Biomedical subjects

A C Cunningham

Publications and source records attributed to A C Cunningham.

At least 19 recordsLinked to original sources

PubMed2006

A GPI-anchored co-receptor for tissue factor pathway inhibitor controls its intracellular trafficking and cell surface expression.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) lacks a membrane attachment signal but it remains associated with the endothelial surface via its association with an, as yet, unidentified glycosyl phosphatidylinositol (GPI)-anchored co-receptor. OBJECTIVES/METHODS: Cellular trafficking of TFPI within aerolysin-resistant ECV304 and EA.hy926 cells, which do not express GPI-anchored proteins on their surface, was compared with their wild-type counterparts. RESULTS AND CONCLUSIONS: Although aerolysin-resistant cells produce normal amounts of TFPI mRNA, TFPI is not expressed on the cell surface and total cellular TFPI is greatly decreased compared with wild-type cells. Additionally, normal, not increased, amounts of TFPI are secreted into conditioned media indicating that TFPI is degraded within the aerolysin-resistant cells. Confocal microscopy and studies using metabolic inhibitors demonstrate that aerolysin-resistant cells produce TFPI and transport it into the Golgi with subsequent degradation in lysosomes. The experimental results provide no evidence that cell surface TFPI originates from secreted TFPI that binds back to a GPI-anchored protein. Instead, the data suggest that TFPI tightly, but reversibly, binds to a GPI anchored co-receptor in the ER/Golgi. The co-receptor then acts as a molecular chaperone for TFPI by trafficking it to the cell surface of wild-type cells or to lysosomes of aerolysin-resistant cells. TFPI that escapes co-receptor binding is secreted through the same pathway in both wild-type and aerolysin-resistant cells. The data provide a framework for understanding how TFPI is expressed on endothelium.

Bacterial Toxins↗

PubMed2001

Genomic organization of the CC chemokine mip-3alpha/CCL20/larc/exodus/SCYA20, showing gene structure, splice variants, and chromosome localization.

We describe the genomic organization of a recently identified CC chemokine, MIP3alpha/CCL20 (HGMW-approved symbol SCYA20). The MIP-3alpha/CCL20 gene was cloned and sequenced, revealing a four exon, three intron structure, and was localized by FISH analysis to 2q35-q36. Two distinct cDNAs were identified, encoding two forms of MIP-3alpha/CCL20, Ala MIP-3alpha/CCL20 and Ser MIP-3alpha/CCL20, that differ by one amino acid at the predicted signal peptide cleavage site. Examination of the sequence around the boundary of intron 1 and exon 2 showed that use of alternative splice acceptor sites could give rise to Ala MIP-3alpha/CCL20 or Ser MIP-3alpha/CCL20. Both forms of MIP-3alpha/CCL20 were chemically synthesized and tested for biological activity. Both flu antigen plus IL-2-activated CD4(+) and CD8(+) T lymphoblasts and cord blood-derived dendritic cells responded to Ser and Ala MIP-3alpha/CCL20. T lymphocytes exposed only to IL-2 responded inconsistently, while no response was detected in naive T lymphocytes, monocytes, or neutrophils. The biological activity of Ser MIP-3alpha/CCL20 and Ala MIP-3alpha/CCL20 and the tissue-specific preference of different splice acceptor sites are not yet known.

Alternative Splicing↗

PubMed2001

Primary human alveolar epithelial cells can elicit the transendothelial migration of CD14+ monocytes and CD3+ lymphocytes.

The ability of freshly isolated primary human alveolar epithelial cells (type II pneumocytes) to induce leucocyte migration across an endothelial monolayer was investigated. Three-way factorial analysis of variance (ANOVA) demonstrated that resting alveolar endothelial cells (AEC) could produce detectable quantities of monocyte chemoattractant protein 1 (MCP-1), which was upregulated in response to tumour necrosis factor-alpha (TNF-alpha) in a dose- and time-dependent fashion. Interferon-gamma (IFN-gamma) had no significant effect on this process. TNF-alpha and IFN-gamma both induced AEC to provoke migration of CD14+ monocytes and CD3+ lymphocytes across endothelium. IFN-gamma and TNF-alpha synergized in their ability to induce production of T lymphocyte, but not monocyte, chemoattractants from AEC. Leucocyte transendothelial migration was inhibited by anti-MCP-1 neutralizing antibody and by heparin, a polyanionic glycosaminoglycan (GAG). These data suggest that human AEC play a role in the multiple mechanisms that facilitate monocyte and T lymphocyte migration into the alveolar compartment of the lung under homeostasis and inflammatory conditions. One of these mechanisms is mediated via constitutive MCP-1 production by alveolar epithelial cells, which is upregulated by TNF-alpha.

