The effect of in vivo dexamethasone on lymphocyte subpopulations: Differential response of EAhu rosette-forming cells

The purpose of this study was to compare the differential effect of dexamethasone on “third population” lymphocytes with its effect on B and T lymphocytes. Five human volunteers were given a single oral dose of dexamethasone (6–8 mg). Samples were evaluated prior to the drug, 5 hr after drug ingestion and 24 hr following the poststeroid sample. Surface marker studies included SIg, EAC rosettes, mouse rosettes, EA_hu (Ripley) rosettes, total E-rosette-forming lymphocytes, Tμ lymphocytes, and Tγ lymphocytes. Functional studies included mitogen stimulation with phytohemagglutinin, concanavalin A, and pokeweed and antigen stimulation with herpes simplex virus and Candida albicans. At the nadir of the steroid-induced lymphopenia absolute counts for all B-cell markers were decreased. No differential effects were noted in SIg using polyvalent, IgM, IgG, κ, or λ antisera. Total T lymphocytes decreased 55% (P < 0.05). There was a differential effect on Tμ and Tγ subsets and the former were significantly decreased. Unlike other lymphocyte subpopulations, cells with high avidity Fc receptors for IgG molecules, i.e., EA_hu (Ripley)-rosette-forming and Tγ lymphocytes, were unresponsive to steroid treatment. EA_hu (Ripley)-rosette-forming lymphocytes increased from a baseline value of 15 to 28% after dexamethasone ingestion but absolute cell counts remained essentially unchanged with a mean baseline value of 287/μl compared with a poststeroid count of 250/μl. A rebound phenomenon was noted for B, T, Tμ, and Tγ lymphocytes (P < 0.05). Functional studies of the peripheral blood mixture of lymphocytes at the nadir of the lymphopenia showed significantly suppressed responses to both mitogens and antigens.     

Molecular characterization and phylogenetic study of velogenic Newcastle disease virus isolates in Iran

The pathogenicity and genetic characterizations of six Newcastle disease virus (NDV) isolates obtained from chicken farms in six different regions in Iran were carried out using conventional and molecular techniques. Based on the pathogenicity indices (MDT, ICPI, and IVPI), all of these isolates were found to be velogenic (highly virulent) strains. A sequence analysis of the full-length mRNA encoding the fusion glycoprotein precursor (F₀) of the NDV’s fusion proteins F₁ and F₂ in these six isolates showed the presence of point mutations in form of nucleic acid substitutions at positions 82^(C→T), 83^(T→C), 736^(A→G), and 1,633^(G→A). However, the nucleic acid residues at positions 330–347 of the precursor F₀ gene, corresponding to the cleavage site of the F₀ protein, were found to have remained conserved among the six NDV isolates. A phylogenetic comparison between the six Iranian isolates and the NDVs whose F₀ gene sequences were previously deposited in GenBank Database showed that all of the newly characterized Iranian NDV isolates belonged to genotype VII.     

Immunodominance with progenitor B cell diversity in the neutralizing antibody repertoire to influenza infection

We report striking immunodominance in the neutralizing antibody responses of major histocompatibility complex congenic mice to natural infection with influenza virus (H3N2 subtype), as deduced by sequencing the hemagglutinin (HA) genes of monoclonal antibody (mAb)-selected mutant viruses. A majority of mAb, established from individual BALB/c (H-2^d) mice, select mutant viruses containing the same single amino acid substitution in the membrane distal ectodomain, HA1 198 A→E, whereas changes at either HA1 158 G→E or HA1 198 A→E are selected for by mAb from BALB.K (H-2^k) donors. The structural basis for immunodominance, and potential diversity of progenitor B cells, was investigated by sequence analysis of H and L chain gene rearrangements in mAb specific for HA1 158 or HA1 198. No correlation was found between antibody specificity and V_H or V_L gene usage, and a minimum of three to six progenitor cells contributed to the individual's repertoire for a single antigenic site. However, in a further analysis of the HA1 158-specific antibody response of CBA/Ca (H-2^k) donors, there was highly restricted light chain gene usage. Focusing of the immune repertoire to limited regions of the HA molecule during a primary viral infection may be a significant factor in immune pressure for antigenic variation, particularly since there is no evident restriction in the antibody response to immunization.     

