London bombings July 2005: the immediate pre-hospital medical response.

On July 7th 2005 a series of terrorist bombs exploded in London. The transport system was targeted and at least 54 passengers were killed and around 700 injured. This paper describes the immediate pre-hospital medical response to the four scenes. From the perspective of the London Helicopter Emergency Medical Service the deployment, difficulties on scene and the initial lessons learned are discussed.

Development of a closed-system process for clinical-scale generation of DCs: evaluation of two monocyte-enrichment methods and two culture containers.

BACKGROUND: Clinical immunotherapy trials using DCs depend on large-scale methods for DC generation that fulfil current good manufacturing practice requirements. Our goal was to develop data on two variables, monocyte-enrichment method and culture container, which could be used to design a closed-system process for ex vivo generation of immature DCs. METHODS: Mononuclear cells were collected by leukapheresis and enriched for monocytes by either counterflow centrifugal elutriation, or immunomagnetic selection using Isolex, an automated closed-system device. Monocytes were cultured for 7 days in serum-free medium with GM-CSF and IL-4, using either plastic flasks or gas-permeable Stericell bags. Monocytes and cultured DCs were evaluated for yield, flow cytometric phenotype, and in vitro function in MLR, and autologous recall responses to tetanus toxoid and influenza virus. RESULTS: Enriched monocyte products from elutriation and immunomagnetic selection were equivalent in yield and purity, and were capable of generating immature DCs in either flasks or bags. DCs from all four culture conditions were equivalent in yield, phenotype, and in vitro function. Mean DC yield was 67-80% per seeding monocyte, and 11-13% per starting mononuclear cell (MNC). A leukapheresis product containing 5 x 10(9) MNCs processed by this method could therefore yield approximately 5 x 10(8) immature DCs. DISCUSSION: In this manufacturing process, the Isolex system was equivalent to elutriation, and Stericell bags were equivalent to flasks. Together, the Isolex system and Stericell bags can be incorporated into a closed-system process to generate immature DCs.

Development of a clinical-scale method for generation of dendritic cells from PBMC for use in cancer immunotherapy.

BACKGROUND: There is growing interest in the use of dendritic cells (DCs) for treatment of malignancy and infectious disease. Our goal was to develop a clinical scale method to prepare autologous DCs for cancer clinical trials. METHODS: PBMC were collected from normal donors or cancer patients by automated leukapheresis, purified by counterflow centrifugal elutriation and placed into culture in polystyrene flasks at 1 x 10(6) cells/mL for 5-7 days at 37 degrees C, with 5% CO(2), with IL-4 and GM-CSF. Conditions investigated included media formulation, supplementation with heat in activated allogeneic AB serum or autologous plasma and time to harvest (Day 5 or Day 7). DCs were evaluated for morphology, quantitative yield, viability, phenotype and function, including mixed leukocyte response and recall response to tetanus toxoid and influenza virus. RESULTS: DCs with a typical immature phenotype (CD14-negative, CD1a-positive, mannose receptor-positive, CD80-positive, CD83-negative) were generated most consistently in RPMI 1640 supplemented with 10% allogeneic AB serum or 10% autologous plasma. Cell yield was higher at Day 5 than Day 7, without detectable differences in phenotype or function. In pediatric sarcoma patients, autologous DCs had enhanced function compared with monocytes from which they were generated. In this patient group, starting with 8.0 +/- 3.7 x 10(8) fresh or cryopreserved autologous monocytes, DC yield was 2.1 +/- 1.0 x 10(8) cells, or 29% of the starting monocyte number. DISCUSSION: In the optimized clinical-scale method, purified peripheral monocytes are cultured for 5 days in flasks at 1 x 10(6) cells/mL in RPMI 1640, 10% allogeneic AB serum or autologous plasma, IL-4 and GM-CSF. This method avoids the use of FBS and results in immature DCs suitable for clinical trials.

Paclitaxel chemotherapy after autologous stem-cell transplantation and engraftment of hematopoietic cells transduced with a retrovirus containing the multidrug resistance complementary DNA (MDR1) in metastatic breast cancer patients.

