SEARCH · PubMed Health

Results for “Functional characterization”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 703 records · Page 39Linked to original sources

PubMed2004

Molecular and functional characterization of novel CRFR1 isoforms from the skin.

In our continued studies on corticotropin releasing factor receptor (CRFR1) signaling in the skin, we tested functional activity of CRFR1alpha, e, f, g and h isoforms after transfection to COS cells. Both membrane-bound and soluble variants are translated in vivo into final protein products that undergo further post-translational modifications. CRFR1alpha was the only isoform coupled directly to adenylate cyclase with the exception of an artificial isoform (CRFR1h2) with the insertion of 37 amino acids between the ligand binding domain and the first extracellular loop that was capable of producing detectable levels of cyclic AMP (cAMP). Soluble isoforms could modulate cell response with CRFR1e attenuating and CRFR1h amplifying CRFR1alpha-coupled cAMP production stimulated by urocortin. Testing with plasmids containing the luciferase reporter gene, and inducible cis-elements (CRE, CaRE, SRE, AP1 or NF-kappaB) demonstrated that only CRFR1alpha was involved directly in the transcriptional regulation, while CRFR1g inhibited CRE activity. Significantly higher reporter gene expression by CRF was observed than that mediated by 4beta-phorbol 12-myristate 13-acetate and forskolin alone, being compatible with the concomitant treatment by phorbol 12-myristate 13-acetate and forskolin. This suggests that both protein kinase A and C can be involved in CRF-dependent signal transduction.

Adenylyl Cyclases↗

PubMed2003

Functional characterization of a composite bacteriocin locus from malt isolate Lactobacillus sakei 5.

Lactobacillus sakei 5, isolated from malted barley, produces three bacteriocins. Genetic and functional analysis of the purified bacteriocins showed that this strain produces a plasmid-encoded bacteriocin that is identical to sakacin P, as well as two novel, chromosomally encoded bacteriocins, which were designated sakacin T and sakacin X. The structural genes specifying sakacin T and sakacin X are part of the sakacin TX locus, which consists of two adjacent but divergently oriented gene clusters. The first gene cluster includes stxP, stxR, stxK, and stxT, which, based on functional and comparative sequence analysis, are believed to encode an inducing peptide and proteins involved in regulation and secretion of these bacteriocins. The second gene cluster includes the structural and immunity genes for sakacin T, a class IIb two-peptide bacteriocin composed of SakTalpha and SakTbeta, and sakacin X, a class IIa bacteriocin. Interestingly, a so-called transport accessory protein was absent from the locus, and based on our results it appears that a dedicated accessory protein is not required for processing and transport of sakacin T and sakacin X.

Amino Acid Sequence↗

PubMed1997

Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]o regulation.

The neuronal K-Cl cotransporter isoform (KCC2) was functionally expressed in human embryonic kidney (HEK-293) cell lines. Two stably transfected HEK-293 cell lines were prepared: one expressing an epitope-tagged KCC2 (KCC2-22T) and another expressing the unaltered KCC2 (KCC2-9). The KCC2-22T cells produced a glycoprotein of approximately 150 kDa that was absent from HEK-293 control cells. The 86Rb influx in both cell lines was significantly greater than untransfected control HEK-293 cells. The KCC2-9 cells displayed a constitutively active 86Rb influx that could be increased further by 1 mM N-ethylmaleimide (NEM) but not by cell swelling. Both furosemide [inhibition constant (Ki) approximately 25 microM] and bumetanide (Ki approximately 55 microM) inhibited the NEM-stimulated 86Rb influx in the KCC2-9 cells. This diuretic-sensitive 86Rb influx in the KCC2-9 cells, operationally defined as KCC2 mediated, required external Cl- but not external Na+ and exhibited a high apparent affinity for external Rb+(K+) [Michaelis constant (Km) = 5.2 +/- 0.9 (SE) mM; n = 5] but a low apparent affinity for external Cl- (Km > 50 mM). On the basis of thermodynamic considerations as well as the unique kinetic properties of the KCC2 isoform, it is hypothesized that KCC2 may serve a dual function in neurons: 1) the maintenance of low intracellular Cl- concentration so as to allow Cl- influx via ligand-gated Cl- channels and 2) the buffering of external K+ concentration ([K+]o) in the brain.