CD3 Complex↗

PubMed2000

Intragraft proliferating T lymphocytes are associated with moderate acute pulmonary rejection.

Acute allograft rejection is characterized by infiltration of the donor organ by host lymphoid cells, predominantly T lymphocytes. However, the site of proliferation and clonal expansion of alloreactive T lymphocytes is not well defined in man. A group of normal transbronchial biopsies (TBB, n=9) from clinically well lung transplant recipients was compared to TBB showing acute rejection (at least grade A2, n=9), using CD3- and Ki67-specific antibodies to double-label proliferating T lymphocytes. Few double-labeled lymphocytes were present in the normal biopsies (range, 0-3 cells). However, five of the rejection biopsies contained significant numbers of proliferating T lymphocytes (range, 19-47; Fisher's exact test; P=0.029). Furthermore, this positive group contained all three cases of grade A3 rejection in the study, as well as a case with persistent grade A2 rejection on follow-up biopsy. These data demonstrate that T lymphocytes do proliferate in transplanted human lungs; such proliferation is associated with more severe rejection.

Acute Disease↗

PubMed1999

UK-78,282, a novel piperidine compound that potently blocks the Kv1.3 voltage-gated potassium channel and inhibits human T cell activation.

1. UK-78,282, a novel piperidine blocker of the T lymphocyte voltage-gated K+ channel, Kv1.3, was discovered by screening a large compound file using a high-throughput 86Rb efflux assay. This compound blocks Kv1.3 with a IC50 of approximately 200 nM and 1:1 stoichiometry. A closely related compound, CP-190,325, containing a benzyl moiety in place of the benzhydryl in UK-78,282, is significantly less potent. 2 Three lines of evidence indicate that UK-78,282 inhibits Kv1.3 in a use-dependent manner by preferentially blocking and binding to the C-type inactivated state of the channel. Increasing the fraction of inactivated channels by holding the membrane potential at - 50 mV enhances the channel's sensitivity to UK-78,282. Decreasing the number of inactivated channels by exposure to approximately 160 mM external K+ decreases the sensitivity to UK-78,282. Mutations that alter the rate of C-type inactivation also change the channel's sensitivity to UK-78,282 and there is a direct correlation between tau(h) and IC50 values. 3. Competition experiments suggest that UK-78,282 binds to residues at the inner surface of the channel overlapping the site of action of verapamil. Internal tetraethylammonium and external charybdotoxin do not compete UK-78,282's action on the channel. 4. UK-78,282 displays marked selectivity for Kv1.3 over several other closely related K+ channels, the only exception being the rapidly inactivating voltage-gated K+ channel, Kv1.4. 5. UK-78,282 effectively suppresses human T-lymphocyte activation.

Animals↗

PubMed1998

Differential effects of cyclosporin A and tacrolimus on the production of TGF-beta: implications for the development of obliterative bronchiolitis after lung transplantation.

The development of obliterative bronchiolitis is a common cause for failure of lung allografts. Fibrinogenesis can occur for a number of different reasons but some groups have suggested that cyclosporin A (CsA) and tacrolimus (FK506) have different effects on the cytokines which induce fibrinogenesis. We investigated the effect of tacrolimus and CsA in tissue culture and found that there was indeed a negative effect on human lung small airway epithelial cell proliferation by recombinant transforming growth factor-beta (TGF-beta), which was reversed by anti-TGF-beta. The same effect was seen with CsA at immunosuppressive concentrations, which was also reversed by anti-TGF-beta, whereas no such inhibition was seen with tacrolimus at immunosuppressive doses unless high concentrations were used. Free TGF-beta was confirmed as being elevated in the supernatant of cell culture wells with standard dose CsA as opposed to low dose CsA or tacrolimus using an ELISA assay.

Bronchiolitis Obliterans↗

PubMed1997

A comparison of the antigen-presenting capabilities of class II MHC-expressing human lung epithelial and endothelial cells.

