Analysis of a natural immune response against tumor antigens in a melanoma survivor: lessons applicable to clinical trial evaluations.

The long-term survival of some patients with metastatic melanoma may be attributable in part to cellular immune responses to melanoma antigens. However, little is known about the level of CTL reactivity in vivo that is required for immunological control of tumor progression. In the present report, T-cell responses were evaluated with lymphocytes obtained from tumor-involved nodes and peripheral blood of a long-term melanoma survivor. Using an ELISPOT assay, naturally occurring functional T cells, which recognize the peptide ALLAVGATK (gp100(17-25)) plus two other HLA-A3 restricted peptides, were detected in a tumor-involved lymph node. The ALLAVGATK-reactive T cells were also evaluated by MHC-tetramers staining and were found to be CD8+ CD45RO+ L-selectin(-) CD11a+, suggesting that they are antigen experienced and have a memory phenotype. Unstimulated peripheral blood lymphocytes from the same patient demonstrated no detectable T-cell responses; however, a single stimulation with ALLAVGATK peptide in vitro resulted in a dramatic expansion of peptide-reactive CTLs. This patient, with evidence of tumor-reactive CTLs targeted to several tumor antigens in a tumor-involved lymph node and with evidence of a circulating memory T-cell response, has remained disease-free for 6 years, despite prior bulky nodal metastasis. In contrast, three HLA-A3+ patients with rapidly progressive metastatic melanoma had no detectable T-cell response in tumor-involved nodes or peripheral blood lymphocytes, even after peptide stimulation ex vivo. The presented data are consistent with a systemic polyvalent immune response against tumor in this long-term survivor. These data provide an estimate of the level of CTL response that may be associated with protection from tumor recurrence.

Phase I trial of a melanoma vaccine with gp100(280-288) peptide and tetanus helper peptide in adjuvant: immunologic and clinical outcomes.

A melanoma vaccine composed of HLA-A2-restricted peptide YLEPGPVTA (gp100(280)), with or without a modified T-helper epitope from tetanus toxoid AQYIKANSKFIGITEL, has been evaluated in a Phase I trial to assess safety and immunological response. The vaccines were administered s.c. in either of two adjuvants, Montanide ISA-51 or QS-21, to 22 patients with high-risk resected melanoma (stage IIB-IV). Local and systemic toxicities were mild and transient. We detected CTL responses to the gp100(280) peptide in peripheral blood in 14% of patients. Helper T-cell responses to the tetanus helper peptide were detected in 79% of patients and had a Th1 cytokine profile. One patient with a CTL response to gp100 had a recurrence in a lymph node 2 years later; her nodes contained CD8+ cells reactive to gp100(280) (0.24%), which proliferated in response to peptide. The overall survival of patients is 75% (95% confidence interval, 57-94%) at 4.7 years follow-up, which compares favorably with expected survival. Four of 14 patients who completed at least six vaccines subsequently developed metastases, all of which were solitary and surgically resectable. They remain alive and clinically free of disease at last follow-up. Data from this trial demonstrate immunogenicity of the gp100(280) peptide and suggest that immune responses may persist long-term in some patients. The frequency and magnitude of the CTL response may be improved with more aggressive vaccination regimens. Although this Phase I study was not intended to evaluate clinical benefit, the excellent survival of patients on this protocol suggests the possibility of a benefit that should be assessed in future studies.

Attenuation of acute lung injury and oxygen radical production by the 21-aminosteroid, U-78518F.

