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Biomedical subjects

J F George

Publications and source records attributed to J F George.

At least 19 recordsLinked to original sources
PubMed2006

Rejection with hemodynamic compromise: objective evidence for efficacy of photopheresis.

BACKGROUND: Photopheresis therapy (photo) has been advocated as a therapy to improve outcome after recalcitrant or severe rejection, but objective evidence of a beneficial effect has been elusive. This study examined the hypothesis that photo provides protection against rejection, rejection with hemodynamic compromise (HC), and death from rejection after cardiac transplantation. METHODS: Between 1990 and 2003, 36 adult patients (from 343 adult transplant recipients) received at least 3 months of photo (2-day treatment every 3 to 6 weeks for a target of 18 months) after HC rejection (n = 12), recurrent/recalcitrant rejection (n = 20), or as prophylaxis in the presence of anti-donor antibodies (n = 4). Survival and risk factors were examined by analysis using multivariate hazard function modulated renewal function. RESULTS: Patients selected for photo were at greater risk for rejection (p < 0.0001) and HC rejection (p < 0.0001) than non-photo patients. After 3 months of photo therapy, rejection risk was decreased (p = 0.04). More importantly, the hazard for subsequent HC rejection or rejection death was significantly reduced toward the risk-adjusted level of lower-risk non-photo patients (p = 0.006). CONCLUSIONS: This study provides objective evidence that photo reduces the risk of subsequent HC rejection and/or death from rejection when initiated for patients with high rejection risk. Photopheresis is recommended as an important therapeutic modality after rejection with hemodynamic compromise, although further studies are needed to define the precise mechanism of the effect and the potential for benefit in other patient sub-sets.

Adult↗
PubMed2001

An essential role for natural killer cells in augmentation of allograft survival mediated by donor spleen cells.

BACKGROUND: Previous studies have shown that skin allograft survival can be augmented by the administration of donor spleen or donor bone marrow in antithymocyte serum (ATS) treated recipients. Because natural killer cells (NK) have been reported to possess immunoregulatory properties, we investigated whether the ability of donor spleen or bone marrow cells to enhance allograft survival was dependent on the presence of donor NK cells. METHODS: Recipient (C57BL/6 x A/J)F1 strain mice (H2 haplotypes Kb/k, Ab/k, E-/k, Db/d) were treated with ATS on days -1 and +2 relative transplantation of a C3H (H-2k) skin allograft. On day +7, each recipient was randomly assigned to one of the following groups that received i.v. donor C3H cell infusions via the tail vein: 1) 5.0x10(7) wild-type donor spleen cells (SPC); 2) 5.0x10(7) spleen cells from C3H/HeJ-Lystbg-2J/+ mice (commonly called beige mice and have selectively impaired NK cell function); 3) 2.5x10(7) wild-type donor bone marrow cells (BMC); 4) 2.5x10(7)beige C3H bone marrow cells; and 5) no donor cell infusion (ATS controls). In another experiment, each recipient was randomly assigned to one of the following groups that received injections of: 1) 4.75x10(7) spleen cells depleted of NK cells; 2) 2.5x10(6) purified splenic NK cells; 3) a coinfusion of 5.0x10(7) beige spleen cells and 2.5x10(6) purified wild-type splenic NK cells. RESULTS: Recipients infused with wild-type SPC exhibited significant augmentation of allograft survival compared with ATS controls. However, graft survival was reduced in recipients that were infused with spleen cells from beige mice compared with recipients infused with wild-type SPC (median survival time (MST): 38 vs. 92 days, P=0.02). In contrast, infusions of beige BMC augmented allograft survival as well as wild-type BMC (MST: 47 vs. 49 days, P=0.76). Furthermore, the ability of wild-type SPC to augment allograft survival was abrogated by the depletion of NK cells (MST=92 vs. 34 days, respectively, P=0.005). The co-infusion of beige SPC and purified splenic NK cells enhanced allograft survival as well as wild-type SPC (MST=56 days, P=0.65). Finally, recipients infused with purified NK cells did not experience increased graft survival compared to recipients that received no infusion (MST=29 vs. 33 days, respectively, P=0.6). CONCLUSIONS: Donor splenic NK cells are necessary, but not sufficient, for the extension of graft survival by infusion of donor splenocytes, suggesting that they may work in concert with another cell-type. In contrast, the extension of graft survival by donor bone marrow does not depend on the presence of donor NK cells.

