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P Alard

Publications and source records attributed to P Alard.

At least 37 records · Page 2Linked to original sources
PubMed1995

Detection of donor-derived cells by polymerase chain reaction in neonatally tolerant mice. Microchimerism fails to predict tolerance.

Although it has long been appreciated that establishment of chimerism is important in the acquisition and maintenance of allograft tolerance, the importance of this relationship has been reemphasized recently. Using the exquisite sensitivity of the polymerase chain reaction we have studied qualitatively and quantitatively the presence of donor-derived chimeric cells in relation to the ability of neonatally injected mice to display skin graft tolerance or rejection. We have found that virtually all mice that receive neonatal injections of F1 hematopoietic cells acquire donor-derived chimerism that is detectable in blood, spleen, lymph nodes, and thymus. Surprisingly, neither the presence nor the quantity of chimeric cells predicts whether a particular neonatally injected mouse will accept or reject donor-specific skin allografts. Moreover, whether the test skin allograft is accepted (tolerance) or rejected by neonatally injected mice, chimerism typically remains detectable within recipient lymphoid tissues. In functionally tolerant mice, challenge with a test skin allograft actually leads to a remarkable expansion in donor-derived genetic sequences, implying that donor-derived cells have been induced by the graft to undergo proliferation. Since persistence of chimerism without proliferation after test grafting is characteristic of mice that fail to display tolerance, we believe that achievement of a threshold level of donor-derived alloantigen may be necessary to retain the tolerant state. We conclude that chimerism is essential for the induction of neonatally induced tolerance, and its expansion may play an important role in the maintenance of that tolerance, when challenged by an allograft.

Animals↗
PubMed1994

Induction of peripheral T cell tolerance and allo/xenoimmunity.

It is theoretically impossible or at least difficult to tolerize animals in xenogeneic situation, particularly in non-concordant species. Both humoral defense and several cellular components (T and non T) are offensive weapons which can be directed at myriad of self-antigens in a normal situation, at allo-antigens in abnormal situations, at xeno-antigens in exceptional situations. In either cases, the fine epitopic definition of allo/xeno antigens seen by the TCR is far from being totally known. From recent studies showing the precise requirement of peptides assembly composition in the MHC class I and II groove it should be theoretically possible to tolerize any group of peptides if they are presented, some other, because not presented, may remain ignored, thus apparently tolerized. It is also know, that transplantation tolerance is not a law of "either nothing or all" but a multiple process depending of both the affinity of the TCR and the nature/compositions of the target. In view of the complex array of factors influencing the pathway of T cell activation, three forms of T cell non responsiveness may be suggested in the context of xenogeneic recognition: physical deletion of potentially reactive T cells occurring predominantly in the thymus, non reactivity of T cells resulting from their failure to be influenced by antigens, and anergy possibly due to inappropriate signals from non professional antigen presenting cells. Future investigations must elucidate the requirements for inducing these events in a purpose of xenogeneic organ transplantation.

Clonal Anergy↗
PubMed1993

A possible role for specific "anergy" in immunologic hyporeactivity to donor stimulation in human kidney allograft recipients.

The low immunological reactivity toward donor cells usually observed in transplant recipients has been linked to clonal deletion or suppression of alloreactive cells. However, the anergy of donor-specific reactive cells is another possibility not extensively tested until now in humans. In this case, donor-specific reactive cells would be present and eventually be activated without becoming effector cells (i.e., without secreting IL-2 or becoming cytotoxic) after donor-specific stimulation. We studied 8 patients under low-dose immunosuppressive drugs who displayed hyporeactivity toward donor stimulation. IL-2 production, proliferative response, and cytotoxic activity toward donor cell stimulation was decreased (respectively 22, 53, and 19% of response toward third-party stimulation). In order to evidence anergy, we studied two activation markers (cell size increase and expression of IL-2 receptor [CD25]) in allografted recipient T cells after autologous (background), donor (experimental), and third-party cell stimulation (positive control). We showed that the percentage of CD25+ cells and the cell size increase were similar after donor or third-party cell stimulation and clearly above the background as early as days 1-2 after the beginning of the mixed lymphocyte culture. Moreover, CD4+ and CD8+ cells similarly expressed CD25 after donor or third-party stimulation. Thus, donor-specific reactive cells not only were present but could be activated without becoming effector cells. These data suggest that anergy may be an important phenomenon in allograft tolerance.