Human lung alveolar epithelial cells constitutively express class II major histocompatibility complex (MHC). Human lung microvascular endothelial and small airway epithelial cells can be induced to express class II MHC by stimulation with the pro-inflammatory cytokine interferon-gamma. The levels of class II MHC on lung epithelial and endothelial cells were comparable to those seen on an Epstein-Barr virus (EBV)-transformed B-cell line. However, the costimulatory molecules B7-1 and B7-2 were not expressed. The ability of the class II MHC expressing human lung parenchymal cells to present alloantigen to CD4+ T lymphocytes was investigated. Freshly isolated human alveolar epithelial cells (type II pneumocytes) and monolayers of interferon-gamma-stimulated small airway epithelial and lung microvascular endothelial cells were co-cultured with allogeneic CD4+ T lymphocytes and proliferation determined by [3H]thymidine incorporation. A clear difference was observed between effects of the epithelial and endothelial cells on CD4+ T-lymphocyte activation. Alveolar and small airway epithelial cells failed to stimulate the proliferation of allogeneic CD4+ T lymphocytes whereas lung microvascular endothelial cells did stimulate proliferation. This difference could not be explained by the levels of class II MHC or the lack of B7-1 and B7-2 solely. Microvascular endothelial cells, and not alveolar or small airway epithelial cells, possess B7-independent costimulatory pathways.

Antigen Presentation↗

PubMed1996

Human CD8 alpha expression in NK cells but not cytotoxic T cells of transgenic mice.

In our previous work, DNase hypersensitivity mapping was used to identify an enhancer within the human CD8 alpha (hCD8 alpha) gene which allowed T cell-specific expression of a reporter construct in transiently transfected cell lines. To study the role of this intronic enhancer in vivo, transgenic mice were made using human CD8 genomic constructs. We found that while a 14 kb wild-type human CD8 alpha (WThCD8) genomic construct did not lead to expression in mature peripheral CD8+ T cells, this transgene was consistently expressed in small populations of T cells and B cells, and in a subset of mouse NK cells. While murine CD8 is not normally expressed on resting NK cells, expression of the human CD8 transgene on mouse NK cells is appropriate since CD8 is expressed on a subset of human NK cells. Deletion of the intronic enhancer resulted in a complete loss of transgene expression in most lines and a loss of expression only in NK cells in one line. Our results indicate, firstly, that cis-acting sequences within the 14 kb genomic fragment are sufficient for NK cell-specific expression. In addition, our results suggest that the enhancer may have dual roles in regulation of transgene expression. It may enhance general expression of the transgene and may also be required for NK cell-specific expression.

Animals↗

PubMed1995

Regulation and function of adhesion molecule expression by human alveolar epithelial cells.

The role of major histocompatibility complex (MHC) and adhesion molecule expression by alveolar epithelium on the modulation of immune responses in the lung is not understood. We have developed efficient methods to isolate, purify and culture human alveolar epithelial cells (type II pneumocytes) in vitro. The expression of MHC and adhesion molecules by isolated, cultured and cytokine-stimulated alveolar epithelial cells was quantified by flow cytometry, and demonstrated the presence of T-cell ligands including class I MHC, HLA-DR and HLA-DP, intracellular adhesion molecule-1 (ICAM-1; CD54) and lymphocyte function-associated antigen (LFA-3; CD58), but not vascular cell adhesion molecule-1 (VCAM-1) (CD106) or B7 (CD80). The proinflammatory cytokine interferon-gamma (IFN-gamma) caused an up-regulation of class I MHC and ICAM-1. In contrast, tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) had little effect on the expression of these surface antigens by human alveolar epithelial cells. The functional activity of alveolar epithelial adhesion molecules was then studied by determining their ability to bind allogeneic lymphocytes. An increase in lymphocyte adherence to monolayers of alveolar epithelial cells was observed following in vitro activation. However, up-regulation of alveolar epithelial counter receptors with the proinflammatory cytokine gamma-IFN did not enhance adhesion. The adhesive interaction between CD18 on lymphocytes and ICAM-1 on alveolar epithelial cells was demonstrated by the use of blocking antibodies specific for both ligands. Blockade of LFA-3 on alveolar monolayers also suppressed lymphocyte adherence. In conclusion, alveolar epithelial cells expressed MHC HLA-A, B, C, HLA-DR and -DP, and functional adhesion molecules including ICAM-1 and LFA-3.

