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Biomedical subjects

J M Johnston

Publications and source records attributed to J M Johnston.

At least 19 recordsLinked to original sources
PubMed1997

Effect of dexamethasone on rat plasma platelet activating factor acetylhydrolase during the perinatal period.

It has been previously reported that the administration of dexamethasone (DEX) to adult rats increases the activity of plasma platelet-activating factor acetylhydrolase (PAF-AH) and prevents the development of intestinal necrosis caused by platelet activating factor (PAF) injection. In this report, we examined the effect of DEX administration on plasma PAF-AH activity during the perinatal period. Timed-pregnant rats received DEX (0.2-1.0 mg/kg/d) or normal saline (controls) on days 16-18 (early group) or days 18-20 (late group) of gestation. Maternal plasma PAF-AH activity was lower in late gestation than in postpartum period (P < 0.001). Fetal and neonatal plasma PAF-AH activity was higher than maternal values (P < 0.05). No changes of PAF-AH activity were seen in maternal, fetal or neonatal plasma after prenatal DEX administration at the aforementioned doses. A higher dose of DEX (1.3 mg/kg/d x 4d) or cortisone (200 mg/kg/d) produced an elevation of maternal plasma PAF-AH activity (DEX 79.2+/-3.0, cortisone 70.5+/-1.9 vs. controls 49.4+/-2.3 nmol/min/ml, P < 0.01), but resulted in a high fetal mortality. Treatment of newborn rats with DEX (0.5 mg/kg/d) on days 1-3 after birth, increased plasma PAF-AH activity on day 4 (DEX 292+/-5 versus controls 140+/-9 nmol/min/ml, P < 0.001) and day 6 (DEX 302+/-12 versus controls 136+/-6 nmol/min/ml, P < 0.001). Postnatal administration of DEX increases the plasma PAF-AH activity in the rat. Only high doses of prenatal corticosteroids that cause fetal death can elevate maternal plasma PAF-AH activity.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1997

The central role of PAF in necrotizing enterocolitis development.

We have addressed two critical questions concerning NEC development. 1) Why is the neonatal intestine particularly susceptible to necrosis? and 2) Does PAF play a critical role in NEC development? We have found that intestinal tissue of the newborn has the highest specific activity for the acetyltransferase of the de novo pathway. It is suggested that the high capacity of this tissue to synthesize PAF may contribute to the fact that the necrosis of the newborn is more prevalent in this tissue. We have previously reported that dexamethasone lowers the activity of acetyl-CoA:lyso-PAF acetyltransferase in liver and spleen. This hormone also cause an increase in plasma PAF-acetylhydrolase activity and an increased secretion of PAF-acetylhydrolase by various macrophages. It would, therefore, appear that the beneficial effects of glucocorticoids on the prevention of NEC may be due to both increased inactivation of PAF as caused by the increase in PAF-acetylhydrolase as well as a decrease in PAF synthesis. We are presently investigating the effect of glucocorticoids on acetyl-CoA: alkyl-lyso-sn-glycero-3-phosphate acetyltransferase. The reported studies in which NEC was prevented by intravenous infusion of recombinant PAF-acetylhydrolase provides further documentation as to the importance of PAF in the development of NEC. The specific activity of PAF-acetylhydrolase required for protection by dexamethasone was similar. This finding would be suggestive of the fact that the mechanisms by which dexamethasone causes a complete protection against NEC may be mediated by increasing the plasma activity. Other mechanisms have been proposed such as facilitating the maturation of the small bowel. As discussed, other factors such as hypoxia, endotoxins, TNF alpha, and enternal feeding have been suggested to be contributing agents of NEC development. Many of these factors and procedures are known to increase in PAF. We have suggested a mechanism to explain the increase in PAF formation as caused LPS, TNF alpha, and interleukins being the inhibition of the secretion of PAF-AH by macrophages. Our previous reports on the mechanisms involve in the prevention of NEC by glucocorticoids and the reported findings that human recombinant PAF-acetylhydrolase can prevent NEC provide further support for a central role for PAF in NEC development. Furthermore, the presence of a high PAF biosynthetic activity in the neonatal intestine affords an explanation as to why this tissue is highly susceptible to this disease.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1997

Metabolism of platelet-activating factor in rat epididymal spermatozoa.