Seven novel mutations in mut methylmalonic aciduria

Methylmalonic aciduria (MMA) is an autosomal recessive inborn error of metabolism that results from functional defects in methylmalonyl CoA mutase (MCM), a nuclear-encoded, mitochondrial enzyme that uses the vitamin B₁₂ derivative, adenosylcobalamin (AdoCbl) as a cofactor. To date, 23 mutations have been identified at the MUT locus on the short arm of chromosome 6, causing the mut forms of MMA (mut complementation group; mut MMA, McKusick #251000). We now report seven novel mutations. Three were found in mut⁰ patients: R228Q (c759G→A) was found as a heterozygous change; G312V (c1011G→T) and 346delL (c1112delCTT) were both found as homozygous changes. Four mutations were found in mut^– patients: A191E (c648C→A) and V633G (c1974T→G) were found in the same patient; 684insL (c2128insCTC) and L685R (c2130T→G) were both found as homozygous changes. The recent modelling of the human methylmalonyl CoA mutase allowed for an interpretation of the identified mutations. Hum Mutat 11:270–274, 1998. © 1998 Wiley-Liss, Inc.     

The Genetic Basis of Human Vh4 Gene Family-Associated Cross-Reactive Idiotype Expression in CD5+ and CD5- Cord Blood B-Lymphocyte Clones

In a recent study we have observed a high frequency expression of cross-reactive idiotypes encoded by genes from the relatively small V_H4 family of immunoglobulin heavy chain genes in cord blood B-lymphocyte lines. Furthermore, we have demonstrated a selective pattern of expression of two V_H4-associated cross-reactive idiotype (CRI) in B-lytnphocyte lines established from CD5⁺ and CD5^- cord blood B-lymphocytes. There was a restricted expression of one CRI marker recognized by the 9G4 monoclonal antibody in lines established from CD5⁺ B-lymphocytes but not in those established from the CD5^- population. In the current study we examine the molecular basis for the selective pattern of CRI expression. Nucleotide-sequence analysis of functional immunoglobulin heavy chain (IgH) gene rearrangements in three CD5 ⁺ lines expressing the CRI recognised by 9G4 reveal that all use a single gene from the V_h4 family, the V4.21 gene. However, all three lines have distinct third complementarity determining regions (CDR3) implying different clonal origins. In contrast, four cord blood cell lines (two established from CD5⁺ B-lymphocytes) expressing the CRI recognized by MoAb Lcl have functional IgH gene rearrangements involving two ditferent genes from the V_h4 family, the V71–4, and V2–1 genes. Antigen specificity analysis reveals that all three 9G4-reactive lines produce antibodies that react with the I and/or i red blood cell carbohydrate antigens. These data suggest that the distinction in V_H4 gene use in CD5⁺ B-lymphocytes in cord blood results from a selection process in vivo that shapes the repertoire of CD5⁺ B-lymphocytes. This study extends recent observations that the monoclonal anti-CRI antibodies 9G4and Lc1 are markers of two distinct subgroups of proteins encoded by two subsets of genes within the V_H4 family. Furthermore, it appears that amino acid residues in framework region one and complementarity determining region two are critical for the expression of the cross reactive idiotypes and the serological distinction between the two subgroups of proteins.     