Humans↗
PubMed1993

A versatile ELISA-PCR assay for mRNA quantitation from a few cells.

Gene expression studies require a sensitive and quantitative assay of mRNA amounts present in small samples. We describe a general method of quantifying specific mRNA quickly and easily from purified RNA or directly from a few cells by PCR and enzyme-linked immunosorbent assay (ELISA) revelation of the resulting products (sensitivity of the last step: < 0.1 fmol). Cells are digested and the total cellular RNA is reverse-transcribed and then amplified with 5' and 3' primers; the former being 5' biotinylated. The amplification product is captured on avidin-coated microplates and quantified by hybridization with a digoxigenin-labeled internal oligonucleotide probe. After revelation with an anti-digoxigenin alkaline phosphatase coupled antibody (anti-DIG-AP1), the amount of hybridized probe is determined by optical reading. The results can be easily converted to absolute values by comparison with an external DNA standard curve. An internal DNA or RNA standard can also be used. The method we describe can be adapted to any cellular or viral gene of known sequence in a matter of days. Since it uses nonradioactive probes, commercially available reagents and standard microplate readers, it is inexpensive and could be automated easily. In this study, interleukin-2 mRNA expression could be studied in as few as 40 Jurkat cells. It was also possible to quantify human immunodeficiency virus (HIV) DNA from 1500 to 1.5 copies out of 1.5 x 10(5) human genomic DNA copies.

Avidin↗
PubMed1993

[Current concepts on the alloreactive response].

Many new data have been obtained in the last 4 years about the structure and the functions of Major Histocompatibility Complex (MHC) molecules. Structural data are summarized and the functions of MHC molecules in the presentation of peptides to T cells is described. The high selectivity of peptides binding to MHC molecules is explained. The negative and positive selection of T cells in the thymus is described and the notion of repertoire introduced. The consequences of these new data on the understanding of H2 restriction and alloreactivity are explained. The 4 potential types of alloreactivity are defined: 1) House keeping gene (HKG) peptides bound to allogenic MHC molecules, 2) allogenic peptides (derived from allogenic MHC molecules) bound to allogenic MHC molecules, 3) empty allogenic MHC molecules, 4) allogenic peptides bound to autologous MHC molecules. In fact, the allogenic response is mostly directed toward HKG peptides bound to the allogenic MHC molecules of the graft cells (type 1). The potential role of type 4 alloreactivity in rejection is discussed.

Animals↗
PubMed1992

Prophylactic use of the IL-2 receptor-specific monoclonal antibody LO-Tact-1 with cyclosporin A and steroids in renal transplantation.

LO-Tact-1 is a rat anti-human monoclonal antibody which is directed to the 55-kDa alpha-chain of the interleukin 2 (IL2) receptor. We conducted a pilot trial in 15 first-time cadaveric renal transplant patients undergoing for immunosuppression a 14-day course of LO-Tact-1 (10 mg i.v. daily) together with cyclosporine, low dose steroids (0.5 mg/kg) and azathioprine. Results showed a good immunosuppressive effect, as measured by the similar incidence of acute rejection episodes (0.6 per patient) when compared with 20 patients treated during the same period with our standard quadruple prophylactic combination with higher initial doses of steroids (2 mg/kg) and antilymphocyte globulin (ALG) instead of LO-Tact-1 (0.4 per patient). At 2 years post-transplant, graft survival was 93%, and only 1 patient lost his kidney by rejection. No local or general adverse effect of antibody administration was encountered, and haematological changes remained of minor importance. Local bacterial infection was observed in 3 patients, but viral diseases (including cytomegalovirus, CMV) remained exceptional. In contrast, severe clinical CMV infections occurred in 3 patients (15%) treated by ALG. Nine of 15 patients developed rat-specific antibodies, but only 4 before the completion of LO-Tact-1 treatment, without any correlation with the further development of acute rejection. Patients who suffered rejection had lower LO-Tact-1 levels and higher soluble IL2 receptor levels during the period of infusion, suggesting the crucial importance of pharmacokinetic monitoring to adjust individual doses.

