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PubMed1998

A genetic screen for temperature-sensitive cell-division mutants of Caenorhabditis elegans.

A novel screen to isolate conditional cell-division mutants in Caenorhabditis elegans has been developed. The screen is based on the phenotypes associated with existing cell-division mutations: some disrupt postembryonic divisions and affect formation of the gonad and ventral nerve cord-resulting in sterile, uncoordinated animals-while others affect embryonic divisions and result in lethality. We obtained 19 conditional mutants that displayed these phenotypes when shifted to the restrictive temperature at the appropriate developmental stage. Eighteen of these mutations have been mapped; 17 proved to be single alleles of newly identified genes, while 1 proved to be an allele of a previously identified gene. Genetic tests on the embryonic lethal phenotypes indicated that for 13 genes, embryogenesis required maternal expression, while for 6, zygotic expression could suffice. In all cases, maternal expression of wild-type activity was found to be largely sufficient for embryogenesis. Cytological analysis revealed that 10 mutants possessed embryonic cell-division defects, including failure to properly segregate DNA, failure to assemble a mitotic spindle, late cytokinesis defects, prolonged cell cycles, and improperly oriented mitotic spindles. We conclude that this approach can be used to identify mutations that affect various aspects of the cell-division cycle.

Animals↗

PubMedDate not supplied

Immunotherapy of bovine ocular squamous cell carcinoma: isolation, culture and characterization of lymphocytes present in the tumor.

Bovine ocular squamous cell carcinoma (BOSCC) is sensitive to intralesional immunotherapy with BCG or recombinant human IL-2 (rhIL-2). The mechanism of tumor regression is as yet unclear. Alterations in the concentration of IL-2 (and possibly other factors) in the tumor, due to regional injection or induction by BCG, may induce killer cell activity and thus tumor regression. To investigate this, lymphocytes were isolated by mechanical fractionation of biopsies of BOSCC. Growth, phenotypical, and functional characteristics were studied. TIL could be isolated and grown from all biopsies of BOSCC. An estimated increase in cell number of 50-150 fold was observed during 5-7 weeks of culture. FACS analysis of a limited number of the TIL cultures showed a characteristic shift in phenotypes until day 28 of culture. CD2+ cells (50-70%), and as a consequence of this CD2- cells, remained stable in number. The number of CD8+ cells increased. CD4+ cells were detected in low numbers by day 28. Prolonged culture resulted in an increase of CD2- gamma delta + cells, CD2+4-8- cells, and occasionally of both CD8+ and CD2+ cells. In 51Cr release assays TIL showed cytotoxicity for BOSCC-derived tumor cell lines in general, which increased transiently by cocultivation with tumor cells. Killing of YAC-1, and P815 was far less efficient. Preferential killing of autologous cell lines was not seen. In conclusion, TIL from bovine ocular squamous cell carcinomas can be cultured in the presence of rhIL-2, which induces cytotoxic activity for BOSCC-derived tumor cells. Cells responsible for killing in vitro and potentially for regression of the tumor after immunotherapy with BCG or rhIL2 cannot yet be identified. Depletion and blocking experiments are being conducted in order to identify the cells (CD2+8+, CD2-gamma delta + or other CD2 +/-) responsible for killing.

Animals↗

PubMed1991

Negative regulation of globin gene expression during megakaryocytic differentiation of a human erythroleukemic cell line.

