Localization of murine Ig-1b and Ig-1a (IgG 2a) allotypic determinants detected with monoclonal antibodies

We have used hybridoma anti-allotype antibodies to define six allotypic determinants: four on Ig-1b molecules and two on Ig-1a molecules. By locating these determinants on fragments obtained by limited proteolysis of IgG_2a, molecules and by hybridoma antibody blocking assays, five distinct allotypic sites have been defined on mouse γ_2a heavy chains. These sites are located in the hinge region and the C_H2 and C_H3 domains. We have not been able to show genetic recombination between these sites by genetic linkage studies.     

Synthesis and ring-opening polymerization of α-chloromethyl-α-methyl-β-propiolactone

α-Chloromethyl-α-methyl-β-propiolactone (CMMPL) was synthesized by dehydrohalogenation of α,α-dichloromethyl-β-propionic acid which was obtained by chlorination of α,α-hydroxymethyl-β-propionic acid (DMPA). Due to high strain of the four-numbered ring, CMMPL can be polymerized by ring-opening with or without an initiator. Both electrophiles like trifluoroacetic acid (TFAA) and nucleophiles like triethylamine (TEA) and pyridine, as well as organometallic compounds such as stannous octoate [Sn(Oct)₂)], aluminium triisopropoxide [Al(OⁱPr)₃] and tetrabutyl orthotitanate [Ti(OC₄H₉)₄], were found to be effective initiators. The polymerization can be conducted by either solution or bulk polymerization. P(CMMPL) is insoluble in almost all organic solvents at room temperature. An endothermic peak (ca. 214 ˜ 250°C) attributed to the melting transition of P(CMMPL) was observed in DSC curves. P(CMMPL) tends to have high crystallinity (40% ˜ 60%) as demonstrated by its X-ray diffraction patterns, and the crystallinity was found to vary with the types of initiator used.     

Structure of the T cell receptor in a TiαVβ2,αVβ8-positive T cell line

The T cell receptor (TcR) is composed of at least six different polypeptide chains consisting of the clonotypic Ti heterodimer (Tiαβ or Tiγδ) and the noncovalently associated CD3 chains (CD3γδ?ζ). The exact number of subunits constituting the TcR is still not known; however, it has been suggested that each TcR contains two Ti dimers. To gain insight into the structure of the TcR we constructed a TiαVβ2,αVβ8-positive T cell line which expressed the endogenous human TiVβ8 and the transfected mouse TiVβ2 both in association with the endogenous Tiα and CD3 chains at the cell surface. Preclearing experiments with radioiodinated cell lysate prepared with digitonin lysis buffer demonstrated that depleting the lysate of TiαVβ8 by immunoprecipitation with anti-Vβ8 monoclonal antibody (mAb) did not reduce the amount of TiαVβ2 in the lysate, and likewise, depleting the lysate of TiαVβ2 with anti-Vβ2 mAb did not reduce the amount of TiαVβ8. Comodulation experiments showed that Vβ8 and Vβ2 did not comodulate with each other. Furthermore, functional tests demonstrated that TcR containing Vβ8 and TcR containing Vβ2 mediated transmembrane activation signals independently of each other. These data demonstrate that mouse Vβ2 and human Vβ8 were not expressed in the same TcR in agreement with a TcR model where each TcR contains only one Ti dimer.     

Purification of rabbit T-derived (Ig-) lymphocytes.

Rabbit peripheral blood cells were rosetted by means of mixed agglutination technique described by Coombs et al. and the unrosetted Ig-, T-derived lymphocytes were separated in Ficoll-Triosil gradient. The cells which did not rosette in mixed agglutination reaction represented highly purified T-derived (Ig-) lymphocytes, as evidence by their response to T and B rabbit cell mitogens, their cytotoxic reactivity versus rabbit thymus lymphocyte antiserum (RTLA), and by failure to be stained with labelled anti-rabbit immunoglobulins antibodies and antibodies against allotypic specificities of the a and b series.     

