Receptor for α1-Microglobulin on T Lymphocytes: Inhibition of Antigen-Induced Interleukin-2 Production

The human plasma protein α₁-microglobulin (α₁m) was found to inhibit the antigen-induced interleukin-2 (IL-2) production of two different mouse T-helper cell hybridomas. α₁m isolated from human plasma and recombinant α₁m isolated from baculovirus-infected insect cell cultures had similar inhibitory effects. Flow cytometric analysis showed a binding of plasma and recombinant α₁m to the T-cell hybridomas as well as to a human T-cell line. Radiolabelled plasma and recombinant α₁m bound to the T-cell hybridomas in a saturable manner and the binding could be eliminated by trypsination of the cells. The affinity constants for the cell binding were calculated to be 0.4–1 × 10⁵  M ⁻¹ using Scatchard plotting, and the number of binding sites per cell was estimated to be 5 × 10⁵–1 × 10⁶. The cell-surface proteins of one of the T-cell hybridomas were radiolabelled, the cells lysed and α₁m-binding proteins isolated by affinity chromatography. SDS–PAGE and autoradiography analysis of the eluate revealed major bands with M _r-values around 70, 35 and 15 kDa. The results thus suggest that α₁m binds to a specific receptor on T cells and that the binding leads to inhibition of antigen-stimulated IL-2 production by T-helper cells.     

Intestinal colonization of a human subject by vancomycin-resistant Enterococcus faecium

Objective: To study the ability of two strains of vancomycin-resistant Enterococcus faecium to colonize the human intestine.
Methods: A single human subject ingested separately two strains of vancomycin-resistant E. faecium isolated from a pig and a chicken. The feces were cultured on selective medium. Prior to ingestion no vancomycin-resistant cocci were present in the feces. Ingestion of 10⁴-10⁵ CFU resulted in either no colonization or isolation only after enrichment. Ingestion of 10⁷ CFU of one strain resulted in colonization for a period of nearly 3 weeks, with fecal counts at times in excess of 10⁶ CFU/g. Ingestion of similar numbers of the other strain and reingestion of the first strain resulted in excretion in the feces for much shorter periods. When the fecal count of the ingested strains was greater than 10⁴-10⁵ CFU/g, the strains were isolated from swabs taken from perianal skin.
Conclusions: Vancomycin-resistant E. faecium strains from pigs and poultry are able to colonize the human gut and the perianal skin.     

Increased Number of IgG Fc Receptors on Monocyte-Enriched Peripheral Blood Leucocytes from Patients with Rheumatoid Arthritis

The number of free Fc receptors (FcR) per cell and the association constant (K_ass) for the binding of monomeric IgG were determined for monocyte-enriched peripheral blood mononuclear cells, isolated from 16 patients with active classical rheumatoid arthritis (RA) and from 15 normal healthy donors. The assay system was based on binding under equili brium conditions of ¹²⁵I-labelled monomeric rabbit IgG to monocytes purified from peripheral blood on a continuous gradient of Petcoll. Monocytes from 14 untreated RA patients (6 seropositive, 8 seronegative) expressed on the average 4.8±1.3 × 10⁴ FcR/cell. This number was significantly higher (P<0.01) than that found in the control group (3.46±0.7 × 10⁴ FcR/cell). There was also a significant difference between the mean K _ass of the RA group and the control group-2.1±0.7 × 10³1/mol and 2.6±1.0 × 10³ 1/mol, respectively (0.05 >P> 0.01). Two seropositive RA patients receiving systemic treatment with penicillamine expressed the same number of FcR/cell as the mean of the control group (3.6 ± 10⁴). Levels of circulating immune complexes (CIC) and of the complement-factor C3 split product C3d were also measured. No correlation was found between the number of FcR/cell and the concentration of C3d, but there was a weak correlation between the number of FcR/ccll and the level of CIC.     