Amino Acid Sequence↗

PubMed1999

Molecular cloning and functional characterization of a polyspecific organic anion transporter from Caenorhabditis elegans.

We have cloned a polyspecific organic anion transporter from Caenorhabditis elegans and elucidated its functional characteristics. The C. elegans anion transporter (CeOAT1) codes for a protein of 526 amino acids containing 12 putative transmembrane domains. It exhibits significant homology at the level of amino acid sequence to the C. elegans organic cation transporter and to the mammalian organic cation and anion transporters. The function of CeOAT1 was investigated by expressing the transporter heterologously in mammalian cells. CeOAT1 transports p-aminohippurate (PAH) in a Na(+)-independent manner. The transport mechanism appears to involve anion exchange because CeOAT1-mediated PAH transport is stimulated by a cell-to-medium concentration gradient of alpha-ketoglutarate or fumarate generated by coexpression in the cells of a mammalian Na(+)-coupled dicarboxylate transporter. CeOAT1 exhibits broad specificity, accepting anions such as folate, indomethacin, furosemide, probenecid, and benzylpenicillin as substrates. The Michaelis-Menten constant for the prototypical organic anion PAH is 0.43+/-0.07 mM. This constitutes the first report of the molecular and functional identification of a polyspecific organic anion transporter in C. elegans.

Animals↗

PubMed1986

Functional characterization of mast cells generated in vitro from the mesenteric lymph node of rats infected with Nippostrongylus brasiliensis.

We have examined the biochemical and functional characteristics of mast cells grown in tissue culture from the mesenteric lymph node (MLN) of rats infected with the nematode Nippostrongylus brasiliensis and compared them with mast cells isolated from the small intestinal mucosa and peritoneal cavity of infected animals. Cultured mast cells (MC) and isolated intestinal mucosal mast cells (MMC) had a similar histamine content, and both contained type II protease (RMCP II) which was absent from peritoneal mast cells (PMC). PMC, MMC and cultured MC each responded to immunologically induced histamine secretion, but MMC and cultured MC were hyporesponsive to calcium ionophores and unresponsive to widely used PMC secretagogues including compound 48/80 and bee venom peptide 401. MMC and cultured MC also differed from PMC in their lack of responsiveness to the anti-allergic agent disodium cromoglycate. Thus, MC cultured from the MLN are distinct from PMC but have a biochemical and functional phenotype similar to that of intestinal MMC.

Animals↗

PubMed1989

[Sodium conductin of the human brain: purification and functional characterization].

A sodium channel enriched preparation was obtained from human brain. Human sodium channel appeared as a heterocomplex peptide alpha beta 1 beta 2. Functional properties of the protein were maintained since, after reconstitution into liposomes, ion fluxes were sensitive to sodium channel specific toxins and to membrane potential. Moreover, the reconstituted protein showed a well defined ionic selectivity.

Brain Chemistry↗

PubMed1996

Immunotherapy for cancer: construction, expression and functional characterization of chimeric antibodies.

Monoclonal antibodies (Mabs) are a potential key component for the treatment of cancer, because of their specificity and multiple effector functions. Hybridoma technology and progress in genetic engineering made it possible to customize antibody molecules, rendering them more suitable for selective application. A widely used technique is the construction of mouse-human hybrid molecules by recombinant DNA techniques. These so-called chimeric antibodies contain the murine variable (V) regions fused to the human constant (C) regions. In this report, a general approach is described for the production of chimeric antibodies. The gene segments encoding the murine variable heavy and light chain are isolated by the polymerase chain reaction and cloned into expression vectors containing the human gamma 1 heavy chain gene and the human K light chain gene, respectively. Subsequently, these constructs are transfected into a non-Ig-producing murine hybridoma, eg SP2/0 cells. The in vitro study of the functional characteristics and biological properties of the thus obtained chimeric antibodies are discussed.

