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Biomedical subjects

J M Thomas

Publications and source records attributed to J M Thomas.

At least 181 records · Page 10Linked to original sources
PubMed1995

Phosphorylation of cyclic AMP response element-binding protein and induction of c-fos gene expression on withdrawal from chronic treatment with carbachol in NG108-15 cells.

Alterations in adenylyl cyclase activity in cultured cells after prolonged exposure to drugs such as morphine have been extensively studied as models for drug tolerance and withdrawal. NG108-15 cells develop increased intracellular cAMP concentrations after abrupt withdrawal from chronic treatment with the muscarinic cholinergic agonist carbachol. To determine whether this withdrawal-induced increase in cAMP modifies gene expression, we studied phosphorylation of the cAMP response element-binding protein (CREB) and expression of the c-fos gene, known to contain a cAMP response element, in NG108-15 cells after abrupt withdrawal from chronic treatment with carbachol. Prostaglandin E1, which activates adenylyl cyclase, caused concentration-dependent increases in the phosphorylation of CREB and in the abundance of c-fos mRNA. These changes occurred with small increments in cAMP accumulation. In cells treated with carbachol for 48 hr, induction of withdrawal with the muscarinic antagonist atropine led to a small increase in intracellular cAMP concentration but an 11.6-fold increase in the phosphorylation of CREB and a 3.4-fold increase in accumulation of c-fos mRNA. The adenylyl cyclase inhibitor 2',5'-dideoxyadenosine, which attenuated the chronic carbachol-induced increase in cAMP concentration, prevented the increased phosphorylation of CREB and the enhanced accumulation of c-fos mRNA during atropine-induced withdrawal. These results indicate that expression of the c-fos gene is induced by the small increments in cAMP concentration that can occur in cells on withdrawal from chronic treatment with drugs such as muscarinic agonists.

Alprostadil↗
PubMed1995

A gene transfer method for the study of chimerism derived from donor bone marrow in organ transplant recipients.

Transduction of DBMC with the neo gene did not negatively affect the viability of DBMC or the graft-prolonging effect of DBMC infusion and provided a sensitive method to monitor DBMC-derived chimerism at a microchimeric level for at least 6 months by molecular and immunohistochemical techniques. Selection of neo-transduced DBMC with G418 prior to infusion will enable more uniform neo expression and will provide novel opportunities to further investigate chimerism in primates.

Animals↗
PubMed1995

A gene transfer method for the study of chimerism derived from donor bone marrow in organ transplant recipients.

Transduction of DBMC with the neo gene did not negatively affect the viability of DBMC or the graft-prolonging effect of DBMC infusion and provided a sensitive method to monitor DBMC-derived chimerism at a microchimeric level for at least 6 months by molecular and immunohistochemical techniques. Selection of neo-transduced DBMC with G418 prior to infusion will enable more uniform neo expression and will provide novel opportunities to further investigate chimerism in primates.

Animals↗
PubMed1994

Assessment of donor bone marrow cell-derived chimerism in transplantation tolerance using transgenic mice.

Allospecific skin graft prolongation can be induced in mice using antithymocyte globulin and allospecific donor bone marrow cells (DBMC). This enhancement may be due to the persistence of chimeric cells of donor origin in the host. In this study, we systematically investigated DBMC-derived chimerism in various lymphoid and nonlymphoid tissues over time. To do this, transgenic mice were used as a source of DBMC to clearly distinguish chimerism due only to the injected DBMC. Chimerism in various tissues was assessed at several times points after DBMC infusion by polymerase chain reaction amplification of tissue DNA using transgene specific primers. A cDNA probe specific for the transgene was used to demonstrate DBMC-derived chimerism in polymerase chain reaction products by the method of Southern. Although chimerism was initially detectable in most tissues tested 1 day after DBMC infusion, the presence of chimeric cells generally diminished over time. At 4 weeks or longer, chimerism was consistently confined to recipient skin. Furthermore, the chimeric cells in recipient skin persisted even after the allograft was rejected. In contrast to chimerism in recipient skin, chimerism became undetectable in donor skin as early as 2 weeks after DBMC infusion. The loss of chimerism in donor skin showed a temporal correlation with a reduction of chimerism in host bone marrow and lymphoid tissues that preceded rejection in all experiments by a range of 7-14 days. The use of DBMC from transgenic mice allowed a unique opportunity to monitor the kinetics of DBMC-derived chimerism. The presence of chimerism in the skin of mice in temporal association with chronic allograft rejection suggests that chimerism per se is not a reliable index of allogeneic unresponsiveness.