Screening the human bradykinin B2 receptor gene in patients with cardiovascular diseases: Identification of a functional mutation in the promoter and a new coding variant (T21M)

To elucidate if genetic variants in the bradykinin B₂ receptor (B₂) gene occur that could affect receptor expression and function, we screened for mutations in the promoter and in the coding region of the human B₂ gene. In our initial study we analyzed 92 consecutive, unrelated subjects (including 25 patients with hypertrophic cardiomyopathy, 18 patients with dilated cardiomyopathy (DCM), 25 patients with hypertension, 18 patients with coronary heart disease, and 6 patients with valvular heart disease) using nonradioactive polymerase chain reaction–single-strand conformation polymorphism analysis as mutation screening method. We detected eight as yet unknown polymorphic sites in the promoter region of the B₂ gene (−845 C/T, −704C/T, −649 insG, −640 T/C, −536 C/T, −412 C/G, −143 C/T and −78 C/T) with allele frequencies between 0.5 and 13%. One of them (−412 C/G) destroys a Sp1 binding site and abolishes protein binding to this Sp1 site in human umbilical vein endothelial cells and human vascular smooth muscle cells. In the protein-coding region one new coding variant (T21M) with the potential to create a truncated receptor isoform was detected. We determined the frequency of the promoter variant at position −412 (C → G) and the newly identified coding variant (T21M) in extended samples of 69 patients with HCM, 163 patients with DCM, 109 patients with hypertension, and 173 healthy anonymous blood donors. The promoter variant (−412C/G) was found in one blood donor and the T21M mutation was not found in the control population. Therefore, it appears that these mutations are rare events and the determination of clinical significance will be a demanding task in the future. Am. J. Med. Genet. 80:521–525, 1998. © 1998 Wiley-Liss, Inc.     

Clinical,In Silico,and Experimental Evidence for Pathogenicity of Two Novel Splice Site Mutations in the SH3TC2 Gene

Abstract: Charcot-Marie-Tooth (CMT) neuropathy is the most common inherited neuromuscular disorder. CMT is genetically very heterogeneous. Mutations in the SH3TC2 gene cause Charcot-Marie-Tooth neuropathy type 4C (CMT4C), a demyelinating form with autosomal recessive inheritance. In this study, two novel splice site mutations in the SH3TC2 gene have been studied (c.279G → A, c.3676–8G → A). Mutation c.279G → A was detected on one allele in two unrelated families with CMT4C in combination with a known pathogenic mutation (c.2860 C →T in one family, c.505T → C in the other) on the second allele of SH3TC2 gene. Variant c.3676–8G → A was detected in two patients from unrelated families on one allele of the SH3TC2 gene in combination with c.2860C →T mutation on the other allele. Several in silico tests were performed and exon trap experiments were undertaken in order to prove the effect of both mutations on proper splicing of SH3TC2. Fragments of SH3TC2 were subcloned into pET01 exon trap vector (Mobitec) and transfected into COS-7 cells. Aberrant splicing was predicted in silico for both mutations, which was confirmed by exon trap analysis. For c.279G → A mutation, 19 bases from intron 3 are retained in cDNA. The mutation c.3676–8G→ A produces a novel splice acceptor site for exon 17 and complex changes in splicing were observed. We present evidence that mutations c.279G → A and c.3676–8G →A in the SH3TC2 gene cause aberrant splicing and are therefore pathogenic and causal for CMT4C.     

Tissue plasminogen activator mutants lacking the growth factor domain and the first kringle domain: I: DNA Constructions,Expression in Mammalian Cells,Protein Structure,Fibrin Affinity and Enzymatic Properties

