Biochemical and Biosynthetic Studies of a Crystallizable Human γ1 Heavy-Chain Disease Protein

We have studied the structure of a crystallizable gamma 1 heavy-chain disease protein that lacks the entire VH and C gamma 1 domains. The protein starts within the hinge region at aspartic acid 221 (Eu numbering). The native protein is a disulphide-linked dimer with an apparent molecular weight of 52,000, consistent with the biochemical data obtained on the whole protein and its cyanogen bromide fragments. The carbohydrate content of this protein was 6.8%. As shown by biosynthesis experiments intracytoplasmic gamma chains synthesized by neoplastic cells had an apparent molecular weight similar to that of the serum heavy-chain disease protein. These data are compared with those obtained for other gamma 1 heavy-chain disease proteins beginning in the hinge region, and the mechanisms leading to those abnormal Ig products are discussed.     

Serial Measurements of Pregnancy-specific β1-glycoprotein (β1SP1) in Patients with Trophoblastic Disease

A radioimmunoassay for pregnancy-specific-β₁-glycoprotein (Bohn. 1971) has been established with a limit of detection of 2 μMg/litre in serum. The assay has been used to measure serial levels of pregnancy-specific-β₁-glycoprotein (β₁SP₁) in the serum of patients with choriocarcinoma and teratoma for comparison with measurements of the β-subunit of human chorionic gonadotrophin. The value of the assay for β₁SP₁, in the management of these patients is discussed.     

Ganglioside GM2 N-acetyl-ß-D-gafactosaminidase and asialo GM2 (GA2) N-acetyl-ß-D-galactosaminidase; studies in human skin fibroblasts

Ganglioside G_M2 and its asialo-derivative, G_A2 were radiolabeled in their N-acetyl-D-galactosaminyl moieties by oxidation with galactose oxidase and reduction with tritiated sodium borohydride. Specific activities of 6 × 10⁴ dpm/nmol (G_M2) and 1.8 × 10⁶ dpm/nmol (G_A2) were achieved. About 98% of the label was in N-acetyl-D-galactosamine. Using these substrates, an assay was developed for G_M2-N-acetyl-β-D-galactosaminidase (E.C.3.2.1.30) and G_A2-N-acetyl-β-D-galactosaminidase (E.C.3.2.1.30) activities in human cultured skin fibroblasts. The products of the G_M2 cleaving reaction were identified as N-acetylgalactosamine and ganglioside G_M3- Both G_M2 and G_A2 cleaving activities were stimulated about 5-fold by purified sodium taurocholate, and this stimulation was inhibited by neutral detergents, lipids and albumin at low concentrations. Addition of various salts, reducing agents and a protein activator factor from human liver of Li et al. (1973) did not stimulate G_M2-N-acetyl-β-D-galactosaminidase activity beyond that found with sodium taurocholate. Under optimal conditions, control fibroblast supernates cleaved ganglioside G_M2 at a rate of 3.7 nmol/mg protein/h compared to 1100 for G_A2-N-acetyl-β-D-galactosaminidase and 4700 for 4-methylumbelliferyl-N-acetyl-β-D-glucosaminidase. Supernates from two patients with Tay-Sachs disease had markedly reduced activity levels for G_M2-N-acetyl-β-D-galactosaminidase but not for the other two substrates. Supernates from two patients with Sandhoff's disease had reduced activities for all three substrates. A supernate from one patient with juvenile G_M2 gangliosidosis cleaved G_M2 at a somewhat faster rate than those from Tay-Sachs or Sandhoff's patients. Two healthy adult women with markedly reduced hexosaminidase A activities using 4MU-N-acetyl-β-D-glucosaminide as substrate had approximately half-normal activities using G_M2 as substrate. A patient with the Tay-Sachs phenotype but with a partial deficiency of hexosaminidase A using the 4-MU substrate had a profound deficiency using G_M2 as substrate. In such unusual hexosaminidase mutants, assays using G_M2 as substrate are better indicators of phenotype than those using synthetic substrates.     

Intraepithelial T Cells of the TcRγ/δ+CD8− and Vδ1/Jδ1+ Phenotypes are Increased in Coeliac Disease

Expression of the gamma/delta T-cell receptor (TcR) for antigen on CD3+ intraepithelial lymphocytes (IEL) was studied in situ by two-colour immunofluorescence on jejunal tissue sections from 24 patients with coeliac disease and 17 controls. The proportion of intraepithelial TcR gamma/delta+ cells was significantly increased (P less than 0.002) in untreated (median 20%, range 11-53%) as well as in treated (gluten-free diet) coeliac disease (median 23%, range 16-55%) compared with controls (median 2%, range 0-39%). Although TcR alpha/beta+ IEL dominated both in controls and coeliac disease, T cells expressing the TcR gamma/delta were preferentially located within the epithelium rather than in the lamina propria. Paired staining for TcR gamma/delta and CD8 revealed that most (approximately 90%) intraepithelial TcR gamma/delta+ lymphocytes in coeliac disease were CD8-. A remarkably large fraction (median 67%, range 58-94%) of intraepithelial TcR gamma/delta+ cells expressed the V delta 1/J delta 1-encoded epitope revealed by monoclonal antibody delta TCS1. Our results suggested that increase of the intraepithelial TcR gamma/delta+ CD8- subset of T cells is particularly related to coeliac disease.     

