X-SCID B cell responses to interleukin-4 and interleukin-13 are mediated by a receptor complex that includes the interleukin-4 receptor α chain (p140) but not the γ chain

This study investigates the effect of interleukin (IL)-4 mutant proteins and a monoclonal antibody to the IL-4 receptor α chain on IL-4 and IL-13 response by B cells from X-linked severe combined immunodeficiency (X-SCID) patients in which the common γ chain (γc chain) gene mutations have been fully characterized and no γc chain expression was detected. In this γc chain gene knockout model, it was confirmed that the γc chain is essential for B cell responses to IL-2 but not for IL-4 or IL-13. Dose-response curves for X-SCID and normal B cell responses to IL-4 were indistinguishable, showing that the loss of the γc chain did not diminish the sensitivity of B cells to IL-4. The mutant protein IL-4_Y124D and an antibody to the IL-4R α chain both inhibited responses of X-SCID B cells to IL-4 and IL-13, showing that X-SCID B cell responses to these cytokines are mediated by a receptor complex that includes the IL-4R α chain but not the γc chain. Another mutant protein, IL-4_R88D, which has greatly reduced affinity for IL-4Rα, was found to inhibit responses by normal B cells to IL-4 but not to IL-13. IL-4_R88D did not, however, inhibit X-SCID B cell responses to IL-4. This result is consistent with IL-4_R88D inhibition of responses mediated by receptor complexes that include the γc chain. We propose that X-SCID B cells responses to IL-4 are mediated by an IL-13 receptor complex comprised of the IL-4R α chain associated with the recently cloned IL-13R binding protein. This model has major implications for understanding normal B cell responses to IL-4.     

Synthesis and Characterization of Comb‐Like Copolymers Based on Poly(ε‐caprolactone) and Poly(α‐olefin)

Comb‐like copolymers based on a polyolefin backbone of poly(10‐undecene‐1‐ol) (PUol) with poly(ε‐caprolactone) (PCL) side chains are synthesized in two steps. After synthesis of PUol by metallocene‐catalyzed polymerization, the side‐chain hydroxyl functionalities of this polar polyolefin are used as an initiator for the ring‐opening polymerization (ROP) of ε‐caprolactone (CL). In this context, copolymers with different lengths of PCL grafts are prepared. The chemical structure and the composition of the synthesized copolymers are characterized by ¹H and ¹³C NMR spectroscopy. It is shown that the hydroxyl end groups of PUol act effectively as initiating sites for the CL ROP. Size‐exclusion chromatography (SEC) measurements confirm the absence of non‐attached PCL and the expected increase in molar mass after grafting. The thermal and decomposition behaviors are investigated by DSC and thermogravimetric analysis (TGA). The effect of the length of the PCL grafts on the crystallization behavior of the comb‐like copolymers is investigated by DSC and wide‐angle X‐ray scattering (WAXS).

     

The nature of the signals regulating CD8 T cell proliferative responses to CD8α+ or CD8α− dendritic cells

The CD8α^?-expressing dendritic cells (DC) of mouse spleen have been shown to be poor inducers of interleukin (IL)-2 production by CD8 T cells when compared to the CD8^? DC. As a consequence, CD8 T cells give a more prolonged proliferative response to CD8^? DC than to CD8⁺ DC. The possible mechanisms underlying these functional differences in DC subtype have been investigated. Inadequate co-stimulation did not underlie the poor T cell response to allogeneic CD8⁺ DC. Equivalent levels of B7-1 (CD80) and B7-2 (CD86) were found on the two DC subtypes and co-stimulator assays did not reveal any functional differences between them. Although CD8⁺ DC were found to die more rapidly in culture than CD8^? DC, this did not explain their reduced stimulatory ability. Neither prolonging DC survival in culture nor renewing the stimulator cells by repeated addition of freshly isolated DC had any significant effect on the T cell responses. Furthermore, later addition to the cultures of DC of the opposite type to the initiating DC did not reverse or eliminate the differential response to the initiating DC. The role of DC-derived soluble factors was examined by addition to the cultures of supernatants derived from freshly isolated or stimulated DC of the opposite type. This neither enhanced the poor stimulatory capacity of CD8⁺ DC nor inhibited the stimulation by CD8^? DC. Furthermore, addition of a series of cytokines that might have been produced by the DC did not eliminate the differences in T cell proliferation. Only the addition to the cultures of the growth factors IL-2 and IL-4 overcame the stimulatory difference between the two DC populations, confirming that the difference in T cell proliferative responses was a consequence of differences in induced cytokine production. The difference in the response of CD8 T cells to CD8⁺ and CD8^? DC is therefore determined by direct DC-T cell contact during the earliest stages of the culture and involves an undetermined and possibly new signaling system.     

αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

T cell receptor and peptide-contacting residues in the HLA-DR17(3) β1 chain

Previously, we have proposed that the β1 residues 9–13, 26, 28 and 86 in HLA-DR17, the most common subtype of DR3, might be critical for the binding of an immunodominant, mycobacterial epitope (peptide 3–13 of the 65-kDa heat shock protein). In order to examine directly (i) which DR17 residues are involved in peptide binding, (ii) whether the same or other DR17 residues are involved in the binding of different peptides, and (iii) whether subtle differences in the mode of peptide binding can influence T cell stimulation, we have now systematically mutated 15 highly polymorphic DR17β1 residues, located in the proposed peptide binding groove of DR17, and examined the effect thereof on binding and presentation of two peptides, hsp65 p3–13 and p56–65 of the 30/31-kDa secreted mycobacterial protein. Mutations in residues 28 (D → H) and 86 (V → G) completely eliminated binding of p3–13 and significantly reduced binding of p56–65. A mutation in residue 26 (Y → F) decreased binding of p3–13 but did not affect binding of p56–65. Substitutions of amino acid residues 28, 67, 71 and 86 in the DR17β1 chain abrogated peptide-specific stimulation of both the p3–13- and the p56–65-specific T cell clones, while specific stimulation by only one peptide was eliminated by substitution at positions 26 and 74 (p3–13) and by substitution of residues 11 and 37 (p56–65). The observation that substitution of several other peptide-contacting DR17β1 chain residues does not significantly affect peptide binding but does affect T cell stimulation, suggests that these substitutions alter the conformation of the bound peptide.     

Abatacept (cytotoxic T lymphocyte antigen 4‐immunoglobulin) improves B cell function and regulatory T cell inhibitory capacity in rheumatoid arthritis patients non‐responding to anti‐tumour necrosis factor‐α agents

The use of biological agents combined with methotrexate (MTX) in rheumatoid arthritis (RA) patients has strongly improved disease outcome. In this study, the effects of abatacept on the size and function of circulating B and T cells in RA patients not responding to anti‐tumour necrosis factor (TNF)‐α have been analysed, with the aim of identifying immunological parameters helpful to choosing suitable tailored therapies. We analysed the frequency of peripheral B and T cell subsets, B cell function and T regulatory cell (T_reg) inhibitory function in 20 moderate/severe RA patients, according to the European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) criteria, primary non‐responders to one TNF‐α blocking agent, who received abatacept + MTX. Patients were studied before and 6 months after therapy. We found that abatacept therapy significantly reduced disease activity score on 44 joints (DAS)/erythrocyte sedimentation rate (ESR) values without causing severe side effects. The size of the circulating B and T cell compartments in RA patients was not significantly different from healthy donors, but B cell proliferation and plasma cell differentiation was impaired before therapy and restored by abatacept. While T_reg cell frequency was normal, its inhibitory function was absent before therapy and was partially recovered 6 months after abatacept. B and T_reg cell function is impaired in RA patients not responding to the first anti‐TNF‐α agent. Abatacept therapy was able to rescue immune function and led to an effective and safe clinical outcome, suggesting that RA patients, in whom anti‐TNF‐α failed, are immunologically prone to benefit from an agent targeting a different pathway.     