Animals↗
PubMed2001

Indefinite allograft survival mediated by donor bone marrow is dependent on the presence of a functional CD95 (Fas) gene in recipients.

It is well documented that donor bone marrow in combination with peri-transplant anti-thymocyte globulin (ATG) administration induces transplantation tolerance in a variety of animal models. Our previous work showed that the ability of donor marrow to induce tolerance was dependent on the presence of CD95 ligand (Fas-ligand) on the donor cells. In this study we investigate whether CD95 (Fas) on the recipient cells is required. By comparing skin allograft survival times between wild-type C57BL/6 ATG-treated recipients and C57BL/6(lpr/lpr) ATG-treated recipients (which do not have a functional CD95 gene), we show that donor bone marrow could induce indefinite transplant survival (median survival time >200 days) only in recipients with a functional CD95 gene. Thus, we conclude that the CD95 ligand-CD95 apoptotic pathway plays a major role in donor bone marrow-induced transplantation tolerance.

Animals↗
PubMed2000

A differential requirement for CD8+ donor cells in the augmentation of allograft survival by posttransplantation administration of donor spleen cells and donor bone marrow cells.

BACKGROUND: Peritransplant treatment with antithymocyte serum (ATS) and posttransplantation administration of donor bone marrow or donor splenocytes results in extended skin allograft survival. In this study, we examined the molecular basis of the tolerance promoting effect of donor bone marrow (BMC) cells and splenocytes with emphasis on the role of CD8 expression on the donor cells. METHODS: (C57BL/6J x A/J)F1 mice were treated on days -1 and +2 with ATS relative to transplantation with C3H/HeJ skin. On day +7, they were infused with CD8+ BMC, CD8- BMC, CD8+ splenocytes, or CD8- splenocyte donor subpopulations isolated by magnetic or fluorescence-based sorting. In additional experiments, B10.D2(R107) mice were treated in the same manner with C57BL/6 skin and BMC or splenocytes from C57BL/6 mice in which the CD8alpha gene had been inactivated. RESULTS: CD8+ donor bone marrow cells induced operational tolerance (defined as graft acceptance in the absence of chronic immunosuppression) in skin graft recipients at a dose that was reduced by 250-fold relative to unfractionated bone marrow cells (1.0x10(5) cells per recipient, median survival time (MST)=41 days vs. 2.5x10(7) cells per recipient, MST=49 days, P=0.40). Similarly, donor bone marrow cells from CD8 knockout mice did not promote graft acceptance (MST=98 days vs. animals not treated with bone marrow cells, MST=70 days, P=0.16). In contrast, the extension of graft survival by donor splenocytes did not require the presence of CD8+ donor cells because splenocytes depleted of CD8+ cells extended graft survival (MST=55 days) as well as unsorted splenocytes (44 days, P=0.2), and splenocytes from CD8 knockout animals (MST=145 days) extended graft survival at least as well as unsorted splenocytes (MST=74 days, P=0.4) CONCLUSIONS: These results suggest that the prolongation of graft survival by donor bone marrow is dependent on the presence of the CD8 molecule, whereas prolongation by donor splenocytes is not. Therefore, we suggest that the prolongation of graft survival by these cell types occurs via distinct molecular mechanisms probably mediated by different cell types.

Animals↗
PubMed2000

Enhanced allograft survival induced by posttransplant donor spleen cell infusion occurs via a mechanism that is distinct from the mechanism of enhancement by donor bone marrow.