Six variants of tissue-type plasminogen activator (t-PA) were produced in mouse C127 cells using a bovine papilloma virus expression vector. All variants lacked the growth factor (G) domain and the first kringle domain (K1) and three of the variants also lacked the finger domain (F). Furthermore, the specific changes, Lys₂₇₇ → Val and Asn₄₄₈ → Gln were introduced into some of the molecules. The variants were denoted K2P, K2P(Val277), K2P(Gln448), FK2P, FK2P(Va1277), and FK2P(Gln448). Amino acid sequence analyses revealed that the variants were proteolytically processed in the amino terminus and at Arg₂₇₅-Ile₂₇₆ in the same way as the full sized molecule. A proteolytically sensitive site was identified in the F domain at Arg₂₇-Ser₂₈. The two variants that lacked glycosylation at Asn₄₄₈, K2P(GIn448) and FK2P(GIn448), were cleaved at Arg₄₄₉-Thr₄₅₀, indicating that the oligosaccharide normally present at Asn₄₄₈ protects this site against proteolysis. The fibrin affinity for all variants was markedly reduced compared with normal t-PA. The plasminogen activator activity of all variants was stimulated by cyanogen bromide fragments of fibrinogen. In an indirect chromogenic assay K2P and K2P(Val277) showed specific activities that were 23% and 36%, respectively, of that of wild type t-PA, while the corresponding non-glycosylated variant K2P(Gln448) was as active as t-PA. The activity of the three F domain-containing variants were between 88 and 98% of the value determined for t-PA. When the specific activity was determined with the fibrin plate assay all variants were found to have higher specific activities than t-PA (1.8–4.7 fold). The lack of correlation between the activity of t-PA and the variants in these two assays indicate that the reaction mechanism may differ between the variants and wild type t-PA. The kinetic constants K_m and k_cat were determined for two-chain forms of t-PA and the variants with the chromogenic peptide substrate DIleProArgpNA. The results show that the t-PA heavy chain is not affecting the reaction with small peptide substrates as the K_m and k_cat values were essentially identical for t-PA, K2P, and FK2P (K_m, 0.18–0.21 mM and k_cat, 8.8–11.1s⁻¹). The values for the two non-glycosylated variants K2P(Gln448) and FK2P(Gln448) were 0.28mM, 9.4s⁻¹ and 0.24mM, 5.3s⁻¹, respectively. Interestingly, the Val277 variants showed significantly reduced K_m values, suggesting that Lys²⁷⁷ is important for the substrate interaction. For the variant K2P(Val277) K_m and k_cat were 0.06mM and 6.5s⁻¹, respectively, and for FK2P(Val277) the corresponding values were 0.06mM and 6.0s⁻¹.     

Laser light scattering studies of soluble high performance fluorine-containing polyimides, 1. Polyimide synthesized from 2,2′-bis(3,4-dicarboxyphenyl)-hexafluoropropane dianhydride and 2,2′-(trifluoromethyl)-4,4′-biphenyldiamine

Five fractions of a polyimide synthesized from 2,2′-bis(3,4-dicarboxyphenyl)hexafluoropropane dianhydride (6FDA) and 2,2′-(trifluoromethyl)-4,4′-biphenyldiamine (PFMB) (6FDA/PFMB) in tetrahydrofuran (THF) at 30°C were investigated by a combination of static and dynamic laser light scattering (LLS). The relations of 〈R_h〉 (nm) = 2.38 x 10^?2 M _w^0.560 and A₂ (mol · cm^?3 · g^?2) = 2.1 × 10^?1 M _w^?0.43 were established, where M _w, 〈R_h〉 and A₂ are weight-average molecular weight, average hydrodynamic radius and second virial coefficient, respectively. A combination of M _w and the translational diffusion coefficient distribution G(D) leads to a relation of D(cm²/s) = 2.41 × 10^?4 M ^0.564. With this relation, we successfully convert each G(D) into a corresponding molecular weight distribution (MWD). On the basis of the Benoit-Doty theory, we found that the persistence length and the Flory characteristic ratio C_∞ of the 6FDA-PFMB chain are ～3.3 nm and ～40, respectively, indicating that the 6FDA-PFMB chain is more extended than typical random-coil chains. On the other hand, the ratio of the radius of gyration to the hydrodynamic radius, i.e., 〈R_g〉/〈R_h〉 ～ 1.8, together with the values of the exponents (～0.56) indicate that the 6FDA-PFMB chain has a coil chain conformation. Therefore, the 6FDA-PFMB chain has an extended coil conformation in THF at 30°C.     

Homozygous EXOSC3 Mutation c.92G→C,p.G31A is a Founder Mutation Causing Severe Pontocerebellar Hypoplasia Type 1 Among the Czech Roma

Abstract

Pontocerebellar hypoplasia type 1 (PCH1) is characterized by cerebellar and anterior horn motor neuron degeneration and loss, signs of spinal muscular atrophy plus. Patients manifest severe perinatal weakness, hypotonia, and respiratory insufficiency, causing death frequently before the age of 1 year. Recently, causative mutations in EXOSC3 were reported in a majority of PCH1 patients, but the detailed clinical phenotype caused by EXOSC3 mutations, genotype-phenotype correlations, and prevalent mutations in specific ethnic groups is not yet known.