The MDR1 multidrug resistance gene confers resistance to natural-product anticancer drugs including paclitaxel. We conducted a clinical gene therapy study to determine whether retroviral-mediated transfer of MDR1 in human hematopoietic cells would result in stable engraftment, and possibly expansion, of cells containing this gene after treatment with myelosuppressive doses of paclitaxel. Patients with metastatic breast cancer who achieved a complete or partial remission after standard chemotherapy were eligible for the study. Hematopoietic stem cells (HSCs) were collected by both peripheral blood apheresis and bone marrow harvest after mobilization with a single dose of cyclophosphamide (4 g/m2) and daily filgrastim therapy (10 microg/kg/day). After enrichment for CD34+ cells, one-third of each collection was incubated ex vivo for 72 h with a replication-incompetent retrovirus containing the MDR1 gene (G1MD) in the presence of stem-cell factor, interleukin 3, and interleukin 6. The remaining CD34+ cells were stored without further manipulation. All of the CD34+ cells were reinfused for hematopoietic rescue after conditioning chemotherapy with ifosfamide, carboplatin, and etoposide regimen. After hematopoietic recovery, patients received six cycles of paclitaxel (175 mg/m2 every 3 weeks). Bone marrow and serial peripheral blood samples were obtained and tested for the presence of the MDR1 transgene using a PCR assay. Six patients were enrolled in the study and four patients received infusion of genetically altered cells. The ex vivo transduction efficiency, estimated by the PCR assay, ranged from 0.1 to 0.5%. Three of the four patients demonstrated engraftment of cells containing the MDR1 transgene. The estimated percentage of granulocytes containing the MDR1 transgene ranged from a maximum of 9% of circulating nucleated cells down to the limit of detection of 0.01%. One patient remained positive for the MDR1 transgene throughout all six cycles of paclitaxel therapy, whereas the other 2 patients showed a decrease in the number of cells containing the transgene to undetectable levels. Despite the low level of engraftment of MDR1-marked cells, a correlation was observed between the relative number of granulocytes containing the MDR1 transgene and the granulocyte nadir after paclitaxel therapy. No adverse reactions to the genetic manipulation procedures were detected. Therefore, engraftment of human HSCs transduced with the MDR1 gene can be achieved. However, the overall transduction efficiency and stable engraftment of gene-modified HSCs must be improved before MDR1 gene therapy and in vivo selection with anticancer drugs can be reliably used to protect cancer patients from drug-related myelosuppression.

Engraftment of MDR1 and NeoR gene-transduced hematopoietic cells after breast cancer chemotherapy.

To determine whether the multidrug resistance gene MDR1 could act as a selectable marker in human subjects, we studied engraftment of peripheral blood progenitor cells (PBPCs) transduced with either MDR1 or the bacterial NeoR gene in six breast cancer patients. This study differed from previous MDR1 gene therapy studies in that patients received only PBPCs incubated in retroviral supernatants (no nonmanipulated PBPCs were infused), transduction of PBPCs was supported with autologous bone marrow stroma without additional cytokines, and a control gene (NeoR) was used for comparison with MDR1. Transduced PBPCs were infused after high-dose alkylating agent therapy and before chemotherapy with MDR-substrate drugs. We found that hematopoietic reconstitution can occur using only PBPCs incubated ex vivo, that the MDR1 gene product may play a role in engraftment, and that chemotherapy may selectively expand MDR1 gene-transduced hematopoietic cells relative to NeoR transduced cells in some patients.

Retroviral gene transduction of adult peripheral blood or marrow-derived CD34+ cells for six hours without growth factors or on autologous stroma does not improve marking efficiency assessed in vivo.

Our previous work in patients undergoing autologous transplant for multiple myeloma (MM) or breast cancer (BC) has shown that retroviral transduction of adult CD34+ cells for 72 hours in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) resulted in .01% to 1% long-term marking of peripheral blood and marrow cells (Blood 85:3948, 1995). In this study we compare these previous studies to transduction with no added growth factors, previously shown to result in higher levels of marking in children (Lancet 342:1134, 1993) or transduction in the presence of an autologous stromal layer. Peripheral blood (PB) mononuclear cells were collected via apheresis after high-dose cyclophosphamide and granulocyte colony-stimulating factor. Bone marrow (BM) was also harvested in all patients. One third of both BM and PB collections were enriched for CD34+ cells and transduced with one of two marking vectors containing the neomycin-resistance gene to distinguish cells originating from BM and PB posttransplantation. Cells from 3 MM and 2 BC patients were transduced without growth factors for 6 hours and cells from 2 MM and 2 BC patients were transduced in the presence of autologous marrow stroma. Immediately posttransduction, the percentage of Neo-resistant PB and BM progenitors (colony-forming units) were: 0% to 19% in the 6-hour no growth factor group and 0% to 36% in the autologous stroma group. After conditioning therapy, both transduced and untransduced PB and BM fractions were infused into the patients. Semi-quantitative nested DNA polymerase chain reaction was performed on total, mononuclear, and granulocyte fractions of PB and BM at 1, 3, 6, 9, 12, and 18 months. Poor marking has been observed in both groups, with no consistently positive patients. These results compare unfavorably with our prior experience using growth factors during transduction. Further optimization of transduction conditions and vectors needs to be developed to improve transduction efficiency of adult human repopulating hematopoietic cells.