Reorganization of Lamellar Diblock Copolymer Poly(ε‐caprolactone)‐block‐poly(4‐vinylpyridine) in the Melting Temperature Range

The reorganization kinetics of the “original” lamellar diblock copolymer poly(ε‐caprolactone)‐block‐poly(4‐vinylpyridine) crystals formed at 260 K is studied in the melting region from 270 K (10 K below the onset of the melting peak of original crystals) to 310 K (the melting peak temperature) on the time scale starting from 10^?4 to 10² s by ultrafast differential scanning calorimetry. Different reorganization pathways are observed in this temperature range. Annealing at temperatures below 295 K leads to further stabilization of original crystals by secondary crystallization. At annealing temperatures higher than 295 K, crystals partially melt and the reorganization occurs via the melting–recrystallization. For even higher temperature, such as 310 K, the melting is completed within a few milliseconds and recrystallization starts from the nuclei formation. The sigmoidal recrystallization kinetics is analyzed by the Avrami equation. It is found that the copolymer experiences about one order of magnitude slower recrystallization rate and has higher melting peak temperatures of crystals formed after recrystallization than the homopolymer. The slower recrystallization kinetics in the copolymer is discussed from the viewpoint of the nanoscale spatial constraint and the intermediate state prior to the recrystallization.

     

Newly diagnosed multiple myeloma patients carrying monoallelic deletion of the whole locus of immunoglobulin heavy chain gene have a better prognosis compared to those with t(4;14) and t(14;16)

The current study evaluated the prognostic significance of the monoallelic deletion of the whole locus of the immunoglobulin heavy‐chain (w_del(IGH)) gene compared to translocations t(4;14) and t(14;16) among newly diagnosed multiple myeloma (MM) patients. We retrospectively analyzed clinical (age, gender, and staging) and laboratory data at diagnosis and the overall survival (OS) of 255 newly diagnosed MM patients carrying w_del(IGH) or translocations t(4;14) or t(14;16). Bone marrow samples were examined by morphological and sequential interphase fluorescense in situ hybridization analyses. Among 255 patients, 117 (45.8%) had w_del(IGH), 99 (38.8%) had t(4;14), and 39 (15.3%) had t(14;16). Mean age was 61.6 ± 11.6 years. Groups did not differ significantly in age, gender, or lactate dehydrogenase levels. Patients in the w_del(IGH) group presented more frequently at International Staging System stage I than at stage II/III. Patients in the w_del(IGH) group had significantly fewer additional chromosomal aberrations (1.58) than the other two groups (2.3 and 2.13 in the del(IGH), t(14;16) and t(4;14) groups, respectively, P < 0.0001). Furthermore, the w_del(IGH) group had significantly longer estimated median OS (9.47 years) compared to those with translocations t(14;16) (3.02 years, P = 0.002) or t(4;14) (4.18 years, P = 0.001), respectively. These findings suggest a potential prognostic significance of monoallelic deletion of IGH among these patients. Additional studies are needed to better understand the nature and mechanism of this prognostic factor.     

Association between the GABAA receptor α5 subunit gene locus (GABRA5) and bipolar affective disorder

Genetic factors seem to play an important role in the pathogenesis of affective disorder. The candidate gene strategies are being used, among others, to identify the genes conferring vulnerability to the disease. The genes coding for the receptors of gamma-aminobutyric acid (GABA) have been proposed as candidates for affective disorder, since the GABA neurotransmitter system has been implicated in the pathogenesis of the illness. We examined the possible genetic association between the GABA_A receptor α5 subunit gene locus (GABRA5) on chromosome 15 and affective disorder, in 48 bipolar patients (BP), 40 unipolar patients (UP), and 50 healthy individuals, age- and sex-matched to the patients. All patients and controls were unrelated Greeks. Diagnoses were made after direct interviews according to the DSM-IV and ICD-10 criteria. For the genotyping, a dinucleotide (CA) repeat marker was used. The polymerase chain reaction (PCR) products found were nine alleles with lengths between 272 and 290 base pairs (bp). The distribution of allelic frequencies of the GABRA5 locus differed significantly between BP patients and controls with the 282-bp allele found to be associated with BP affective disorder, while no such difference was observed between the groups of UP patients and controls nor between the two patient groups. The presence or absence of the 282-bp allele in the genotype of BP patients was not shown to influence the age of onset and the overall clinical severity, but was found to be associated with a preponderance of manic over depressive episodes in the course of the illness. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81:73–80, 1998. © 1998 Wiley-Liss, Inc.     

