Constitutive and mitogen-stimulated cytokine mRNA expression by peripheral blood mononuclear cells from most autologous and allogeneic bone marrow transplant recipients is intact.

The immunodeficiency that occurs after human bone marrow transplantation (BMT) leaves BMT recipients susceptible to fatal infections. Although cytokines are critical for coordinating immune responses and immune reconstitution after BMT, there are still gaps in our knowledge about the expression of mRNA for cytokines in peripheral blood mononuclear cells (PBMC) after BMT. Therefore, we systematically studied cytokine gene expression by PBMC from 11 allogeneic and four autologous BMT recipients from 111 to 837 days after BMT and compared the results with PBMC from seven normal controls tested in parallel. PBMC were examined for mRNA expression for IL-2r alpha, IL-2, IL-3, IL-4, IL-6, and IL-7 using reverse transcription polymerase chain reaction (RT/PCR). PBMC from 11 allogeneic recipients constitutively expressed mRNA for IL-2r alpha in 2 of 11 and IL-2 in 1 of 9 samples tested whereas the same PBMC constitutively expressed mRNA for IL-3 in 8 of 11, IL-4 in 3 of 7, IL-6 in 6 of 7 and IL-7 in 3 of 6 samples tested. After PHA/PMA stimulation, PBMC from the same recipients frequently expressed mRNA for IL-2r alpha in 9 of 11, IL-2 in 8 of 9, IL-4 in 3 of 7 and IL-6 in 7 of 7. PBMC from four autologous recipients (two short-term and two long-term) frequently constitutively expressed mRNA for IL-2r alpha (3 of 4) IL-2 (3 of 4), and IL-3 (4 of 4). Stimulation of PBMC from the autologous recipients did not alter cytokine expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Coactivation with anti-CD28 monoclonal antibody enhances anti-CD3 monoclonal antibody-induced proliferation and IL-2 synthesis in T cells from autologous bone marrow transplant recipients.

Recent studies show that costimulation of T cells with anti-CD28 Mab (anti-CD28) enhances anti-CD3 Mab (anti-CD3)-induced proliferative responses and cytokine production. This study determines if coactivation with anti-CD3 and anti-CD28 corrects defects in proliferation and IL-2 secretion in peripheral blood lymphocytes (PBL) from bone marrow transplant (BMT) recipients. PBL or T cells from 5 of 16 autologous and 5 of 22 allogeneic recipients increased their anti-CD3-induced proliferation responses by > 50% after coactivation. In short-term (< 180 days after BMT) autologous recipients, the group mean response increased after anti-CD3 activation from 62,900 to 97,800 cpm after coactivation. In long-term (> 180 day after BMT) autologous recipients, the group mean response after anti-CD3 activation increased from 62,600 to 78,400 cpm after coactivation. The long-term autologous recipient group had costimulated responses from PBL that were significantly higher than the paired anti-CD3-induced responses (p < 0.01); in contrast, such differences were not seen in allogeneic recipient groups. After anti-CD3 stimulation, the mean response of 88,000 cpm for PBL from short-term allogeneic recipients and the mean response of 83,600 cpm for PBL from long-term allogeneic recipients were higher than those in PBL from autologous recipients were higher than those in PBL from autologous recipient groups. The amount of IL-2 secreted by T cells from three autologous and three allogeneic recipients was enhanced 0.9-25-fold by coactivation. Coactivation of PBL from selected recipients increased proliferation into the normal range and increased IL-2 secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Preclinical studies for adoptive immunotherapy in bone marrow transplantation. Generation of anti-CD3 activated cytotoxic T cells from normal donors and autologous bone marrow transplant candidates.