Detection of a Single Viable Cryptosporidium parvum Oocyst in Environmental Water Concentrates by Reverse Transcription-PCR.

Volume 62, no. 9, p. 3386, column 1, last line: "(specific gravity, 2.10) and centrifuging the mixture at 2,000 x g for 10 min" should read "(specific gravity, 1.10) and centrifuging the mixture at 1,050 x g for 10 min." Page 3387, column 1, line 16: "25 cycles at 90(deg)C for 5 min" should read "25 cycles at 90(deg)C for 5 s." [This corrects the article on p. 3385 in vol. 62.].

Detection of a single viable Cryptosporidium parvum oocyst in environmental water concentrates by reverse transcription-PCR.

Current methods for detection of Cryptosporidium parvum oocysts in water are time-consuming and difficult. We have developed a reverse transcription (RT)-PCR which can detect the presence of a single viable oocyst spiked into concentrated environmental water samples. The test is based on the detection of mRNA from a C. parvum heat shock protein (hsp). The synthesis of hsp was induced by a short 45 degrees C incubation followed by oocyst lysis by a freeze-thaw process. Hsp70 mRNA, produced only from viable oocysts, was then isolated by hybridization to oligo(dT)25-coated magnetic beads. Detection was achieved by RT-PCR amplification of a 590-bp region of hsp70 mRNA specific for C. parvum. To test the method, samples of reticulated, reservoir, bore, and river water were concentrated by chemical flocculation and Percoll-sucrose gradient centrifugation and then spiked with dilutions of oocysts. In all four of the water types examined, the detection of single oocysts was possible by RT-PCR combined with Southern hybridization. RT-PCR products were not obtained from formalin-inactivated oocysts. An RNA internal positive control fragment was synthesized that was included with each reaction to guard against RT-PCR false-negative results that may be caused by the presence of inhibitory substances. However, when the magnetic beads were used to extract and concentrate mRNA, no inhibition was observed. The technique is versatile, straightforward, and rapid (1 day) and provides a sensitive and economic means of screening concentrated water samples for the presence of C. parvum.

Growth of tumor-infiltrating lymphocytes from human solid cancers: summary of a 5-year experience.

Between 1989 and 1993, 255 tumor biopsies representing 4 tumor histologies (melanoma, breast cancer, colon cancer and renal cell cancer) were received by the Surgery Branch of the National Cancer Institute. Tumor-infiltrating lymphocytes (TIL) were grown from single-cell suspensions of tumor biopsies over the course of 30-45 days. The TIL were grown in medium containing IL-2. To obtain numbers suitable for therapy (>10(11)), TIL were expanded using a large-scale system of cell culture and harvesting. While the largest number of biopsies was obtained from melanoma patients, TIL were successfully grown from 160 of 255 tumor biopsies representing all 4 histologies. Under the culture conditions employed, several characteristics of TIL expansion were observed. The cell surface phenotype of TIL which grew out from the tumor biopsies was generally a mix of CD3+/CD4+ or CD3+/CD8+ lymphocytes. Only TIL from melanoma biopsies were found to be consistently cytolytic and, in many cases, lysed autologous tumor cells preferentially. Interestingly, TIL derived from extra-nodal sites of metastatic melanoma biopsies (subcutaneous, lung, bowel; 36 of 67, 54%) were more likely to have these cytolytic characteristics than TIL derived from tumor-involved lymph node biopsies (7 of 39, 18%). The present study summarizes 5 years of laboratory effort and validates the technologies developed for the large-scale growth and harvesting of TIL. In addition, it summarizes the laboratory effort supporting previously published clinical reports on TIL from our group.

5-Azacytidine-induced 6-thioguanine resistance at the gpt locus in AS52 cells: cellular response.

