Lysosomal enzymes in human lymphoblastoid lines: unusual characteristics of RAJI and DAUDI

The lysosomal enzymes N-acetyl hexosaminidase, α-galactosidase and α-mannosidase vary in electrophoretic mobility in different human lymphoblastoid (lymphoid) lines. The relative mobilities of these three enzymes and three out of four other lysosomal enzymes tested correlate well with each other. The patterns appear to be relatively stable characteristics of each line. The lines RAJI and DAUDI show a strikingly fast electrophoretic mobility for all of these enzymes. N-acetyl hexosaminidase is also markedly deficient in DAUDI.     

Induction of an antiviral state by interferon in the absence of elevated levels of 2,5-oligo(A) synthetase and eIF-2 kinase

A series of clones has been derived from an interferon-resistant murine cell line, Ltk- aprt-, and their antiviral properties have been characterized. In the parental Ltk- aprt- line interferon is unable to establish antiviral properties or to increase the levels of 2,5-oligo(A) synthetase, the 2,5-oligo(A)-activated endonuclease F, 2',5'-phosphodiesterase, or eIF-2 kinase. However, interferon did prevent replication of vesicular stomatitis, Mengo virus, and reovirus in some of the derivative cell lines. The effect of interferon on the levels of the enzymes of the 2,5-oligo(A) and eIF-2 kinase pathways did not correlate directly with the antiviral properties of these cell clones. Greatly increased levels of 2,5-oligo(A) synthetase occurred in one clone without activation of an antiviral state. Another clone exhibited antiviral activity without detectably increased 2,5-oligo(A) synthetase activity. Changes in the levels of endonuclease F and 2',5'-phosphodiesterase were slight in all the clones examined. Neither 2,5-oligo(A) synthetase nor eIF-2 kinase levels were altered by interferon in another clone and yet an antiviral state was established and prevented replication of vesicular stomatitis, Mengo virus, and reovirus. The results show that mechanisms other than the 2,5-oligo(A) and eIF-2 kinase pathways are likely to contribute to the antiviral effects of interferon.     

Characterization and tissue distribution of N-acetyl hexosaminidase C: suggestive evidence for a separate hexosaminidase locus

1. An electrophoretic system in which N-acetyl hexosaminidase C (HEX(C)) MIGRATES LESS ANODALLY THAN N-acetyl hexosaminidase A (HEX(A)) is described. 2. HEX(C) is shown to differ from HEX(A) and HEX(B) in substrate specificity, molecular size and affinity for Concanavalin-A. 3. HEX(C) is present in a wide range of adult and foetal tissues and in tissues from patients with Tay-Sachs and Sandhoff's diseases. It is particularly prominent in brain, testis, thymus and lymphoblastoid cell extracts and in several foetal tissues. 4. It is suggested that HEX(C) is coded at a separate gene locus from HEX(A) and HEX(B).     

Pathogenicity of T helper 2 T-cell clones from T-cell receptor transgenic non-obese diabetic mice is determined by tumour necrosis factor-alpha

Autoimmune diabetes is predominated by a T helper 1 (Th1) response at the expense of an impaired Th2 response. Although T cells producing Th2 cytokines are generally thought to counter a Th1 response, there have been reports of Th2 T-cell clones with pathogenic activity, including one previously reported by us in which the Th2 T-cell clone was derived from a T-cell receptor transgenic (TCR-Tg) mouse bearing pathogenic TCR. In this study, our goal was to determine whether Th2 T-cell clones derived from a TCR-Tg in which the autoantigen was absent would be pathogenic and if so, to investigate possible mechanisms by which the Th2 T-cell clone could promote disease. We found that a Th2 T-cell clone derived from the 6.9 TCR-Tg/non-obese diabetic (NOD).C6 mouse in which 6.9 T cells do not encounter autoantigen, produced Th2 cytokines but not interferon-gamma. This Th2 T-cell clone, like the previous one we had isolated from the 2.5 TCR-Tg/NOD mouse, also turned out to be pathogenic. Intracellular staining revealed that these Th2 T-cell clones produce low levels of tumour necrosis factor-alpha (TNF-alpha) in vitro, and after adoptive transfer, they migrate to the pancreas where they produce TNF-alpha as well as Th2 cytokines (interleukin (IL)-4, IL-10). Induction of disease was prevented by administration of soluble TNF-alpha receptor to recipient mice, suggesting that the diabetogenicity of these Th2 T-cell clones is caused by their low level production of TNF-alpha.     

