Mannan-Binding Protein Forms Complexes with α-2-Macroglobulin. A Proposed Model for the Interaction

We report that α-2-macroglobulin (α₂M) can form complexes with a high molecular weight porcine mannan-binding protein (pMBP-28). The α₂M/pMBP-28 complexes were isolated by PEG-precipitation and affinity chromatography on mannan-Sepharose, protein A-Sepharose and anti-IgM Sepharose. The occurrence of α₂M/pMBP-28 complexes was further indicated by crossed immunoelectrophoresis and by use of an anti-α₂M affinity column and chelating Sepharose loaded with Zn²⁺. The eluates from these affinity columns showed α₂M subunits (94 and 180 kDa) and pMBP subunits (28 kDa) in SDS-PAGE, which reacted with antibodies against α₂M and pMBP-28, respectively, in Western blotting. Furthermore, the α₂M/pMBP-28 complexes were demonstrated by electron microscopy, Fractionation of pMBP-containing D-mannose eluate from mannan-Sepharose on Superose 6 showed two protein peaks which reacted with anti-C1 s antibodies in ELISA, one of about 650–800 kDa, which in addition contained pMBP-28 and anti-α₂M reactive material, the other with an M_r of 100–150 kDa. The latter peak revealed rhomboid molecules (7 × 15 nm) in the electron microscope and a 67 kDa band in SDS-PAGE under reducing conditions. This band was also seen in eluates from the anti-α₂M and chelating Sepharose columns. Based on these observations and previous findings by other investigators of a serine protease with about 67 kDa subunits which copurifies with human MBP we propose a model for the interaction of pMBP-28 with α₂M.     

Purification and Characterization of Mannan-Binding Protein from Mouse Serum

Mouse mannan-binding protein (MBP) was identified in serum by its Ca²⁺ -dependent binding to mannan. On gel permeation chromatography, the protein eluted corresponding to a molecular weight of approximately 750 kDa. Analysed on SDS-PAGE under reducing conditions, the polypeptide showed an apparent molecular weight of 28 kDa, while several high molecular weight bands were seen under non-reducing conditions. The presence of collagen-like domains within the molecule was indicated by a high glycine content (14.9%) and substantiated by sensitivity to collagenase. Rabbit anti-mouse MBP antisera were raised. The concentration of MBP in serum from normal mice was measured by rocket immunoelectrophoresis and found to be from below 1 μg/ml to 100 μg/ml (average 50 μg/ml, n = 60). The binding of mouse MBP to mannan could be inhibited by mono- and disaccharides in the following order of potency: L-fucose > D-mannose > N-acetyl-D-glucosamine > maltose > D-mannoheptulose > D-glucose > N-acetyl-D-mannosamine ≫ lactose > D-galactose ≫ N-acetyl-D-galactosamine. Mouse MBP was shown to activate the classical complement cascade after binding to mannan. The sequence of 14 NH_2- terminal amino acid residues of the molecule showed 93% identity to rat MBP-A and complete identity to the translated cDNA sequences for mouse MBP-A and mouse Ra-reactive factor component P28b (RaRF P28b) published previously. The amino acid composition of mouse MBP showed a high degree of homology to MBPs from other species and mouse RaRF P28b.     

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.     

Interferon gamma receptor 1 gene polymorphism in patients with tuberculosis in China

The aim of the study was to examine the influence of the CA microsatellite polymorphisms of interferon gamma receptor 1 on patients with tuberculosis (TB) in the south-eastern Chinese population. Genomic DNA from patients with TB ( n  = 155) and ethnically matched controls ( n  = 89) were genotyped by short tandem repeat-PCR method. The allele frequency of (CA)₂₅ was 1.70-fold higher among patients than that among controls (95% CI 1.07–2.70) ( P  = 0.025). Compared with the non-(CA)₂₅/non-(CA)₂₅ reference group, the risk to TB of the carriers of (CA)₂₅/(CA)₂₅ genotypes were 6.46-fold (95% CI 1.40–29.74) ( P  = 0.0017) higher. On the contrary, the allele frequency of (CA)₂₆ was 0.29-fold lower in patients than that in controls (95% CI 0.11–0.76) ( P  = 0.012). Genotypes with (CA)₂₆ allele were at 0.35-fold (95% CI 0.13–0.98) ( P  = 0.045) lower to the risk of TB, compared with that of the non-(CA)₂₆/non-(CA)₂₆ in the reference group. The above results indicated that the allele (CA)₂₅ appeared to be susceptible to TB, while the allele (CA)₂₆ to be protective towards TB. Our data also suggest that the CA repeat was a highly polymorphic marker and could be used for linkage and association analysis.     