Treatment of AS52 cells with 5-azacytidine resulted in an induction of 6-thioguanine-resistant [6TG] colonies, which reached a maximum by an expression time of 9 days. Dose responses for both cytotoxicity and mutation induction were determined following treatment with 5-azacytidine. At 20 microM treatment, 5-azacytidine exposure resulted in about 50% survival. Mutant frequency reached a maximum of 10 microM. At concentrations between 10 and 20 microM, 5-azacytidine was a potent mutagen but did not exhibit a dose response. Although many compounds both induce cell death and affect the growth rate of cells, 5-azacytidine specifically induced cell death and did not affect the doubling time of the surviving treated cell population.

An immunofiltration apparatus for accelerating the visualization of antigen on membrane supports.

Although proteins can be transferred and bound to a membrane with several different methods such as capillary transfer, electroblotting, etc., only the "shaker/incubation" method is commonly used for visualization of the proteins. We have tested an apparatus for the immunofiltration of solutions through nitrocellulose membrane which greatly accelerates the kinetics for the visualization of proteins. As a model system, avidin, bound on nitrocellulose membrane, was detected with 5-min filtering steps of a 1:2000 dilution of ascites of murine monoclonal antibody against avidin followed by a 1:5000 dilution of goat anti-mouse IgG-horseradish peroxidase and 0.5 mg/ml chloronapthol in the presence of 0.01% peroxide. The same solutions used with 5-min incubation steps with the shaker/incubation method could not detect avidin at almost 10 times that amount. Further studies with monoclonal antibodies specific for native C-reactive protein and modified-CRP, 15.1D6 and 13.3H12 respectively, showed that immunofiltration did not result in altered specificity compared to the shaker/incubation method. Also, data are presented showing the advantages of a 10-slot top for the immunofiltration of solutions through distinct areas of a membrane.

Immune dysregulation in TGF-beta 1-deficient mice.

Approximately 2 wk after birth, mice having a TGF-beta 1 null mutation (TGF-beta 1(-/-)) exhibit a progressive wasting syndrome and death. Associated with this phenotype is a multifocal infiltration of lymphocytes and macrophages into target organs, especially the heart, lungs, and salivary glands. To explore the consequences of TGF-beta 1 deficiency on the immune system, lymphocyte phenotype and function were analyzed. Initially, lymphoid organ architecture seemed to be normal and, as symptoms developed, the thymus decreased in size, whereas lymph nodes were enlarged. Phenotypically, the TGF-beta 1(-/-) lymphoid cells seemed to be more differentiated in the thymus and activated in the lymph nodes, but remarkably unaffected in the spleen. Moreover, TGF-beta 1(-/-) spleen and lymph nodes displayed enhanced numbers of proliferating cells, as measured by proliferating cell nuclear Ag and/or cyclin-dependent kinase levels. Consistent with this hyperproliferative response, constitutive levels of IL-2 mRNA were elevated in the thymus and both IL-2 and IL-2R mRNA were increased in the lymph nodes. In contrast with the activation profile of TGF-beta 1(-/-) lymphoid cells in vivo, mitogen challenge of these cells in vitro revealed suppressed proliferation that was associated with a defect in inducible IL-2 mRNA expression and IL-2 secretion. Moreover, the addition of rIL-2 restored the deficient mitogen-induced proliferation. The mechanism leading to T cell anergy remains unclear; however, these data confirm the essential role for TGF-beta 1 in maintaining normal immune function.

Synthetic fibronectin peptides suppress arthritis in rats by interrupting leukocyte adhesion and recruitment.

In an experimental model of arthritis, increased leukocyte adhesion is associated with the evolution of acute and chronic synovial inflammation. Whereas peripheral blood mononuclear cells (PBMC) from control animals bind minimally to fibronectin matrices, PBMC from animals receiving arthropathic doses of bacterial cell walls demonstrate increased integrin mRNA expression and enhanced adhesion. To determine whether this augmented adhesion was causal in the development of synovial pathology, peptides synthesized from several fibronectin domains which inhibited leukocyte adhesion in vitro were administered to arthritic animals either as free peptides or coupled to a carrier molecule. Not only were peptides containing either the RGD or CS-1 cell-binding domains inhibitory to chronic synovial pathology (articular index = 10.5 +/- 0.3 for untreated animals compared to 1.25 +/- 0.25 for RGD and 2.5 +/- 0.7 for CS-1), but three peptides synthesized from the carboxy-terminal 33-kD heparin-binding domain of fibronectin were also found to significantly inhibit leukocyte recruitment and the evolution of arthritis. Based on these data, which are the first to explore the therapeutic potential of heparin-binding fibronectin peptides in chronic inflammation, it appears that antagonism of cellular adhesion and recruitment by fibronectin peptides may provide an important mechanism for modulating the multi-step adhesion process and attenuating aberrant inflammatory responses.