Mitogenic Activation of Human Lymphocytes: a Protein A Plaque Assay Evaluation of Polyclonal B-Cell Activators

Using the protein A plaque assay, the capacity of various polyclonal B cell activators to induce differentiation in human B lymphocytes was investigated. Dextran sulphate and native Dextran were both virtually devoid of mitogenic properties, Lipopolysaccharide, however, was round to be a potent mitogen in human cells that, although giving rise to low DNA synthetic response, induced high numbers of immunoglobulin-synthesizing cells. Mean plaque-forming cell (PFC) numbers in healthy blood donors assayed on the optimal day (days 5–7) were 23, 493 IgM/10⁴ cells, 11, 288 IgG/10⁶ cells, and 2643 IgA/10⁶ cells. Values obtained in spleen cells, peaking at days 4–6, were slightly higher. Purified protein derivative (PPD) was equally or oven more-effective than lipopolysaccharide (LPS) in generating PFC of different subclasses in peripheral blood with mean of 29, 241 IgM/10⁶, 21, 269 IgG/10⁶, and 3681 IgA/10⁶. PPD furthermore induced a marked DNA synthetic response in human lymphocytes. These data suggest that LPS and PPD may both be used as functional markers in human cells when analysing patients with a suspected immunodeficiency state. It is suggested that cultures should be assayed using the protein A plaque assay, thereby being able not only to investigate the individual immunoglobulin classes but also to avoid the possible hazards involved in measuring antigen-specific responses in patients whose prior immunization to the antigen tested can never be totally excluded     

GLUCOCORTICOID-INDUCED GROWTH INHIBITION OF HUMAN NEOPLASTIC SALIVARY GLAND DUCT CELL LINE (HSG)

The purpose of this study was to examine the effect of glucocorticoid on human neoplastic salivary duct epithelial cell line (HSG). Dexamethasone was found to inhibit cell growth and to increase cell size and the ratio of protein content to DNA content in a cell. The inhibition of cell growth was dose-dependent; in comparison to the control (33.8±3.1 h), the population doubling time was 1.57-fold longer in 10⁵ M dexamethasone (P<0.01, N-K test). [³H] thymidine incorporation was inhibited in 45.5% of the control at 10^-5 M. Plating efficiency was 20.5±3.0% in 10⁵ M and 47.0±4.4% in the absence of dexamethasone. Cell diameters increased 1.29 fold in 10^-5 M dexamethasone in comparison to the control size (16.0±2.1 μm). The ratio of total protein content of DNA content increased 1.46 fold in 10^-5 M dexamethasone-treated cells on the seventh day of cultivation. Scatchard plot analysis using [6, 7-³H] -triamcinolone revealed that the HSG cells had apparent cytosolic glucocorticoid receptors with an equilibrium dissociation constant (Kd value) of 6.48 nM, whose number of binding sites (NBS) was 57.8fmol/mg protein.     

The Interaction between Beta 2-Microglobulin (ßm) and Purified Class-I Major Histocompatibility (MHC) Antigen

The function of MHC class-I molecules is to sample peptides from the intracellular environment and present them to CD8⁺ cytotoxic T lymphocytes. To understand the molecular details of the assembly (and disassembly) of peptide-ß₂m-class-I complexes a biochemical peptidc-class-I binding assay has been generated recently and this paper reports on a similar assay for the interaction between ß₂m and class I. As a model system human ß₂m binding to mouse class I was used. The assay is strictly biochemical using purified reagents which interact in solution and complex formation is determined by size separation. It is specific and highly sensitive. The observed affinity of the interaction, K_D, is close to 0.4 nw. The rate of association at 37 C is very fasi (the k_a is around 5 × 10⁴/M/s) whereas the dissociation is slow (the k_d is around 8 × 10⁻⁶/s); the ratio of dissociation to association yields a calculated K_D close to the observed value. At 37° C almost all of the purified class I participates in binding of the exogenously offered ß₂m showing that a considerable exchange of the endogenous ß₂m occurs. Finally, it was demonstrated that exogenous ß₂m enhances binding to MHC class-I of short perfectly-matching peplides as well as longer peptides.     

Binding of Helix Pomatia A Hemagglutinin to Human Erythrocytes and their Cells. Influence of Multivalent Interaction on Affinity

The binding of ¹²⁵I-labelled intact (hexavalent) and partially reduced (divalent) Helix pomatia A hemagglutinin to human A.₁, A.₂, A.₃, A₁B and A₂B erythrocytes, human lymphocytes, human lymphoblastoid and other tumor cell lines was investigated. The essential finding was that the association constants calculated for the interaction between hemagglutinin and A-erythrocytes were many orders of magnitude higher than the intrinsic association constant for the interaction between the hemagglutinin and the blood group A determinant. The latter value was 5–10³1/mole. The K-values for intact hemagglutinin and A and AB erythrocytes were in the order of 10¹⁰1/mole at 18–22 °C and pH 7.3. For partially reduced hemagglutinin the K-values were in the order of 5–10⁷ 1/mole. Multivalent interaction would seem to be the essential factor responsible for the high K-values in the cell binding experiments. Intact hemagglutinin reacted against A₁ and A₂ erythrocytes or against a human osteogenic sarcoma cell tine (2T) gave homogeneous binding curves in Scatchard's plot. Human lymphocytes and most lymphoblastoid cell lines lacked hemagglutinin receptors.     