The human erythroleukemic cell line K562 was used as a model for analysis of the mechanisms responsible for alterations in gene expression during differentiation. K562 cells normally synthesize fetal hemoglobin (gamma-globin), but treatment with tumor-promoting phorbol esters (phorbol myristate acetate and tetradecanoyl phorbol acetate) results in the loss of the erythroid phenotype of the cells and causes a shift toward a megakaryocytic phenotype. This shift involves markedly decreased production of fetal hemoglobin and de novo synthesis of a number of proteins specific for megakaryocytes. The results of this work indicate that negative regulation of fetal hemoglobin during megakaryocytic differentiation of K562 cells occurs at the level of down regulation of gamma-globin mRNA accumulation. This effect consists of at least two components: reduction in the rate of transcription of the gamma-globin gene and decrease in stability of the normally very stable gamma-globin mRNA. We have developed two assay systems that permit investigation of the transcriptional and posttranscriptional effects of phorbol myristate acetate independently from each other. These assay systems make use of a heterologous reporter gene for the transcriptional analysis and a marked gamma-globin gene for the analysis of mRNA stability. The DNA sequences located in the 3' flanking region of the A gamma-globin gene were found to be responsible for the decrease in transcription rate.

Cell Differentiation↗

PubMed2004

Truncated RON tyrosine kinase drives tumor cell progression and abrogates cell-cell adhesion through E-cadherin transcriptional repression.

RON is a tyrosine kinase receptor that triggers scattering of normal cells and invasive growth of cancer cells on ligand binding. We identified a short RON mRNA, which is expressed in human lung, ovary, tissues of the gastrointestinal tract, and also in several human cancers, including ovarian carcinomas and cell lines from pancreatic carcinomas and leukemias. This transcript encodes a truncated protein (short-form RON; sf-RON), lacking most of the RON receptor extracellular domain but retaining the whole transmembrane and intracellular domains. Sf-RON shows strong intrinsic tyrosine kinase activity and is constitutively phosphorylated. Epithelial cells transduced with sf-RON display an aggressive phenotype; they shift to a nonepithelial morphology, are unable to form aggregates, grow faster in monolayer cultures, show anchorage-independent growth, and become motile. We show that in these cells, E-cadherin expression is lost through a dominant transcriptional repression pathway likely mediated by the transcriptional factor SLUG. Altogether, these data show that expression of a naturally occurring, constitutively active truncated RON kinase results in loss of epithelial phenotype and aggressive behavior and, thus, it might contribute to tumor progression.

Cadherins↗

PubMed1982

Temperature-sensitive changes in the structure of globin chromatin in lines of red cell precursors transformed by ts-AEV.

Chicken bone marrow cells infected in vitro with a temperature-sensitive avian erythroblastosis virus fall to produce hemoglobin at 36 degrees C. When the product or products of the transforming gene (erb) are inactivated by a temperature shift to 42 degrees C in culture, several different cloned lines of cells infected with the temperature-sensitive avian erythroblastosis virus begin to make hemoglobin. This shift in phenotype correlates with an increase in hemoglobin mRNA specific to both adult and embryonic alpha and beta globin. The switch is accompanied by the acquisition of DNAase I-hypersensitive sites in one cell line (clone 2); however, a hypothetically more mature line (clone 3) has already acquired globin DNAase-hypersensitive sites but does not express hemoglobin until the temperature shift. Several (but not all) specific restriction sites associated with both the alpha and beta domains become unmethylated after the switch from 36 degrees C to 42 degrees C. The magnitude of these methylation switches is small compared with changes that occur in these genes during normal avian erythropoiesis. The results suggest that changes in chromosomal structure precede transcription and are not a consequence of transcription. Since (presumptive) precursor cloned lines can be established with some, but not all, of the structural properties of active globin chromatin, it is likely that many of these properties can be independently established and are not obligatorily related.

Alpharetrovirus↗

PubMed2000

Overexpression of GATA-2 inhibits erythroid and promotes megakaryocyte differentiation.