BACKGROUND: Our previous studies have shown that the ability of donor bone marrow to augment skin graft survival in antithymocyte serum (ATS)-treated recipients is dependent on the presence of functional CD95-ligand (Fas-ligand) molecules on donor cells. Because donor spleen cells can augment graft survival to a similar degree in the same model, we investigated whether the donor spleen cell effect was also dependent on the presence of CD95-ligand on donor cells and CD95 on recipient cells. METHODS: Mutant mice bearing defects in the expression of CD95 (lpr mutation) and CD95-ligand (gld mutation) were used as recipients and cell donors, respectively. Recipients were injected with rabbit ATS on days -1 and +2, and then were injected with 5x10(7) spleen cells on day +7. Skin graft survival was compared and correlated with the use of mutant mice as recipients and cell donors. RESULTS: The combination of ATS and infusions of wild-type [median survival (MST)=44 days, P=0.0004] and gld (mutant CD95-ligand, MST=37 days, P=0.02) donor spleen cells enhanced C3H graft survival, compared with (C57BL/6 x A)F1 recipients treated with ATS alone (MST=27 days). Furthermore, C57BL/6 lpr (CD95-deficient) strain recipients treated with ATS and donor spleen cells demonstrated enhanced B10.D2(R107) strain skin graft survival (MST=44 days, P=0.003), compared with C57BL/6 lpr recipients treated with ATS alone (MST=31 days). Wild-type C57BL/6 recipients treated in the same manner also exhibited an extension of graft survival (MST=64 days) versus controls treated with ATS alone (MST=31 days). CONCLUSION: The data demonstrate that the ability of donor spleen cells to augment allograft survival is not dependent on the CD95/CD95-ligand pathway; therefore the deletion of allospecific cells by donor spleen cells may be induced via a pathway other than deletion by donor bone marrow cells.

Animals↗
PubMed1999

Immunoregulatory role of CD8alpha in the veto effect.

BACKGROUND: Allogeneic bone marrow cell (allo-BMC) infusion induces tolerance to incompatible renal allografts in rhesus macaques after depletion of peripheral T lymphocytes with cytolytic anti-T cell antibodies. The tolerogenic effect of allo-BMC, ascribed to a veto mechanism, associates with specific functional deletion of antidonor cytotoxic T lymphocyte precursor (CTLp), and is dependent on a CD8+ donor BMC subset. In previous studies, the CD8 molecule was implicated by loss of suppression after blocking interaction between CD8 on allo-BMC and major histocompatibility complex class Ialpha3 domain on CTLp. CD8 cross-linking on BMC induced secretion of active transforming growth factor-beta1 (TGF-beta1), suggesting a regulatory mechanism(s) operating via a CD8-mediated signaling pathway. METHODS: CD8 on rhesus cells was cross-linked using IgG-conjugated beads, and TGF-beta1 mRNA and protein were quantified. CD8+ cells were tested for veto activity by mixed lymphocyte reaction (MLR)-induced cell-mediated lymphocytotoxicity (CML) assay. Activated rhesus T cells exposed to TGF-beta1 were examined for apoptosis by TdT-mediated end-labeling and annexin staining. RESULTS: CD8 cross-linking induces accumulation of TGF-beta1 mRNA and protein. Both CD3- CD8+CD16+ and CD3+ CD8+CD16- subsets of allo-BMC up-regulate TGF-beta1 mRNA after CD8 cross-linking, and exhibit veto activity. The CD3-CD8+CD16+ subset expresses more TGF-beta1 mRNA and increased veto activity at low BMC/CTLp ratios. Exposure of activated T cells to TGF-beta1 induces apoptosis. CONCLUSIONS: CD8+ allo-BMC are enriched for veto activity and activation via CD8 induces TGF-beta1 mRNA and protein accumulation. These results agree with the hypothesis that paracrine TGF-beta1 may be involved in peripheral deletion of alloreactive CTLp by CD8+ allo-BMC. We suggest that TGF-beta1 overexpression by donor lymphohematopoietic cells may enhance tolerance induction.

Animals↗
PubMed1999

The molecular mechanisms of veto mediated regulation of alloresponsiveness.

Transplantation tolerance, as defined by the specific acceptance of allogeneic tissue without chronic immunosuppression, can be induced in adults by a variety of methods. One method that is close to clinical implementation involves transient peritransplant depletion of host T cells followed by intravenous administration of a low dose of donor bone marrow cells (BMC). Current evidence suggests that BMC play an active role in tolerance induction by establishing residence in the host and effecting deletion of graft reactive T cells. This activity, in which the BMC inactivate T cells that recognize them, has been called the "veto effect," and cells that mediate such activity have been called "veto cells." The specificity of veto activity is conferred by the T cell receptor of the donor reactive T cells that are to be deleted. Other molecules that have been directly implicated in the mechanism of deletion of graft reactive T cells by BMC include the CD8 accessory molecule, major histocompatibility class I gene products, CD95/CD95 ligand (Fas/Fas ligand), and transforming growth factor-beta. These molecules react with their respective ligands by distinct, non-exclusive pathways among which the relative contribution to the deletion of graft reactive T cells may depend on the relative abundance of different cellular subpopulations capable of mediating veto activity.