Use of Lightly Haptenated Phage to Detect High-Affinity Antibody

We coupled bacteriophage T4 with standard (0.16%) or low (0.005%) concentrations of NIP-azide. The low concentration produced haptenated phage (type B) that was inactivated by the IgG anti-NIP of some rabbits but not by that of other rabbits similarly immunized. The most active IgG fraction at an anti-NIP concentration of 100 μg/ml inactivated the type B phage with a titre of 2400, while the same anti-NIP concentration of the least active IgG fraction from another rabbit had a titre of 0.3. With the standard haptenated phage titres only varied within a 20-fold range. IgM fractions of a few rabbits tested did not appear to exhibit a great variation with the type B phage. The IgG fractions with high titres against the type B phage bound more NIP-hapten at low (∼ 10⁻¹⁰ M) hapten concentrations than those with a low titre, but this superiority was greatly reduced by an increase of hapten concentration to 10⁻⁸ M. The same phenomenon could be demonstrated by an inhibition technique, lnactivation of type B phage appeared to detect exclusively antibodies with association constants considerably higher than 10⁸. Its affinity threshold seems to be higher than the threshold of two other methods employed, haemagglutination of haptenated erythrocytes and inactivation of the standard haptenated phage.     

Control of T-cell activation by CD4+ CD25+ suppressor T cells

Summary: Depletion of the minor (∼10%) subpopulation of CD4⁺ T cells that co-expresses CD25 (interleukin (IL)-2 receptor α-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4⁺ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4⁺ CD25⁺ cells. CD4⁺ CD25⁺-mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-β. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4⁺ CD25⁺ T cells are also powerful suppressors of the activation of both CD4⁺ and CD8⁺ T cells in vitro . Suppression is mediated by a cell contact-dependent, cytokine-independent T–T interaction. Activation of CD4⁺ CD25⁺ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4⁺ or CD8⁺ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4⁺ CD25⁺ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4⁺ or CD8⁺ effector cells to suppress the development of autoimmune disease.     

Structural Difference in the Complement Activation Site of Human IgG1 and IgG3

The C1q binding epicentre on IgG molecules involves residues Asp²⁷⁰, Lys³²², Pro³²⁹ and Pro³³¹ in the C_H2 domain. IgG1 and IgG3 are usually the most efficient of the four human IgG subclasses in activating complement and they both share all these residues. To reveal possible differences in the structural requirement for complement activation, we created a number of NIP (5-iodo-4-hydroxy-3-nitro-phenacetyl) specific IgG1 and IgG3 antibodies with parallel mutations in or near the putative C1q binding site. The mutants were tested simultaneously for antibody induced, antibody-dependent complement-mediated lysis (ADCML) at high and low antigen concentration on the target cells using sera of human, rabbit and guinea pig as complement source. In addition, we tested the antibodies against target cells decorated with the NP hapten, which has 10-fold lower affinity for the antibodies compared to the NIP hapten. We also used ELISA methods to measure complement activation. We observed a clear difference between IgG1 and IgG3 localized to residues Asp²⁷⁰, Leu³³⁴, Leu³³⁵. For all these residues, and especially for Asp²⁷⁰, IgG1 was heavily reduced in complement activation, while IgG3 was only moderated reduced, by alanine substitution. This difference was independent of the long hinge region of IgG3, demonstrated by hinge region truncation of this isotype such that it resembles that of IgG1. This report indicates the presence of structural differences between human IgG1 and IgG3 in the C1q binding site, and points to a specialization of the two isotypes with respect to complement activation.     