α1-Microglobulin Glycopeptides Inhibit Antigen-Specific Stimulation of Human Peripheral Blood Leucocytes

Glycopeptides were prepared from proteolytically digested human and guinea pig α₁ microglobulin (α₁-m) by gel chromatography on Sephadex G-100 and affinity chromatography on concanavalin A-Sepharose. Amino acid analysis showed that the average glycopeptide was a tripeptide containing the amino acids aspartic acid/asparagine, isoleucine, and serine and/or threonine. It has earlier been shown that human α₁-m carries two or three N-glycosidically linked oligosaccharides of the high-mannose type. These preparations of glycopeptides inhibited the proliferative response of peripheral human blood leucocytes in the antigen purified protein derivative. The dose-response relationship of the inhibitions showed a greater specific activity of the glycopeptides than of whole α₁-m. Mild alkali treatment of human α₁-m did not affect its specific inhibitory activity, suggesting that the peptide backbone is of little importance for the immunosuppressive effect of α₁-m. No difference in inhibitory activity was seen between human and guinea pig native α₁-m or α₁-m glycopeptides. Human asialo α₁-m exerted a suppressive effect on the antigen-specific leucocyte proliferation similar to that of native α₁-m.     

Transforming growth factor-β production during the development of renal fibrosis in rats with subtotal renal ablation

The histologic changes observed in the remnant kidney model include progressive mesangial expansion with collapse of capillary lumina, interstitial fibrosis and mononuclear cellular infiltration. Transforming growth factor-beta (TGF-β₁) is an important regulator of extracellular matrix formation. The purpose of this study was to investigate the production and distribution of TGF-β₁ in the kidney during the development of glomerulosclerosis and renal fibrosis in rats with subtotal renal ablation. Eighty-two female Wistar rats weighing 180–220 g were divided into two groups: 49 rats were subjected to 5/6 renal ablation and 33 to sham operation. Urinary albumin excretion, blood pressure and glomerular filtration rate (GFR) were evaluated after the surgical procedure. We also performed histology and immunohistochemistry and determined mRNA for TGF-β₁ in the kidneys of these rats 8, 15, 30 and 90 days after operation. The results showed progressively higher immunohistochemical TGF-β₁ staining in rats with subtotal renal ablation. Cortical renal content of TGF-β₁ mRNA was also higher in these animals and peaked at day 15.
The existence of a temporal association between glomerulosclerosis, interstitial fibrosis and intense mononuclear cellular infiltration on the one hand and higher immunohistochemical TGF-β₁ staining in the renal cortex on the other show that this polypeptide may contribute to the development of renal fibrosis in this model.     

Further Characterization of the Alternative Protein-A Interaction of Immunoglobulins: Demonstration of an Fc-binding Fragment of Protein A Expressing the Alternative Reactivity

Intact IgG and fragments F(ab')₂γ, Fabγ and Fcγ from a rabbit anti-protein-A serum (RapA) and corresponding preparations from normal rabbit IgG (NRG) were tested for their inhibitory effect on the binding of protein-A-reactive ¹²⁵I-IgE and ¹²⁵I-Fcγ, respectively, to protein-A–Sepharose. Intact IgG, F(ab')₂γ and Fabγ of RapA inhibited the binding of protein-A-reactive ¹²⁵I-IgE, whereas only intact IgG and Fcγ fragments from both RapA and NRG inhibited the binding of ¹²⁵Fcγ to protein A-Sepharose. Further, the functional relationship between RapA and human polyclonal IgG was studied in a nephelometric test system. Intat IgG or fragments of IgG from human polyclonal IgG and rabbit anti-protein-A were found to affect the precipitation between human IgG and protein A in a similar way. Thus F(ab')₂γ fragments and intact IgG enhanced the precipitation, whereas Fabγ and Fcγ fragments inhibited the precipitation. Protein A and an Fc-binding fragment of protein A (fragment B) were tested for their abilities to link different radiolabelled immunoglobulin preparations expressing the alternative and the classical protein-A reactivity to immobilized Fc fragments. All proteins expressing the alternative reactivity were efficiently bound both by fragment B and by protein A, indicating that fragment B, in addition to its classical Fc-binding activity, also expresses the alternative protein-A reactivity.     