Animals↗
PubMed1999

An automated method for the analysis of T-cell receptor repertoires. Rapid RT-PCR fragment length analysis of the T-cell receptor beta chain complementarity-determining region 3.

The examination of T-cell receptor (TCR) repertoires has an important role in the study of lymphoproliferative disorders and autoimmune diseases. Analysis of the complementarity-determining region 3 (CDR3) of the TCR beta chain is used to assess the clonality of T-cell populations. We developed a rapid fluorescence-based method for CDR3 length analysis of expressed TCR gene families. TCR beta chain complementary DNA is amplified by a nested polymerase chain reaction with V beta family-specific oligonucleotide primers and a fluorochrome-labeled C beta primer. The polymerase chain reaction products were analyzed on a compact automated DNA sequencing system (OpenGene system, Visible Genetics, Toronto, Ontario). To demonstrate the usefulness of our technique, we examined the CDR3 length distribution of peripheral blood T cells from a healthy subject, intestinal T cells from a patient with ulcerative colitis, and the T-cell leukemia cell line Jurkat. The analysis revealed polyclonal, oligoclonal, and monoclonal CDR3 distributions, respectively, for the 3 T-cell populations. Our new method shows virtually identical CDR3 length patterns compared with the traditional radioisotope-based method. The new technique offers the convenience of rapid throughput, nonradioactive labeling, and quality data analysis.

Automation↗
PubMed1999

A developmental bias in reading frame usage by human fetal thymic TCRBDJ transcripts is not present in genomic TCRBDJ rearrangements.

We have previously reported that reading-frame usage and functional diversification is developmentally regulated, with virtually all TCRB DJ mRNA transcripts using a single reading frame at 8 weeks of gestational age, tapering to 50% by adult life. We used the polymerase chain reaction to create genomic libraries of DJ rearrangements in the TCRB locus from thymuses at 7.7, 10, and 16 weeks of gestational age, and from adult thymuses. Clones were randomly picked and sequenced to determine junctional sequences and reading-frame utilization. The resulting data address the hypothesis that cells bearing genomic joints in reading frame one are preferentially selected during fetal life. This hypothesis predicts that reading-frame bias would also be observed among genomic DJ joints. Instead, we observed random utilization of the three possible D-region reading frames among genomic D1s1 ==> J1s1 joints during fetal life. Similar results were obtained at 7.7 weeks of gestational age in a second thymus in which both RNA and DNA were simultaneously isolated and used to create libraries of TCRBDJ transcripts or rearrangements. We conclude that reading-frame utilization is random among genomic D1s1-JB1s1 rearrangements and that the preferential usage of a single reading frame among mRNA transcripts of TCRB DJ transcripts is the result of preferential transcription of genomic TCRB DJ joints in a single reading frame, or that TCRB DJ transcripts have a longer half-life than transcripts in reading frames two or three.

Embryonic and Fetal Development↗
PubMed1999

Donor bone marrow and transplantation tolerance: historical perspectives, molecular mechanisms and future directions (review).

The induction of transplantation tolerance, defined as the indefinite existence of an intact, functioning allograft, has been a goal of transplantation immunologists for decades because of the morbid effects of immunosuppressive drugs required to maintain graft function. One promising method of tolerance induction involves the peritransplant administration of cytoablative drugs followed by administration of a low dose of donor bone marrow cells. A subpopulation of donor bone marrow cells bearing the CD8 accessory molecule cause the functional deletion of graft reactive T cells via a CD95 dependent mechanism that also involves the cytokine TGFbeta. This interaction is bi-directional in which the bone marrow cell deletes the graft reactive T cell that recognizes it. Therefore, only T cells that recognize the donor antigens are deleted, leaving the immune response to other antigens intact. This protocol has been successfully used in rodents and an outbred large animal model. Clinical trials of donor bone marrow administration are now underway and initially indicate that administration of donor bone marrow is clinically safe.