Ultrastructure of the conjunctiva, skin, and gingiva: a case of Sandhoff's disease in a Jewish patient

Pleomorphic membranous cytoplasmic bodies that indicated glycolipid storage were found in the conjunctiva, skin, and gingiva of a Jewish patient with Sandhoff's disease. The clinical symptoms were typical of GM2 gangliosidosis. Both hexosaminidase A and hexosaminidase B activities were deficient in the leukocytes and serum. Glycosaminoglycan levels in cultured fibroblasts were elevated. Membranous cytoplasmic bodies were observed in high concentrations in a large proportion of the vascular endothelial cells, pericytes, and Schwann cells and to a somewhat lesser extent in the fibrocytes of all tissues studied. Ultrastructural analysis of the conjunctiva, skin, and gingiva as an aid for the diagnosis of Sandhoff's disease is suggested.     

Rapid tissue culture and microbiochemical methods for analyzing colonially grown fibroblasts from normal, Lesch-Nyhan and Tay-Sachs patients and amniotic fluid cells

A means of reducing the time interval between amniocentesis and biochemical analysis for the prenatal diagnosis of biochemical diseases is presented. Fibroblasts from nomal individuals, patients with Lesch-Nyhan syndrome and patients with Tay-Sachs disease, and normal amniotic fluid cells, were grown in isolation in MicroTest II tissue culture plates. Hypoxanthine-guanine phosphoribosyl transferase (HGPRTase) and hexosaminidase A were studied in independently derived colonies arising after a median culture period of 14 days. Colony six ranged from 2,000 to 8,000 cells. HGPRTase activity, expressed as the ratio of hypoxanthine-C14 to adenine-113 uptake, was assayed using differential scintillation spectrometry. Hexosaminidase A was investigated by micro-electrophoresis. Discrimination of enzyme deficient from non-deficient colonies was accomplished in each case and both enzymes were detectable in amniotic fluid cell colonies. The potential of the method for prenatal diagnosis of the two diseases studied and of biochemical diseases in general is discussed.     

Isolation and characterization of infective and non-infective clones of Leishmania tropica

Cloning by limit dilution of an isolate of Leishmania tropica (LRC-L137) that is infective for mice resulted in 7 stable clones, only one of which was infective in BALB/c mice. Three of the non-infective clones that were examined for survival in BALB/c macrophages in vitro seemed to be killed more readily, suggesting failure to establish in macrophages as the basis for non-infectivity in vivo. Promastigotes from three non-infective clones and one infective clone were biosynthetically labelled or surface radioiodinated, and the detergent lysates were analyzed by 2-dimensional gel electrophoresis. The pattern of the radiolabelled cytoplasmic and membrane proteins of promastigotes from all L. tropica clones was similar, with minor differences. All clones as well as the uncloned population bound to the same extent to a series of lectins with galactose and N-acetylgalactosamine as specificities. They also bound in a solid-phase radioimmunossay to 9 monoclonal antibodies raised against the uncloned L. tropica (LRC-L137). The genetic characterization of four L. tropica clones was attempted by analysis of their isolated kinetoplast DNA. The clones form two schizodemes since they possess kinetoplast DNAs which exhibit similar restriction endonuclease fingerprints and show extensive DNA sequence homology, suggesting that the four clones are closely related and that the noninfective variants may be derived from the infective presumptive parental clone L137-7-121. Further characterization of the clones of L. tropica should allow a better understanding of the genetic basis of parasite virulence in cutaneous leishmaniasis.     