Cross-linking of Both Types of IgG Fc Receptors, FcγyRI and FcγyRII, Enhances Intracellular Free Ca2+ in the Monocytic Cell Line U937

We have studied the effects of Fc receptor triggering on the free cytosolic Ca²⁺ concentration, [Ca²⁺] _i , in U937. These cells express two types of IgG Fc receptors, FcγRI and FcγRII. Binding of several anti-FcγRI and anti-FcγRII mouse monoclonal antibodies (MoAb) to Quin2- or Indo-I-loaded U937 cells had no direct effect on [Ca²⁺] _i . After addition of a bridging anti-mouse Ig antibody however, transient increases in [Ca²⁺] _i were observed for both types of FcγR. One of the anti-FcγRII MoAb, CIKM5, was exceptional in that it could induce Ca²⁺ increases in U937 cells by itself. Studies with F(ab')₂ fragments of CIK M5 revealed that this MoAb simultaneously binds to FcγRII, via both its Fab and Fc fragments, which might induce cross-linking of two FcγRII molecules. One anti-FcγRI MoAb, 197, a mouse (m)IgG2a antibody directed to an epitope outside the IgG-binding region, remarkably also caused an immediate increase in [Ca²⁺] _i , but only when added to U937 precultured with gamma interferon (IFN-γ). FcγRI can bind monomeric human IgG as well as mIgG2a, and cross-linking of cytophilic Ig induced an increase in [Ca²⁺] _i . Our results show that [Ca²⁺] _i increases can be induced only after cross-linking of FcγR, either via anti-FcγR MoAb or via Fc-FcR interactions. Furthermore, we show that FcγR cross-linking results in activation of the Ca²⁺/phosphatidylinositol (PI) signal transduction pathway.     

Characterization of Monoclonal Antibodies to the αIIbβ3 Integrin on Bovine Platelets

A set of monoclonal antibodies (MoAbs) to leucocyte antigens is an essential tool to identify different cell types and functional membrane molecules involved in immune responses. Since no MoAbs existed to bovine integrins, except against the β₂ subfamily, we generated MoAbs to β₃ integrin after the immunization of mice with bovine platelets. Two MoAbs, IL-A164 (IgG_2a) and IL-A166 (IgG₁), were selected that reacted specifically with bovine platelets and detected the same membrane molecule. The antigen was a heterodimer of two polypeptide chains of 122 kDa and 95 kDa as resolved by SDS-PAGE under reducing conditions. Although the Mr of the smaller subunit is identical to that of β₂ integrin, pre-absorption with an antibody to β₂ (or CD18) did not remove the bovine antigen. Comparing the molecular masses of the two subunits in reduced and non-reduced forms showed a pattern that was similar to that of human GPIIb/IIIa (also called α_IIbβ₃ or CD41a). Reduction of the bovine molecule increased the apparent Mr of the light chain from 76 kDa to 95 kDa, while the heavy subunit changed from 136 kDa to 122 kDa. As with human GPIIb, the decrease in Mr of the α-subunit is probably a result of a small disulphide-linked polypeptide, although no additional evidence for this was detected for the bovine integrin. Sequencing of the N-terminal amino acids of both bovine polypeptides showed identity of the bovine integrin with human GPIIb/IIIa.     

Low Molecular Weight CEA, Isolation and Characterization

A low molecular weight CEA-fraction (CEA_low) was purified from liver metastases of colorectal cancer. The following purification sequence was used: homogenization—PCA extraction—ion-exchange chromatography— Con A affinity chromatography—gel filtration on Sephadex G-200 (x2)—affinity chromatography on specific anti-CEA immunoadsorbent—gel filtration (G-200).
CEA_low was homogeneous on gel filtration in 6M guanidine-HCl and gave a single band on SDS-PAGE. CEA_low and CEA gave a reaction of identity in immunodiffusion with 7/9 monkey anti-CEA sera. 2/9 monkey anti-CEA sera showed a weak spur, with CEA spurring over CEA_low. CEA_low gave a reaction of partial identity with NCA and BGP-1 with unabsorbed rabbit and sheep anti-CEA sera. CEA_low consisted of a single polypeptide chain and had a molecular weight of 125,000 as compared to 175,000 ± 8,000 for CEA (SDS-PAGE). CEA and CEA_low contained the same sugars and aminoacids in approximately the same molar proportions. However the carbohydrate content of CEA_low was lower (30–40% as compared to 45–50% for CEA). The peptide relatedness (expressed in SΔQ-units) of CEA, CEA_low, NCA and BGP-I was compared. CEA_low was most closely related to CEA (SΔQ-values of 5–7) followed by NCA (SΔQ values of 12–17) and BGP-I (SΔQ-values of 35–40).
It is clear from these studies that CEA_low is closely related to CEA but contains less carbohydrate. Whether the polypeptide chain is shorter remains to be established.     