Antibodies, Monoclonal↗

PubMedDate not supplied

Structural and functional characterization of the human NBC3 sodium/bicarbonate co-transporter carboxyl-terminal cytoplasmic domain.

The sodium bicarbonate co-transporter, NBC3, is expressed in a range of tissues including heart, skeletal muscle and kidney, where it modulates intracellular pH and bicarbonate levels. NBC3 has a three-domain structure: 67 kDa N-terminal cytoplasmic domain, 57 kDa membrane domain and an 11 kDa C-terminal cytoplasmic domain (NBC3Ct). The role of C-terminal domains as important regulatory regions is an emerging theme in bicarbonate transporter physiology. This study determined the functional role of human NBC3Ct and characterized its structure using biochemical techniques. The NBC3 C-terminal domain deletion mutant (NBC3DeltaCt) had only 12 +/- 5% of wild-type transport activity. This low activity is attributable to low steady-state levels of NBC3DeltaCt and almost complete retention inside the cell, as assessed by immunoblots and confocal microscopy, suggesting a role of NBC3Ct in cell surface processing. To characterize the structure of NBC3Ct, amino acids 1127-1214 of NBC3 were expressed as a GST fusion protein (GST.NBC3Ct). GST.NBC3Ct was cleaved with PreScission Protease and native NBC3Ct could be purified to 94% homogeneity. Gel permeation chromatography and sedimentation velocity ultracentrifugation of NBC3Ct indicated a Stokes radius of 26 and 30 angstroms, respectively. Shape modelling revealed NBC3Ct as a prolate shape with long and short axes of 19 and 2 nm, respectively. The circular dichroism spectra of NBC3Ct did not change over the pH 6.2-7.8 range, which rules out a large change of secondary structure as a component of pH sensor function. Proteolysis with trypsin and chymotrypsin identified two proteolytically sensitive regions, R1129 and K1183-K1186, which could form protein interaction sites.

Amino Acid Sequence↗

PubMed1994

Functional characterization of the rat GAP-43 promoter.

GAP-43 is a highly conserved neuronal protein whose expression is spatially and temporally regulated. Because this regulation may occur, at least in part, at the level of transcription, we have begun to characterize the regions upstream of the GAP-43 transcription unit which direct its neuronal-specific expression. Functional analyses of GAP-43 promoter-reporter constructs have been performed in stably transfected cell lines, including PC12, C6 and RAT2. These data indicate that as little as 600 bp of GAP-43 5'-flanking DNA sequence directs the expression in a neuronal-specific manner. A shorter construct containing 230 bp of 5'-flanking DNA sequence defines a GAP-43 minimal promoter that is active in both neuronal and glial but not in non-neural cell lines. An upstream region, previously shown by other investigators to have promoter activity, was able to stimulate transcription when linked to the downstream minimal promoter. However, this upstream region was by itself unable to direct transcription of the reporting gene. In addition, we have demonstrated that two polypurine tracts within the 5'-flanking DNA sequence of the GAP-43 gene adopt a non-duplex configuration in plasmids, and, when studied in the context of chromosomal integration, these regions have a stimulatory effect on transcription.

Animals↗

PubMed2003

Molecular and functional characterization of the first nucleobase transporter gene from African trypanosomes.