Cell Adhesion↗

PubMed1995

Alloantigen presentation by B cells: analysis of the requirement for B-cell activation.

This paper describes a model for investigation of the functional implications of B-cell activation for antigen presentation. Mixed lymphocyte cultures were used to assess the ability of freshly isolated B cells, mitogen-activated B cells and Epstein-Barr virus (EBV)-transformed B-cell lines to stimulate the activation and proliferation of allogeneic T cells under a variety of experimental conditions. It was found that resting B cells presented antigen poorly, while activated cells were highly immunogenic. Paraformaldehyde fixation completely eliminated antigen presentation by resting B cells, despite constitutive expression of class II MHC antigens. However, fixation had little effect on antigen presentation by activated B cells that expressed B7-1 and B7-2 in addition to class II major histocompatibility complex (MHC) molecules. Arrest of B-cell activation by serial fixation after treatment with F(ab')2 fragments of goat anti-human IgM produced cells with variable antigen-presenting capacity. Optimal antigen presentation was observed for cells fixed 72 hr after the initiation of B-cell activation. Although both B7-1 and B7-2 antigen expression increased after B-cell activation, it was found that the rate of T-cell proliferation correlated most closely with B7-2 expression. Stimulation of T cells by fixed activated B lymphocytes could be blocked by antibodies directed at class II MHC molecules, indicating involvement of the T-cell antigen receptor. In addition, T-cell proliferation was inhibited by antibodies specific for B7-1 and B7-2 and by the fusion protein CTLA4-Ig, demonstrating a requirement for CD28 signal transduction. The sole requirement of B7 family expression for antigen presentation by B lymphocytes was shown by demonstration of T-cell stimulation by fixed resting B cells in the presence of CD28 antibody as a source of artificial costimulation.

Antigen Presentation↗

PubMed1994

Limiting dilution analysis: quantification of IL-2 producing allospecific lymphocytes after renal and cardiac transplantation.

A rapid and robust limiting dilution assay was developed to measure the frequency of donor-reactive, IL-2 (interleukin 2) producing, helper T lymphocytes in the peripheral T cell population of organ allograft recipients. The IL-2 bioassay was performed using two methodologies to assess the response of CTLL-2 indicator cells. The first depended on spectrophotometric detection of bioreduced XTT whilst the second involved measurement of [3H]thymidine incorporation. The radioisotopic method was slightly more sensitive but both assays could be used for analysis of limiting dilution culture supernatants after primary incubation of recipient lymphocytes with donor splenic cells for 48 hours. All the assays produced results which conformed to single hit kinetics, indicating that IL-2 was production was dependent on a single limiting cell type. The frequency of allospecific helper lymphocytes in the peripheral T cell population of normal volunteers did not vary significantly during a 28-day period. It was found that immunosuppressed allograft recipients had a significantly reduced proportion of T cells in their peripheral blood mononuclear cell population. However, it was possible to measure the frequency of donor-reactive helper cells in the T cell population of transplant patients. These frequency values were very low in two renal allograft recipients who were HLA-DR matched to their donor organs. Three of four HLA-DR mismatched cardiac recipients showed a significant decrease in the frequency of their donor-reactive helper lymphocytes during the period of monitoring. The fourth patient, who received antilymphocyte antibodies for the first three days after transplantation, showed significant fluctuations in the frequency of these cells. The four cardiac recipients showed little histopathological evidence of acute graft rejection with only one patient experiencing a brief episode of moderate rejection; this patient showed a high frequency of donor-reactive helper cells when assayed immediately after this episode but the frequency subsequently declined.

DNA↗

PubMed1994

Development of immunological assays to monitor pulmonary allograft rejection.