Temperature change does not affect force between regulated actin filaments and heavy meromyosin in single-molecule experiments

The temperature dependence of sliding velocity, force and the number of cross-bridges was studied on regulated actin filaments (reconstituted thin filaments) when they were placed on heavy meromyosin (HMM) attached to a glass surface. The regulated actin filaments were used because our previous study on muscle fibres demonstrated that the temperature effect was much reduced in the absence of regulatory proteins. A fluorescently labelled thin filament was attached to the gelsolin-coated surface of a polystyrene bead. The bead was trapped by optical tweezers, and HMM–thin filament interaction was performed at 20–35°C to study the temperature dependence of force at the single-molecule level. Our experiments showed that there was a small increase in force with temperature  ( Q ₁₀= 1.43)  and sliding velocity  ( Q ₁₀= 1.46)  . The small increase in force was correlated with the small increase in the number of cross-bridges  ( Q ₁₀= 1.49)  , and when force was divided by the number of cross-bridges, the result did not depend on the temperature  ( Q ₁₀= 1.03)  . These results demonstrate that the force each cross-bridge generates is fixed and independent of temperature. Our additional experiments demonstrate that tropomyosin (Tm) in the presence of troponin (Tn) and Ca²⁺ enhances both force and velocity, and a truncated mutant, Δ23Tm, diminishes force and velocity. These results are consistent with the hypothesis that Tm in the presence of Tn and Ca²⁺ exerts a positive allosteric effect on actin to make actomyosin linkage more secure so that larger forces can be generated.     

β-Endorphin Enhances Phagocytosis of Latex Particles in Mouse Peritoneal Macrophages

The effects of β-endorphin (βEnd) on phagocytosis in peritoneal macrophages were examined by using flow cytometry (FCM). βEnd enhanced phagocytosis in a dose-dependent manner. Leucine—enkephalin (Leu-Enk), methionine—enkephalin (Met—Enk), α-endorphin (αEnd), γ-endorphin (γEnd), αEnd (18–31) and βEnd (28–31) had no such activity. βEnd (1–27) and βEnd (6–31) enhanced phagocytosis less effectively than βEnd did. Naloxone did not inhibit the enhancement of phagocytosis induced by βEnd. Unstimulated control phagocytosis was partially suppressed in Ca²⁺-free EGTA-containing solution and even in this solution βEnd enhanced phagocytosis. However, the enhancement was suppressed in the solution containing BAPTA-AM. The present study showed that βEnd enhanced extracellular Ca²⁺ ([Ca²⁺]_o)-dependent and -independent phagocytosis and that the enhancement is largely dependent on intracellular Ca²⁺ ([Ca²⁺]_i). These results support the contention that βEnd is one of the mediators that modulates the immune system.     

Biochemical and Allergenic Properties of the House Dust Mite Extract, Dermatophagoides pteronyssinus

A Further study has been made on the house dust mite extract, Dermatophagoides pteronyssinus , with emphasis on gel-filtrated fraction 2 (F₂). The crude mite extract showed at least nine discs on polyacrylamide disc electrophoresis and contained less than 0.25 % sialic acid and less than 0.5 mM hexosamine and no detectable uronic acid. From gel filtration of the crude extract a No. 2 fraction (F₂) with allergenic activity showed at least five components on SDS disc electrophoresis covering a molecular weight range of between 15,000 and 70,000. The major allergenic activity of F₂ dissolved in pH 7–8 and 4–5 on an isoelectric focusing column. Affinity chromatography of lectins showed that allergenic activity did not relate to structures of N -acetyl-D-glucosamine or N -acetyl-D-galactosamine. Allergenic activity of the crude extract was not affected by peptic digestion and the mite digest prepared by trypsin and pronase showed a similar fraction-action and activity profile as crude extract.     