In order to obtain T cells for adoptive immunotherapy after autologous bone marrow transplantation (ABMT) for patients with resistant hematological malignancies, a culture system was developed for growing T cells and inducing non-MHC-restricted cytotoxicity using anti-CD3 monoclonal antibody (OKT3) activation. In this investigation, we show that (1) peripheral blood lymphocytes or bone marrow mononuclear cells (BMMNC) from normal donors and cancer patients can be activated with OKT3 and grown in interleukin-2; (2) normal BMMNC activated with OKT3/IL-2 exhibited non-MHC-restricted cytotoxicity and surface markers comparable to that exhibited by normal PBL activated with OKT3/IL-2; (3) both proliferation and cytotoxic functions were IL-2-dependent; (4) PBL activated with OKT3/IL-2 after cryogenic storage grew and killed comparable to PBL activated with OKT3/IL-2 prior to cryopreservation; (5) OKT3/IL-2-activated PBL and BMMNC obtained from 5 patients with non-Hodgkin's lymphomas (NHL) and 1 patient with acute myelogenous leukemia (AML) increased cell numbers 41-75-fold in 2 weeks of culture; 5 of 6 patients with NHL or AML had PBL and BMMNC that exhibited cytotoxic activity; and (6) contaminating leukemic cells did not overgrow in OKT3/IL-2-activated cultures and could no longer be detected on cytospin specimens after 3 weeks of culture. These data show that T cells in PBL or BMMNC from ABMT candidates can be activated with OKT3/IL-2 for adoptive immunotherapy in combination with ABMT.

End-tidal CO2 and tissue pH in the monitoring of acid-base changes: a composite technique for continuous, minimally invasive monitoring.

The infrared CO2 analyzer continuously monitors the CO2 tension in exhaled air at end-tidal expiration. In experimental animals, we found a consistent relationship between PaCO2 and end-tidal CO2 (ET.CO2) in the normal steady state, and in acid-base disturbances (respiratory acidosis and alkalosis, and hypoperfusion acidosis). Paired data analyses of PaCO2 (X) and ET.CO2 (Y) yielded correlation coefficients of r = 0.98 (Y = 0.96X + 4.43) during progressive hypercarbia (PaCO2: 32----110 torr), and r = 0.93 (Y = 0.89X + 0.93) during hyperventilation hypocapnia (PaCO2: 41----14 torr). The relationship between PaCO2 and ET.CO2 was seen during hypovolemic shock if pulmonary perfusion was maintained uniform in all areas of lung. The ability of the ET.CO2 sensor to predict instantaneously the PaCO2 makes it attractive enough to be used in conjunction with the subcutaneous tissue pH(pHe) sensor in the management of acid-base disturbances. After hypercarbia (FiCO2 0.15 X 40 min; PaCO2/ET.CO2: 100/101 torr), when the dogs were returned to room air, abruptly both the ET.CO2 and pHe sensors were sensitive to the changes in Fi.CO2. But the response of the ET.CO2 was swifter. The advent of transcutaneous gas monitors has shown that intermittent blood gas analyses, however frequent, are inadequate for the monitoring of the rapidly altering blood gas status in the acutely ill. The ability of the pHe sensor to identify whole-body acidosis and alkalosis combined with the speed and ease of the ET.CO2 monitor in pinpointing hypercarbic and hypocarbic states makes this two-parameter system suitable for the continuous, noninvasive monitoring of the critically ill.

Continuous monitoring of pH in the tissue mode: evaluation of a miniature sensor during acidosis and tissue hypoperfusion.