GATA-1 and GATA-2 transcription factors are required for effective hematopoiesis. These regulatory proteins present overlapping yet distinct patterns of expression in hematopoietic cells. Absence of GATA-2 leads to defective hematopoiesis and an embryonic lethal phenotype. Disruption of GATA-1 results in a compensatory increase in GATA-2 in early erythroid cells and incomplete erythropoiesis with embryos dying at 11.5 days. We examine the specific role of GATA-2 later in hematopoiesis, during erythroid differentiation. Stable K562 cell lines expressing various levels of GATA-2 were generated using a GATA-2 expression plasmid. Overexpression of GATA-2 transcripts was determined by quantitative polymerase chain reaction (PCR). Cytospin smears, growth curve analysis, PCR, and flow cytometry were used to examine the effects of increased levels of GATA-2 in altering cell phenotype and activation of megakaryocytic markers. Human progenitor erythroid cells also were transfected with a GATA-2 expression vector. Growth curve analysis, benzidine staining, and high-performance liquid chromatographic analysis were used to study the effects of GATA-2 on erythroid maturation and proliferation.K562/GATA-2 cell lines expressing high levels of GATA-2 mRNA showed a marked decrease in proliferation and a shift in phenotype toward the megakaryocyte lineage. Ploidy analyses showed that these cell lines developed a multinuclear phenotype, including tetraploids and octaploids. PCR analysis showed activation of megakaryocyte-specific genes including thrombopoietin receptor (c-mpl). Surface expression of platelet glycoprotein receptors Ib/IX (CD42b/CD42a) and IIb/IIIa (CD41/CD61) also was demonstrated by flow cytometry. In primary human adult erythroid cultures transfected with a GATA-2 expression vector, production of total hemoglobin and cell proliferation decreased in a dose-dependent manner.GATA-2 plays an important role in deciding cell lineage throughout hematopoiesis, and increased expression of GATA-2 determines megakaryocytic differentiation. Downregulation of GATA-2 is required for erythroid differentiation.

Adult↗

PubMed1985

Spreading of non-transformed and transformed cells.

Mechanisms of cellular reactions responsible for the spreading of non-transformed cultured tissue cells on the surface of various substrata and relationships of these reactions to the control of cell proliferation are reviewed; the special role of the membrane-cytoskeleton interactions leading to extension and attachment of pseudopods is stressed. Transition of cells from non-transformed to transformed phenotype is characterized by decreased spreading and by decreased dependence of proliferation on spreading. Manifestations of both of these spreading-associated changes are reviewed and their possible mechanisms are discussed. It is suggested that cell transition to transformed phenotype involves shift of an equilibrium between the reactions induced by the two groups of membrane-bound ligands: those attached and those not attached to the substratum.

Animals↗

PubMed2001

Intervertebral disc cells exhibit differences in gene expression in alginate and monolayer culture.

STUDY DESIGN: The mRNA levels of aggrecan and collagen were quantified in intervertebral disc cells cultured under three conditions: primary alginate culture, monolayer culture, and re-encapsulation in alginate after monolayer culture. OBJECTIVES: To compare the phenotype of intervertebral disc cells under different culture conditions and to investigate the reversibility of cell phenotype after re-encapsulation in alginate after monolayer culture. SUMMARY OF BACKGROUND DATA: The intervertebral disc contains heterogeneous populations of cells that vary with anatomic region. These cells possess significant differences in phenotype that can be preserved in vitro, although the effect of culture conditions on the phenotype of these cells is poorly understood. METHODS: The intervertebral disc cells of 4-5-month-old pigs were isolated enzymatically from three anatomic zones: anulus fibrosus (AF), transition zone (TZ), and nucleus pulposus (NP). Gene expression levels of aggrecan and collagen Types I and II were measured using a quantitative reverse transcriptase--polymerase chain reaction. RESULTS: Gene expression levels of anulus fibrosus and transition zone cells were shifted in monolayer compared with alginate, although the shift was partially reversed when re-encapsulated in alginate. However, NP cells appeared to be insensitive to culture conditions. Furthermore, characteristic patterns of gene expression among AF, TZ, and NP cells in primary alginate culture did not exist in monolayer culture, but they were also observed after re-encapsulation in alginate. CONCLUSION: The findings of this study suggest that anulus fibrosus and transition zone cells undergo a reversible shift in phenotype when cultured in monolayer compared with alginate. These differences suggest that the culture system exerts a strong influence on cell phenotype and may play a role in the response of these cells to biophysical and biochemical stimuli in vitro.