African trypanosomes are unable to synthesize purines and depend upon purine nucleoside and nucleobase transporters to salvage these compounds from their hosts. To understand the crucial role of purine salvage in the survival of these parasites, a central objective is to identify and characterize all of the purine permeases that mediate uptake of these essential nutrients. We have cloned and functionally expressed in a purine nucleobase transport deficient strain of Saccharomyces cerevisiae a novel nucleobase transporter gene, TbNT8.1, from Trypanosoma brucei. The permease encoded by this gene mediates the uptake of hypoxanthine, adenine, guanine, and xanthine with Kms in the low micromolar range. The TbNT8.1 protein is a member of the equilibrative nucleoside transporter (ENT) family of permeases that occur in organisms as diverse as protozoa and mammals. TbNT8.1 is distinct from other ENT permeases that have been identified in trypanosomes in utilizing multiple purine nucleobases, rather than purine nucleosides, as substrates and is hence the first bona fide nucleobase permease identified in these parasites. Furthermore, unlike the mRNAs for other purine transporters, TbNT8.1 mRNA is significantly more abundant in insect stage procyclic forms than in mammalian stage bloodstream forms, and the TbNT8.1 permease thus may represent a major route for purine nucleobase uptake in procyclic trypanosomes.

Amino Acid Sequence↗

PubMed1998

Molecular and functional characterization of a recombinant protein of Trichuris trichiura.

The pore-forming protein of the human whipworm, Trichuris trichiura, has been postulated to facilitate invasion of the host gut and enable the parasite to maintain its syncytial environment. The data presented here describe the first, to our knowledge, molecular characterization of a pore-forming protein in any helminth and provide a unique demonstration of the functional interaction between a parasite antigen and host molecules. Immunological screening of a T. trichiura cDNA library with T. trichiura infection sera identified a clone of 1.4 kB, the cDNA consisting of 1495 base pairs encoding a protein of 50 kDa. The sequence has a highly repetitive nature containing nine four-disulphide-bonded core domains. Structural prediction analyses reveals an amphipathic nature. TT50 induced pore formation in bilayers in a manner identical to that of the native protein. IgG antibody isolated from T. trichiura infection serum was observed to abolish channel activity.

Amino Acid Sequence↗

PubMed1975

[Significance of some coefficients resulting from dye photometry in the functional characterization of mitral defects].

One hundred and eighty six patients with mitral defects were examined and subdivided into four groups according to the basic clinical and instrumental tests: 59 with pure, 46 with prevailing mitral stenosis, 37 with prevailing and 44 with pure mitral insufficiency. The patients, within the pointed group were subdivided into subgroups, depending on the character of the auricular rhythm, the presence of accompanying tricuspid insufficiency with hemodynamic significance and cardiac decompensation. The circilatory times, TA, TD and TC were calculated for all the patients and especially the coefficients (see article) from the dye dilution curves. The analysis covers only patients from the different groups without other defects and cardiac decompensation. Fluctuations of the minimal and maximal values of TA, K1, K2, and K3 are presented. Their average values are calculated as well as the square deviations. In sinus rhythm and compensated heart, the combination of the values for K1 over 1,80 and K2 over 1,50 in prevailing and for K1 over 1,90 and K2 over 1,60 in pure mitral insufficiency might be used as a criterion for differentiation of pure and prevailing mitral stenosis. The close values in pure and prevailing mitral stenosis, as well as in pure and prevailing mitral insufficiency do not present a possibility to be used in the differentiation of the groups with hemodynamic significance of stenosis and insufficiency. In arrhythmia absoluta, accompanying tricuspid insufficiency and cardiac decompensation, the coefficient K1 and K2 have no value in mitral defect characterization. The close average values of TA and K3 for all patient groups devaluate their importance as functional indices of mitral defects.

Arrhythmias, Cardiac↗

PubMed1998

Functional characterization of PM6/13, a beta3-specific (GPIIIa/CD61) monoclonal antibody that shows preferential inhibition of fibrinogen binding over fibronectin binding to activated human platelets.

We report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 microg/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 microg/ml) and U46619 (3 microM) with EC50 = 4, 8 and 4 microg/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GPIIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55 kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

Antibodies, Monoclonal↗

PubMed1987

Phenotypic and functional characterization of recombinant interleukin 2 (rIL 2)-induced activated killer cells: analysis at the population and clonal levels.