The in vivo performance of a 20G copolymer pH sensor, needlelike in configuration, was studied in the normal dog, and dogs made acidotic by the constant infusion of lactic acid, or by the induction of tissue perfusion defects. Sensors were placed at two extravascular sites in the leg, deep subcutaneous (pHe/sc), and intramuscular in the adductor (pHe/im). This pH sensor is a silver wire capped by a H+-specific polymer; it has a built-in reference system. Its electrochemical characteristics and in vivo performance are similar to those of glass pH electrodes. The continuously monitored values were compared with discrete arterial blood gas analyses at 10 to 20 minute intervals. The baseline values in 15 dogs under general anesthesia were: pH/art 7.331 +/- .042, pHe/sc 7.291 +/- .076, and pHe/im 7.265 +/- .102 (mean +/- SD; n = 45 observations each). During metabolic acidosis (lactic acid infusion), the direction and rates of change were similar in pHe/sc and pHe/im. Tissue perfusion defects were induced by moderate-to-severe hemorrhage (single or repeated bleeds) or operative shock (splenectomy and exteriorization of bowel). Both pHe/sc and pHe/im fell sharply, with a more gradual drop in pH/art. In those who survived after infusion of shed blood or dextran-40, pHe recovered rapidly. In the moribund, pHe continued to deteriorate. This pH sensor is a sensitive prognosticator of acid-base changes in the tissue. The in vivo drift is small: 0.008 pH per hour. The placement of the sensor via an intracath cannula in the subcutaneous tissue of the thigh is recommended.(ABSTRACT TRUNCATED AT 250 WORDS)

Postnatal development of lipid-clearing enzymes in the suckling animal.

Age-related changes in the enzyme activities in the storage and secretory pools of extrahepatic lipoprotein lipase and hepatic triglyceride lipase were determined during a primed/constant infusion of heparin for 2 hr in puppies between birth and 18 wk of life. Lipoprotein lipase activity was low in the first 4 wk of suckling. Its storage pool increased 6-fold in the next 14 wk, with a less dramatic rise in the secretory pool. Sustained increases in the activity of this enzyme were seen late when (1) the pup was being weaned, and (2) the insulin-dependent glucoregulatory mechanism had matured. Both phases of hepatic triglyceride lipase were well developed at birth reflecting the metabolic maturity of the liver at birth. The ability to clear intravenous lipid during a 2-hr infusion was also measured in the pups. The rate of Intralipid utilization increased from 198 +/- 43 mg/kg/hr in the first week of life to 424 +/- 20 mg/kg/hr at 12 wk. Blood Intralipid homeostasis was causally related to increased activity in the storage and secretory pools of lipoprotein lipase. The progressive increase in lipoprotein lipase activity and the maintenance of the secretory capacity was dependent on the growth of the muscle mass and its capillary bed. Endogenous insulin rather than the fat content of the feed appeared significant in the postnatal development of the heparin-releasable lipid clearing enzymes.

The storage and synthetic pools of heparin-releasable lipoprotein lipase and hepatic triacylglycerol lipase in the growing puppy.

Age-related changes in the activities of extrahepatic lipoprotein lipase and hepatic triacylglycerol lipase were determined during a primed/constant-rate infusion of heparin for 2 h in puppies between birth and 18 weeks of age. The early (storage) and late (synthetic) phases were measured. Both phases of hepatic triacylglycerol lipase activity were well developed in the first week, reflecting the metabolic maturity of the liver at birth. During the 18 weeks of study, the activity remained relatively unchanged except for a sharp peak at 12 weeks. Extrahepatic lipoprotein lipase activity was low in the first 4 weeks of suckling. Its storage pool increased 6-fold in the next 14 weeks, with a less marked rise in its late (synthetic) pool. Sustained increases in the activity of this enzyme were first noticed during weaning, when the insulin-secretory response matured. Endogenous insulin-secretory capacity rather than the fat content of the feed appeared significant in the postnatal development of lipoprotein lipase (Clearing-factor lipase) activity.

Depression of glucose utilization by Intralipid in the post-traumatic period: an experimental study.

We investigated the accelerated clearance of Intralipid (IL) in the immediate post-traumatic period and the influence of the concomitant rise in fatty acid ((FFA) metabolism on carbohydrate tolerance. As a result we postulate that intermediary products of fatty acid oxidation inhibit key enzymes in the glycolytic pathway. The fatty acidemia and its metabolic sequlae can be avoided by intermittent Intralipid supplementation (at low rates) during TPN. It will assure (1) a larger carbohydrate-to-fat caloric ratio during infusion and (2) cyclical regeneration of the enzyme systems involved in lipid metabolism.