Oxygen radicals play an important role in the mechanism of acute lung injury. The 21-aminosteroid lazaroid, U-78518F, is a potent antioxidant. We examined the effect of intravenous U-78518F on acute lung injury in septic guinea pigs over 8 h. The experimental groups (n = 6) were 1) saline control, 2) Escherichia coli (2 x 10(9)/kg i.v.), 3) pretreatment (U-78518F 5 mg/kg bolus + 1 mg.kg-1 x h-1, 15 min before E. coli injection), and 4) posttreatment (U-78518F 30 min after E. coli injection). We measured wet-to-dry weight ratio (W/D) as an index of pulmonary edema and concentration ratios of 125I-labeled albumin in lung tissue and bronchoalveolar lavage fluid compared with plasma (L/P and BAL/P, respectively) as indexes of lung protein fluxes. In septic guinea pigs, pretreatment with U-78518F attenuated W/D, L/P, and BAL/P and posttreatment attenuated W/D and BAL/P (P < 0.05 for each). Furthermore, we studied the effect of U-78518F on human neutrophil oxygen radical production (ORP) by using flow cytometry to assess intracellular ORP and lucigenin-dependent chemiluminescence to assess extracellular ORP. Neutrophils (5 x 10(5) were stimulated with 0.5 micrograms/ml of phorbol myristate acetate. With flow cytometry, we measured intracellular ORP, cross-sectional cell area, and degranulation in neutrophils. U-78518F (minimum concn 1.0 microM) decreased intracellular ORP (n = 4; P < 0.05) when the dihydrorhodamine 123 assay was used. U-78518F (minimum concn 1.0 microM) inhibited phorbol myristate acetate-induced neutrophil chemiluminescence (n = 4; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

TGF-beta 1 causes increased endothelial ICAM-1 expression and lung injury.

Neutrophil adherence to vascular endothelium is partially mediated by adhesion molecules, including intracellular adhesion molecule 1 (ICAM-1), on endothelial cells. We examined the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of ICAM-1 in human umbilical vein endothelial cells (HUVEC). TGF-beta 1 (1 ng/ml) increased ICAM-1 and ICAM-1 mRNA expression in HUVEC, as assessed by flow cytometry and Northern blot analysis, respectively. In addition, we investigated whether exogenous recombinant TGF-beta 1 can cause neutrophil-mediated lung injury in guinea pigs. The plasma half-life of 125I-labeled TGF-beta 1 in guinea pigs was 4.6 +/- 0.1 min, and the 125I activity was 2.8 +/- 0.2% 8 h after injection. The ratio of 125I-labeled albumin concentration in lung tissue and bronchoalveolar lavage (BAL) fluid to that in plasma, lung wet-to-dry weight ratio, numbers of neutrophils in BAL fluid, and numbers of neutrophils per alveolus in fixed lung sections increased in guinea pigs that received a high dose of TGF-beta 1 (25 micrograms i.v. followed by 2 micrograms/h for 8 h) compared with the control group. These results suggest that TGF-beta 1 causes neutrophil-mediated lung injury, possibly through upregulation of ICAM-1 on endothelial cells, and might be important in the pathogenesis of lung injury.

A2A adenosine receptor (AR) activation inhibits pro-inflammatory cytokine production by human CD4+ helper T cells and regulates Helicobacter-induced gastritis and bacterial persistence.

Helicobacter pylori causes a lifelong infection and provides a model of bacterial adaptation and persistent colonization. Adenosine is an anti-inflammatory mediator that limits tissue damage during inflammation. We studied the role of adenosine in the T-cell-mediated regulation of gastritis and bacterial persistence. After 4 h of activation, human T helper (Th) cells increased A(2A) adenosine receptor (A(2A)AR) mRNA level (sevenfold). A(2A)AR was the predominant subtype expressed in resting and stimulated gastric or peripheral Th cells. Stimulation with ATL313, an A(2A)AR agonist, increased cyclic AMP (cAMP) accumulation and reduced interleukin-2 (IL-2) production by 20-50%. ATL313 also attenuated tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production, which was inhibited by an A(2A)AR antagonist. Infection of IL-10-deficient mice with H. pylori is cleared spontaneously due to the marked inflammation. Administration of ATL313 during infection reduced gastritis and pro-inflammatory cytokine responses while bacterial load increased. In contrast, infection of A(2A)AR-deficient mice enhanced gastritis. Thus, A(2A)AR limits the pro-inflammatory effects of Th cells and favor chronic Helicobacter infection.

Polyethylene glycol-conjugated superoxide dismutase attenuates septic lung injury in guinea pigs.