Animals↗
PubMed1998

Gene polymorphisms for plasminogen activator inhibitor-1/tissue plasminogen activator and development of allograft coronary artery disease.

BACKGROUND: Impaired fibrinolytic activity has been linked to the presence and severity of allograft vasculopathy (Tx CAD). This impairment may be associated with the presence of certain fibrinolytic protein gene polymorphisms. METHODS AND RESULTS: To investigate the relation between donor-specific fibrinolytic protein genotypes and Tx CAD, we identified donor plasminogen activator inhibitor-1 (PAI-1) HindIII and tissue plasminogen activator (TPA) EcoRI restriction fragment length polymorphisms-based genotypes by Southern blot analysis in 48 recipients of cardiac allografts and correlated these genotypes with the development of CAD. No association was found between donor TPA genotypes and the presence of Tx CAD. Among the 48 patients, 17% were homozygous for the 1/1 PAI-1 genotype, 51% for the 2/2 PAI-1 genotype, and 32% for the 1/2 PAI-1 genotype. The actuarial freedom from any CAD for the recipients with each respective donor PAI-1 genotype at 12 and 24 months was 100% and 100% for the 1/1 PAI-1 genotype, 92% and 92% for the 1/2 PAI-1 genotype, and 75% and 45% for the 2/2 PAI-1 genotype (P=0.03). Recipients with a diseased 2/2 PAI-1 genotyped allograft had longer ischemic times (P=0.02) than those recipients with a Tx CAD-free allograft. CONCLUSIONS: These data suggest that recipients with a 2/2 PAI-1 genotype are at a significant risk of developing Tx CAD. This genotype may serve as a useful screening tool for predicting the future development of Tx CAD.

Adult↗
PubMed1998

DR non-B1 mismatches influence allogeneic MLR-induced TH1- or TH2-like cytokine responses in rhesus monkeys.

Human and nonhuman primates have multiple DR B1 and non-B1 alleles. However, the role of mismatched DR non-B1 alleles in primary alloimmune responses is not well understood. Macaques, which share close DNA homologies with human MHC genes and have a high number of beta-chain genes in the DR subregion, are preeminent preclinical models for immunologic studies of transplant tolerance and immunosuppression. In this study, we examined the effect of allogeneic MHC Class II DRB mismatches in Th1- and Th2-like cytokine responses elicited in one-way MLR cultures in rhesus macaques. An ELISPOT method was used to estimate cytokine secretion at the single cell level. Molecular typing for DRB1 and DR non-B1 alleles was performed by a moderate-high resolution PCR-SSP method using a panel of 55 primer pairs covering 74 DRB alleles and clusters. Of 35 unrelated combinations, 66% had multiple (> or = 2) allelic MM at DRB1 and DR non-B1 with no significant correlation between numbers of DRB1 and DR non-B1 mismatches. Pairs with 1 or 0 MM were assigned to a mono/null MM group to obtain sufficient numbers for statistical analysis. The pairs differing by multiple vs. mono/null DRB1 MM showed no significant difference in cytokine prevalence (P = 0.69). In contrast, high IFN-gamma/ IL4 SFC ratios were noted in pairs with multiple vs. mono/null DR non-B1 MM (p = 0.0009). IFN-gamma/IL-10 spot forming cell (SFC) ratios were consistent with IFN-gamma/IL-4 SFC ratios (r = 0.98). Multiple DR non-B1 mismatches showed a trend towards higher MLR proliferative responses, although the stimulation index did not reflect the dominant cytokine response. These observations suggest a bias towards Th1-like cytokine production under allostimulation with multiple DR non-B1 gene products. Further study of the primary structure of DR non-B1 determinants may be helpful in understanding the fine molecular mechanisms governing the regulation of cytokine profiles during allostimulation in primates.

Animals↗
PubMed1998

Molecular cloning, expression and characterization of rhesus macaque Fas ligand cDNA.