Cross talk between αvβ3 and α4β1 integrins regulates lymphocyte migration on vascular cell adhesion molecule 1

Local inflammation leads to increased expression of the vascular cell adhesion molecule (VCAM)-1 on vascular endothelium which contributes to the encapture of leukocytes from the circulating blood through the leukocyte ligand α4β1 integrin. Inflammatory vascular endothelium expresses VCAM-1 at high density. We found that the speed of locomotion of activated lymphocytes migrating along surfaces coated with recombinant VCAM-1 at a comparable density to that found on inflammatory endothelium was slow. However, lymphocytes do migrate and extravasate rapidly under inflammatory conditions, indicating that there must be mechanisms that regulate the interaction between α4β1 and VCAM-1 in vivo. Here we show that the lymphocyte αβ3 integrin and integrin-associated protein (IAP) is able to regulate this interaction. The occupancy of lymphocyte αvβ3 integrin by platelet cell adhesion molecule-1 or vitronectin regulated the speed of α4β3 integrin-dependent locomotion of lymphocytes on recombinant VCAM-1. This allowed rapid lymphocyte migration at VCAM-1 densities which are typical of inflammatory vessels. This αvβ3-mediated enhanced migration of lymphocytes via α4β1 is likely to depend on the interaction of αvβ3 integrin with the IAP. Furthermore, this motile process correlates with polarization of the actin cytoskeleton in lymphocytes. Our results suggest that cross talk between αvβ3 integrin and α4β1 integrin is a mechanism in the regulation of lymphocyte locomotion along inflammatory endothelium and subsequent transendothelial migration. This can explain how lymphocytes overcome tight adhesion to the vascular endothelium and start rapid migration along and through the endothelial lining of blood vessels into inflammatory tissue.     

Appearance and distribution of laminin a chain isoforms and integrin α2, α3, α6, β1, and β4 subunits in the developing human small intestinal mucosa

Background: Laminin, a major component of basement membranes, is well known in its classical heterotrimeric form (B1-A-B2) to regulate diverse biological functions, including cell polarization and differentiation. However, the role of merosin, a laminin-like molecule in which an M chain is substituted for its homologous A chain, remains largely unknown. Methods: In the present study, we analyzed by indirect immunofluorescence the expression and distribution of these four laminin chains as well as the integrins α2β1, α3β1,α6β1, and α6β4, four potential recptors, at the epithelial-mesenchymal interface of the developing human small intestine, with a panel of specific monoclonal antibodies. Results: Beginning at 7 weeks of gestation and throughout mucosal organogenesis, the B1 and B2 chains were uniformly detected at the epithelial basement membrane. The A chain also was detected beginning at 7 weeks, and its distribution at the basement membrane remained uniform throughout villus (9+ weeks) and crypt (16+ weeks) formation. In contrast, M chain expression was not observed until 16 weeks; between 16 and 20 weeks, it was exclusively associated with the base of epithelial cells that comprised the forming crypts. Integrins α6β1 and α6β4, as determined by their subunit immunolocalization, appeared to be expressed by all enterocytes from 7 to 20 weeks. In contrast, the expression of the α2β1 and α3β1 integrins was found time- and site-restricted. The α2 subunit was predominantly detected in the epithelial cells of the intervillous area and its derivative, the crypt, whereas the α3 subunit was strongly expressed by all epithelial cells except those located at the bottom of 19–20-week-old crypts. Conclusions: Taken together, these observations demonstrate that both compositional changes in the basement membrane and differential expression of receptors occur during human intestinal organogenesis, suggesting that epithelial cell-matrix interactions play a role during development. © 1995 Wiley-Liss, Inc.     