Real-Time Biospecific Interaction Analysis of a Natural Human Polyreactive Monoclonal IgM Antibody and its Fab and scFv Fragments with Several Antigens

Surface plasmon resonance (SPR) was used to investigate the kinetics of interactions between the human monoclonal polyreactive IgM antibody CB03, its Fab as well as its single-chain variable fragment (scFv) and different antigens. From these experiments apparent binding constants were determined and compared with binding constants obtained by ELISA experiments. In SPR studies with the complete antibody, the polyreactivity of the CB03 antibody as derived from ELISA experiments was confirmed.
Interaction of scFv with k casein and human myoglobin is strong evidence for the location of polyreactivity within the variable domains of the antibody.
Apparent binding constants of the complete antibody to immobilized K casein (9.2 × 10⁷ M⁻¹) and to human myoglobin (1.6 × 10⁷ M ⁻¹) are up to 83 times higher than those of Fab. The binding constants of the scFv to the above mentioned antigens are again about 10 times lower when compared with Fab, which is mainly due to the lower association rates of the complexes formed by the scFv.     

The Role of the Different CD34 Epitopes in Detection and Positive Selection of CD34+ Bone Marrow and Peripheral Blood Stem Cells

Positive selection of CD34⁺ cells is an attractive approach to reduce tumour cell contamination in bone marrow (BM) and peripheral blood progenitor cell (PBPC) autografts in malignancies not expressing CD34. All current selection methods use monoclonal antibodies (MoAbs) specific for the class I or class II CD34 epitopes, while for detection most investigators use class III MoAbs. Since the distribution of the different CD34 epitopes on haematopoietic progenitors differs, we studied their significance in CD34⁺ selection procedures. Testing MoAbs against class I, II and III CD34 epitopes on normal BM we observed that ± 23% of class III positive cells was class I negative. A higher expression of the class III epitope compared with classes I or II was observed on the KG1 cell line, whereas no differences in binding capability were found. The class III epitope anti-CD34, 561, was compared with the class I epitope anti-CD34, BI-3C5, both coupled to M450 Dynabeads. The yield of CD34⁺ cells obtained with the 561 beads was 1.7% of the mononuclear cells versus 0.95% using the class I epitope, a 1.95-fold increase (1.3–2.7), whereas the purity was similar (96% in both cases). The absolute number of CD34⁺ cells was therefore twofold higher after 561 selection, including cells with a more mature phenotype. In single cell assay comparable numbers of highly proliferative progenitors but higher numbers of differentiated colonies per phenotypic subfraction were measured. In conclusion, M450 beads coated with the 561 anti-class III CD34 epitope are more efficient in isolating CD34⁺ cells from bone marrow, probably due to a broader distribution of the class III epitope.     

Purification of Human Interferon by Antibody Affinity Chromatography, using Highly Absorbed Anti-interferon

Antibodies against partially purified human leucocyte interferon (PIF) were bound to Sepharose 4B and crude interferons applied on this affinity column were purified, up to 8 × 10⁵ interferon units (IFU) per mg protein in one step. Antibodies against PIF were absorbed with immobilized crude human leucocyte interferon bound to Sepharose, whereby antibodies against impurities were predominantly removed. Extensively absorbed antisera were coupled to Sepharose and used for antibody affinity chromatography of crude interferon preparations. Leucocyte and fibroblast interferons were purified in one step with around 100% recovery, up to 1 × 10⁸ IFU per mg protein, and Namalva interferon up to 2 × 10⁷ IFU/mg. SDS electrophoresis of affinity-purified leucocyte interferon revealed that the interferon activity appeared in two bands (19,000 and 23,000 D).     

Recognition of HLA-A1 by murine monoclonal antibodies

Abstract: Balb/c mice were immunized with cells from the mouse mastocytoma line P815 transfected with an HLA-A1 gene. The splenocytes of the immunized mice were fused with cells from the murine myeloma NS-1. In an initial screening, supernatants of growing cultures were tested for their binding capacity to the immunizing P815/A1⁺ cells as well as to P815/A2⁺ cells. Three out of 756 hybrids produced antibodies which bound to P815/A1⁺ cells only. They were cloned and further analyzed for their binding reactivity to reference B-lymphoblastoid cells from the Tenth International Histocompatibility Workshop. One monoclonal antibody, designated 6B11, reacted only with HLA-A1⁺ cells, while the two other antibodies, 3G3 and 7F10, appeared to detect antigenic determinants shared by HLA-A1, A3, A11, A26, and A30 (3G3) and by HLA-A1, A3, A11, A26, A28 and A30 (7F10). Flow cytometric studies on B-lymphoblastoid cell lines as well as on a series of tumor cell lines, including melanoma and colon carcinoma lines, confirmed the specificity of these antibodies. Monoclonal antibodies 7F10 and 6B11 were found to be of the IgM class and 3G3 of the IgG1 class. By complement-dependent ⁵¹Cr release experiments it was further shown that the two IgM antibodies 7F10 and 6B11 were able to lyse all cell lines of the HLA-A1 haplotype tested.     