Adrenal Cortex Hormones↗
PubMed1992

Allogenic recognition in 1992.

Many new data have been obtained in the last 4 years concerning the structure and functions of major histocompatibility complex (MHC) molecules. Structural data are summarized and the functions of MHC molecules in the presentation of peptides to T cells are described. The high selectivity of peptides binding to MHC molecules is discussed. The negative and positive selection of T cells in the thymus is described and the notion of repertoire introduced. The consequences of these new data on the understanding of H2 restriction and alloreactivity are described. The four potential types of alloreactivity are defined: i) house keeping gene (HKG) peptides bound to allogenic MHC molecules; ii) allogenic peptides (derived from allogenic MHC molecules) bound to allogenic MHC molecules; iii) empty allogenic MHC molecules; iv) allogenic peptides bound to autologous MHC molecules. In fact, the allogenic response is mostly directed toward HKG peptides bound to the allogenic MHC molecules of the graft cells (type 1). The potential role of type four alloreactivity in rejection is discussed.

Humans↗
PubMed1991

EBV cell-lines (LCL) and E- cells as stimulator cells for limiting dilution analysis (LDA) of alloreactive IL-2-secreting cells (IL-2-SC) and cytotoxic precursors (CTLp). Comparison of their stimulator capacity and ability to generate specific alloreactivity.

The choice of stimulator cells is crucial in determining the frequency of alloreactive cells by limiting dilution analysis (LDA). In humans, E- cells or EBV-lymphoblastoid cell lines (LCL) are available for this purpose. The E- cells have been mostly used until now, but they appear to stimulate much less than LCL in other models. Consequently, we undertook a systematic comparison of the efficiency of both types of cell for the LDA of alloreactive IL-2-secreting cells (IL-2-SC) and cytotoxic precursors (CTLp). We show that LCL are stronger stimulator cells both for IL-2 secretion and for cytotoxicity induction. However, high levels of non-allogeneic reactivity were also induced. Therefore, to measure the frequency of specific alloreactive IL-2-SC and CTLp, T cells were depleted of such irrelevant reactive cells by a suicide technique: culture with autologous LCL in the presence of BUDR and use of the remaining live cells as responding effectors in LDA. Using this methodology, no non-allogeneic reactive T cells remain in the responding cells: after restimulation by autologous LCL, no IL-2-SC could be seen and no cytotoxic activity could be observed against autologous, irrelevant or LAK sensitive targets. In contrast, the number of IL-2-SC and CTLp against allogeneic targets was preserved. Therefore with this methodology, the strong stimulator capacity of LCL could be used whereas non-specific activation could be avoided in order to assess the frequency of allo-specific IL-2-SC and CTLp.

Bromodeoxyuridine↗
PubMed1990

Structural interpretation of the amino acid sequence of a second domain from the Artemia covalent polymer globin.

Artemia has a complex extracellular hemoglobin of Mr 260,000 comprising two globin chains (Mr 130,000) each of which is a polymer of eight covalently linked domains of Mr 16,000. The primary structure of this polymeric globin was studied to understand how globin folded domains are ordered within a globin chain and, in turn, how the latter associate into a functional hemoglobin molecule. Here we report the amino acid sequence of a second domain, E7 (Mr 16,081, excluding the heme), and interpretations of sequence data by computer-assisted alignment and modeling. This clearly shows that, as with domain E1 (Moens, L., Van Hauwaert, M.-L., De Smet, K., Geelen, D., Verpooten, G., Van Beeumen, J., Wodak, S., Alard, P., & Trotman, C. (1988) J. Biol. Chem. 263, 4679-4685), domain E7 is compatible with a globin folded structure of the beta-type chain. Several specific differences of domains E7 and E1 from the classic globins are identified. They possibly can be interpreted in terms of specific requirements for a double octameric functional molecule.