Molecular characterization and clinical presentation of HKαα and anti‐HKαα alleles in southern Chinese subjects

The HKαα allele is a rearrangement occurring in the α‐globin gene cluster containing both the ‐α^3.7 and ααα^anti4.2 unequal crossover junctions. The anti‐HKαα allele is the reciprocal product containing both the ‐α^4.2 and ααα^anti3.7 unequal crossover junctions, which had been predicted but had not been detected previously. The phenotypic feature and population frequency of these two unusual alleles were not described. We report the identification of nine individuals carrying the HKαα allele and two individuals carrying the anti‐HKαα allele in southern China and describe their phenotype and haplotype data. The molecular structures of HKαα allele and anti‐HKαα allele were confirmed by two‐round nested polymerase chain reaction assay. The mechanism of origin of both alleles is related to probably simultaneous double crossover. Heterozygotes of HKαα or anti‐HKαα allele show a normal hematological phenotype. Finally, we report the carrier rates of these both alleles in the Guangxi Zhuang Autonomous Region of southern China, namely, ∼0.07% for the HKαα allele and ∼0.02% for the anti‐HKαα allele.     

Preparation and investigation of optically active polyesters from α-ethyl-α-phenyl-β-propiolactone, 1

α-Ethyl-α-phenyl-β-propiolactone ( 7 ) of an optical purity up to 95% was prepared; the resolution of the two optical antipodes was carried out with the optical active precursor ethyl 2-aminomethyl-2-phenylbutyrate ( 5 ), and the optical purity was determined by ¹H NMR analysis using europium salt shift reagents. The solution and thermal properties of the corresponding polyesters 8 , as obtained by anionic ring opening polymerization, were investigated. It was found that the inherent viscosity increased with the optical purity of the lactone. Distinct differences in the melting behaviour were found between the racemic and the optically active polymers; the ideal melting temperature T_M⁰ for the pure enantiomeric polymer was estimated to about 535 K.     

Presence of common idiotypes on antibodies induced by glutamic acid-lysine-containing terpolymers in responder and nonresponder mice with the Ig-1b heavy chain allotype.

A B10.A (5R) responder mouse to the random linear terpolymer, poly--(Glu, Lys, Phe), GLphi, can produce immunoglobulins which bind poly-L (Glu, Lys), GL, that share idiotypic determinants with GL-binding antibodies produced by other members of the same strain. Expression of these common idiotypic determinants, termed BGL, is independent of the H-2 halotype and closely linked to the Ig-lb heavy chain allotype. Moreover, nonresponder mice with the Ig-lb heavy chain allotype, when immunized with GLphi that has been chemically coupled to an immunogenic carrier, chicken IgG, can produce GL-binding antibodies that share BGL idiotypic specificities with anti-GLphi antibodies produced by responder animals. Also, the responses to other GL-containing polymers, such as poly-L (Glu, Lys, Ala) and poly-L (Glu, Lys, Pro), which are under the control of distinct Ir genes, can stimulate the production of GL-binding antibodies that share common BGL idiotypic determinants with antibodies induced with GLphi. These findings are discussed with respect to their implications concerning the mechanism(s) of Ir gene control.     

Synthesis of 3-amino-ribofuranans having 1,5-α and -β structures by selective ring-opening polymerization of a 1,4-anhydro-3-azido-3-deoxy-α-D-ribopyranose derivative

