Proteolipid protein is necessary in peripheral as well as central myelin.

Alternative products of the proteolipid protein gene (PLP), proteolipid protein (PLP) and DM20, are major components of compact myelin in the central nervous system, but quantitatively minor constituents of Schwann cells. A family with a null allele of PLP has a less severe CNS phenotype than those with other types of PLP mutations. Moreover, individuals with PLP null mutations have a demyelinating peripheral neuropathy, not seen with other PLP mutations of humans or animals. Direct analysis of normal peripheral nerve demonstrates that PLP is localized to compact myelin. This and the clinical and pathologic observations of the PLP null phenotype indicate that PLP/DM20 is necessary for proper myelin function both in the central and peripheral nervous systems.

Efficacy of treatment with antisense oligonucleotides complementary to immunoglobulin sequences of bcl-2/immunoglobulin fusion transcript in a t(14;18) human lymphoma-scid mouse model.

In t(14;18)-positive lymphoma cells, bcl-2 is expressed from a fusion mRNA transcript containing the full coding sequence of bcl-2 and 3' immunoglobulin sequences. We reported previously that antisense oligodeoxyribonucleotides directed at the bcl-2 translational start site, as well as those targeted to immunoglobulin sequences 3' of the translocation breakpoint, down-regulate bcl-2 and inhibit growth of the t(14;18)-positive lymphoma line WSU-FSCCL in vitro. We have developed a scid mouse model with this human cell line and demonstrate that antisense oligodeoxyribonucleotides targeted to immunoglobulin c(mu) sequences down-regulate bcl-2 protein expression and induce apoptosis of WSU-FSCCL cells in vivo. This leads to prolonged survival of the mice. Targeting non-oncogenic sequences outside of the breakpoints of fusion transcripts may be a clinically useful therapeutic strategy.

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.

Duplications and de novo deletions of the SMNt gene demonstrated by fluorescence-based carrier testing for spinal muscular atrophy.

Approximately 95% of individuals with spinal muscular atrophy (SMA) lack both copies of the SMNt gene at 5q13. The presence of a nearly identical centromeric homolog of the SMNt gene, SMNc, necessitates a quantitative polymerase chain reaction approach to direct carrier testing. Adapting a radioactivity-based method described previously, multiplex polymerase chain reaction was performed using fluorescently labeled primers followed by analysis on an ABI 373a DNA sequencer. The SMNt copy number was calculated from ratios of peak areas using both internal and genomic standards. Samples from 60 presumed carriers (50 parents of affected individuals and 10 relatives implicated by linkage analysis) and 40 normal control individuals were tested. Normalized results (to the mean of five or more control samples harboring two copies of the SMNt gene) were consistently within the ranges of 0.4 to 0.6 for carriers (one copy) and 0.8 to 1.2 for normal controls (two copies), without overlap. Combining linkage analyses with direct carrier test results demonstrated de novo deletions associated with crossovers, unaffected individuals carrying two SMNt gene copies on one chromosome and zero SMNt gene copies on the other chromosome, and unaffected individuals with three copies of the SMNt gene. This report demonstrates that fluorescence-based carrier testing for SMA is accurate, reproducible, and useful for genetic risk assessment, and that carrier testing may need to be combined with linkage analysis in certain circumstances.

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)

Effect of corticosterone on serotonin and catecholamine receptors and uptake sites in rat frontal cortex.

The effects of corticosterone (1 mg/kg per day for 7 days) on serotonin 5-HT1A, 5-HT2A, 5-HT uptake sites, and alpha 2-adrenergic receptor sites were measured. Corticosterone treatment significantly decreased the number of 5-HT1A receptor sites (Bmax = 108 +/- 8.20 fmol/mg protein and 152.31 +/- 13.36 fmol/mg protein in corticosterone- and vehicle-treated rats, respectively). No significant differences were found in other measures. It is possible that corticosteroids exert some of their behavioral effects via regulation of 5-HT1A sites in frontal cortex.

A Novel Missense Mutation, R1283S, of the Cystic Fibrosis Transmembrane Conductance Regulator Gene in a 47-Year-Old African-American Patient.

Background: More than 600 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been described; however, at least 50% of the disease-associated mutations in the African-American population remain unknown. Reported here is a novel missense mutation, R1283S, in a 47-year-old African-American patient with mild cystic fibrosis. Methods and Results: The patient was screened for 27 common and less common CFTR mutations and 2 mutations were detected. Direct sequencing confirmed the presence of the DeltaF508 mutation and revealed the presence of a novel missense mutation, R1283S. Conclusions: R1283S appears to be a cystic fibrosis mutation associated with mild disease, and adds to the number of known mutations in African-Americans. R1283S can be confused with the more common mutation, W1282X, when polymerase chain reaction-restriction fragment length polymorphism analysis is used for detection.