The Fas/Fas ligand (FasL) interaction is pivotal in apoptosis-mediated regulation of the immune system. As such, it has relevance in areas of transplantation, gene therapy, AIDS, etc., all of which utilize the rhesus macaque as a preclinical animal model. In order to examine rhesus Fas/FasL, we cloned the rhesus FasL cDNA and have analyzed the function of the cloned gene. Our findings indicate that the rhesus FasL is highly homologous to the human but not the mouse (97% for human, 85% for mouse). In addition, soluble rhesus FasL can induce apoptosis, a property shared with the human soluble protein but not the mouse protein. The deduced protein sequence is 280 amino acids with a calculated Mr. of 31,646. Transfection of COS cells with the full-length cDNA yielded a 40 KD protein, which is in agreement with the size of human FasL. COS cells expressing rhesus FasL induced apoptosis in rhesus PHA blasts and human Fas+ CEM-6 cells. Thus, the cloned rhesus macaque FasL is functional and cross-reacts with human Fas. The cloned functional rhesus FasL cDNA will enable studies of its regulatory role in the nonhuman primate immune system.

Amino Acid Sequence↗
PubMed1998

An essential role for Fas ligand in transplantation tolerance induced by donor bone marrow.

Medawar and co-workers originally demonstrated that injection of donor bone marrow (DBM) into immuno-incompetent neonatal rodents could induce tolerance to grafts from animals of the same strain as the bone marrow donor. Induction of tolerance in this manner can also be accomplished in mature mice, dogs and monkeys if the resident T-cell populations in the recipient are depleted by a polyclonal antithymocyte globulin or an anti-T cell immunotoxin. The molecular mechanisms by which bone marrow cells mediate the induction of tolerance remain uncertain. Here we examined a well-established adult mouse model of antithymocyte globulin and DBM treatment and show that expression of functional Fas ligand (FasL, also CD95L) on the injected bone marrow cells is required for tolerance induction. The results indicate that a state of microchimerism per se is insufficient for the induction of tolerance in T cell-depleted transplant recipients. Moreover, the results are consistent with the hypothesis that tolerance induced by DBM involves an apoptotic process leading to deletion of graft-reactive cells.

Animals↗
PubMed1997

Serial measurements of interleukin-6, interleukin-8, tumor necrosis factor-alpha, and soluble vascular cell adhesion molecule-1 in the peripheral blood plasma of human cardiac allograft recipients.

BACKGROUND: Cardiac allograft rejection is largely an inflammatory response that, if allowed to proceed unchecked, will result in hemodynamic compromise or cardiogenic shock. Soluble mediators produced during an inflammatory response could potentially provide information regarding the initiation, progression, and outcome of a rejection episode. To test this hypothesis, we investigated the use of plasma cytokine measurements for interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF alpha) in combination with measurements of soluble vascular cell adhesion molecule-1 (VCAM-1), an adhesion molecule, as a means for the detection of cardiac allograft rejection. METHODS: Serial enzyme-linked immunosorbent assays were performed on plasma samples collected from 29 patients three times per week during the first 8 weeks after transplantation. RESULTS: IL-6 plasma concentrations increased fivefold in the first week after transplantation (p < 0.001 vs pretransplantation levels) and thereafter remained at low levels for the next 6 weeks, with a small increase during the 8 weeks after transplantation (p = 0.006). In contrast, TNF-alpha, IL-8, and VCAM-1 levels remained low during the first 6 weeks after transplantation followed by a rise in mean VCAM-1 levels from 841 +/- 38 to 979 +/- 52 ng/ml at week 8. To determine the relationship of levels of each of the four soluble factors with rejection, the mean values obtained during the time interval 1 to 5 days before rejection were compared to mean values obtained during rejection and at other periods of no rejection (baseline). Cytokine levels were not predictive of rejection (no difference in levels 0 to 5 days before rejection versus baseline, p > 0.3 for IL-6, IL-8, TNF-alpha). However, VCAM-1 levels increased 0 to 5 days before rejection compared with baseline (914 +/- 40 vs 844 +/- 30 ng/ml, p = 0.06). CONCLUSIONS: IL-6 levels are increased immediately after heart transplantation. Circulating IL-6, IL-8, and TNF alpha levels do not predict rejection during the first 8 weeks after transplantation. Soluble VCAM-1 increases within 5 days before rejection and may potentially serve as a noninvasive marker for early rejection.