A role for platelet-activating factor (PAF) in sperm function has been proposed. In the present investigation the metabolism of PAF was examined in sperm and epididymal tissue. The major regulatory enzymes for the synthesis of PAF via the de novo pathway have been established in spermatozoa; these include acetyltransferase and cholinephosphotransferase specific for PAF biosynthesis. De novo acetyltransferase activity for PAF biosynthesis in spermatozoa was significantly higher than that of the acetyltransferase of the remodeling pathway. A metabolic pathway was described for the catabolism of PAF in sperm, involving PAF-acetylhydrolase (-AH), lysophospholipase D, and a phosphohydrolase. An isozyme of PAF-AH similar to that reported in sera was also demonstrated in epididymal fluid and tissue. This isozyme was distinctly different from that found in the spermatozoa. The partial inactivation of PAF-AH by the vaginal pH, and/or its detachment from sperm during migration to the site of fertilization, may allow increased motility and migration to the site of fertilization. It is suggested that a decapacitation factor previously described may be related to PAF-AH.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1997

Platelet-activating factor (PAF) and PAF acetylhydrolase activity in rat uterus and placenta during the late stages of pregnancy.

We evaluated the roles of platelet-activating factor (PAF) and PAF-acetylhydrolase (PAF-AH) activity in late pregnancy. Uterine and placental concentrations of PAF were determined by the washed rabbit platelet aggregation bioassay. Uterine, placental, and plasma PAF-AH activities were also assayed. PAF concentration in the uterus increased 4-fold between Days 15 and 21 of pregnancy. PAF was also determined in the placenta on Days 15 and 21. In contrast to findings in the uterus, the concentration in the placenta was decreased by 75%. Platelet aggregation caused by uterine and placental PAF was inhibited by the PAF receptor antagonists CV-3988 and TCV-309. Plasma and uterine PAF-AH activities decreased significantly between Days 15 and 21. In contrast, the placental PAF-AH activity significantly increased during this same time period. On the basis of these findings, it is suggested that the PAF concentration in the uterus and placenta may be regulated by intracellular PAF-AH and/or plasma PAF-AH activities. Increased PAF activity in the pregnant rat uterus may be related to the initiation of labor due to its known effect on myometrial contraction. Decreased PAF concentration in the placenta may contribute to the fetoplacental circulation due to its known hypotensive activity and the increase in vascular permeability.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1997

Subcutaneous administration of CAMPATH-1H: clinical and biological outcomes.

OBJECTIVE: A 24 week study of subcutaneous (sq) dosing with titration of CAMPATH-1H (C1H) dose against the circulating CD4+ T cell count in patients with rheumatoid arthritis (RA) was undertaken to examine the safety, biologic activity, and clinical efficacy of this approach. METHODS: All patients met American Rheumatism Association (ARA) criteria for active RA. Patients received either 0.5 or 1.0 mg of C1H subcutaneously twice per week; dosing could be doubled after the first 8 weeks of treatment and subsequently following 4 week dose intervals for lack of clinical efficacy, but was discontinued any time the CD4+ T cell count fell below 400/mm3. Patients were evaluated weekly for 2 weeks and then biweekly for clinical and laboratory variables of safety, biological activity, and disease activity. RESULTS: Ten patients were treated, 6 in the 0.5 mg cohort and 4 in the 1.0 mg cohort. Four of ten patients had a 20% modified Paulus response (2 in each cohort) while taking drug; there were minimal side effects, primarily limited to local reaction at the injection site. All patients had a > 50% drop in circulating CD4+ T cells within the first 2 weeks of therapy, with no further significant reduction; only 1/6 patients in the 0.5 mg cohort had dose limiting CD4+ T cell depression vs 2/4 in the 1.0 mg cohort. All patients developed antibodies to C1H. Appearance of anti-C1H was temporarily associated with a halt in further reduction of CD4+ T cell count despite continued C1H administration. CONCLUSION: Subcutaneous administration of C1H in low doses (0.5 mg biweekly) was well tolerated and did not result in dose limiting CD4+ T cell depletion in 5 of 6 patients. Clinical efficacy was observed in some patients but could not be maintained, possibly due to the production of anti-C1H antibodies.

Alemtuzumab↗
PubMed1997

Determinants of plasma platelet-activating factor acetylhydrolase: heritability and relationship to plasma lipoproteins.