Amino Acid Sequence↗
PubMed1990

Persistence of donor-specific IL-2-secreting cells and cytotoxic T lymphocyte precursors in human kidney transplant recipients evidenced by limiting dilution analysis.

The low reactivity to donor alloantigens reported in PBL from kidney transplant recipients might be related to clonal deletion and/or suppression of donor-specific alloreactive cells. To discriminate between these two hypotheses, we quantified the number of IL-2 secreting cells (IL-2-SC) and of cytotoxic precursors (CTLp) in the T cells from tolerant recipients when stimulated with either donor specific or nonrelated third-party LCL. To eliminate the irrelevant reactivity, we used as responding cells high-density T cells that had been depleted of such reactivity by 4 days preculture with autologous lymphoblastoid cell line in the presence of bromodeoxyuridine. Thus, frequencies of IL-2-SC and CTLp specifically directed at alloantigens could be measured. In 11 recipients, there was no strong decrease in the frequency of donor-reactive T cells when compared to the frequency of those directed at a third-party lymphoblastoid cell line, either for IL-2-SC (tested in 11 patients) or for CTLp (tested in 6 patients). In three cases of seven, a suppression was observed only when T cells were stimulated by donor cells. These data suggest that donor-reactive cells are still present in PBL of kidney-transplant recipients tested from 6 mo to 4 y posttransplantation. Moreover, suppression of donor-specific cells can be demonstrated in peripheral T cells of some recipients, which may account in part for the absence of rejection.

Histocompatibility↗
PubMed1990

Comparative molecular modeling of Amphioxus calcium vector protein with calmodulin and troponin C.

Calcium vector protein (CaVP), a new protein isolated from Amphioxus muscle, binds in a Ca2(+)-regulated manner to a 27 kd target protein, named CaVPT, whose function has not been elucidated yet. CaVP bears significant sequence homology to both calmodulin and skeletal muscle troponin C, especially in the C-terminal half of the molecule, which presumably contains the two functional Ca2(+)-binding sites. The N-terminal half contains two abortive EF-hands and is intramolecularly crosslinked with a disulfide bond. Using the crystallographic structures of calmodulin and striated muscle troponin C as a framework, we constructed two different three-dimensional models of CaVP and modeled the intramolecular disulfide bridge. The modeling based upon the coordinates of calmodulin yields a Ca2(+)-filled sites configuration in the N-terminal half of the molecule, even though no Ca2+ is bound in this half, whereas the troponin C-derived model generates a Ca2(+)-empty sites configuration. The models predict that neither is the Ca2(+)-filled nor in the Ca2(+)-empty sites conformation is there any steric and/or energetic obstacle for the formation of the disulfide bridge and that the disulfide bond is poorly accessible to reducing reagents. The optical properties of the Trp and Tyr residues of CaVP indicate that the calmodulin-derived model represents the most plausible prediction.

Amino Acid Sequence↗
PubMed1990

Decreased lymphokine-activated killer cells in kidney transplant recipients. Correlation with a diminished number of CD3-/NKH1+ cells.