Interactions between Ricinus Agglutinin and Human Plasma Proteins

Ricinus agglutinin purified to homogeneity precipitates certain serum glyco-proteins containing terminal non-reducing galactose residues. Immunoelec-trophoresis of human sera in agarose gel with ricinus agglutinin in the antibody trough gave 2 fusing precipitin lines due to reaction with IgM and haptoglobin. All of 50 monoclonal IgM proteins precipitated with ricinus agglutinin, and the reactive sites were localized to the Fc μ fragment. By affinity chromatography on Sepharose columns containing insolubilized ricinus agglutinin prealbumin, albumin, α₁-lipoprotein, α₁-antitrypsin, α₁-acid glycoprotein, α₁-antichymotrypsin, α₂HS glycoprotein, Ge-globulin, caerulo-plasmin, β-lipoprotein, β₁ C-globulin, and transferrin were not retained, whereas α₂-macroglobulin, haptoglobin, IgM, IgA, and IgG were retained and could be eluted with lactose. Fused rocket immunoelectrophoresis revealed varying degrees of microheterogeneity among the latter proteins; almost all of IgM but only a small fraction of polyclonal IgG was retained on the ricinus column.     

T-Cell Epitope Mapping of the Borrelia garinii Outer Surface Protein A in Lyme Neuroborreliosis

We studied the T-cell reactivity to overlapping peptides of B. garinii OspA, in order to locate possible immunodominant T-cell epitopes in neuroborreliosis. Cells from cerebrospinal fluid (CSF) and blood from 39 patients with neuroborreliosis and 31 controls were stimulated with 31 overlapping peptides, and interferon-γ secreting cells were detected by ELISPOT. The peptides OspA_17–36, OspA_49–68, OspA_105–124, OspA_137–156, OspA_193–212 and OspA_233–252 showed the highest frequency of positive responses, being positive in CSF from 38% to 50% of patients with neuroborreliosis. These peptides also elicited higher responses in CSF compared with controls ( P  = 0.004). CSF cells more often showed positive responses to these peptides than blood cells ( P  = 0.001), in line with a compartmentalization to the central nervous system. Thus, a set of potential T-cell epitopes were identified in CSF cells from patients with neuroborreliosis. Further studies may reveal whether these epitopes can be used diagnostically and studies involving HLA interactions may show their possible pathogenetic importance.     

Are Anti-Beta2-Glycoprotein-I Antibodies Markers for Recurrent Pregnancy Loss in Lupus Anticoagulant/Anticardiolipin Seronegative Women?

Problem  Anti-beta₂-Glicoprotein-1 antibodies (anti-β₂GPI-ab) have been related to recurrent miscarriage (RM) with conflicting results. The aim was to evaluate the role of anti-β₂-GPI-ab as unique biological marker in RM related to antiphospholipid (aPL).
Method of study  A cohort study that included 59 cases, divided in two groups, was designed: group 1 comprised 43 pregnant women with 'obstetric' antiphospholipid syndrome (APS) and group 2 included 16 cases with similar complaints but only having repeatedly anti-β₂-GPI-ab. Previous thrombosis and/or inherited thrombophilia were excluded. Lupus anticoagulant, anticardiolipin antibodies (aCA), anti-β₂-GPI-ab, and other autoantibodies were analyzed. Miscarriages, premature births, pre-eclampsia, live births, placental and systemic thromboses were studied.
Results  No differences in previous obstetric complications were detected ( P  =   1.00–0.164). After the treatment, differences in number of obstetric complications were not seen ( P  =   1.00). Live births were similar in two groups (88.4% and 93.7%; P  =   1.00). Placental thrombosis was equal in both groups, 93.3% versus 80% ( P  =   1.00).
Conclusion  These results suggest that anti-β₂-GPI-ab may be considered a biological marker for obstetric APS.     

Response linearity in primary auditory cortex of the ferret

The responses of neurons within the primary auditory cortex (A1) of the ferret elicited by broadband dynamic spectral ripple stimuli were examined over a range of ripple spectral densities and ripple velocities. The large majority of neurons showed modulated responses to these stimuli and responded most strongly at low ripple densities and velocities. The period histograms of their responses were subjected to Fourier analysis, and the ratio of the magnitudes of the f ₁ and f ₀ (DC) components of these responses were calculated to give a quantitative index of response linearity. For 82 out of 396 neurons tested (20.7%) this ratio remained above 1.0 over the entire range of ripple densities and velocities. These neurons were classified as 'consistently linear'. A further 134/396 (33.8%) of neurons maintained an f ₁/ f ₀ ratio above 1.0 for either a range of ripple densities at a fixed ripple velocity, or over a range of ripple velocities at a specific ripple density, and were classified as 'locally linear'. Interestingly, for the superficial layers of the primary auditory cortex, consistently linear and locally linear neurons outnumbered nonlinear neurons by a 2:1 ratio. The converse was true for the deep layers. Unlike in primary visual cortex, where f ₁/ f ₀ ratios have been reported to exhibit a bimodal distribution with a minimum at   f ₁/ f ₀≈ 1  , f ₁/ f ₀ ratios for A1 are unimodally distributed with a peak at   f ₁/ f ₀≈ 1  .     