Three unrelated Czech Roma patients with PCH1 were investigated clinically, electrophysiologically, neuroradiologically, and neuropathologically (patients 1 and 2). The entire coding region of the EXOSC3 gene, including the adjacent intron sequences, was sequenced in all three patients. The same mutation c.92G→C, p.G31A in EXOSC3 was found in all three affected patients in homozygous state and in heterozygous state in the parents from two of the families. Haplotype analysis with four flanking microsatellite markers showed identical haplotype in 9 out of 11 haplotypes carrying the c.92G→C, p.G31A mutation. Furthermore, four heterozygotes for this mutation were found in anonymous DNA samples from 90 unrelated Roma individuals. All four of these samples shared the same haplotype. No heterozygous sample was found among 120 anonymous DNA samples from Czech non-Roma individuals with no familial relation. It may therefore be concluded that EXOSC3 c.92G→C, p.G31A mutation is a founder mutation with high prevalence among the Czech Roma causing a similar and particularly severe phenotype of PCH1. These observations from the Czech Roma may have consequences also for other Roma from other countries.

PCH1 caused by EXOSC3 founder mutation c.92G→C, p.G31A extends the list of autosomal recessive disorders rare among the general population but more frequent among Roma at least in the Czech Republic.     

Interferon-γ arrests proliferation and causes apoptosis in stromal cell/interleukin-7-dependent normal murine pre-B cell lines and clones in vitro,but does not induce differentiation to surface immunoglobulin-positive B cells

Normal pre-B cells from fetal liver or bone marrow of the mouse proliferate for long periods of time in tissue culture on stromal cells in the presence of interleukin-7 (IL-7). Their IgH loci are partly in germ-line, partly in D_HJ_H-rearranged configuration, while their light chain loci are in germ-line configuration. They express the pre-B cell-specific genes V_preB and λ₅. Proliferation of these pre-B cells is inhibited by interferon (IFN)-γ, with half-maximal inhibition at concentrations between 0.1 and 1 unit/ml. Normal pre-B cells exposed to IFN-γ die by apoptosis, as is evidenced by the disintegration of pre-B cell DNA into oligonucleosomal multimers of 180-200 bp. While the proliferation of pre-B cells from Eμ-bcl-2 transgenic (tg) mice is inhibited by IFN-γ, these cells do not die by apoptosis. IFN-γ does not induce differentiation to more mature B lineage cells. In the absence of IL-7 normal pre-B cells differentiate to V_HD_HJ_H/V_LJ_L-rearranged, surface immunoglobulin-positive B cells expressing the a chain of the IL-2 receptor. They also down-regulate the expression of V_preB and X.5, and lose the capacity to proliferate on stromal cells in the presence of IL-7. In contrast, both normal and Eμ-bcl-2 tg pre-B cells exposed to IFN-γ in the presence of stromal cells and IL-7 fail to differentiate, i.e. do not express surface immunoglobulin, retain expression of V_preB and λ₅, do not express the α chain of the IL-2 receptor, and retain the capacity to proliferate on stromal cells in the presence of IL-7, once IFN-γ is removed. The potential usefulness of a treatment of acute lymphocytic leukemia of the B cell lineage (pre B-ALL) with IFN-γ is discussed.     