In these studies we investigated the phenotypic and functional characteristics of human rIL 2-activated killer cells (LAK). By FACS sorting we separated PBL into Leu-11- and Leu-11+ cell fractions and cultured them for 4 days in 100 U/ml rIL 2. Under these culture conditions, cells of the Leu-11+ fraction acquired a stronger LAK activity against fresh autologous or allogeneic melanoma cells as compared with Leu-11- cells or unfractionated PBL. To better characterize the cells responsible for this cytolytic activity, we directly cloned Leu-11+ and Leu-11- FACS-sorted cells in the presence of 1% PHA, irradiated spleen feeder cells, and rIL 2. From 6 to 10% of the Leu-11+ cells and from 42 to 66% of the Leu-11- cells plated gave rise to clonal progenies that were tested simultaneously for cytolytic activity against fresh melanoma cells and NK-sensitive K562 target cells in a 4-hr 51Cr-release assay. Most of the Leu-11+ microcultures lysed fresh melanoma target cells (35 out of 38 and 26 out of 34 in two separate experiments), whereas only a few clones derived from the Leu-11- cell fraction had this capability (four out of 45 and one out of 41). All the clones lysing fresh melanoma cells also efficiently killed K562 target cells, whereas other clones lysing only K562 could be found among Leu-11+ and Leu-11- clones. Nine clones expressing LAK activity were tested for their reactivity against a panel of different tumor target cells. All clones were able to lyse a broad panel of target cells including NK-sensitive and NK-resistant cultured or noncultured human tumor target cells, as well as mouse tumor cell lines. Surface marker analysis of 14 clones displaying LAK activity, all derived from Leu-11+ cells, showed that they were all T3 (CD3)-, whereas 10 out of 14 expressed the T11 (CD2) antigen and only four were weakly stained by an anti-T8 (CD8) mAb. All 14 clones expressed the T40 (CD7) T cell marker and DR and LFA-1 antigens. Cytolysis inhibition experiments performed on a rIL 2-activated Leu-11+ population and on two LAK cell clones, both expressing T11 antigen, showed that anti-LFA-1 but not anti-T11 mAb could inhibit cytolysis of freshly derived tumor target cells.

Antibodies, Monoclonal↗

PubMed2003

Functional characterization of phytochrome interacting factor 3 in phytochrome-mediated light signal transduction.

Phytochromes regulate various light responses through their interactions with different signaling proteins, such as phytochrome interacting factor 3 (PIF3). However, the physiological functions of PIF3 in light signaling are not yet fully understood. To increase our understanding of these roles, we characterized a T-DNA insertional pif3 mutant and transgenic plants overexpressing the full-length PIF3. Transgenic overexpressing lines displayed longer hypocotyls and smaller cotyledons under red light and reduced cotyledon opening under both red and far-red light, whereas the pif3 mutant showed the opposite phenotypes. The accumulation of anthocyanin and chlorophyll further indicated complicated features of PIF3 function. The accumulation of anthocyanin was increased and the content of chlorophyll was decreased in the overexpression lines. Our data indicate that PIF3 plays complex roles depending on the type of light response and the light conditions.

Arabidopsis↗

PubMed2006

In vitro identification and functional characterization of glial precursor cells in human gliomas.