Kinetics of the Reaction between a Monoclonal IgM Anti-I and Rabbit Red Cells

Covalently linked subunits, subunits aggregated from two half subunits, half subunits, F(ab'μ)₂ and Fab'μ were prepared. K-values 1900–5800 times lower than those for intact IgM and rabbit red cells were found for the covalently linked subunits, the aggregated subunits, and F(ab'μ)₂ and 12,500–38,500 times lower for half subunits and Fab'μ. Only whole subunits and F(ab'μ)₂ agglutinated rabbit red cells; compared to intact IgM the titre was reduced markedly, but from the calculated amount of molecules bound per red cell, IgM was found to be on a weight basis only twice as effective as the subunits and F(ab'μ)₂ in agglutinating the cells.     

A Highly Conserved Phenylalanine in the α, β-T Cell Receptor (TCR) Constant Region Determines the Integrity of TCR/CD3 Complexes

In the present study, we have investigated the importance of a phenylalanine (phe¹⁹⁵) in the Tcr-Cα region on Tcr-α, β/CD3 membrane expression. An exchange of phe¹⁹⁵ with a tyrosine residue does not affect Tcr/CD3 membrane expression; however, exchange with aspartic acid, histidine or valine prohibit completely Tcr/CD3 membrane expression. This seems to be due to a lack of interaction between mutated Tcr-α, β/CD3-γɛ, δɛ complexes and ζ₂ homodimers. The Tcr-Cα region around phe¹⁹⁵ seems together with the same region in the Tcr-Cβ region to constitute an interaction site for ζ₂ homodimers. The presence of phe¹⁹⁵ on both Tcr-Cα and Tcr-Cβ causes high avidity interaction with ζ₂ homodimers, whereas his¹⁹⁵ in both Tcr-Cγ and Tcr-Cδ results in an apparently lower avidity interaction with ζ₂ homodimers. It is suggested that the phe¹⁹⁵ region (on β-strand F) and eventually adjacent aromatic amino acid residues on β-strand B region may play an important role in Tcr-α, β/CD3 membrane expression, in Tcr-α, β/CD3 competition with Tcr-γ, δ/CD3 complexes for ζ₂ homodimers and in the control of formation of 'mixed' Tcr heterodimers.     

Variation in Gut-Homing CD27-Negative Lymphocytes in Inflammatory Colon Disease

Prolonged antigenic stimulation results in lymphocyte shedding of CD27, a member of the tumour necrosis factor receptor (TNFR) family, and transformation to a stable phenotype capable of synthesizing interleukin-4 (IL-4). Co-expression of α4β7 identifies those cells with gut-homing potential. We have investigated these cell populations in patients with inflammatory colonic disease. Circulating and lamina propria mononuclear cells were isolated from patients with Crohn's disease (CD), ulcerative colitis (UC), non-inflammatory bowel disease (non-IBD) colonic inflammation and healthy controls. Double and triple colour flow cytometry for CD3, CD4, CD27, α4β7 and intracellular cytokines was performed. Circulating CD4+CD27– populations were increased in patients with CD (8.8 ± 0.8%, P  < 0.001), UC (12.2 ± 1.9%, P  < 0.001) and non-IBD colitis (10.5 ± 1.3%, P  < 0.01) as compared with controls (6.1 ± 0.5%). CD4⁺CD27⁻α4β7⁺ cells were increased in CD ( P  < 0.01). Lamina propria CD4⁺CD27⁻ populations were depressed significantly in CD ( P  < 0.05), UC ( P  < 0.02) and non-IBD colitis ( P  < 0.03). Mucosal CD4⁺CD27⁻ cells synthesized IL-4 in preference to interferon-γ. Thus, colonic inflammation is associated with alterations in gut-tropic circulating and mucosal populations of differentiated memory T cells with the phenotype of predominantly IL-4-synthesizing cells.