Missense mutation of the cholecystokinin B receptor gene: Lack of association with panic disorder

Cholecystokinin tetrapeptide (CCK₄) is known to induce panic attacks in patients with panic disorder at a lower dose than in normal controls. Therefore, the cholecystokinin B (CCK_B) receptor gene is a candidate gene for panic disorder. We searched for mutations in the CCK_B gene in 22 probands of panic disorder pedigrees, using single-strand conformation polymorphism (SSCP) analysis. Two polymorphisms were detected. A polymorphism in an intron (2491 C→A) between exons 4 and 5 was observed in 10 of 22 probands. A missense mutation in the extracellular loop of exon 2 (1550 G→A, Val¹²⁵→Ile) was found in only one proband. This mutation was also examined in additional 34 unrelated patients with panic disorder and 112 controls. The prevalence rate of this mutation was 8.8% in patients with panic disorder (3/34) and 4.4% in controls (5/112). The mutation did not segregate with panic disorder in two families where this could be tested. These results suggest no pathophysiological significance of this mutation in panic disorder. © 1996 Wiley-Liss, Inc.     

Expression of pro-inflammatory cytokines by TCRαβ+ T and TCRγδ+ T cells in an experimental model of colitis

An inflammatory bowel disease (IBD) comparable to human ulcerative colitis is induced upon transfer of T cell-depleted wild-type (F1) bone marrow into syngeneic T cell-deficient (tgε26) mice (F1 → tgε26). Previously we have shown that activated CD4⁺ T cells predominate in transplanted tgε26 mice, and adoptive transfer experiments verified the potential of these cells to cause disease in immunodeficient recipient mice. Using flow cytometry for the detection of intracellular cytokine expression, we demonstrate in the present study that large numbers of CD4⁺ and CD8⁺ TCRαβ⁺ T cells from the intraepithelial region and lamina propria of the colon of diseased, but not from disease-free mice, produced interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Large numbers of T cells from peripheral lymphoid tissues of these animals also expressed IFN-α and TNF-α, but few expressed interleukin-4, demonstrating g strong bias towards Th1-type T cell responses in these animals. TCRγδ⁺ T cells, typically minor constituents of the inflammatory infiltrate of the colon in F1 → tgε26 mice, also expressed IFN-γ at a high frequency upon CD3 stimulation. In light of these findings we examined the potential involvement of TCRγδ⁺ T cells by testing their ability to induce colitis in tgε26 mice. We report here that tgε26 mice transplanted with T cell-depleted bone marrow from TCRα^null and TCRβ^null animals developed IBD. Furthermore, disease in these mice correlated with the development of peripheral and colonic TCRαδ⁺ T cells capable of IFN-γ production. These results suggest that IFN-γ may be a common mediator of IBD utilized by pathogenic T cells of distinct phenotype.     

Bacterial expression and characterization of an anti-desipramine single-chain antibody fragment

Tricyclic antidepressant toxicity is a leading cause of death from intentional drug overdose. Monoclonal antibody Fab' fragments specific for the tricyclic antidepressant, desipramine, reverse acute drug toxicity but may themselves have adverse effects at therapeutic doses. To evaluate the characteristics of smaller antibody fragments, we cloned, expressed and characterized a 26 kD single chain Fv fragment (G5-sFv). A DNA sequence encoding V_H-linker-V_L was constructed from hybridoma mRNA encoding a high affinity monoclonal desipramine-specific IgG₁ and expressed in E. coli. G5-sFv was produced at high levels as insoluble inclusion bodies. Single chain Fv was solubilized, folded in a redox buffer and affinity purified on desipramine-Sepharose. The affinity of G5-sFv for desipramine was similar to that of the corresponding monoclonal Fab' as measured by surface plasmon resonance (Fab′ 5.5±0.5 × 10⁸M⁻¹, sFv 2.3±0.5 × 10⁸ M⁻¹). G5-sFv administered to rats after a tracer dose of ³H-desipramine produced rapid and marked redistribution of drug from tissues into serum. G5-sFv was stable at 4°C for greater than 6 months but lost activity at higher temperatures.We conclude that desipramine-specific-single chain Fv expressed in E. coli retains the affinity of the parent antibody for desipramine. The pharmacokinetic effect of G5-sFv on desipramine distribution suggests that it may be useful as an antidote for desipramine overdose.     