Synthesis of Functional Poly(ε‐caprolactone)s via Living Ring‐Opening Polymerization of ε‐Caprolactone Using Functionalized Aluminum Alkoxides as Initiators

The new aluminum compounds 1–3 modified by unsaturated alcohol, Me_3−n Al(O(CH₂)₄OCHCH₂)_n (n = 1 ( 1 ), 2 ( 2 ), 3 ( 3 )), are synthesized and investigated by multinuclear (¹H, ¹³C, ²⁷Al) NMR spectroscopy. The compounds 1 – 3 initiate living ring‐opening polymerization of ε‐caprolactone in bulk at 40–80 °C to afford polyesters with controlled molecular weight (M _n up to 35 000 g mol⁻¹) and relatively narrow molecular weight distribution (M _w/M _n < 1.8). Among initiators studied here, aluminum trialkoxide shows the highest activity, whereas aluminum dialkoxide is a less active. In all cases, the fragment of unsaturated alcohol is transferred to the end of the polymeric chain with high degree of functionality (>85%) yielding macromonomers. These macromonomers are copolymerized with maleic anhydride to give poly(vinyl ether‐co‐maleic anhydride)‐g‐poly(ε‐caprolactone) graft copolymers.

     

The protein interactions of the immunoglobulin receptor family tyrosine-based activation motifs present in the T cell receptor ζ subunits and the CD3 γ,δ and ϵ chains

Immunoglobulin family tyrosine-based activation motifs (ITAM), which define the conserved signaling sequence EX₂YX₂L/IX₇YX₂L/I, couple the T cell antigen receptor (TCR) to cellular proteins including protein tyrosine kinases (PTK) and adapter molecules. The TCR is a multichain complex with four invariant chains CD3γ, δ and ? that each contain a single ITAM and the TCR ζ chain that contains three ITAM. The present study explores the protein interactions of the doubly phosphorylated CD3 γ, δ, ? ITAM to determine whether they have common or unique biochemical properties. The data show that the doubly phosphorylated ITAM all bind the PTK ZAP-70, but the ITAM also variably bind the PTK p59fyn and the adapters Shc, Grb-2 and the p85 regulatory subunit of phosphoinositol 3′ kinase. The CD3 and ζ ITAM display a hierarchy of ZAP-70 binding: ζ1 = γ = δ > ζ3 > ζ2 = ?. Shc, Grb-2 and p85 could bind the ζ ITAM and the CD3 γ and δ ITAM, but not the CD3 ? ITAM. There were also subtle differences in the hierarchy of reactivity of these adapters for the CD3 γ,δ and ζ ITAM that show that the ζ, CD3 γ, δ and ? ITAM have different binding properties. The present study thus shows that the different ITAM of the TCR/CD3 complex can interact with different cytosolic effectors, indicating that differential ITAM phosphorylation during T cell activation could be a mechanism to generate signaling diversity by the TCR complex.     

Ternary Thermosetting Blends of Epoxy Resin,Poly(ethylene oxide) and Poly(ε‐caprolactone)

Summary: The ternary thermosetting blends composed of epoxy resin, poly(ethylene oxide) (PEO) and poly(ε‐caprolactone) (PCL) were prepared via in situ polymerization of epoxy monomers in the presence of the two crystalline polymers, PEO and PCL. DSC results showed that the binary blends of epoxy with PEO (and/or PCL) are fully miscible in the entire composition in the amorphous state. FTIR indicates that there were interchain specific interactions between the crosslinked epoxy and the linear polymers in the binary blends and the hydrogen bonding interactions between epoxy and PCL are much weaker than those between epoxy and PEO. The difference in the strength of interchain specific interactions gives rise to the competitive hydrogen bonding interactions in the ternary blends of epoxy, PEO and PCL, which were evidenced by the results of FTIR. The results of optical microscopy and DSC showed that in the ternary blends PCL component separated out with inclusion of PEO. The formation of the specific phase structures is ascribed to the competitive interchain specific interactions among the crosslinked epoxy, PEO and PCL.

Phase boundary diagram of epoxy, PEO and PCL ternary blends.     