Cultured skin fibroblasts in lipidoses. Enzymatic, histochemical, and ultrastructural relationship in Fabry's Tay-Sachs, and Sandhoff's diseases

Enzymatic, histochemical, and ultrastructural studies were performed on cultured skin fibroblasts from patients with Fabry's disease, Tay-Sach's disease, and Sandhoff's disease and from their families (carriers). alpha-Galactosidase activity was deficient in the proband with Fabry's disease (lower in the homozygotes than in the heterozygote). Levels of hexosaminidase A in the patient with Tay-Sachs disease and hexosaminidase A and B in the patient with Sandhoff's disease were deficient and were lower in her mother than in the control subject. Lysosome-like structures were observed in cultured fibroblasts from the patients with each disease, as well as in the heterozygote with Fabry's disease and the carrier with Tay-Sach's disease. The amount of the accumulating arrays in the lysosome-like structures was related to low level of enzymatic activity.     

Molecular studies of the differential replication at pyrexial temperatures of two influenza viruses differing in virulence for ferrets

Replication of a virulent clone (7a) of the reassortant influenza virus A/Puerto Rico/8/34-A/England/939/69 (H3N2) in ferret nasal turbinate tissue is less affected than that of an attenuated clone (64d) by temperatures which occur during pyrexia in ferrets. This is a factor which contributes to the difference in virulence of the two clones. The differential replication of the two clones at pyrexial temperatures has been reproduced in allantois-on-shell (egg-bit) cultures, and the synthesis of viral polypeptides and RNA species examined. This virus-host system was chosen because it was more convenient to use than organ cultures but, like the latter, might provide information relevant to the in vivo situation. With this system it was not possible to achieve single cycle replication: the observed effects are cumulative over several (2 to 3) cycles of replication (24 h) and therefore conclusions from them may not be as definitive as those from single cycle conditions. However, in cells infected with clone 64d both A(+) cRNA and polypeptide synthesis were little affected at 40 degrees C but levels were decreased by about 70-80% at 41 degrees C; A(+) cRNA and polypeptide levels were unaffected even at 41 degrees C with clone 7a. These reductions seem insufficient to account for the 10-fold reduction in infectious yields of clone 64d at 40 degrees C or the 100-fold and 10-fold reductions in yields of clones 64d and 7a respectively at 41 degrees C. There was no evidence of increased production of non-infectious virus at elevated temperatures by either clone. Levels of vRNA were considerably reduced at 40 and 41 degrees C for both clones, but the levels were considerably greater at all temperatures in clone 7a-infected cells than in those infected with clone 64d; vRNA levels were higher for clone 7a at 41 degrees C than for clone 64d at 37 degrees C. The different levels of vRNA do not reflect differences in the availability of template A(-) cRNAs since levels of these were similar for both clones at 37 and 40 degrees C and only reduced for clone 64d at 41 degrees C. Although the interpretation of these data is complicated by multiple cycles of replication it appears that limited availability of vRNA could be an important constraint on the ability of clone 64d to replicate at pyrexial temperatures.     

Biochemical identification of I-J as a novel dimeric surface molecule on mouse helper and suppressor T cell clones

A monoclonal anti-I-Jk antibody JK10-23 was capable of precipitating the putative I-Jk molecule from NP-40 lysates of 125I-surface labelled mouse T cell clones with either helper or suppressor functions. The I-J molecule detected by specific immunoprecipitation and subsequent one- or two-dimensional gel analysis was a Mr 84-90 K dimer composed of 42-46 K glycopeptide subunits having isoelectric point pH 5.3 to 6.4. A monomeric form of I-J also existed in some of the T cell clones. The I-J subunit was a glycosylated polypeptide with a 41 K backbone having at least two glycosylation sites. I-J was distinguishable from other known dimeric T cell surface molecules with comparable molecular size, that is, T cell receptor alpha beta heterodimer, A1 and YE molecules expressed on a T cell leukemia EL4, and mouse CD28. The I-Jk molecule was precipitable from T cell clones with I-Ak and I-Ek restriction specificities including a clone derived from an H-2b----H-2bxkF1 radiation bone marrow chimera. None of the H-2b-restricted T cell clones from H-2b and its F1 showed the I-Jk immunoreactivity. T cell clones having either I-Ab or I-Ek restriction specificities derived from intra-H-2 recombinant mouse B10.A(5R) were positive for the I-Jk, while an I-Ab-restricted T cell clone from B10.A(3R) was negative in the I-Jk immunoprecipitation. The results indicate that I-J is a novel dimeric surface molecule, most likely to be a homodimer, expressed on T cells according to the major histocompatibility complex.     