The activity of lymphokine-activated killer cells, measured either by a clonal or polyclonal technique, was assessed in 30 kidney transplant recipients (TX), in 13 hemodialyzed patients (HD-CRI), and in 18 normal (N) controls. A highly significant decrease of the LAK activity in TX in comparison with HD-CRI or N (P = 0.0001) was observed. Moreover, the percentage of CD3-/NKH1+ cells was decreased in TX in comparison with N (P = 0.01). LAK activity was strongly correlated (r = 0.72; P = 0.0001) with the percentage of CD3-/NKH1+ cells and not with that of double-positive CD3+/NKH1+ cells. Multivariate analysis showed that the sole independent variable that determined the LAK activity was the percentage of CD3-/NKH1+ cells: the pathological status (TX, HD-CRI, and N) variable was statistically not significant. On the other hand, two T cell-specific functions (IL-2 secretion and specific cytotoxic activity) were, on the whole, preserved in TX. Altogether, these results suggest that TX are LAK deficient predominantly because they have a decreased number of CD3-/NKH1+ cells. The normality of T cell functions suggests that the high rate of malignancies seen in TX is related to this LAK deficiency. Moreover, our study indicates that, in vivo, CD3+ cells do not significantly contribute to the LAK precursors.

Antigens, CD↗
PubMed1988

A structural domain of the covalent polymer globin chains of artemia. Interpretation of amino acid sequence data.

Artemia is unusual in having extracellular hemoglobins of Mr 260,000 comprising two globin chains (Mr 130,000), each of which is a polymer of eight covalently linked domains of about Mr 16,000. The amino acid sequence of one of these domains (E1) has been determined. It has 147 residues and Mr of 17,574 including heme. Sequence alignment revealed 19.0% identity with sperm whale myoglobin, whereas other vertebrate and invertebrate globins had between 13 and 24% identity. However, a much higher percentage of residues has a similar side chain character, suggesting that the domain E1 is very similar to other globins in showing the myoglobin fold. Template model building based on the known three-dimensional structure of myoglobin further supports this conclusion. Conversely, the differences between E1 and other globins are believed to reflect differences in the packing of the domains, first in a covalent polymeric subunit containing eight hemes and subsequently by association of two of these subunits as dimers. These findings provide further evidence for the versatility of the myoglobin fold.

Amino Acid Sequence↗
PubMed1988

Structural analysis of the 2.8 A model of Xylose isomerase from Actinoplanes missouriensis.

The structure of Xylose isomerase (X.I.) from Actinoplanes missouriensis has been solved to 2.8 Angstroms resolution. Phases were determined from a single Eu3+ derivative and from the noncrystallographic 222 symmetry of the tetrameric molecule. An atomic model was built and subjected to restrained crystallographic refinement. The resulting model is shown to be closely similar to the recently reported X.I.'s structures from three other bacterial sources. Each monomer is found to be composed of an eight-stranded alpha/beta "T.I.M." barrel forming an N-terminal domain of 328 residues followed by a large loop of 66 residues embracing an adjacent subunit. Analysis of intersubunit packing shows that the X.I. tetramer is an assembly of two tight dimers. The beta barrel fits a simple hyperboloid model as other T.I.M. barrels do. The active site, identified as the binding site for the inhibitor xylitol, is located at the carboxyl end of the beta strands in the barrel next to a pair of binding sites for Eu3+ ions, which are assumed to be sites for the divalent ions involved in catalysis. Active sites in the tetramer are oriented towards the interface between dimers. It is suggested that subunit interfaces might stabilize the active site region and this might explain the oligomeric nature of other alpha/beta barrel enzymes.

Actinomycetales↗
PubMed1988

Structural principles of parallel beta-barrels in proteins.

Eight-stranded beta-sheets in nine protein structures containing "TIM (triose phosphate isomerase) barrels" are shown to be fitted satisfactorily by hyperboloids, the generating lines of which pass through the beta-strands. Simple parameterizations of the hyperboloid model are then used to determine the constraints that govern key parameters, such as the number of strands in the barrel, and to rationalize the remarkable conservation of strand number, observed to be eight, in nearly all the known examples of parallel beta-barrels. It is shown that the requirement to exclude solvent from the barrel interior, while at the same time keeping an upper limit on strand twist and interstrand distance so as to foster extensive hydrogen bonding interactions within the sheet, imposes strong constraints on barrel geometry. A formal description of the relationships between beta-sheet twist, strand number, and barrel dimensions is given here. It could have important implications for studies of protein folding and design.

Computer Simulation↗