Plasma platelet-activating factor acetylhydrolase (PAF-AH) is the enzyme that inactivates PAF (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine). We determined the relative contributions of genetic and environmental factors to variation in plasma PAF-AH activity in 240 individuals from 60 nuclear families. Regression of mean-offspring PAF-AH activity on the mid-parent value indicated that 62% of the variation in plasma PAF-AH activity was heritable. Spousal values were weakly negatively correlated, indicating that familial aggregation of PAF-AH activity is due to genetic rather than to environmental factors. Among normolipidemic individuals, plasma PAF-AH activity was strongly correlated with the plasma concentration of low density lipoprotein cholesterol (LDL-C), and treatment with lovastatin resulted in proportional decreases in plasma PAF-AH activity and LDL-C concentrations. To further elucidate the relationship between PAF-AH and plasma concentrations of LDL, plasma PAF-AH activity was measured in families with well-defined, monogenic disorders of LDL metabolism. Plasma PAF-AH activity cosegregated with plasma LDL-C concentrations in familial hypercholesterolemia, but not in familial hypobetalipoproteinemia. We speculate that the rate of removal of LDL from the circulation may determine the clearance rate of PAF-AH, thereby modulating the activity of PAF-AH in blood.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1996

Identification and neuron specific expression of the S182/presenilin I protein in human and rodent brains.

Many individuals with familial Alzheimer disease (FAD) have mutations in a gene termed S182 or presenilin I (PS-I). Currently, the PS-I gene product has not been identified and its function remains unknown. Here we report that affinity purified antibodies against the predicted amino acid sequence of the PS-I gene product detected in homogenates of human, mouse, and rat brains a single antigen of approximately 48 kDa. This antigen was also present in immortalized human and mouse neuronal cell cultures. Brain tissue fractionation showed that all PS-I antigen was found in the membrane fraction. In stained tissue sections of mouse central nervous system (CNS), PS-I antigen was found only in neurons throughout brain and spinal cord and was located within cell bodies, axons, and dendrites. Remarkably the relative partition among these three compartments varied dramatically. A striking feature of PS-I expression was its intense concentration in some (but not all) dendrites, at levels substantially above those in the parent perikarya. In most of the cerebrum, PS-I staining in axons was very weak or undetectable. By contrast, many axons in portions of the brainstem and in the spinal cord showed marked PS-I immunoreactivity. Similarly, staining of sections from human temporal cortex showed that PS-I was present mainly in neuronal cell bodies and dendrites. These data show that in the CNS, PS-I is expressed mainly in neurons and suggests that this protein may perform a neuron specific function. The pattern of PS-I expression in the CNS would suggest that the premature neurodegeneration associated with PS-I mutations involves a primary neuronal process rather than a secondary effect of PS-I produced in non-neuronal cells.

Animals↗
PubMed1996

Analysis of hypermutation in immunoglobulin heavy chain passenger transgenes.

Somatic hypermutation of immunoglobulin (Ig) genes plays a critical role in the maturation of the human antibody response. The molecular basis of this important process is, however, unknown. To identify cis-acting sequences that initiate and target hypermutation, we have made three minitransgenes containing different portions of an Ig heavy chain (IgH) locus. Each transgene is a passenger, bearing a nonsense mutation preventing its translation; thus, transgene mutations reflect the endogenous mutational process and are not subject to affinity selection. To study transgenes after their circulation through the compartment associated with hypermutation in vivo, we rescued B cells as hybridomas after hyperimmunizing mice with the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). Hybridoma transgene and endogenous variable regions were amplified by polymerase chain reaction, subcloned, and sequenced. Endogenous anti-NP VDJ regions show the expected, at times extensive degree of base substitution. In mice bearing the smallest construct, which includes 2.4 kb of 5' IgH sequences, a rearranged VDJ region, the 5' matrix attachment region, and the intron enhancer, one of four evaluable hybridomas demonstrates two base substitutions in the V segment of one transgene copy. The two larger constructs include additional 3' IgH sequences (an alpha constant region and the 3' enhancer) and either the original VDJ segment or a substituted T cell receptor beta segment. Ten hybridomas derived from mice bearing these larger constructs demonstrate no evidence of targeted mutation, despite demonstrable transgene transcription in all hybridomas. In our system, mutation of a rearranged VDJ segment and surrounding promoter/enhancer regions is not increased by the juxtaposition of a constant region segment and the IgH 3' enhancer.

Animals↗
PubMed1996

Platelet-activating factor acetylhydrolase activity in human follicular fluid.