Ring-opening polymerization of a new anhydro ribose-type monomer, 1,4-anhydro-3-azido-3-deoxy-2-O-tert-butyldimethylsilyl-α-D -ribopyranose (A3ASR), was investigated. The monomer was synthesized from 1,4-anhyro-α-D -xylopyranose by three steps comprising Walden inversion at the C3 position into ribose configuration. Ring-opening polymerization of A3ASR by Lewis acid catalysts such as boron trifluoride etherate and stannic chloride gave a stereoregular 3-azido-3-deoxy-2-O-tert-butyldimethylsilyl-(1→5)-α-D -ribofuranan having specific rotations of +246 ～ +271 deg · dm^?1 · g^?1 · cm³ and number-average molecular weights of 18,7 × 10³ ～ 25,1 × 10³. When the polymerization was carried out by antimony pentachloride at 0°C, the resulting polymer exhibited a negative specific rotation of ?6 deg · dm^?1 · g^?1 · cm³ and the C1 absorption in the ¹³C NMR spectrum shifted downfield to 107,5 ppm, suggesting that the polymer might consist of 1,5-β furanosidic unit. The reduction of the azido group of the 1,5-α and 1,5-β furanosidic polymers into amino group and subsequent desilylation gave 3-amino-3-deoxy-(1→5)-α- and -β-D -ribofuranans, respectively. In addition, copolymerization of A3ASR with 1,4-anhydro-2,3-di-O-tert-butyldimethylsilyl-α-D -ribopyranose (ADSR) in various feeds was performed by boron trifluoride etherate as catalyst to give copolymers with different monomeric components. The structural analysis of the homopolymers and copolymers was examined by means of ¹H and ¹³C NMR spectroscopies, IR spectroscopy, and optical rotation.     

Synthesis and X-ray photoelectron spectroscopy of poly(α-ethyl-α-methyl-β-propiolactone) terminated with a perfluoroalkyl group

Perfluorinated carboxylates are efficient initiators of the α-ethyl-α-methyl-β-propiolactone polymerization resulting in perfluoroalkyl-terminated polyester. The X-ray photoelectron spectra of the C₈F₁₇-end-capped polyester agree with an enrichment in fluorine atoms of the surface layer corresponding to an average number of 7 perfluoro groups per chain or to 36,6 wt.-% of C₈F₁₇-groups instead of 8,4 wt.-% as calculated for a random distribution.     

Human γδ T Cells Express a Higher TCR/CD3 Complex Density Than αβ T Cells

The aim of our study was to compare CD3 expression on γδ T cells and αβ T cells in human patients. The antigen density of TCR and CD3 on both subsets was assessed by a quantitative method in eight patients. In parallel, we developed and validated a reliable direct tricolor staining protocol that we tested on samples from hospitalized and healthy individuals (n = 60). Our results demonstrate that human γδ T cells constitutively express approximately twofold more of the TCR/CD3 complex than αβ T cells. We suggest that this enhanced expression of the TCR/CD3 complex could contribute to the higher reactivity of γδ T cells compared to αβ T cells. These clinical laboratory results confirm the fundamental data described elsewhere. γδ T cells deserve further clinical investigations to understand their precise role in human immunity.     

The production of 3aα-H-4α-(3′-propionic acid)-5α-hydroxy-7aβ-methylhexahydro-1-indanone-δ-lactone by Nocardia restricta mutants

Two mutants of Nocardia restricta breakdown androst-4-ene-3,17-dione into 3aα-H-4α-(3′-propionic acid)-5α-hydroxy-7aβ-methylhexahydro-l-indanone-δ-lactone. The metabolic intermediates formed are very different in each mutant. One uses certain aromatic compounds very slowly. A degradation pathway for steroids by Nocardia restricta has been proposed.     

Scanning of estrogen receptor α (ERα) and thyroid hormone receptor α (TRα) genes in patients with psychiatric diseases: Four missense mutations identified in ERα gene