Phenotypical and Functional Characterization of Small Intestinal TcRγδ+ T Cells in Coeliac Disease

Increased numbers of TcR gamma delta + T cells are present in the small intestinal epithelium of patients with coeliac disease (CoD). Their function, however, is unknown. In order to facilitate detailed functional studies, intestinal gamma delta T cells have been isolated from small intestinal biopsies of patients with CoD (n = 18) and controls (n = 14). As expected, increased numbers of V delta 1+ TcR gamma delta + T cells were detected in freshly isolated intraepithelial cell suspensions (IEL) from CoD patients. Also, in the in vitro expanded IEL T-cell populations from CoD patients the numbers of V delta 1+ TcR gamma delta + T cells were increased compared with similar cell cultures from control patients. From IEL cultures derived from six CoD patients, 107 T-cell clones were generated by limiting dilution and analysed. Sixty of these clones were either CD4 or CD8 positive TcR alpha beta + clones. The remaining 47 clones expressed the TcR gamma delta. Further phenotypical analysis of the gamma delta T-cell clones indicated that the TcR gamma delta + T-cell population in the small intestinal epithelium of CoD patients is heterogeneous: four TcR gamma delta phenotypes could be detected and, although the majority of the TcR gamma delta + T cells were CD4 CD8, gamma delta T-cell clones expressing either a CD8 alpha alpha homodimer, a CD8 alpha beta heterodimer or CD4 were also identified. In contrast to the TCR alpha beta + IEL, most TcR gamma delta + IEL were CD5 negative. Furthermore, biochemical analysis indicated that the increase in V delta 1+ gamma delta T cells in the small intestinal epithelium of CoD patients was not the result of a monoclonal expansion. The small intestinal epithelium-derived gamma delta T-cell clones were functional in vitro since the majority of these clones were able to lyse target cell lines such as K562. Molt4 and Daudi. These novel findings therefore indicate that the gamma delta T cells in the small intestine of CoD patients represent a heterogeneous population and that such cells are functional in vitro. The isolation and the in vitro propagation and cloning of these cells may open new avenues for the study of the putative immune mechanisms leading to coeliac disease.     

Concanavalin A Induces Suppressor Cell Activity in Both TgM and Tnon-γ Cells: Most of the Suppressor Cells Do Not Carry HLA-DR Antigens

Concanavalin A (Con A) stimulation of T lymphocytes for 48 h induced a dose-dependent increase in the percentage of T gamma cells, and proliferation of these cells was dependent on pre-existing T gamma cells. Con A stimulation also induced a dose-dependent increase in suppressor cell activity as measured in a Con A/mixed lymphocyte culture assay. Removal of T gamma cells before stimulation with Con A did not influence suppressor cell activity, whereas depletion of T gamma cells after Con A stimulation significantly reduced the suppression. Treatment of T lymphocytes with monoclonal anti-HLA-DR antibodies and complement significantly increased the percentage of T gamma cells and also increased the suppressor cell activity, both when the cells were treated before and when they were treated after Con A stimulation. Similarly, increased suppressor cell activity was also seen when T mu cells were removed. The results indicate that Con-A-induced suppressor cells are recruited from both T gamma and T non-gamma cells and also suggest that most of the Con-A-induced suppressor cell activity is exerted by HLA-DR-negative cells.     

Reactivity to sodium tetrachloropalladate (Na2[PdCl4]) compared to PdCl2 and NiCl2 in lymphocyte proliferation tests

Background: For patch testing, replacement of the commonly used palladium dichloride (PdCl₂) by sodium tetrachloropalladate (Na₂[PdCl₄]) was recently demonstrated to improve test accuracy and show a significant correlation with nickel (Ni), supporting the concept of cross‐reactivity between Pd and Ni. A promising alternative to metal allergy patch testing is the in vitro lymphocyte proliferation test (LTT). Objectives: The aim of this study was to test whether Na₂[PdCl₄] is also more sensitive for diagnosing Pd allergy with a standardized LTT. Patients/methods: After determining optimal nontoxic and nonmitogenic concentrations for Na₂[PdCl₄], blood samples from 105 patients with clinical suspicion of metal allergy were tested with an LTT called memory lymphocyte immuno stimulation assay for Na₂[PdCl₄], PdCl₂ and NiCl₂. Reaction profiles were analysed for concordant positive reactions. Results: Using the conventional cut‐off of stimulation index ≥ 3, 74.3% showed a positive reaction to NiCl₂, 15.2% to PdCl₂ and 28.6% to Na₂[PdCl₄]. All positive results to PdCl₂ were covered by Na₂[PdCl₄]. From the 30 positive reactions to Na₂[PdCl₄], 26 (87%) were concordant for NiCl₂ reactivity. Conclusion: In LTT, the use of Na₂[PdCl₄] results in more positive reactions in Pd allergy testing which are in concordance with positive reactions to PdCl₂ and NiCl₂.     