Aggrecans↗

PubMed2003

Stage-dependent effects of oocytes and growth differentiation factor 9 on mouse granulosa cell development: advance programming and subsequent control of the transition from preantral secondary follicles to early antral tertiary follicles.

The development of an ovarian follicle requires a complex set of reciprocal interactions between the oocyte and granulosa cells in order for both types of cells to develop properly. These interactions are largely orchestrated by the oocyte via paracrine factors such as growth differentiation factor 9 (GDF9). To examine these interactions further, a study was conducted of the effects of oocytes at different stages of development on proteins synthesized by mouse granulosa cells during the transition of granulosa cells (GCs) from preantral, secondary (2 degrees ) follicles (2 degrees GCs) to mural granulosa cells (3 degrees GCs) of antral tertiary (3 degrees ) follicles. The ability of recombinant GDF9 to mimic the effects of oocytes was also determined. Effects were evaluated by high- resolution, two-dimensional protein gel electrophoresis coupled to computer-assisted, quantitative gel image analysis. Coculture of the 2 degrees GCs with growing oocytes (GOs) from 2 degrees follicles brought about many of the changes in granulosa cell phenotype associated with the 2 degrees to 3 degrees follicle transition. GDF9 likewise brought about many of these changes, but only a subset of GDF9-affected protein spots were also affected by coculture with GOs. Coculture of 2 degrees GCs with the nearly fully grown oocytes (FGOs) from 3 degrees follicles had a reduced effect on 2 degrees GC phenotype, in comparison with coculture with GOs. For some proteins, oocyte coculture or GDF9 treatment appeared to have opposite effects on 2 degrees GCs and 3 degrees GCs. Additional effects of GDF9 and oocytes were seen in cultures of 2 degrees GCs for proteins other than those that differed between untreated control 2 degrees and 3 degrees GCs. These results indicate that GOs and GDF9 can each induce 2 degrees GCs to shift their phenotype toward that of 3 degrees GCs. The ability of the oocyte to produce this effect is diminished with oocyte development. The transition in the GC phenotype promoted by oocytes appears stable because differences in 2 degrees GCs promoted by oocytes and GDF9 were observed in untreated 3 degrees GCs. We conclude that the influence of the oocyte on GCs changes with the progression of their development, and so too does the response of the GCs to the oocyte. Moreover, by acting on the 2 degrees GCs, GOs are able to influence stably the phenotype of 3 degrees GCs. Thus, at or near the 2 degrees to 3 degrees follicle transition, signals from the growing oocyte contribute to the development of the mural GC phenotype.

Animals↗

PubMed2006

Biological relevance of colony morphology and phenotypic switching by Burkholderia pseudomallei.

Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42 degrees C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.

Animals↗

PubMed1995

Experimental human gonococcal urethritis: 250 Neisseria gonorrhoeae MS11mkC are infective.

Neisseria gonorrhoeae MS11mkA (mkA) expresses one 3.6-kDa lipooligosaccharide (LOS). Variant MS11mkC (mkC), expressing four larger LOSs, occurs in vitro among mkA at a frequency of 10(-3). Infectivity of these variants was compared in 2 groups of volunteers inoculated with approximately 40,000 piliated, Opa- gonococci of either strain. The mkC variant infected 5 of 5 while mkA infected only 2 (40%) of 5. Gonococci recovered from the mkA infections showed a transition toward the mkC LOS phenotype. The mkA inoculum contained approximately 40 mkC gonococci. These data confirmed earlier studies and suggested that small numbers of mkC gonococci would be infective. This hypothesis was tested in three more experiments. In two, volunteers were inoculated with 250 or 1250 mkC, infecting 3 of 7 in each group, and in the third, 1600 mkC infected 2 of 6, resulting in a total of 8 of 20 infected by < or = 1600 mkC. Gonococci shed by infected volunteers maintained the mkC LOS phenotype but shifted from Opa- to Opa+. Thus, LOS and opacity protein, as well as pilus, are gonococcal virulence factors.