Synthetic fibronectin peptides interrupt inflammatory cell infiltration in transforming growth factor beta 1 knockout mice.

Pronounced mononuclear leukocyte (MNL) infiltration occurs in multiple organs of mice homozygous for a transforming growth factor beta 1 (TGF-beta 1) loss-of-function gene mutation [TGF-beta 1 (-/-)], followed by cachexia and eventually death. Consistent with the increased leukocyte adhesion and tissue infiltration, MNLs isolated from spleen, thymus, and peripheral blood of symptomatic TGF-beta 1 (-/-) mice, as compared to littermate controls, exhibited increased adhesion to extracellular matrix proteins and to endothelial cells in vitro. Incubation of TGF-beta 1 (-/-) MNLs with selected synthetic peptides corresponding to cell- and heparin-binding sequences of fibronectin (FN) significantly attenuated adhesion of these cells not only to FN but also to endothelial cells in vitro. Based on these observations, mice were treated with the FN peptides in an attempt to rescue them from tissue inflammation and cardiopulmonary failure. Daily injections of a combination of four synthetic FN peptides that interact with beta 1-integrins and/or cell surface proteoglycans blocked the massive infiltration of MNLs into the heart and lungs of TGF-beta 1 (-/-) mice. Peptide treatment initiated on day 8, coincident with the first evidence of increased leukocyte-endothelial cell interactions, not only blocked tissue infiltration but also moderated the lethal wasting syndrome.

An in situ protocol for measuring the expression of chemically-induced mutations in mammalian cells.

The generation of expression curves and the evaluation of mutagenic responses of mammalian cells using standard mutagenesis assays can be inaccurate because mutant and wild-type cells are usually mixed during the expression phase. If some mutant progenitors or mutants grow more slowly than the wild-type cells during the expression period, there will be a decrease in the mutant to wild-type ratio with time and the mutant fraction will not accurately represent the number of mutational events that occurred. The mutant fraction may also inaccurately assess the number of mutations if these mutations are expressed over a number of generations during the time before selection. We previously showed that recovery of L5178Y mouse cell mutants is not complete when mutations are allowed to express in suspension because slowly growing mutants and/or mutant progenitors are diluted out during this time (Rudd et al., 1990). In order to more accurately quantitate the mutagenic response of the cells, we developed an in situ procedure which segregates and immobilizes cells during expression. Because of this immobilization, slowly growing mutant progenitors and mutants expressed at different times will have an equal probability of being scored as mutants. Thus, one mutation leads to one mutant colony and the measurement of the mutagenic response of the cells to the chemical accurately reflects the mutational events that occurred. We plated L5178Y tk+/- mouse cells in semisolid medium immediately after treatment. As the cells grew and formed microcolonies, the selective agent TFT was added as an overlay at specified times, permitting only TFTr cells to survive. In this procedure, each mutation was captured as an individual colony; consequently, the measured mutation fraction accurately reflected the mutational events that occurred at the selected locus. In addition, the induced mutant colonies arising in the agar are the result of independent mutational events. We previously described the in situ protocol for L5178Y cells and showed that the spontaneous mutation rate measured was 50-fold greater than when the cells expressed the phenotype in suspension (Rudd et al., 1990). From this we concluded that the slow growth phenotype was expressed before TFT resistance. In the present paper, we evaluate the effect of chemical treatment on the mutation fraction as a function of the time to TFT addition. Using the in situ protocol, we generated expression curves for three nucleotide analogs, 5-azacytidine, TFT and AraC. The numbers of TFTr colonies produced at various times after treatment indicated that chemically-treated cultures had higher mutation fractions than the solvent controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Monitoring of intracellular free calcium in perfused rat liver.