Estrogen and thyroid hormones exert effects on growth, development, and differentiation of the nervous system. Hormone administration can lead to changes in behavior, suggesting that genetic variants of the estrogen receptor α (ERα) and the thyroid hormone receptor α (TRα) genes may predispose to psychiatric diseases. To investigate this possibility, regions of likely functional significance (all coding exons and flanking splice junctions) of the ERα and TRα genes were scanned in patients with schizophrenia (113), along with pilot studies in patients with bipolar illness (BPI), puerperal psychosis, autism, attention‐deficit hyperactivity disorder (ADHD), and alcoholism. A total of 1.18 megabases of the ERα gene and 1.16 megabases of the TRα gene were scanned with Detection of Virtually All Mutations‐SSCP (DOVAM‐S), a method that detects virtually all mutations. Four missense mutations, seven silent mutations and one deletion were identified in the ERα gene, while only four silent mutations were present in the TRα gene. Two of the missense mutations in ERα are conserved in the six available mammalian and bird species (H6Y, K299R) and a third sequence variant (P146Q) is conserved in mammals, birds, and Xenopus laevis, hinting that these sequence changes will be of functional significance. These changes were found in one patient each with BPI, puerperal psychosis, and alcoholism, respectively. Analysis of the ERα and TRα genes in 240 subjects reveals that missense changes and splice site variants are uncommon (1.7% and 0%, respectively). Further analyses are necessary to determine if the missense mutations identified in this study are associated with predisposition or outcome for either psychiatric or nonpsychiatric diseases. © 2001 Wiley‐Liss, Inc.     

Monocyte activation: rapid induction of α1/β1 (VLA-1) integrin expression by lipopolysaccharide and interferon-γ

Monocytes play a key role in inflammation, tissue injury and remodelling and wound healing, and most monocyte effector functions are dependent on adhesive interactions. We have analyzed the changes in the pattern of β1 integrin expression that take place during monocyte activation and demonstrated that lipopolysaccharide (LPS) and interferon (IFN)-γ specifically induce the expression of the α1/β1 integrin, which was detectable on the monocyte membrane as early as 12 h after monocyte activation. The up-regulated α1/β1 expression was not dependent on monocyte adherence to solid surfaces, and Northern blot analysis revealed that LPS and IFN-γ induce the α1 mRNA de novo. Monocyte deactivating cytokines such as interleukin (IL)-4 or IL-10, could only minimally inhibit the LPS- or IFN-γ mediated up-regulation of α1/β1, suggesting that cytokine release subsequent to monocyte activation does not play a major role in the integrin induction. Interestingly, the LPS-induced expression of α1/β1 was found to be dependent on the redox state of the cell, since it was inhibited by antioxidants which also altered the morphological changes that take place during monocyte culture in vitro. The rapid induction of α1 in LPS-activated monocytes suggests that α1/β1 might be involved not only in monocyte/extracellular matrix interactions during inflammatory reactions, but also in contributing to further monocyte activation and cytokine production during septic shock syndrome.     

α-thalassaemia due to a single codon deletion in the α-1-globin gene. Computational structural analysis of the new α-chain variant

A new unstable α-globin chain associated with α-thalassemia phenotype has been found in a Spanish patient. Molecular analysis of the α-globin gene complex using PCR and non-radioactive single-strand conformation analysis, allowed to identify a new mutation in the second exon of the α-globin gene. Direct sequencing of the abnormal fragment revealed a 3 bp deletion, which led to the loss of a single codon corresponding to a Lys (K) residue at position 60 or 61 DK60 or DK61. Theoretical structural analysis, performed by computational methods, indicated that the loss of an amino acid residue at this position disturbed the contact region between the B and E-helices, affecting the overall stability of the molecule. Therefore, the DK60 or DK61 results in a structurally abnormal α-globin chain, not previously described, named Hb Clinic, which leads to the α-thalassemia phenotype in the heterozygote patient. No abnormal hemoglobin was detected by standard electrophoretic procedures, suggesting that this α-globin chain variant is so unstable that it may be catabolized immediately after its synthesis. This mutation was confirmed by PCR using an allele specific primer. Hum Mutat 11:412, 1998. © 1998 Wiley-Liss, Inc.     