BACKGROUND: At present the diagnosis of pulmonary allograft rejection is made after examination of transbronchial biopsy specimens; this method is highly invasive. A study was performed to determine whether immunological parameters measured in peripheral blood or bronchoalveolar lavage samples correlate with the histological diagnosis of rejection. METHODS: Left unilateral pulmonary allotransplantation was performed between dogs. The animals were immunosuppressed with cyclosporin A after transplantation but the dose of this drug was gradually reduced to allow controlled rejection to take place. Rejection was diagnosed histologically. Four immunological parameters were investigated: measurement of lavage derived T cell proliferation in response to limited culture with interleukin 2; measurement of changes in the frequency of donor reactive cytotoxic T lymphocytes; assay of the level of donor cell binding IgG antibody in recipient plasma; and measurement of the antibody dependent cell mediated cytotoxic response to donor cells after labelling with recipient plasma. RESULTS: Assays based on measurement of the function of T cells produced significant results at a time later than the histological diagnosis of severe rejection. The level of donor reactive IgG antibody increased at a time that corresponded closely with the diagnosis of severe rejection. This IgG did not activate the antibody dependent cell mediated cytotoxic effector mechanism to a significant extent. CONCLUSIONS: Measurement of parameters of donor specific immunoreactivity can yield data which are indicative of severe pulmonary allograft rejection. These methods make use of samples which can be obtained by minimally invasive methods. Measurement of the plasma level of donor reactive IgG antibody appears to be the most useful assay. However, each of the in vitro assays used during this series of experiments was less sensitive to the onset of rejection than was routine histological examination.

Animals↗

PubMed1994

Constitutive expression of MHC and adhesion molecules by alveolar epithelial cells (type II pneumocytes) isolated from human lung and comparison with immunocytochemical findings.

Highly purified populations of alveolar epithelial cells (type II pneumocytes) were isolated from human lung specimens. These cells were characterised histochemically, by demonstrating the presence of intracellular alkaline phosphatase, and morphologically, by electron microscopic demonstration of lamellar bodies and microvilli. Expression of the epithelial glycoprotein HEA-125, of MHC class I and class II (HLA-DR, -DP and -DQ) antigens and of the intercellular adhesion molecules ICAM-1, VCAM-1, LFA-3 and B7 was quantified by flow cytometry. Comparison was made between the expression of these molecules by isolated type II cells and by alveolar epithelium in normal human lung tissue after immunocytochemical staining of frozen sections of donor lung. Isolated type II pneumocytes expressed HEA-125 and class I MHC molecules and the class II MHC molecules HLA-DR and -DP; HLA-DQ was not detected. The intercellular adhesion molecule ICAM-1 was expressed constitutively at low levels but there was minimal expression of VCAM-1, LFA-3 and B7. It was not possible to differentiate type II cells from the predominant type I pneumocytes on frozen sections. Alveolar epithelium expressed HEA-125, class I MHC antigens, the class II molecules HLA-DR, and -DP and the intercellular adhesion molecule LFA-3. Expression of the adhesion molecules ICAM-1, VCAM-1 and B7 was variable. As with the isolates, HLA-DQ was not observed on alveolar epithelium. In conclusion, a reproducible method for the isolation of pure populations of human type II pneumocytes has been developed. These cells were not damaged by the isolation procedure. It is not known whether alveolar epithelium can present antigens to T lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Demonstration of direct xenorecognition of porcine cells by human cytotoxic T lymphocytes.

It is not known whether human cytotoxic T cells can recognize porcine major histocompatibility antigens directly, or whether recognition occurs by co-operation with syngeneic human antigen-presenting cells (APC). Limiting dilution assays were used to quantify human anti-pig precursor cytotoxic T-cell (CTLp) frequencies and to analyse the 'kinetics' of the interaction between human lymphoid cells and porcine splenic cells. Single-hit kinetics are demonstrative of direct recognition, as only one cell type, the CTLp, is diluted out, whereas multi-hit kinetics indicate that more than one cell is limiting and provide evidence for co-operative recognition of xenoantigens. Initial assays indicated that the frequency of CTLp reactive with alloantigens on human splenic targets (mean 1/1845; n = 3) was approximately sixfold greater than the frequency of CTLp reactive with porcine splenic cells (1/12,082; n = 3). However, not all of the assays performed using the xenogeneic combination produced single-hit kinetics. Subsequent assays were performed by mixing limiting numbers of human peripheral blood mononuclear cells (PBMC) or APC-depleted PBMC preparations with porcine splenocytes. There was a significant difference in the frequency of xenospecific CTLp between PBMC and APC-depleted preparations (P = 0.034). The overall frequency increased in the APC-depleted group. Variation between the seven human donors was also significant (P = 0.006). There was no significant difference in frequency between the two cell preparations after correction for the proportion of CD3+ cells (P = 0.13). There was, however, a significant departure from single-hit kinetics in the PBMC group (P = 0.004) which was not observed in the APC-depleted group (P = 0.052). It is concluded that human cytotoxic T cells can be activated by porcine xenoantigens directly. However, the direct recognition mechanism can be altered in the presence of human APC.