Human autoreactive (Th0) CD4(+) T-cell clones with cytolytic activity recognizing autologous activated T cells as the target

In attempt to obtain a clue to understanding possible physiological roles played by autoreactive T cells, autoreactive T-cell clones originally derived from an allogeneic mixed lymphocyte culture have been analyzed for their target spectrum, lytic function and cytokine profiles. Five CD4(+) T-cell clones established from allogeneic MLR, in which the stimulator cells shared certain class II MHC antigens with the responder, turned out to be reactive to autologous PBL. Among these, three clones were cytolytic against autologous B-cell line. These three cytolytic autoreactive clones were shown to be capable of specifically lysing autologous activated T cells expressing class II MHC molecules, raising possibility that such autoreactive clones might play a role in negatively regulating T cell responses. Cytolysis by an autoreactive clone 21C5 was inhibited completely by concanamycin A (CMA) known as a specific inhibitor of perforin, suggesting an involvement of the perforin/granzyme system. T-cell clones derived from the same MLC showed distinct correlation between their specificity and lymphokine profiles. Thus, the three cytolytic autoreactive clones belonged to Th0, whereas the two noncytolytic autoreactive clones belonged to Th2 and three alloreactive CD4(+) clones derived from the same culture were of Th1 type.     

Cytotoxic T cell clone-specific monoclonal antibodies used to select clonotypic antigen-specific cytotoxic T cells

Two rat monoclonal antibodies (mAb) have been produced which recognize a clone-specific determinant on the alloreactive cytotoxic T lymphocyte (CTL) clone 3 F9. CTL clone 3F9 of BALB/c origin is specific for H-2Db and can be grown by weekly restimulation with irradiated stimulator spleen cells expressing H-2Db in the presence of interleukin 2. Two mAb against T cell clone 3F9, 44-22-1(IgG2a) and 46-6 B5(IgM), have been proven to be clone specific: they inhibit cytotoxic activity of 3F9 only and bind specifically to 3 F9 when compared in a panel of different CTL clones, or cells from different mixed lymphocyte cultures (MLC), BALB/c thymus and spleen cells. The mAb 44-22-1 has been used to sort cells from a primary MLC BALB/c anti-H-2Db by fluorescence-activated cell sorter (FACS) to select CTL expressing 3 F9 clonotype-specific determinants. The lymphocytes reactive with 44-22-1 represent a minor subpopulation of the CTL of the primary MLC. The specific alloreactive cytotoxicity of unsorted lymphocytes of the bulk primary MLC could not be inhibited by the mAb 44-22-1 and 46-6 B5 whereas the sorted 3 F9 clonotype-positive cultures could be inhibited very effectively. All the CTL clones derived from the FACS-sorted clonotype-positive culture show all the same properties and are identical with clone 3 F9 with respect to antigen-specific cytotoxicity, inhibition of cytotoxicity by the mAb and surface markers.     

The effects of carnosine on oxidative DNA damage levels and in vitro lifespan in human peripheral blood derived CD4+T cell clones

Carnosine (β-alanyl-l-histidine), an abundant naturally-occurring dipeptide has been shown to exhibit anti-ageing properties towards cultured cells, possibly due in part to its antioxidant/free radical scavenging abilities. In this paper the results of an investigation on the effects of carnosine, at the physiological concentration of 20 mM, on oxidative DNA damage levels and in vitro lifespan in peripheral blood derived human CD4+ T cell clones are reported. Under the culture conditions used (20% O₂) long term culture with carnosine resulted in a significant increase in the lifespan of a clone derived from a healthy young subject. No such extension was observed when a T cell clone from a healthy old SENIEUR donor was similarly cultured. Culture with carnosine from the midpoint of each clone's lifespan did not have any effect on longevity, independent of donor age. Oxidative DNA damage levels were measured in the clones at various points in their lifespans. Carnosine acted as a weak antioxidant, with levels of oxidative DNA damage being lower in T cells grown long term in the presence of carnosine. The possibility that carnosine might confer anti-ageing effects to T cells under physiological oxygen tensions would appear to be worthy of further investigation.     

Th1 cells specific for HIV-1 gag p24 are less efficient than Th0 cells in supporting HIV replication, and inhibit virus replication in Th0 cells.