Developmental expression of the αIELβ7 integrin on T cell receptor γδ and T cell receptor αβ T cells

A novel monoclonal antibody, 2E7, was shown by immunoprecipitation to be reactive with the α_IELβ7 integrin and was employed to analyze the expression of this integrin in lymphocyte subsets and during T cell ontogeny. In adult lymph nodes, α_IEL was expressed at low levels by 40–70% of CD8⁺ T cells and < 5% of CD4⁺ T cells. However, virtually all intestinal intraepithelial lymphocytes and ?20% of lamina propria CD4⁺ T cells were 2E7⁺, indicating a preferential expression of this integrin on mucosal T cells. Examination of α_IEL integrin expression during thymus ontogeny revealed that ?3–5% of fetal or adult thymocytes were 2E7⁺. Interestingly, early in fetal thymus ontogeny, ?40% of 2E7⁺ cells expressed T cell receptor (TcR)-γδ and this subset persisted through birth. A developmental switch occurred such that 2E7⁺ TcR^? CD4^?8⁺ cells detected on fetal day 19 were followed by 2E7⁺ TcR-αβ CD4^?8⁺ cells in the neonatal thymus. The latter population persisted throughout thymus ontogeny into adulthood. Interestingly, a subset of TcR-γδ Vγ3⁺ day 16 fetal thymocyte dendritic epidermal cell (DEC) precursors were 2E7⁺, but all mature DEC expressed high levels of α_IEL integrin, suggesting that the α_IEL integrin was acquired late in DEC maturation. This possibility was strenghthened by immunohistochemical localization of the majority of 2E7⁺ γδ and αβ T cells to the medullary regions of the thymus. Overall, the results demonstrate a developmentally ordered expression pattern of the α_IELβ₇ integrin that suggests a common function for this integrin during TcR-γδ and -αβ CD4^?8⁺ T cell thymocyte development or perhaps in effector functions for these subsets.     

Kinetic determination of stereoselectivity in the anionic polymerization of α-ethyl-α-phenyl-β-propiolactone

The kinetics of the anionic polymerization of optically active α-ethyl-α-phenyl-β-propiolactone (optical purity 16,8%) initiated with bis(triphenylphosphine)iminium acetate was investigated and the rate constants for the homo-(k_ph) and crosspropagation (k_pc) (considering R and S enantiomers as comonomers) were determined. The knowledge of the values of k_ph and k_pc, equal to 1,53·10^?4 and 9,0·10^?51· mol^?1·s^?1, respectively (25°C, CH₂Cl₂ solvent), allowed us to calculate the distribution of homosequences in the polymer prepared from racemic monomer. The concentration of homosequences was slightly higher than calculated for the process with random enchainment of enantiomers. Thus, the content of homodyads, homotriads, and homotetrads equals 63, 40, and 25%, whereas for the random process it was 50, 25, and 13%, respectively. This difference is, however, too small to create homoblocks which could be responsible for the observed crystallinity of these polymers.     

Mice heterozygous for a deletion of the tumor necrosis factor-α and lymphotoxin-α genes: biological importance of a nonlinear response of tumor necrosis factor-α to gene dosage

The tumor necrosis factors (TNF-α and lymphotoxin, or LT-α) are important mediators of the immune and inflammatory responses, and it has been proposed that a positive feedback loop could boost the expression of the TNF to sufficiently high levels to fend off infections. To investigate this phenomenon and its biological consequences, we have generated LT-α/TNF-α knockout mice and compared mice having one or two functional LT-α/TNF-α alleles. In response to lipopolysaccharide (LPS) stimulation, TNF-α levels in the circulation or in the supernatant of macrophage cultures were 20- to 100-fold lower in heterozygous samples than in their wild-type counterparts. This differential increased with the intensity of stimulation and throughout the response, supporting the involvement of a positive feedback loop. Moreover, the heterozygous mice had an increased bacterial load following Listeria monocytogenes infection and exhibited a bimodal response to the association of D -galactosamine and LPS which was similar to that of wild-type mice at low doses of LPS and more like that of homozygous mutants at high doses. These results therefore establish the biological importance of the nonlinear response of TNF-α levels to gene dosage, and these mice provide a unique tool to study how the propensity to produce TNF can determine the immunological fitness of individuals.