Animals↗

PubMed1992

Lung transplantation: pulmonary cell lysis mediated by alveolar mononuclear cells.

Methods were developed to monitor graft rejection in a porcine model of unilateral lung transplantation. The ability of peripheral blood mononuclear cells and lavage-derived mononuclear cells to lyse donor pulmonary tissue was determined by standard chromium release assays at various times after transplantation. Effective antigraft activity was observed in the local environment of a rejecting graft, but not in the periphery. Since transplant rejection is a reversible process, with the administration of suitable immunosuppressive regimes frequently restoring graft function, it was reasoned that immunological assays based on the lysis of individual cells may not be relevant to the in vivo situation. We therefore describe an assay of the lung barrier function; perturbations of the tight intraepithelial junctions which compose the air-blood barrier can be determined in vitro by the measurement of transmonolayer resistance values.

Animals↗

PubMed1992

Humoral responses to Malassezia furfur serovars A, B and C in normal individuals of various ages.

A transferable solid-phase (TSP) ELISA was developed for the determination of antibody titres specific to Malassezia furfur serovars A, B and C in human sera. A survey of levels of class-specific antibodies (IgM, IgG and IgA) to M. furfur serovars A, B and C in relation to age (2-64 years; 60 individuals) demonstrated that individuals had immunity to M. furfur by the age of 2-3 years. There was no difference in either IgM or IgG levels into adulthood. The only age-related differences were lower IgM titres to the three serovars in the 60-64 year age-group compared with younger individuals. There was, however, a difference between titres of antibody specific to the three serovars. The mean reciprocal log2 IgM titre to serovar A (6.9) was significantly higher (P < 0.05) than that to serovar B (mean reciprocal log2 titre of 5.8), but not to serovar C (6.1). In contrast, the mean reciprocal log2 IgG titre to serovar A (6.5) was significantly lower (P < 0.05) than those to serovars B and C (mean reciprocal log2 titre of 8.9 in both cases).

Adult↗

PubMed1990

Cyclophosphamide and 15(S)-15 methyl PGE1 correct the T/B lymphocyte ratios of NZB/NZW mice.

The lupus of NZB/NZW F1 female mice is associated with immune complex glomerulonephritis and premature death. Cyclophosphamide and 15(S)-15 methyl PGE1 therapy halt disease progression. Fluorescein conjugated antibodies were utilized to label specific leukocytes and the subsets were quantitated using a Fluorescence Activated Cell Sorter. Normal outbred CD-1 female mice showed a decrease in absolute T and B cell numbers with age, but the ratio of T and B cells remained essentially constant through 9 months of age. By contrast the NZB/W female mice showed decreased numbers of total lymphocytes relative to CD-1 controls at all ages. Moreover relative to CD-1s, there was a far greater decrease in T cell numbers (7 x for NZB/W versus 2 x for CD-1) and B cell numbers failed to decrease with age. The characteristic decline in T lymphocyte numbers and relative increase in B cell numbers in NZB/W mice were corrected with cyclophosphamide and PGE1 therapy. However, there was no selective modification of T cell subsets (L3T4+ or Ly2+) with therapy. Our investigation suggests correction of the abnormal T/B cell ratio may be a useful marker of therapeutic activity in NZB/W mice.

Aging↗

PubMed1990

Differentiation of three serovars of Malassezia furfur.

Malassezia furfur strains were isolated from the clinically normal skin of 10 volunteers by swabbing four different sites (forehead, ear, back and chest). The strains could be divided into three basic groups on the basis of cultural characteristics. Both unabsorbed and absorbed specific rabbit antisera were prepared against nine of the strains, and both species and group specific antigens could be demonstrated. Serologically, three group specific surface antigens could be identified which corresponded to the three groups identifiable on cultural characteristics. The relevance of these findings to previous in vitro results is discussed.

Adult↗