Adolescent↗

PubMed1997

Mosaicism due to a somatic mutation of the androgen receptor gene determines phenotype in androgen insensitivity syndrome.

Premature stop codons of the human androgen receptor (AR) gene are usually associated with a complete androgen insensitivity syndrome. We, however, identified an adult patient with a 46,XY karyotype carrying a premature stop codon in exon 1 of the AR gene presenting with signs of partial virilization: pubic hair Tanner stage 4 and clitoral enlargement. No other family members were affected. A point mutation at codon position 172 of the AR gene was detected that replaced the original TTA (Leu) with a premature stop codon TGA (opal). Careful examination of the sequencing gel, however, also identified a wild-type allele, indicating a mosaicism. In addition, elimination of the unique AflII recognition site induced by the mutation was incomplete, thus confirming the coexistence of mutant and wild-type AR alleles in the patient. Normal R1881 binding and a normal 110/112-kDa AR doublet in Western immunoblots consolidated the molecular genetic data by demonstrating the expression of the wild-type AR in the patient's genital skin fibroblasts. Transfection analysis revealed that only relatively high plasmid concentrations carrying the mutated AR complementary DNA lead to expression of a shortened AR due to downstream reinitiation at methionine 189. Thus, reinitiation does not play a role in the presentation of the phenotype; rather, the partial virilization is caused by the expression of the wild-type AR due to a somatic mosaic. We conclude that somatic mosaicism of the AR gene can represent a substantial factor for the individual phenotype by shifting it to a higher degree of virilization than expected from the genotype of the mutant allele alone.

Adult↗

PubMed1997

T cell-derived IL-4 and dendritic cell-derived IL-12 regulate the lymphokine-producing phenotype of alloantigen-primed naive human CD4 T cells.

Previous studies on human Th subset development were restricted to the analysis of naive T cells activated with anti-CD3 mAb in the absence of physiologic APC. In this study, we have analyzed the role of cytokines and physiologic APC on T cell maturation in an Ag-specific system, in which naive neonatal CD4 T cells were primed with allogeneic dendritic cells (DC). We found that the cytokine profile of primed cells was dependent upon 1) the ratio between T cells and allogeneic DC and 2) the endogenous production of IL-4 and IL-12. Neutralization of IL-4 during primary MLR increased IFN-gamma production at priming and shifted the phenotype of primed cells from Th0 to Th1. These effects were IL-12 dependent, in that they were suppressed by anti-IL-12 Abs. The production of IL-12 in primary MLR was further evidenced by the presence of IL-12 p40 in the culture supernatant fluids. IL-12 production was suppressed by exogenous IL-4 and increased by anti-IL-4 blocking mAbs, indicating that endogenous IL-4 down-regulated IL-12 production by DC. Finally, IL-12 was produced as a result of T cell/DC interaction involving the CD40/CD40 ligand and CD28/B7 costimulation pathways, as revealed by the inhibitory effect of anti-CD40 ligand mAb and CTLA-4Ig. These observations suggest that in neutral conditions, Ag presentation by DC results in the coordinate production of naive T cell-derived IL-4 and DC-derived IL-12 that in concert shape the cytokine profile of Th cells.