Animals↗
PubMed1994

A role for transforming growth factor-beta in the veto mechanism in transplant tolerance.

We have studied the veto cell-mediated induction of transplant tolerance by allogeneic donor bone marrow cells and have achieved kidney allograft tolerance in a preclinical rhesus monkey model. Here we extend these studies to investigate the veto mechanism of CTLp suppression and the role of CD8 and TGF-beta in these events. Infusion of DR-/dim donor BMC into RATG-treated rhesus monkeys induced functional deletion of donor-specific CTLp and prolongation of kidney allograft survival, whereas depletion of the CD8+ subset from BMC ablated these effects. A role of CD8 in the veto effect was further implicated by rhesus MLR-induced CML experiments in which pretreatment of normal responder cells with MAb to MHC class I, the natural ligand of CD8, blocked the suppressive activity of allogeneic BMC. In addition, pretreatment of the BMC with anti-CD8 MAbs blocked strong veto activity significantly, suggesting that CD8 functions as an accessory or adhesion ligand. In contrast, anti-CD8 treatment significantly enhanced weak BMC-mediated veto activity, suggesting that CD8 might additionally serve as a signal transducer to increase veto activity, perhaps by the induction of cytokine release. The cytokine TGF-beta was studied because it has immunosuppressive properties that are shared by veto cells. Human TGF-beta, like BMC veto cells, inhibited MLR-induced CML in a dose-dependent manner, and anti-TFB-beta Ig relieved the BMC-mediated veto suppressive effect. Active TGF-beta was detected only in the supernatants of CML cultures containing BMC. Pretreatment of BMC with L-leucyl-leucine methyl ester (Leu-leu-OMe), which eliminates cytotoxic precursor and effector lymphocytes and monocytes, did not affect levels of active TGF-beta. In previous studies, the veto effect of BMC was also shown to be Leu-leu-OMe-resistant. Finally, treatment of isolated DR-/dim BMC cultures with anti-CD8 elicited TGF-beta secretion, whereas anti-CD2 or anti-CD3 had no effect. When isolated after stimulation with anti-CD8, only the CD8+ subset of DR-/dim BMC produced detectable levels of active TGF-beta. In summary, these studies demonstrate that CD8 functions as an immunoregulatory molecule in veto effects by freshly isolated rhesus BMC and suggest that CD8-ligand interactions may induce low-level secretion of TGF-beta to mediate or facilitate the veto mechanism of CTLp inactivation in a paracrine manner.

Animals↗
PubMed1994

A comprehensive definition of the major antibody specificities in polyclonal rabbit antithymocyte globulin.

The potent immunosuppressive action of rabbit antithymocyte globulin (RATG) in allotransplant recipients has been recognized for many years. Some of the antibody specificities and immunoregulatory effects of RATG have been described, but a comprehensive definition of RATG components has not been reported previously. In this study, we have identified 23 specificities that are consistent among different clinical RATG batches and represent the major antibody specificities in RATG. These specificities were defined by immunoprecipitation/gel electrophoresis and also antibody blocking/flow cytometry methods. Titration studies performed for semiquantitative analysis of RATG antibodies showed that the antibodies present in highest titer were directed to CD6, CD16, CD18, CD28, CD38, CD40, and CD58 (titer > 1:4000), most of which are not T cell-specific antibodies. In contrast, the RATG antibodies that persisted the longest in vivo in the plasma of rhesus monkeys transplant recipients are antibodies to CD3, CD4, CD8, CD11a, CD40, CD45, CD54, and class I. These antibodies, which are directed at signal transduction and adhesion molecules, were present during the early period of lymphocyte recovery. We suggest that the persistence of these antibodies in vivo is directly related to the prolonged anergy of circulating T cells after RATG treatment and to the unusual potency and complex tapestry of immunological effects in transplantation.

Animals↗
PubMed1994

Effects of prenatal exposure to 50 Hz magnetic fields on development in mice: I. Implantation rate and fetal development.

Pregnant CD1 mice were exposed or sham-exposed from day 0 to day 17 of gestation to a 50 Hz sinusoidal magnetic field at 20 mT (rms). Preimplantation and postimplantation survival were assessed and fetuses examined for the presence of gross external, internal, and skeletal abnormalities. There were no statistically significant field-dependent effects on preimplantation or postimplantation survival, sex ratio, or the incidence of fetuses with internal or skeletal abnormalities. Magnetic field exposure was, however, associated with longer and heavier fetuses at term, with fewer external abnormalities. The results lend no support to suggestions of increased rates of spontaneous abortion or congenital malformation following prenatal exposure to power frequency magnetic fields.