Effects of lipoxin A4 on chemotaxis and degranulation of human eosinophils stimulated by platelet-activating factor and N-formyl-L-methionyl-L-leucyl-L-phenylalanine

Lipoxins are trihydroxytetraene metabolites derived through a double lipoxygenation of arachidonic acid. Lipoxin A₄ (LXA₄) was prepared by total chemical synthesis, and its capacity to modulate eosinophil migration has been evaluated. LXA₄ is a weak and partial chemotactic agent; at 10⁻⁶ M, it achieved about 20% of the response of 10⁻⁶ M platelet-activating factor (PAF). Preincubation of eosinophils with increasing doses of LXA₄ (10⁻¹⁰−10⁻⁵ M) resulted in a concentration-dependent inhibition of cell migration induced by 10^-6 M formyl-methionyl-leucyl-phenylalanine (FMLP) and 10^-6 M PAF. The concentration of LXA₄ which produced 50% inhibition (IC₅₀) of eosinophil migration was approximately 10^-6 M. LXA₄ (10^-10-10^-6 M) did not elicit ECP release or modulate ECP release induced by 10^-6 M FMLP. LXA₄ may have antiallergic properties in preventing eosinophilic migration.     

Antigen Binding by Non-Bursa-Derived Chicken Leukocytes

Antigen-binding peripheral blood leukocytes (PBL) from normal and bursectomized agammaglobulinemic chickens were labeled by incubation in vitro with radioiodinated antigen at 4°C in the presence of sodium azide. [¹²⁵I] TGAL-binding cells could be detected by autoradiography of PBL from normal, unimmunized chickens at a frequency of 1 to 4 labeled cells per 10⁴ leukocytes. No [¹²⁵I] TGAL-binding cells were found in PBL from bursectomized chickens, even after incubation with 25 μg/ml of labeled antigen followed by prolonged autoradiographic exposure. The binding to normal PBL was specific as judged by inhibition with unlabeled TGAL but not with unlabeled TIGAL. The binding was, furthermore, inhibited by preincubation with rabbit anti-chicken L chain antibody but unaffected by normal rabbit IgG [¹²⁵I] TIGAL was, in contrast, found to bind to PBL from both normal and bursectomized chickens at a frequency of 6 to 80 labeled cells per 10⁴ leukocytes. The labeling was specific, since it was inhibited by cold TIGAL but not by cold TGAL. The binding of [¹²⁵I] TIGAL to PBL from bursectomized chickens showed from none to slight inhibition on preincubation of the cells with anti-L chain antibody, whereas preincubation with normal rabbit IgG resulted in almost complete inhibition. To our knowledge this is the first demonstration of antigen binding to PBL from agammaglobulinemic chickens.     

Differential Expression of T-Cell Adhesion Molecules and LFA-1-Dependent Intercellular Adhesion in HgCl2-Induced Autoimmunity and Immune Suppression

Exposure of Brown Norway (BN) rats to HgCl₂ induces Th2-mediated systemic autoimmunity. In contrast, in Lewis rats, HgCl₂ induces immune suppression, mediated by CD8⁺ T cells. HgCl₂ was previously found to enhance expression of LFA-1, ICAM-1 and CD134 (OX40) on T cells in BN rats. In the present study, T cells from Lewis rats were studied at day 4 after injection of HgCl₂. CD8⁺ T lymphoblasts were significantly increased, which were predominantly CD45RC^hi, and which showed enhanced LFA-1 expression. Furthermore, CD4⁺CD45RC^hi T cells showed increased numbers of ICAM-1⁺ cells, whereas expression of CD134 and CD26 was relatively decreased in CD4⁺ T lymphoblasts. Ex vivo experiments demonstrated that HgCl₂- exposure of BN rats, but not of Lewis rats, significantly enhances PMA [phorbol 12-myristate 13-acetate]-induced lymphocyte aggregation, mediated by LFA-1 and ICAM-1. In conclusion, HgCl₂-injected Lewis rats show early signs of T-lymphocyte activation, predominantly on CD8⁺ cells. Strain-dependent effects of HgCl₂ on cell adhesion molecules and expression of CD134 may play an important role in development of either autoimmunity or immune suppression.     