新等位基因HLA-B*40:96的确认及家系遗传

目的 鉴定一个中国汉族人群中发现的人类白细胞抗原(human leukocyte antigen,HLA)新等位基因,调查该基因的家系遗传情况.方法 应用Luminex DNA杂交流式分型技术进行HLA分型,发现1例HLA-B位点反应格局异常,采用DNA测序分型技术进行新等位基因鉴定并对该基因的先证者进行家系调查.结果 DNA序列确定为一个新HLA-B等位基因序列,与同源性最高的HLA-B* 40:06:01相比,在第2外显子有7个碱基不同,分别是302位G→A、309位G→C、311位A→C、313位C→G、314位T→C、317位G→T、319位G→C,并导致相应密码子编码的氨基酸发生了改变,密码子101位由丝氨酸(Ser)→天冬氨酸(Asn)、104位由天冬氨酸(Asn)→苏氨酸(Thr)、105位由亮氨酸(Leu)→丙氨酸(Ala)、106位由精氨酸(Arg)→亮氨酸(Leu)、107位由甘氨酸(Gly)→精氨酸(Arg).家系分析表明该新基因来自先证者父亲.结论 鉴定了一个HLA-B新等位基因,2009年2月被世界卫生组织HLA因子命名委员会正式命名为HLA-B* 40:96.     

The expanding clinical phenotype of the tRNALeu(UUR) A→G mutation at np 3243 of mitochondrial DNA: Diabetic embryopathy associated with mitochondrial cytopathy

We describe a family which demonstrates and expands the extreme clinical variability now known to be associated with the A→G transition at nucleotide position 3243 of the mitochondrial DNA. The propositus presented at birth with clinical manifestations consistent with diabetic embryopathy including anal atresia, caudal dysgenesis, and multicystic dysplastic kidneys. His co-twin was normal at birth, but at 3 months of life, presented with intractable seizures later associated with developmental delay. The twins' mother developed diabetes mellitus type I at the age of 20 years and gastrointestinal problems at 22 years. Since age 19 years, the maternal aunt has had recurrent strokes, seizures, mental deterioration and deafness, later diagnosed as MELAS syndrome due to the tRNA^Leu(UUR) A→G mutation. A maternal uncle had diabetes mellitus type I, deafness, and normal intellect, and died at 35 years after recurrent strokes. This pedigree expands the known clinical phenotype associated with tRNA^Leu(UUR) A→G mutation and raises the possibility that, in some cases, diabetic embryopathy may be due to a mitochondrial cytopathy that affects both the mother's pancreas (and results in diabetes mellitus and the metabolic dysfunction associated with it) and the embryonic/fetal and placental tissues which make the embryo more vulnerable to this insult. © 1996 Wiley-Liss, Inc.     

Two BALB/c anti-arsonate idiotype families: two heavy chain variable regions (Vh) shared with anti-DNP antibodies are used by one family while a Vh similar to anti-GAT antibodies is used by the other

Amino terminal amino acid sequences were obtained for both the heavy (H) and light (L) chains of seven BALB/c anti-arsonate (Ar) monoclonal antibodies representing the 5AF6 and 3C6 idiotype (id) families described in this strain. 5AF6 family H chains showed strong homology to the germ-line gene sequence for the A strain 36–60 family. However, four to five identical H chain sequence differences for two of these antibodies (5AF6 and 95B5), as well as two previously reported related sequences (92D5, 94B10), suggested they were encoded by a different V_h. The 36–60 family V_h genes have been shown to be identical to the V_h gene of the anti-DNP binding myeloma M460 [Dzierzak et al., J. Immun. 136, 1864–1870 (1986)]. H chains amino acid sequences derived from an id-460⁺ anti-DNP hybridoma and a germ-line gene differing from the 30–60-like V_h sequence [Dzierzak et al., J. Immun.136, 1864–1870 (1986)] were found to be virtually identical to the 95B5 and 5AF6 V_h sequences. This suggests that the same two related H chains making up two subsets of the 5AF6 anti-Ar id family are also both used in two subsets of the id-460 anti-DNP response. 5AF6 family L chains were highly homologous to the other V_k2 L chains of the 36–60 family. 3C6 family H chains can be placed in the V_h 1 group, are unrelated to the described anti-Ar H chain families, and have been placed in a new anti-Ar V_h family, Ars-E. The 3C6 H is similar, however, to a V_h used by a BALB/c anti-GAT idiotype family of antibodies. 3C6 L chains were of the murine kappa chain group, V_k 8, and most resembled an L chain from an A strain monoclonal anti-Ar having no defined idiotype.     