Platelet-activating factor (PAF) has been implicated in a number of reproductive processes ranging from ovulation to parturition. To examine the role of PAF in the human periovulatory processes, the PAF-acetylhydrolase (PAF-AH) activity was assayed in the follicular fluid (FF) obtained in conjunction with the in vitro fertilization and embryo transfer (IVF-ET) procedure and the activity related to oocyte maturation. The PAF-AH activity was also related to the concentrations of estradiol (E2) and progesterone (P) in FF. PAF-AH activity was significantly lower in the FFs obtained from follicles of more than 20 mm in diameter. The enzyme activity was significantly lower in the FFs of patients with a successful outcome of their pregnancies. E2 concentrations were negatively correlated with PAF-AH activities in the FFs. No correlation was found between the PAF-AH activity and concentration of P in the FF. Significantly more mature oocytes were recovered in the group who subsequently become pregnant compared to the non-pregnant group. It is suggested that PAF may be increased following follicular maturation. The increase in PAF may contribute to oocyte maturation and to the successful outcome of pregnancy following fertilization. An additional function of the increased PAF in FF may also be the stimulation of the contraction of smooth muscle in the ovary, thereby assisting the extrusion of the oocyte cumulus cell mass and signaling the completion of ovulation.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1996

The biochemical role of platelet-activating factor in reproduction.

The presence of Co-A independent transacylase activity in amnion cells and the preferential transfer of arachidonic acid to acceptor-ethanolamine plasmalogen provide a satisfactory explanation to the questions raised by the observation that arachidonate-enriched ethanolamine plasmalogen increases in amnion late in gestation without alteration in the total amount of ethanolamine glycerophospholipids. The proposed mechanism also serves as a link between the observed changes in glycerophospholipid composition and the generation of PAF. We have emphasized a role for PAF in fetal lung maturation, the initiation and maintenance of parturition, and in certain complications associated with a premature delivery. Although PAF is known to be the most potent lipid mediator yet described and its importance in reproductive biology is well documented, it is our view that these events cannot be attributed solely to PAF and in all likelihood a number of autacoids participate in these processes.

Amnion↗
PubMed1996

Changes in glycerophospholipid profile in experimental nephrotic syndrome.

We investigated changes in the glycerophospholipids in kidney tissue and its various intracellular fractions in rats with nephrotic syndrome induced by puromycin aminonucleoside. The ethanolamine plasmalogen, 1-O-alk-1'-enyl-2-acyl-GPE (EP), was increased in kidney tissue obtained from the puromycin-treated animals. A similar increase was found in the mitochondria and endoplasmic reticulum (microsomes) of this tissue. These increases were not found in the liver. Since platelet-activating factor (PAF) is known to be produced in increased amounts in inflammatory disorders, it is suggested that the higher plasmalogen found in rat kidneys during experimental nephrotic syndrome might be derived from increased levels of this autacoid. The increase in PAF may also result in the elevation of plasma PAF-acetylhydrolase (AH) activity observed in these animals.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1996

Repopulation of blood lymphocyte sub-populations in rheumatoid arthritis patients treated with the depleting humanized monoclonal antibody, CAMPATH-1H.

Patients with severe rheumatoid arthritis who had failed treatment with conventional therapies were treated with a course of five or 10 daily intravenous infusions of CAMPATH-1H, a humanized antibody against the CD52 antigen, resulting in profound depletion of peripheral blood mononuclear cells. During the subsequent 18 months, lymphocytes were analysed for sub-populations by fluorescence-activated cell sorter (FACS) and for proliferation in response to polyclonal T-cell stimulation with anti-CD3 or staphylococcal enterotoxin B (SEB). Treatment resulted in almost complete depletion of lymphocytes from the blood followed by gradual repopulation. CD16+ natural killer (NK) cells and CD14+ monocytes returned to pretreatment levels within 1-2 months. CD19+ B cells returned to within 50% of pre-treatment levels by day 66 and to within normal range by day 150, whereas CD8+ T cells recovered to 50% of pretreatment levels by day 66, but did not show any further increase during the rest of the study period. The most profound effects were on the CD4+ T lymphocyte sub-population, as the mean CD4+ count did not increase above 20% of pre-treatment level at any time during the study period (550 days), at all the doses tested. The T cells which initially repopulated the blood 1-2 months after treatment, nearly all expressed the activation markers human leucocyte antigen (HLA)-DR and CD45RO, although the percentage of T cells expressing these molecules gradually declined to normal levels over time. Proliferative responses to polyclonal T-cell stimulation (anti-CD3 and SEB) were also significantly reduced in the first few months after treatment, but recovered to pre-treatment levels by day 250. The relationship between these observations and the clinical response is discussed.

Antibodies, Monoclonal↗
PubMed1996

CAMPATH-1H in rheumatoid arthritis--an intravenous dose-ranging study.