Sequential Analysis of Distributions of Donor-derived Thymocytes Bearing T-cell Antigen Receptor (TCR) and Donor-derived la+ Cells in Thymuses of Fully Allogeneic Bone Marrow Chimera in Mice

Lethally irradiated SJL/J mice were reconstituted with B10 bone marrow cells, and the process of thymic reconstitution by donor derived cells positive for I- A or Vβ8 molecules was investigated. The donor-derived la⁺ cells appeared in the medulla on day 7 after reconstitution. The la+ cells became confluent up to day 14, and the cellularity in the medulla on day 17 was almost the same as that in the normal thymus. Dull Vβ8⁺ thymocytes were first recognized in the cortex on day 10 and were identifiable in the medulla by day 14. The Vβ8⁺ cells seemed to be mainly CD4⁺8⁺ double-positive. Furthermore, most of the Vg8'cells in the medulla of chimeras given cyclosporin A for 3 weeks after reconstitution appeared to be CD4⁺8⁺. The present findings demonstrate that CD4 8+ thymocytes which bear a low concentration of TCR exist in the thymic medulla at a relatively early stage when donor-derived la⁺ cells have already settled there. The coincidental appearance and coexistence of la⁺ cells and TCR⁺ thymocytes in the medulla suggest that these histological characteristics may be related to the selection of thymocytes in this area.     

Induction of CD8+ CTL Recognizing Mycobacterial Peptides

Mycobacterium tuberculosis is the single, most important cause of morbidity attributable to a single infectious organism. CD8⁺ T cells play an important role in anti-tuberculous immune responses in both mice and humans. Data concerning the identity of mycobacterial antigens recognized by CD8⁺ T cells is limited; consequently, few CTL epitopes have been characterized. The authors identified allele-specific (H-2^{b and d}) MHC class I binding motifs in six prominent M. tuberculosis protein antigens (the 19 and 38 kDa lipoglycoproteins and the 10, 16, 65 and 70 kDa stress proteins). These predicted epitopes were tested for MHC binding as well as their ability to elicit peptide-specific CTL following in vivo priming. The authors were able to identify eight previously undescribed mycobacterial CTL epitopes by using spleen cells from peptide-immunized mice. In addition, CTL specific for at least one of these epitopes also recognized the naturally processed epitope presented on transfected EL4 target cells. These mycobacteria-derived CTL epitopes could be important for future analysis of the involvement of CD8⁺ T cells in M. tuberculosis infection, pathogenesis and vaccine development.     

Histidine-stimulated divalent metal uptake in human erythrocytes and in the erythroleukaemic cell line HEL.92.1.7

The uptake of ⁶⁵Zn by human erythrocytes was investigated in the presence of high (40 m m ) and low (5 m m ) concentrations of histidine and 0–500 μ m cobalt, nickel, manganese and zinc. Varying concentrations of metal mono- and bis-histidine complexes will be formed and the inhibition of ⁶⁵Zn uptake could be correlated with the calculated complex concentrations to investigate competition between metals. For each metal, the calculated concentrations of bis-histidine complex giving 50% inhibition of ⁶⁵Zn uptake were similar at both 5 m m and 40 m m histidine. Manganese–bis-histidine appeared to have a much higher affinity for the binding site than the other metal–bis-histidine complexes, which had similar affinities to each other. Studies of the inhibition of histidine-stimulated ⁵⁴Mn uptake by the addition of manganese confirmed that manganese–bis-histidine does act as a substrate for the transporter in a similar fashion to the other metals studied. In addition, human erythroleukaemic cells (HEL cells) were used as a model for erythroid precursor cells. l -histidine, but not d -histidine, stimulated ⁶⁵Zn uptake in a saturable fashion. The other metals competed with zinc in a similar manner to that seen in erythrocytes, and the affinity for manganese–bis-histidine was much greater than for the bis-histidine complexes of the other three metals. Both the capacity for metal transport per cell, and the affinity of the transporter for the metal–bis-histidine complexes, were much greater in the HEL cells than in the erythrocyte. It is suggested that histidine-stimulated metal transport may play a role in the supply of metals to maturing erythroid cells.