Abnormalities, Radiation-Induced↗
PubMed1994

Superficial parotidectomy in the treatment of cutaneous melanoma of the head and neck.

The preauricular lymph nodes are frequently the first site of metastatic disease from primary malignant melanoma of the upper two-thirds of the face or the anterior scalp. For these patients, or those with adjacent metastatic nodal disease, the prognosis is poor. The median survival of 13 such patients presented was 2 years, with two long-term survivors at 4 and 6 years. Palliative surgery can, however, prevent uncontrolled locoregional disease. Patients with palpable preauricular and cervical node disease should be treated by facial nerve-preserving parotidectomy and radical neck dissection. When no cervical nodes are palpable in patients with preauricular node metastasis, peroperative jugulodigastric node biopsy and frozen-section histopathological examination are useful to select patients for radical neck dissection.

Adolescent↗
PubMed1994

Management of soft tissue sarcoma.

A total of 485 patients with a proven or suspected diagnosis of soft tissue sarcoma were referred over a 3-year period. Of these, 61 were referred for opinion only and 424 for definitive treatment. Overall there were 223 patients with primary soft tissue sarcoma and 84 with recurrent sarcoma, 84 with benign soft tissue tumours and 22 with non-soft tissue sarcoma malignancy; 11 patients were referred with known metastatic disease. The commonest benign tumours mimicking soft tissue sarcoma were fibromatosis and benign intermuscular or intramuscular lipoma. The malignant tumours suspected clinically of being soft tissue sarcoma were bone tumours, lymphoma and metastatic carcinoma. Of the 172 patients with primary soft tissue sarcoma referred with a proven diagnosis this had been established by excision biopsy in 94 and open incision biopsy in 48. By contrast, of those undiagnosed at referral, 44 of 51 patients with soft tissue sarcoma had the diagnosis made by Tru-cut core biopsy. Amputation was performed in four of 165 patients with primary extremity soft tissue sarcoma and in five of 54 with recurrent extremity sarcoma, giving an overall amputation rate of 4 per cent. It is proposed that the amputation rate for extremity soft tissue sarcoma can be reduced by the use of reconstructive procedures, particularly myocutaneous flaps to the proximal limbs and limb girdles, and free vascularized flaps to sites distal to the midthigh and midarm.

Adolescent↗
PubMed1994

Blood flow measurement in extremity soft tissue sarcoma with technetium-99m hexamethyl-propyleneamineoxime and single photon emission computed tomography.

Blood flow measurements were made in 28 patients with soft tissue sarcoma of the extremities to investigate the prognostic significance of tumour vascularity. Four patients with benign tumours also underwent blood flow measurement. Mean and maximum tumour blood flow was calculated from technetium-99m hexamethyl-propyleneamineoxime uptake measured using single photon emission computed tomography (SPECT), tumour volume measured from SPECT transaxial image reconstructions and cardiac output assessed with Doppler ultrasonography. Twenty-seven malignant lesions and one benign tumour showed increased uptake of isotope relative to surrounding tissues. Mean sarcoma blood flow varied between 1 and 33 ml per 100 ml tumour per min, and maximum flow between 5 and 57 ml per 100 ml per min. Fourteen patients developed progressive disease during the first year of follow-up. Eight of 11 patients with a high isotope uptake ratio, eight of 12 with a high mean blood flow and eight of 14 with a high maximum blood flow relative to the respective medians for the series showed disease progression.

Adult↗
PubMed1994

Enhanced transcription of the human alpha 2A-adrenergic receptor gene by cAMP: evidence for multiple cAMP responsive sequences in the promoter region of this gene.

Expression of the human alpha 2A-adrenergic receptor gene is induced by cAMP. The present studies were designed to define potential cAMP-responsive enhancer elements (CREs) in the promoter region of this gene. Regions from the 5'-flanking sequences of the gene were placed in a promoterless vector with a chloramphenicol acetyltransferase (CAT) reporter gene, and cAMP-stimulated CAT activity was assayed in transfected JEG-3 placental carcinoma cells. Enhancer activity responsive to cAMP was located in DNA sequences both upstream and downstream from the endogenous promoter region. Within the upstream sequences there is a putative "core sequence" homologous to the eight base CRE consensus palindrome, but this region did not function independently as a CRE enhancer; additional upstream sequences were required to provide significant enhancer activity in response to cAMP. Regulation of expression of the alpha 2A-adrenergic gene by cAMP is complex and involves multiple and likely novel DNA sequences.