Adolescent↗
PubMed1996

Semiquantitative measurement of cytokine messenger RNA in endomyocardium and peripheral blood mononuclear cells from human heart transplant recipients.

BACKGROUND: Cytokines play a central role in inflammatory responses and in specific immune responses directed toward alloantigens. The pattern and quantity of cytokines produced in graft rejection can yield valuable information regarding the cellular and molecular mechanisms of the antigraft response. METHODS: We used the polymerase chain reaction to semiquantitatively measure changes in the amount of messenger RNA from the interleukin-1 beta, interleukin-2, interleukin-4, interleukin-6, interleukin-10, tumor necrosis factor-alpha, interferon-gamma, interleukin-1 receptor antagonist, and the interleukin-2 receptor genes in the peripheral blood and endomyocardium of cardiac allograft recipients during the first 8 weeks after transplantation. A total of 328 samples of resting (n = 251) and stimulated (n = 77, stimulated with phytohemagglutinin and lipopolysaccharide for 18 hours) peripheral blood mononuclear cells collected from 16 patients were measured. To measure intragraft cytokine levels, we analyzed 150 endomyocardial biopsy specimens from 19 patients. RESULTS: No elevation in expression was seen before injection, but, after the onset of rejection and concomitant with treatment, there was a decrease in detectable mRNA (p < 0.05) for the pro-inflammatory monokines interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha in resting peripheral blood mononuclear cells, and a decrease for the T-cell derived cytokines interleukin-4 and interleukin-10 in stimulated peripheral blood mononuclear cells. These changes in mRNA expression occurred coincidentally with decreases in the percentage of lymphocytes and monocytes in the peripheral blood after administration of rejection therapy. In endomyocardial biopsy specimens, there were no detectable changes in the quantities of cytokine mRNA specimens for the interferon-gamma, interleukin-6, interleukin-10, interleukin-1ra, and interleukin-1 beta genes before rejection. In general, the levels of these cytokines were near the lower limits of detection by our assay in endomyocardial biopsies, mRNA from the interleukin-2, interleukin-4, tumor necrosis factor-alpha, and interleukin-2R genes were undetectable. CONCLUSIONS: We conclude that changes in the expression of cytokine mRNA in both peripheral blood mononuclear cells and endomyocardial biopsy specimens as measured by the semiquantitative polymerase chain reaction method used in this study does not effectively predict rejection. The decline in peripheral blood mononuclear cell cytokine mRNA after rejection treatment is likely due to changes in the proportion of lymphocytes and monocytes in the peripheral blood in concert with a steroid-induced downregulation by cytokine gene transcription.

Biopsy↗
PubMed1994

Arrested rearrangement of TCR V beta genes in thymocytes from children with X-linked severe combined immunodeficiency disease.

Human X-linked severe combined immunodeficiency disease (SCID) is an immunodeficiency disorder in which T cell development is arrested in the thymic cortex. B lymphocytes in children with X-linked SCID seem to differentiate normally. X-linked SCID is associated with a mutation in the gene that encodes the IL-2R gamma-chain. Because TCR-beta gene recombination is a pivotal initial event in T lymphocyte ontogeny within the thymus, we hypothesized that a failure to express normal IL-2R gamma could lead to impaired TCR-beta gene recombination in early thymic development. PCR was used to determine the status of TCR-beta gene-segment rearrangements in thymic DNA that had been obtained from children with X-linked SCID. The initial step in TCR-beta gene rearrangement, that of D beta to J beta recombination, was readily detected in all thymus samples from children with X-linked SCID; in contrast, V beta to DJ beta gene rearrangements were undetectable in the same samples. Both D beta to J beta and V beta to DJ beta TCR genes were rearranged in the thymic tissues obtained from immunologically normal children. We conclude that TCR beta-chain gene rearrangement is arrested in children with X-linked SCID. Our results suggest a causative relationship between the failure of TCR beta-chain gene rearrangements to proceed beyond DJ beta rearrangements and the production of a nonfunctional IL-2R gamma-chain.

Base Sequence↗