Fluorescent calcium indicators have been widely used to assess cytoplasmic calcium concentration in cells. To examine the role of calcium ions on different physiological functions (e.g. in case of liver; bile secretion, glucose metabolism, etc.) there is a need for whole organ studies. We have developed a technique to estimate intracellular free calcium changes in perfused rat liver. Krebs-Henseleit perfused livers were loaded with 7 microM or 35 microM Indo-1/AM. An area 3 mm in diameter and approximately 300 microns in depth was illuminated at 340 nm. Fluorescence was monitored with photomultiplier tubes at 3 wavelengths (400 nm for Ca-bound dye, 504 nm for free dye and 464 nm for NADH). The viability of liver preparations was assessed by measurement of the concentrations of lactate dehydrogenase and alanine aminotransferase in the effluent. Loading of the livers with 7 microM Indo-1/AM via the portal vein resulted in a 5-fold increase of fluorescence at 400 nm. However the dye 'leaked' out of the liver with a half-time of 18 min. Probenecid (a specific anion carrier blocker) inhibited loss of dye in a dose dependent fashion (2.5-10 mM). Transient calcium elevations were observed in response to vasopressin (5-50 nM) at physiological levels, ethanol (0.3-0.8 M) and the calcium ionophore, ionomycin. Certain limitations were apparent with this approach: (1) it was necessary to use an anion carrier blocker to maintain a relatively steady dye concentration; (2) endogenous NADH fluorescence interfered with the calcium signal; and (3) absolute values of calcium concentration could not be determined.

Regulation of hepatic glycogenolysis and vasoconstriction during antigen-induced anaphylaxis.

Effects of sensitizing antigen (ovalbumin) on various physiological and hepatic parameters were investigated in sensitized rats and isolated perfused livers derived from sensitized rats. Administration of ovalbumin (500 micrograms) to the portal venous circulation of sensitized but not nonsensitized rats resulted in a rapid and sustained decrease in systemic arterial pressure, characteristic of antigen-induced anaphylaxis, and pronounced increases in hepatic portal pressure and blood glucose concentration. These antigen-mediated alterations were similar to those observed in response to platelet-activating factor (PAF) (0.1 micrograms/kg) administration to rats and were inhibited significantly by specific PAF receptor antagonist WEB 2086 (250 micrograms/kg). Infusion of ovalbumin (3.8 micrograms/ml) into isolated perfused livers derived from sensitized rats resulted in significant increases in hepatic glucose output and portal pressure and decreases in oxygen consumption, as observed in response to PAF (0.28 nM) infusion into perfused livers. These hepatic responses to ovalbumin were antigen specific and were not observed in nonsensitized rat perfused livers. Hemodynamic and glycogenolytic responses to ovalbumin in perfused livers were inhibited significantly but less effectively than similar responses to PAF by infusion of WEB 2086 (500 nM) into livers. Coinfusion of indomethacin (2.8 microM) and nordihydroguariatic acid (1 microM) with WEB 2086 (500 nM) into perfused livers inhibited further hemodynamic but not glycogenolytic responses to ovalbumin. Infusion of nitric oxide (34 microM) into sensitized rat perfused livers prevented the hemodynamic and glycogenolytic responses to both ovalbumin and PAF. These observations provide evidence that hepatic glycogenolysis and vasoconstriction are stimulated during antigen-induced anaphylaxis and suggest that these responses are mediated in part by PAF.

Significance of an opiate mechanism in the adjustment of cerebrocortical oxygen consumption and blood flow during hypercapnic stress.

The role of adrenal medulla-derived enkephalins in the control of hypercapnic cerebrocortical blood flow (CBF) and oxygen consumption (CMRO2) was investigated in the ketamine anesthetized rat. Three experimental interventions were utilized: inhibition of opioid receptors with naloxone, decrease of adrenal enkephalin production with chronic adrenal medullectomy, and treatment of adrenal demedullated animals with the synthetic enkephalin analog, D-Ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAGO). In intact, untreated animals hypercapnia increased CBF and CMRO2 by approximately 300 and 35%, respectively. Naloxone reduced the hypercapnic increase of CBF, and transformed the hypercapnic increase of CMRO2 into a decrease. The mid-points of the dose-response curves for (1)-naloxone and (d)-naloxone were 10 micrograms/kg and 100 micrograms/kg, respectively. Adrenal demedullation and treatment with (1)-naloxone (0.2 mg/kg) decreased the hypercapnic CBF and CMRO2 by approximately 50%. DAGO treatment of adrenal demedullated animals restored the hypercapnic CBF and CMRO2 to values similar to those found in intact animals. These observations suggest that opioid peptides (most likely adrenal medulla-derived enkephalins) play a significant role in the regulation of CMRO2 and CBF during moderate hypercapnia.