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.

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.

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.

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)

Effects of intrathecal fentanyl as an adjunct to hyperbaric bupivacaine in spinal anesthesia for elective caesarean section.

Hyperbaric bupivacaine is the most common drug used in spinal anesthesia for caesarean section. The aim of this study was to compare the effects of adding fentanyl to intrathecal bupivacaine on the onset and duration of spinal anesthesia and its effect on mother and neonate. Seventy healthy parturients with singleton pregnancy scheduled for elective cesarean section were randomly allocated to receive subarachnoid block with 0.5% bupivacaine heavy 2.4 ml (Group A) or fentanyl 20 microgram (0.4 ml) added to 0.5% bupivacaine heavy 2 ml (Group B). Blood pressure, heart rate, respiratory rate, oxygen saturation, along with characteristics of spinal block were assessed throughout the surgery and in the postoperative ward until the patient requested for analgesia. It was found that duration of sensory block was prolonged in fentanyl group (p < 0.05). Duration of complete analgesia (97 ± 8.23 minutes vs 153 ± 7 minutes; p value = 0.00) and effective analgesia (134 ± 5.6 minutes vs 164 ± 9; p value = 0.00) were also found to be prolonged in Group B. There was not much difference in the occurrence of side effects in both the groups. Addition of fentanyl to intrathecal bupivacaine for cesarean section increases the duration of postoperative analgesia without increasing maternal or neonatal side effects.

Imaging anoxic depolarization during ischemia-like conditions in the mouse hemi-brain slice.

Focal ischemia evokes a sudden loss of membrane potential in neurons and glia of the ischemic core termed the anoxic depolarization (AD). In metabolically compromised regions with partial blood flow, peri-infarct depolarizations (PIDs) further drain energy reserves, promoting acute and delayed neuronal damage. Visualizing and quantifying the AD and PIDs and their acute deleterious effects are difficult in the intact animal. In the present study, we imaged intrinsic optical signals to measure changes in light transmittance in the mouse coronal hemi-brain slice during AD generation. The AD was induced by oxygen/glucose deprivation (OGD) or by ouabain exposure. Potential neuroprotective strategies using glutamate receptor antagonists or reduced temperature were tested. Eight minutes of OGD (n = 18 slices) or 4 min of 100 microM ouabain (n = 14) induced a focal increase of increased light transmittance (LT) in neocortical layers II/III that expanded concentrically to form a wave front coursing through neocortex and independently through striatum. The front was coincident with a negative voltage shift in extracellular potential. Wherever the LT front (denoting cell swelling) propagated, a decrease in LT (denoting dendritic beading) followed in its wake. In addition the evoked field potential was permanently lost, indicating neuronal damage. Glutamate receptor antagonists did not block the onset and propagation of AD or the extent of irreversible damage post-AD. Lowering temperature to 25-30 degrees C protected the tissue from OGD damage by inhibiting AD onset. This study shows that anoxic depolarization evoked by global ischemia-like conditions is a spreading process that is focally initiated at multiple sites in cortical and subcortical gray. The combined energy demands of O(2)/glucose deprivation and the AD greatly exacerbate neuronal damage. Glutamate receptor antagonists neither block the AD in the ischemic core nor, we propose, block recurrent PID arising close to the core.

Affinity isolation of neuron-reactive antibodies in MRL/lpr mice.

Autoantibodies from the MRL/lpr mice react with numerous proteins on neuronal cell surfaces. The purpose of this study was to isolate and characterize a population of autoantibodies reactive preferentially or exclusively with nervous system tissue. Using a purified plasma membrane preparation from brain cortex of balb/c mice coupled to diaminopropylamine agarose gel, we affinity-isolated antineuronal antibodies from pooled MRL/lpr immunoglobulins. The isolated immunoglobulins reacted with brain cortex plasma membranes and neuroblastoma cells (but not liver, kidney, or fibroblasts) by Western blot and indirect immunofluorescence with confocal microscopy. By Western blot, the epitopes in the brain cortex were proteins of apparent molecular weights 101, 63, 53, 43, 39, and 33, kd; the epitopes in the neuroblastoma cells were 63, 57, and 53 kd. Lectin column isolation revealed that the 101 and 63 kd epitopes were glycosylated. Indirect immunofluorescence revealed that the antibodies bound to the cell soma more intensely than to the cell processes of viable cultured neuroblastoma cells. The cell surface localization of this binding was confirmed by confocal microscopy. Within the central nervous system the antibodies bound more intensely to primary cultures of isolated neurons from fetal cortex than to hippocampal or neostriatal cells. With these antibodies we can begin studies of their potential pathogenic effects.

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.