CD4-Positive T-Lymphocytes↗

PubMed2002

2,3,7,8-tetrachlorodibenzo-p-dioxin does not directly alter the phenotype of maturing B cells in a murine coculture system.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), acting through the aromatic hydrocarbon receptor (AhR), elicits numerous toxicological effects, including immunosuppression. Previous work from our laboratory has suggested that TCDD exposure in mice is associated with altered lymphopoietic development, in particular altered B-cell phenotype in the bone marrow. It remains to be determined which specific hematopoietic populations or subpopulations within the marrow cavity are directly targeted by TCDD. To examine the effects of TCDD on developing B cells in vitro, a stromal coculture model was used. Primary bone marrow cells from male, 6- to 7-week-old C57Bl/6 mice were cocultured separately on two AhR-containing stromal cell lines (M2-10B4 and S17) that support B-cell development in the presence of IL-7. The cocultures were treated with 0 to 10 nM TCDD. Shifts in phenotype were quantified by cell surface marker staining and flow cytometry. Four populations in the maturing B cell (very early pre-pro-B, pre-pro-B, pro-B, and pre-B) were defined for quantification. The results show that the only statistically significant effect of TCDD was within the pre-pro-B population in cultures with the S17 stromal cell line. The increase in number of cells with this phenotype was seen in cultures with both wild-type and AhR-/- primary bone marrow cells. These results suggest that the maturing B220+ B cell is not the direct target for TCDD-induced bone marrow B-cell alterations.

Animals↗

PubMed1992

The hepatocyte-specific phenotype of murine liver cells correlates with high expression of connexin32 and connexin26 but very low expression of connexin43.

This investigation was initiated in order to find out whether expression of the hepatocyte-specific phenotype is accompanied by expression of certain connexin genes coding for gap junctional protein subunits. Several clones of mouse embryonic hepatocytes immortalized in serum-free MX83 medium by infection with recombinant retrovirus-expressed transcripts for connexin32, connexin26, albumin, alpha-fetoprotein, tyrosine aminotransferase, as well as aldolase A and B, at more than half of the levels found in primary mouse hepatocytes. In addition the immortalized hepatocyte clones contained low levels of connexin43 mRNA of which only trace amounts were detected in primary embryonic mouse hepatocytes and in rat liver. Two of the immortalized hepatocyte clones were shifted from serum-free MX83 medium to Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum and, after 2, 14, or 180 days, back to MX83 medium. We found that expression of connexin32 and connexin26 mRNAs as well as transcripts of other liver-specific proteins was reversibly decreased in serum-containing medium, whereas the expression level of connexin43 transcripts was increased in serum-containing DMEM compared to serum-free MX83 medium. The expression levels of connexin26, connexin32, or connexin43 mRNAs were altered by the addition of fetal calf serum or arginine or by the absence of hydrocortisone in MX83 medium, all of which contributed to the shift in phenotype. Furthermore several dedifferentiated cell lines derived from rat or mouse liver and cultivated in serum-containing medium were found to express little connexin32 or connexin26 mRNA but relatively high levels of connexin43 mRNA.

Animals↗

PubMed1978

Phenotypic exclusion in mouse melanoma-rat hepatoma hybrid cells: pigment and albumin production are not reexpressed simultaneously.

Hybridization of cells of defined and different histotypes has been carried out to investigate whether the expression (or reexpression) of parental functions is mutually exclusive, as is expected if the generally assumed rule of discreteness of differentiation applies to hybrid cells. A cross of pigmented mouse melanoma cells and albumin-producing rat hepatoma cells gave rise to hybrids containing essentially one set of chromosomes from each parent and producing neither melanin nor albumin. Cells of one hybrid clone are shown to retain the potential to reexpress both parental differentiations. Successive subclonings of this hybrid have shown that cells which reexpress one function may retain the potential to reexpress the other, and that freshly isolated, morphologically homogeneous subclones may produce pigment or albumin, but not both; there successive and exclusive shifts of phenotype are documented, and in these cases, chromosome loss is very slight. The use of immunoadsorbed antisera has revealed that most (if not all) of the albumin produced by the hybrid cells is of the mouse type. We conclude that both parental determinations are retained by the hybrid cells, and that the parental differentiations are reexpressed only in a mutually exclusive fashion.