Forty-one patients with active and refractory rheumatoid arthritis(RA) received a total of 100, 250 or 400 mg of CAMPATH-1H (CAMPATH is a trademark of Glaxo-Wellcome group companies, registered in the US Patent and Trademark Office) over 5 or 10 days in an open, uncontrolled study. Following therapy, patients were monitored for adverse effects and disease activity for 6 months. Therapy was associated with prolonged peripheral blood lymphopenia in all dosing cohorts. During the month immediately following therapy, lymphopenia was most profound in the 400 mg cohorts. The first dose of monoclonal antibody (Mab) was associated with a 'flu'-like syndrome, more pronounced at higher initial doses. One patient developed haemolytic-uraemic syndrome. There were a number of dose-related infections during the early post-treatment period and one fatal opportunistic infection which followed additional immunosuppressive therapy. Antiglobulin responses developed in 9 of 31 patients tested. The majority of patients showed symptomatic improvement following therapy and 20% of patients maintained a 50% Paulus response at 6 months, all of whom were in the 250 or 400 mg cohorts. CAMPATH-1H appears to be an effective treatment for RA. Allowing for the small number of patients treated, infections were more common with higher doses, although this was not true for adverse events overall, and therapeutic responses were more sustained at higher dosing levels. The broad specificity of CAMPATH-1H may be appropriate for the immunotherapy of RA and future studies should aim to define a dose with an optimal therapeutic ratio.

Alemtuzumab↗
PubMed1996

Effect of estrogens on plasma platelet-activating factor acetylhydrolase and the timing of parturition in the rat.

In the present study we examined the effects of estrogens on plasma platelet-activating factor acetylhydrolase (PAF-AH) activity in pregnant rats in an attempt to further define the role of platelet-activating factor (PAF) in parturition. The plasma PAF-AH activity decreased starting on Day 13 of pregnancy and reached its lowest level by Day 19 (36.5 +/- 5.7 nmol.min-1.ml-1 plasma, mean +/- SD). The maternal plasma PAF-AH activity rapidly increased to 67.3 +/- 3.5 nmol.min-1.ml-1 plasma by Day 2 postpartum, to 88.8 +/- 14.4 by Day 7, and to 109.1 +/- 4.4 by Day 21, returning to nonpregnant levels by Day 35 in the dams that were allowed to nurse through Day 21. The PAF-AH activity did not increase in dams from which the pups were removed 2 days after delivery. When 17 alpha-ethynylestradiol (0.25 mg/kg) was administered s.c. to pregnant rats for 3 days starting at Day 17 of pregnancy, the plasma PAF-AH activity in the animals decreased by 80% on Day 19, and the rats delivered prematurely on Day 21. When a similar dose of 17 alpha-ethynylestradiol was injected into the pregnant rats for 3 days starting on Day 15, the enzyme activity again decreased by approximately 80% on Day 17 and Day 19 of pregnancy; however, it returned to a value similar to that seen in the controls by Day 21, and the time at which the treated rats delivered was similar to that for the control animals. These observations provide further support for a role of PAF and PAF-AH and their possible regulation by estrogens in the initiation and maintenance of parturition.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1996

The absence of ongoing immunoglobulin gene hypermutation suggests a distinct mechanism for c-myc mutation in endemic Burkitt's lymphoma.

PURPOSE: Burkitt's lymphoma is a malignancy of mature, immunoglobulin (Ig)-bearing B cells characterized by translocation between c-myc and Ig gene loci. A role for the juxtaposed Ig genes in the mutation and deregulation of c-myc expression typical of endemic Burkitt's lymphoma (eBL) has been proposed, but never proven. Our objective was to determine whether Ig gene hypermutation is ongoing in eBL. METHODS: We isolated Ig heavy-chain sequences from K962 eBL tumor cells using reverse transcription and polymerase chain reaction (PCR) amplification. The PCR product was ligated into Bluescript II vectors. Multiple subclones were sequenced and the variable regions were compared for evidence of ongoing Ig hypermutation. RESULTS: Six total single base substitutions were observed within four of the nine subclones studied. Four substitutions resulted in amino acid changes and two were silent. There was no clustering of mutations in hypervariable regions, or a high incidence of amino acid replacement or link substitutions, all of which are characteristic of Ig hypermutation. The observed mutations occurred at a rate consistent with Taq polymerase error. CONCLUSIONS: Our data indicate that in the eBL tumor sample K962, the mechanism underlying c-myc mutation is distinct from that which gives rise to Ig hypermutation.

Base Sequence↗