Human gliomas including astrocytomas and oligodendrogliomas are defined as being composed of neoplastic astrocytes and oligodendrocytes respectively. Here, on the basis of in vitro functional assays, we show that gliomas contain a mixture of glial progenitor cells and their progeny. We have set up explant cultures from pilocytic astrocytomas, glioblastomas and oligodendrogliomas and studied antigens that characterize glial lineage, from the precursor cells (glial restricted precursors and oligodendrocyte-type2-astrocyte/oligodendrocyte precursor cells expressing the A2B5 ganglioside) to the differentiated cells (oligodendrocyte and type-1 and type-2 astrocytes). All tumoral explants contain A2B5+ cells and can generate migrating cells with distinctive functional properties according to glioma subtypes. In pilocytic astrocytomas, very few migrating cells are dividing and can differentiate in type-2 astrocytes or towards the oligodendrocyte lineage. In glioblastomas, most migrating cells are dividing, express A2B5 or glial fibrillary acid protein (GFAP) and can generate oligodendrocytes and type-1 and type-2 astrocytes in appropriate medium. Oligodendroglioma explants are made by actively dividing glial precursor cells expressing A2B5 or PSA-NCAM. Only few cells can migrate and differentiation towards oligodendrocyte lineage does not occur. Isolated A2B5+ cells from both glioblastomas and oligodendrogliomas showed similar genetic alterations as the whole tumour. Therefore, pilocytic astrocytomas contain slowly dividing oligodendrocyte-type2-astrocyte/oligodendrocyte precursor cells in keeping with their benign behaviour whereas both glioblastomas and oligodendrogliomas contain neoplastic glial restricted precursor cells. In oligodendrogliomas, these cells are trapped in undifferentiated and proliferating state. The precursor cells properties present in gliomas give new insight into their histogenesis and open up new avenues for research in the field of gliomagenesis.

Adult↗

PubMed2006

First identification of a chemotactic receptor in an invertebrate species: structural and functional characterization of Ciona intestinalis C3a receptor.

In mammals, the bioactive fragment C3a, released from C3 during complement activation, is a potent mediator of inflammatory reactions and exerts its functional activity through the specific binding to cell surface G protein-coupled seven-transmembrane receptors. Recently, we demonstrated a Ciona intestinalis C3a (CiC3a)-mediated chemotaxis of hemocytes in the deuterostome invertebrate Ciona intestinalis and suggested an important role for this molecule in inflammatory processes. In the present work, we have cloned and characterized the receptor molecule involved in the CiC3a-mediated chemotaxis and studied its expression profile. The sequence, encoding a 95,394 Da seven-transmembrane domain protein, shows the highest sequence homology with mammalian C3aRs. Northern blot analysis revealed that the CiC3aR is expressed abundantly in the heart and neural complex and to a lesser extent in the ovaries, hemocytes, and larvae. Three polyclonal Abs raised in rabbits against peptides corresponding to CiC3aR regions of the first and second extracellular loop and of the third intracellular loop react specifically in Western blotting with a single band of 98-102 kDa in hemocyte protein extracts. Immunostaining performed on circulating hemocytes with the three specific Abs revealed that CiC3aR is constitutively expressed only in hyaline and granular amoebocytes. In chemotaxis experiments, the Abs against the first and second extracellular loop inhibited directional migration of hemocytes toward the synthetic peptide reproducing the CiC3a C-terminal sequence, thus providing the compelling evidence that C. intestinalis expresses a functional C3aR homologous to the mammalian receptor. These findings further elucidate the evolutionary origin of the vertebrate complement-mediated proinflammatory process.

Amino Acid Sequence↗

PubMed1986

Functional characterization of infiltrating T lymphocytes in human hepatic allografts.

We have employed recently developed techniques in T-cell culturing to study the nature and function of infiltrating hepatic allograft T cells. Using the rationale that intragraft T cells are activated during cell mediated damage to the allograft, we were able to show that these cells would propagate and remain functionally active in the presence of the T-cell growth factor, IL-2. In several instances, phenotypic analysis of cells grown in this manner was very similar to that found within the graft. Both proliferative and cytotoxic responses could be detected from the cultured cell lines. The majority of the proliferative responses were donor-directed and immunogenetic analysis could define donor-directed HLA reactivity, to either class I or class II antigens, or both. Monoclonal anti-HLA antibodies inhibition profiles verified the apparent HLA reactivity. In a smaller percentage of cases, only IL-2 responsiveness could be detected, and no HLA reactivity could be determined. Cytotoxicity could be detected against both class I and class II antigens, however, those cells which demonstrated a greater magnitude of donor-directed cytotoxicity appeared to be directed against class I antigens. A significant correlation between donor-directed proliferation of biopsy cultured lymphocytes and cellular rejection was found. This model appears to be useful in delineating functions of the intragraft T-cell population during rejection.

Adolescent↗