Aquaporin-1 and HCO3−-Cl− transporter-mediated transport of CO2 across the human erythrocyte membrane

Recent studies have suggested that aquaporin-1 (AQP1) as well as the HCO₃⁻-Cl⁻ transporter may be involved in CO₂ transport across biological membranes, but the physiological importance of this route of gas transport remained unknown. We studied CO₂ transport in human red blood cell ghosts at physiological temperatures (37 °C). Replacement of inert with CO₂-containing gas above a stirred cell suspension caused an outside-to-inside directed CO₂ gradient and generated a rapid biphasic intracellular acidification. The gradient of the acidifying gas was kept small to favour high affinity entry of CO₂ passing the membrane. All rates of acidification except that of the approach to physicochemical equilibrium of the uncatalysed reaction were restricted to the intracellular environment. Inhibition of carbonic anhydrase (CA) demonstrated that CO₂-induced acidification required the catalytic activity of CA. Blockade of the function of either AQP1 (by HgCl₂ at 65 μM) or the HCO₃⁻-Cl⁻ transporter (by DIDS at 15 μM) completely prevented fast acidification. These data indicate that, at low chemical gradients for CO₂, nearly the entire CO₂ transport across the red cell membrane is mediated by AQP1 and the HCO₃⁻-Cl⁻ transporter. Therefore, these proteins may function as high affinity sites for CO₂ transport across the erythrocyte membrane.     

A comparison of the biochemical properties of the human diaphorase (DIA3) isozymes determined by the common alleles DIA13, DIA23 and DIA33

(1) Various buffer systems for the starch gel electrophoresis of human diaphorase isozymes have been explored. Electrophoresis in a Tris/Borate system at pH 8.6 which includes 70 micron NADH in the gel and cathodal electrode buffers, provides good resolution of the six DIA3 phenotypes previously resolved by isoelectric focusing. (2) The variant genes DIA13, DIA23 and DIA33 occur with frequencies of about 0.76, 0.23 and 0.01 respectively in the English population. (3) The isozymes determined by the least common gene, DIA33, are markedly different from the isozymes determined by DIA13 and DIA23 in their relatively low heat stability, high affinity for Blue Sepharose and slow anodal electrophoretic mobility in buffer systems containing borate. The DIA3 1 and DIA3 2 isozymes are similar to one another in these characteristics.     

Urinary leukotriene E4 and 11-dehydrothromboxane B2 in patients with aspirin-sensitive asthma

The objective of this study was to define the participation of cysteinyl leukotrienes (LTs) or thromboxane A₂ in the pathogenesis of aspirin-sensitive asthma (ASA). Leukotriene E₄ (LTE₄) and 11-dehydrothromboxane B₂ (11DTXB₂) values in spot urine were measured in 22 asthmatics with a history of aspirin sensitivity and in 17 without such a history of aspirin-sensitive asthma [NASA]) in the outpatient clinic. The urinary LTE₄ value was significantly higher in ASA patients than in NASA (340±47 vs 65±15 pg/mg·cr, P <0.001), but there was no significant difference in urinary 11DTXB2 between the two groups (891±77 vs 657±90 pg/mg·cr). A high value of LTE4 was not associated with type of asthma, severity of disease, oral prednisolone treatment, sex, or age. A higher value of 11DTXB₂ was observed in the atopic type than the nonatopic type in ASA (1086±111 vs 697±147 pg/mg·cr, P<0.05). No correlation was observed between urinary LTE₄ and 11DTXB₂ in either ASA or NASA. In conclusion, LTs may play an important role in the pathogenesis of ASA, and TXA₂ in the pathogenesis of the atopic type in ASA.     

Linkage relationships between the three phosphoglucomutase loci PGM1, PGM2 and PGM3

1. Phosphoglucomutase phenotypes have been studied in several generations of the family of an individual heterozygous at each of the three loci, PGM₁, PGM₂, and PGM₃. 2. PGM₁ and PGM₂ phenotypes were determined using red cells. Fibroblasts grown in tissue culture were used for PGM₃ phenotyping. 3. The family results support the genetical hypothesis based on the analysis of dizygotic twin pairs that the PGM₃ isozyme patterns found in the placenta are determined by two alleles, PGM¹₃ and PGM²₃. 4. Locus PGM₃ is not closely linked to locus PGM₂ 5. The data also support the previous findings that locus PGM₁ is not closely linked to PGM₂ or PGM₃.