This report provides three lines of evidence to suggest that T-helper type 1 (Th1) and type 0 (Th0) cells could play an opposing role in acquired immune deficiency syndrome (AIDS). Using a panel of Th1 and Th0 clones specific for human immunodeficiency virus-1 (HIV-1) gag p24, derived from seronegative volunteers immunized with gag p24: Ty virus-like particles, a Th1 clone specific for tuberculin (PPD), and a Th0 clone derived by random activation from the same volunteer, we have demonstrated the following differences in the capacity of these clones to regulate the in vitro replication of HIV. (1) Th1 clones were less efficient than Th0 clones in supporting HIV replication, both in their resting state (by 10-1000-fold) and after antigen activation (by five to 100-fold). Furthermore, the infectious titre of HIV recovered from the Th0 population was more than 1000-fold higher than virus from the Th1 population, and the number of HIV-infected Th0 cells was five to 16 times higher than the number of infected Th1 cells. (2) Antigen- or mitogen-activated Th1, but not Th0 clones, inhibited HIV in bystander CEM-4 cells. Th1 cells also inhibited HIV in autologous and allogeneic Th0 cells. The level of inhibition in these experiments ranged from 50% to 100% and was three to 10-fold higher and more sustained in the presence of p24-specific clones compared to the PPD-specific Th1 clone. The capacity of Th1 cells to inhibit HIV in neighbouring cells was also reflected in the reduced replication of HIV in the clones immediately after antigen activation compared to unstimulated cells. Kinetic studies of virus production, cytokine release and proliferation showed that inhibition of HIV was associated with peak cytokine release and preceeded proliferation. (3) The Th1 clones had higher cytolytic potential than the Th0 clones. Therefore, the HIV inhibitory activity of Th1 cells could be partly due to cell to cell killing. These data demonstrate the opposing effects of Th1 and Th0 cells on the in vitro replication of HIV, and suggest that Th1 cells might be important in immunity whereas Th0/Th2 cells might lay a role in promoting disease.     

In vitro positive selection of alpha beta TCR transgenic thymocytes by a conditionally immortalized cortical epithelial clone

Development of mature CD4 and CD8 single-positive T cells requires a process known as positive selection, which depends on the specific recognition of self-peptide-MHC complexes on thymic stromal cells by immature CD4+CD8+ thymocytes. We have used an in vitro reaggregate system to study the positive selection of thymocytes by conditionally immortalized thymic epithelial clones. Thymocytes from mice transgenic for the F5 alpha beta TCR, specific for a peptide from the influenza nucleoprotein in the context of H-2Db, are positively selected in the H- 2b MHC background, but fail to mature in mice expressing the H-2q haplotype. Development of embryonic day 15 F5 H-2q transgenic thymocytes was followed in reaggregate cultures supplemented with H-2b- expressing epithelial clones. A conditionally immortalized cortical epithelial clone, derived from H-2Kb-tsA58 transgenic mice, was found to be as efficient as freshly isolated thymic stromal cells in positively selecting CD8 transgenic thymocytes. In contrast, an H-2b- expressing kidney epithelial clone did not augment positive selection above background levels, implying that the effect of the thymic epithelial clone was not merely the presentation of selecting MHC molecules. Mature transgenic thymocytes generated in reaggregate cultures were able to differentiate into functionally competent cytotoxic T cells. This model provides an important in vitro system for the detailed study of the specific molecular interactions leading to positive selection of developing thymocytes.      

The effects of carnosine on oxidative DNA damage levels and in vitro lifespan in human peripheral blood derived CD4+T cell clones

Carnosine (beta-alanyl-L-histidine), an abundant naturally-occurring dipeptide has been shown to exhibit anti-ageing properties towards cultured cells, possibly due in part to its antioxidant/free radical scavenging abilities. In this paper the results of an investigation on the effects of carnosine, at the physiological concentration of 20 mM, on oxidative DNA damage levels and in vitro lifespan in peripheral blood derived human CD4+ T cell clones are reported. Under the culture conditions used (20% O(2)) long term culture with carnosine resulted in a significant increase in the lifespan of a clone derived from a healthy young subject. No such extension was observed when a T cell clone from a healthy old SENIEUR donor was similarly cultured. Culture with carnosine from the midpoint of each clone's lifespan did not have any effect on longevity, independent of donor age. Oxidative DNA damage levels were measured in the clones at various points in their lifespans. Carnosine acted as a weak antioxidant, with levels of oxidative DNA damage being lower in T cells grown long term in the presence of carnosine. The possibility that carnosine might confer anti-ageing effects to T cells under physiological oxygen tensions would appear to be worthy of further investigation.     