Synthesis of a Poly(vinyl alcohol)‐Based Graft Copolymer Having Poly(ε‐caprolactone) Side Chains by Solution Polymerization

Poly(vinyl alcohol)‐graft‐poly(ε‐caprolactone) (PVA‐g‐PCL) was synthesized by ring‐opening polymerization of ε‐caprolactone with poly(vinyl alcohol) in the presence of tin(II) 2‐ethylhexanoate as a catalyst in dimethyl sulfoxide. The relationship between the reaction conditions of the solution polymerization and the chemical structure of the graft copolymer was investigated. The degree of substitution (DS) and degree of polymerization (DP) of the PCL side chains were roughly controlled by varying the reaction periods and feed molar ratios of the monomer and the catalyst to the backbone. PVA‐g‐PCL with a PCL content of 97 wt.‐% (DP = 22.8, DS = 0.54) was obtained in 56 wt.‐% yield. The graft copolymer was soluble in a number of organic solvents, including toluene, tetrahydrofuran, chloroform, and acetonitrile, which are solvents of PCL. The molecular motion of the graft copolymer from ¹H NMR measurements appears to be restricted to some extent at 27–50°C, however the ¹H NMR signal intensities measured at temperatures higher than ca. 50°C reflect the actual chemical structure of the graft copolymer as determined by elemental analysis. The graft copolymer having a short PCL side chain (DP = 4.4, DS = 0.15) was amorphous. The melting temperature of a sample with relatively high PCL content (DP = 22.8, DS = 0.54) was observed at 39°C. Thermogravimetric analysis revealed that the thermal stability of PVA was improved by introducing PCL side chains. The surface free energies of the air‐side of a graft copolymer film, as calculated by Owens' equation using contact angles, were comparable to that of PCL homopolymer.     

Selection of internalization-deficient cells by interleukin-2-Pseudomonas exotoxin chimeric protein: the cytoplasmic domain of the interleukin-2 receptor β chain does not contribute to internalization of interleukin-2

To study the structural basis of ligand-induced receptor-mediated internalization of interleukin-2 (IL-2), a strategy has been developed to generate variant T cells that are deficient in internalization of this cytokine. IL-2 receptor (IL-2R) α- and β-bearing EL4 cells, that express high-affinity IL-2R and internalize IL-2, were treated with low doses of IL-2-Pseudomonas exotoxin chimeric protein (IL-2-PE40). This treatment resulted in isolation of a variant (CX1) that was unable to express high-affinity IL-2R or internalize IL-2. Transfection of CX1 with the IL-2Rβ cDNA led to surface expression of IL-2Rβ and high-affinity IL-2R as well as the ability to internalize IL-2. This finding indicates that the absence of the p subunit was the sole defect in CX1 responsible for its failure to internalize IL-2. By transfecting CX1 with mutated β cDNA, several CX1 transfectants were produced that expressed a β-subunit that lacked all amino acids of the intracytoplasmic region. These transfectants expressed high-affinity IL-2R and internalized IL-2 at a rate comparable to cells expressing wild-type β-chain. These results demonstrate that internalization of IL-2 is independent of any signals contained in the intracytoplasmic tail of the β subunit and raise the possibility that such signals may be entirely contained within the γ subunit.     

Reconstitution of a functional interleukin (IL)-7 receptor demonstrates that the IL-2 receptor γ chain is required for IL-7 signal transduction

The interleukin (IL)-2 receptor γ chain has recently been shown to be a component of the IL-7 and IL-4 receptors. Using a transient transfection assay and the trans-activation of reporter gene constructs which are under the control of cytokine-responsive promoter elements, we have studied signal transduction through the IL-7 receptor (IL-7R). The reporter gene expression was not stimulated by receptors that contained the cytoplasmic domain of the IL-7R, either as intact IL-7R or as part of a chimeric receptor. However, co-expression of the IL-7R with the IL-2 receptor γ chain was able to stimulate gene activation. For maximal stimulation the intact cytoplasmic domains of each chain was required.     

Surface Patterning of Poly(ε‐caprolactone): Epitaxial Crystallization and Enzymatic Degradation

Surface patterning was carried out by the epitaxial crystallization of biodegradable PCL on a HOPG, and the surface morphologies were observed by atomic force microscopy. Edge‐on view lamellae were aligned along the HOPG lattice to display stripe patterns in the threefold symmetry. The intervals of stripe patterns composed of ridges and valleys increased with an increase in the crystallization temperature. Enzymatic degradation of the PCL nanopattern allowed the different depth profiles of the fringed structure. The persistence length of the nanopattern could be tuned by the molecular weight of PCL.