Base Sequence↗
PubMed1994

The effect of social adversity on the fatigue syndrome, psychiatric disorders and physical recovery, following glandular fever.

Two hundred and fifty patients attending primary care with glandular fever or an upper respiratory tract infection were studied prospectively up to 6 months after onset. Of these patients 228 were interviewed with the Life Events and Difficulties Schedule and the Schedule for Affective Disorders and Schzophrenia, giving Research Diagnostic Criteria for psychiatric disorders. The experience of severe social adversity (provoking agents) had a significant association with psychiatric disorder at 2 months (odds ratio = 5.3) and 6 months (odds ratio = 5.8) after onset of infection. This association was especially significant for depressive illness (odds ratio = 9.1 at 2 months and 11.9 at 6 months). In contrast, social adversity had little association with the development of the post-infectious fatigue syndrome, or delayed physical recovery. Social adversity may be an important maintaining factor for psychiatric disorders, especially depressive illness, following acute infections.

Adolescent↗
PubMed1994

Generation of interleukin-2-secreting melanoma cell populations from resected metastatic tumors.

This study aimed to determine the feasibility of producing patient-specific, interleukin-2 (IL-2)-secreting tumor cell vaccines for the treatment of metastatic melanoma. Primary tumor cell cultures were established from 26/33 resected metastatic melanoma samples. Recombinant retroviral gene transfer and expression in these cultures was optimized using an amphotropic, defective retrovirus carrying the LacZ gene. All cell cultures were infectable; those that proliferated more rapidly were infected at a higher frequency. Addition of fibroblast growth factor to the culture medium increased the rate of cell proliferation and the efficiency of infection. A single infection with an identical retrovirus carrying a human IL-2 cDNA resulted in the generation of unselected cell populations secreting up to 300 units IL-2/10(6) cells.48 hr. Multiple infections increased the level of IL-2 secretion to 5,000 units/10(6) cells.48 hr. The recombinant viral genome could be detected at approximately single copy in the multiply infected cells; no helper virus was detected. IL-2 secretion from infected cultures was maintained following cryopreservation and x-irradiation. These data demonstrate that heterogeneous tumor cell populations secreting IL-2 can be generated from individual patients to be used as autologous, irradiated cell vaccines.

DNA, Neoplasm↗
PubMed1994

Further studies of veto activity in rhesus monkey bone marrow in relation to allograft tolerance and chimerism.

Infusing the DR-/dim fraction of bone marrow cells (BMC) from an allogeneic kidney donor into rabbit antithymocyte globulin-treated transplant recipients delivers a tolerogenic signal, leading to functional allograft tolerance in rhesus monkeys without additional drug therapy. Our updated results in an expanded series show a median 131-day graft survival of recipients given DR-/dim donor BMC with a 23% 1-year survival (P < 0.00001 vs. rabbit antithymocyte globulin controls). Removing DRbright cells from donor BMC appeared to have a significant effect (P < 0.05). We have further investigated the tolerogenic mechanism within the experimental framework of the veto hypothesis in this preclinical model. In limiting dilution assays, we demonstrated the donor specificity of clonal inactivation of CTL precursors (CTLp) after in vitro or in vivo exposure to DR-/dim donor BMC, confirming specific tolerance. Additionally, in vitro studies confirmed the allogeneic specificity of CTLp inactivation in 3-cell MLR assays; minimal bystander effects were seen on normal CTLp responses to third party stimulator cells, while CTLp responses to the BMC donor's cells were abrogated in the same cultures. BMC mediating the veto effect were found to be resistant to L-leucyl-L-leucine methyl ester (Leu-leu-OMe), which excluded BMC-mediated cytotoxicity by NK or lymphokine-activated killer cells, CTL, or activated macrophages. In contrast, veto activity was abolished if the BMC were pretreated with either high dose UV-B light irradiation, mitomycin, or gamma-irradiation, indicating that BMC contained a UV-B-sensitive precursor of the veto effector, and that a proliferative step separated the two. Irradiation of DR-/dim donor BMC or administration of cyclophosphamide after infusion of nonirradiated BMC prevented the tolerogenic effect. Only recipients given nonirradiated DR-/dim donor BMC demonstrated PBL chimerism, which associated with functional deletion of antidonor CTLp and duration of graft survival. The Leu-leu-OMe resistance and the other properties of the allogeneic monkey CD3- CD2+ CD8+ BMC subpopulation that exhibits tolerance-promoting activity in vitro and in vivo lead us to postulate that a donor BMC-derived precursor population, possibly a dendritic cell population, may induce allogeneic unresponsiveness in this model.

Animals↗