Allergen-specific human T cell clones: derivation, specificity, and activation requirements

The interaction between allergen and immune cells plays a pivotal role in the development of human allergy. In an attempt to understand this interaction, we have studied allergen-specific T cells in vitro. These T cells are derived from rodent-allergic individuals and are specific for a major allergen found in mouse urine (MA-1). Antigen-specific, major histocompatibility complex-restricted, human T cell clones have been generated by limiting dilution from lines derived from the peripheral blood T cells of allergic individuals. Antigen (Ag)-presenting cells are necessary for this response, and they can be modulated by appropriate agents. These clones can be propagated in vitro under conditions of restimulation with Ag in the presence of Ag-presenting cells without the continuous use of exogenous interleukin-2. Most clones are CD3+ or CD4+, but one clone is CD3+ CD8+ by fluorescence-activated cell sorter and monoclonal antibody-killing data. Ag stimulation of these clones induces them to produce interleukin-2 and proliferate. These T cell clones can provide a basis for studying the structure of allergenic epitopes and the potential role of altered Ag in the induction of T cell tolerance. If the determinants of T cell allergen recognition and tolerance are solved, it might provide a basis for a new approach to the immunotherapy of allergic disease.     

Characterization of human bone marrow-derived closed circular DNA clones

Because of interest in mechanisms of recombination involved in chromosomal deletions in neoplastic disease, and their relation to possible rearrangements in normal tissues, we are studying circular DNA molecules from human tissue with a long-term goal of investigating them as possible by-products of physiologically relevant intrachromosomal recombination events. Covalently closed circular (ccc) DNA from human bone marrow was cloned in bacteriophage vectors, and fourteen clones chosen randomly from the cccDNA-derived library were characterized. Five clones originated from chromosome-specific centromeric α-satellite DNA; two clones carried highly repetitive sequences probably derived from interspersed repetitive elements; six clones were derived from single-copy chromosome-specific sequences which detected homologous rodent sequences; and one clone (EPM10) was derived from a small chromosome 11-specific sequence family which localized to chromosome regions 11cen and 11q14. Oligonucleotide primers derived from the cccDNA clones were used in polymerase chain reaction studies to show that (1) the EPM10 clone carried the circular junction, (2) several of the single-copy products could be detected in three different bone marrow cccDNA preparations, and (3) the Alu-PCR profile for bone marrow cccDNA showed distinct bands which were similar in four bone marrow cccDNA preparations. © 1993 Wiley-Liss, Inc.     

Isolation of two Giardia lamblia (WB strain) clones with distinct surface protein and antigenic profiles and differing infectivity and virulence.

To determine the relationship between antigenic profiles and pathogenicity among Giardia lamblia clones (WB strain), trophozoites were cloned by the technique of limiting dilution. The phenotype of each clone was determined by an indirect immunofluorescence test using a polyclonal rabbit anti-G. lamblia trophozoite serum made against the parent strain. Two clones were chosen for further studies: a highly fluorescent clone, F+, in which more than 95% of the trophozoites fluoresced, and a low-fluorescence clone, F-, in which fewer than 5% fluoresced. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and enzyme-linked immunotransfer blot studies of the membrane fractions of the two clones and parent strain revealed differences in both the total protein and antigenic profiles. A serum cytotoxicity test with the polyclonal serum showed that the F+ clones were more susceptible to immobilization and killing, while the majority of cells of the F- clones were resistant to such killing. Assessment of the infectivity of the two clones in the Mongolian gerbil animal model indicated that the F- clone more readily initiated infections, produced more cysts, had a higher intestinal trophozoite load, and produced a more severe clinical syndrome, while the F+ clone was less phenotypically stable in vivo and in some cases took longer to be cleared from the intestine.