Reactive oxygen species (ROS), including superoxide anions, play an important role in mediating acute lung injury. We examined whether polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) attenuates lung injury in Escherichia coli-treated guinea pigs. Twenty-four guinea pigs were divided into four groups: (1) control group; (2) septic group, in which live E. coli (2 x 10(9)/kg) were injected intravenously; (3) pretreatment group, in which PEG-SOD (2,000 IU/kg) was injected intravenously 15 min before E. coli; and (4) posttreatment group, in which PEG-SOD (2,000 IU/kg) was injected intravenously 30 min after E. coli. Lung injury was assessed by the concentration ratio of 125I-labeled albumin in lung tissue and bronchoalveolar lavage (BAL) fluid relative to plasma (L/P and BAL/P), lung wet-to-dry weight ratio, and the number of neutrophils in BAL fluid. Plasma half-life of PEG-SOD in normal guinea pigs was 13.5 h. L/P, lung wet-to-dry weight ratio, and the number of neutrophils in BAL fluid decreased in both pretreatment and posttreatment groups compared with the septic group. BAL/P decreased in the pretreatment group but not in the posttreatment group compared with the septic group. After the animal model studies, we investigated the effect of PEG-SOD on the human neutrophil extracellular generation of ROS stimulated by phorbol myristate acetate (PMA) in lucigenin-dependent chemiluminescence (CL). PEG-SOD at concentrations greater than or equal to 0.1 U/ml inhibited PMA-induced CL in a dose-dependent manner. We also examined the effect of PEG-SOD on the neutrophil intracellular generation of ROS using flow cytometry to assess intracellular hydroethidine oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

The protein kinase C inhibitor, H-7, induces acute lung injury in guinea pigs.

OBJECTIVES: To determine if the protein kinase C inhibitor, H-7, alone can cause acute lung injury. In cell studies, H-7 inhibited phorbol myristate acetate-induced neutrophil oxygen radical release. Additionally, one animal study demonstrated that H-7 inhibited phorbol myristate acetate-induced lung injury. There have been no studies on the effect of H-7 alone on lung function or on neutrophil release of oxygen radicals. DESIGN: Prospective, randomized, laboratory study along with in vitro studies using flow cytometry and lucigenin-dependent chemiluminescence. SETTING: Experimental laboratory. SUBJECTS: Specific, pathogen-free guinea pigs and isolated human peripheral neutrophils. INTERVENTIONS: Guinea pigs were randomized into three experimental groups: saline control, H-7 low dose (2 mg/kg bolus + 0.2 mg/kg/hr), and H-7 high dose (6 mg/kg bolus + 0.5 mg/kg/hr). Human neutrophils were randomized into control and experimental groups. The effects of H-7 on pulmonary permeability in guinea pigs were examined over an 8-hr period. MEASUREMENTS AND MAIN RESULTS: We measured the wet/dry weight ratio as an index of pulmonary edema and we measured the concentration ratios of 125I-labeled albumin in lung tissue and in bronchoalveolar lavage fluid and compared the ratios with those values in plasma as indices of pulmonary permeability. We also studied the in vitro effect of H-7 on human neutrophil oxygen radical production, using flow cytometry and lucigenin-dependent chemiluminescence. By flow cytometry, we measured oxygen radical production using the 2',7'-dichlorofluorescin and hydroethidine assays. The 2',7'-dichlorofluorescin assay mainly measures hydrogen peroxide, while the hydroethidine assay measures either superoxide anion alone or in combination with other oxygen intermediaries like hydrogen peroxide. Neutrophils (5 x 10(5)) were obtained by Ficoll-Hypaque gradient centrifugation and were incubated with H-7 (5, 25, 100 microM). In the H-7 high-dose group, wet/dry weight ratio, and 125I-labeled albumin ratios in lung/plasma, and bronchoalveolar lavage/plasma were significantly increased (p < .05 for each ratio). Pulmonary endothelial gap and subendothelial bleb formation were demonstrated in the high-dose group by electron microscopy. One hundred micromols of H-7 caused a small, significant decrease (23.3%, p < .05) in neutrophil oxygen radical production assessed by 2',7'-dichlorofluorescin. H-7 had no other effects on neutrophil oxygen radical production. H-7 did not stimulate neutrophil chemiluminescence; it decreased chemiluminescence. CONCLUSIONS: a) Protein kinase C inhibition with high-dose H-7 increased wet/dry weight and albumin in lung/plasma and bronchoalveolar lavage/plasma ratios in guinea pigs; b) the H-7 high-dose group demonstrated damaged pulmonary endothelium by electron microscopy; and c) since neutrophil oxygen radical production was not increased by H-7 as assessed by flow cytometry and chemiluminescence, it appears that H-7-induced acute lung injury and endothelial damage are not mediated by increased neutrophil oxygen radical production.