Interaction of IFN-γ with cholinergic agonists to modulate rat and human goblet cell function

Goblet cells populate wet-surfaced mucosa including the conjunctiva of the eye, intestine, and nose, among others. These cells function as part of the innate immune system by secreting high molecular weight mucins that interact with environmental constituents including pathogens, allergens, and particulate pollutants. Herein, we determined whether interferon gamma (IFN-γ), a Th1 cytokine increased in dry eye, alters goblet cell function. Goblet cells from rat and human conjunctiva were cultured. Changes in intracellular [Ca²⁺] ([Ca²⁺]_i), high molecular weight glycoconjugate secretion, and proliferation were measured after stimulation with IFN-γ with or without the cholinergic agonist carbachol. IFN-γ itself increased [Ca²⁺]_i in rat and human goblet cells and prevented the increase in [Ca²⁺]_i caused by carbachol. Carbachol prevented IFN-γ-mediated increase in [Ca²⁺]_i. This cross-talk between IFN-γ and muscarinic receptors may be partially due to use of the same Ca²⁺_i reservoirs, but also from interaction of signaling pathways proximal to the increase in [Ca²⁺]_i. IFN-γ blocked carbachol-induced high molecular weight glycoconjugate secretion and reduced goblet cell proliferation. We conclude that increased levels of IFN-γ in dry eye disease could explain the lack of goblet cells and mucin deficiency typically found in this pathology. IFN-γ could also function similarly in respiratory and gastrointestinal tracts.     

IMMUNOCHEMICAL OBSERVATIONS ON THE HUMAN BLOOD GROUP P SYSTEM

Haemagglutination inhibition experiments were carried out with anti-P₁, anti-P^k and anti-P sera in an attempt to increase understanding of the chemical, genetical and serological relationships within the P system. The test-substances comprised a glycoprotein with human blood group P₁ and P^k activity isolated from sheep hydatid cyst fluid, fragments isolated from the partial acid hydrolysis products of the P₁P^k active glycoprotein, glycolipids, monosaccharides and di- and oligosaccharides of known structure. The trisaccharide αGal(1→4)βGal(1→4)GlcNAc isolated from the glycoprotein hydrolysis products, and earlier established as the P₁ determinant (Cory et al., 1974), was the only low molecular weight compound that gave strong inhibition with human, rabbit and goat anti-P₁ sera. A disaccharide αGal(1→4)Gal, also isolated from the glycoprotein hydrolysis products, failed to react with anti-P₁ reagents but inhibited human anti-P^k sera as strongly as the trisaccharide. The glycolipid αGal(1→4)βGal(1→4)Glc-Cer (ceramide trihexoside) and other oligosaccharides containing αGal(1→4)Gal at the non-reducing terminal were also strong inhibitors of anti-P^k sera. Oligosaccharides with terminal α-galactosyl residues joined in other positional linkages gave definite, although less strong, inhibition. The inhibition results suggest a close structural relationship between the P₁ and P^k determinants and indicate that the specificity of anti-P^k sera is less closely delineated than that of anti-P₁. Human anti-P sera differed markedly from anti-P₁ and anti-P^k and were not inhibited by any of the compounds containing α-galactosyl residues. The glycolipid βGalNAc(1→3)αGal(1→4)βGal(1→4)Glc-Cer (globoside) strongly inhibited the anti-P sera. The inhibition of anti-P^k and anti-P sera by ceramide trihexoside and globoside, respectively, confirms the observations of Naiki & Marcus (1974) and supports their conclusions that P^k is the precursor of P. The genetic relationship of the P₁ antigen to P and P^k is not clear but biosynthetic pathways are discussed that might explain the absence of P₁, P^k and P antigens in individuals of the p phenotype.