter was demonstrated in both ganciclovir-resistant tumors. Thus, transcription units combining selectable markers and genes of interest allow selection of high producer cells in vitro and efficient elimination of transgene-expressing cells in vivo. However, cells that hypermethylate transfected genes to terminate gene expression in vivo may escape conditional ablation.

Systemic hematological effects of granulocyte colony-stimulating factor produced by irradiated gene-transfected fibroblasts.

Although long-term expression of therapeutic molecules is necessary for the treatment of permanent deficiencies, short-term expression of therapeutic molecules inducing local or systemic effects is preferable in clinical situations where temporary substitution is the goal. One such clinical setting is the administration of hematopoietic growth factors in cancer chemotherapy-induced myelosuppression. Several plasmid vectors containing the human granulocyte colony-stimulating factor (G-CSF) gene under transcriptional control of different regulatory elements were constructed. In vitro production of G-CSF by nonvirally transfected murine fibroblast clones initially increased after lethal irradiation and was detectable for at least 12 days. We also demonstrate that a single injection of irradiated G-CSF-secreting fibroblasts leads to accelerated hematopoietic recovery and mobilization of committed peripheral blood progenitor cells equivalent to that achieved by twice daily s.c. administration of high doses of recombinant human G-CSF. Using dicistronic vectors, high levels of G-CSF secretion were also obtained in human fibroblasts.

Induction of circulating phospholipase A2 activity by intravenous infusion of endotoxin in patients with neoplasia.

The purpose of this study was to evaluate the impact of repeated intravenous infusions of endotoxin (EN) in patients with cancer on the systemic release of extracellular proinflammatory phospholipase A2 (PLA2) and its relationship to the release of tumor necrosis factor (TNF) and interleukin-6 (IL-6). Six patients received 15 infusion of EN isolated from Salmonella abortus equi at a dose of 4 ng/kg. Marked increase in the activity of circulating PLA2 was noted within 3 h after the first EN infusion and reached a maximal level of 20.4-fold greater than baseline 24 h after infusion. In five patients challenged with EN 2 weeks later, PLA2 reached peak levels 15.5-fold greater than baseline. In two patients who received three sequential daily infusions, the incremental increase in PLA2 activity after the second and third challenge reached maximum levels 6 h after EN infusion. PLA2 response followed those of TNF and IL-6 but was quantitatively different. Whereas maximal levels of TNF and IL-6 declined substantially after repeat EN challenges, no such decline occurred in PLA2 activity. Since, in the clinical setting of gram-negative sepsis, there is recurrent increase in circulating EN, our study approximates this clinical situation and shows that extracellular release of PLA2 follows temporally that of proximal cytokines such as TNF and IL-6. These cytokines may be related to PLA2 release and sustained high activity in the systemic circulation.(ABSTRACT TRUNCATED AT 250 WORDS)