Albumins↗

PubMed1983

Impaired immunoregulation in systemic lupus erythematosus: defective adenosine-induced suppressor T lymphocyte generation.

It is currently unclear whether the suppressor cell dysfunction observed during active systemic lupus erythematosus (SLE) reflects a primary T cell disorder or one that results from immunologic modulation of suppressor T cell activity by autoantibodies. To determine whether the suppressor T cell dysfunction of active SLE is the result of a primary T cell disorder, the model of adenosine-induced immunosuppression was utilized to study the suppressor T cell functions of 12 patients with SLE (seven active SLE, five inactive SLE) and 12 matched healthy controls. T lymphocyte phenotyping was performed by utilizing monoclonal antibodies directed against T cell-specific determinants. Suppressor T cell functions were assessed by two assays in parallel. The first technique tested the capacity of two suppressor T cell subsets (spontaneous suppressors, Ts; adenosine-inducible suppressors, TRA) to inhibit pokeweed mitogen- (PWM) induced B cell differentiation. In the second technique, the ability of enriched T cell preparations to suppress mitogen- and alloantigen-induced proliferation was assayed. It was demonstrated that brief treatment of the control theophylline-resistant T lymphocyte (TR) subset possessing inducer/helper activity with adenosine (10(-5) M, 30 min, 37 degrees C) triggered a rapid shift in phenotype (RFC gamma -, T-4+ leads to RFC gamma +, T-8+) in a proportion of the subset, and the development of radioresistant suppressor function. By contrast, exposure of active SLE TR to adenosine failed to induce either the switch of phenotype or suppressor activity. When compared to controls, both the TS and TRA suppressors failed to inhibit B cell differentiation (TS, p less than 0.001; TRA, p less than 0.001). Moreover, enriched T cell preparations incompletely suppressed the proliferative responses to phytohemagglutinin (p less than 0.003), PWM (p less than 0.0003), or alloantigens (p less than 0.01). During inactive SLE, the T cell responses were usually restored. Treatment of the TR subsets with adenosine induced a switch of phenotype in four of five patients and the subsequent expression of effective suppressor function. We conclude that a) during active SLE, there is impaired suppression of proliferation and B cell differentiation; b) the impaired suppression of B cell differentiation results from abnormal spontaneous (TS) and adenosine-inducible (TRA) suppressor functions; c) the defective generation of suppressor T cell function during active disease results, in part, from a block in the transition from inducer/helper to suppressor cell; and, d) the suppressor T cell dysfunction is reversible with disease remission. The investigation of immunopharmacologic events by using the adenosine-induced immuno-suppression model in T cells from normal donors and SLE patients may provide insights into the molecular basis of disordered immunoregulation in SLE.

Adenosine↗

PubMed1998

Experimental murine Trypanosoma congolense infections. I. Administration of anti-IFN-gamma antibodies alters trypanosome-susceptible mice to a resistant-like phenotype.

The mechanisms regulating resistance or susceptibility to African trypanosomes have been enigmatic. In this study, we assessed the production of several cytokines (IL-4, IFN-gamma, and TNF-alpha) in vivo and in vitro using genetically susceptible (BALB/c) or resistant (C57BL/6) mice infected with cloned Trypanosoma congolense and the role of these cytokines in pathogenesis of this infection. Plasma of infected BALB/c mice contained higher levels of IL-4 and IFN-gamma than the plasma of infected C57BL/6 mice. Conversely, plasma TNF-alpha levels were elevated significantly in the resistant mice relative to the susceptible ones. Splenic IFN-gamma mRNA appeared earlier and were maintained at higher levels in infected BALB/c than in C57BL/6 mice. Both spontaneous and Con A-induced secretions of IL-4 and IFN-gamma by splenocytes from infected BALB/c mice were significantly higher than those from their C57BL/6 counterparts. Con A-induced proliferation of splenocytes from infected BALB/c mice was progressively suppressed. Nitric oxide was not involved in this suppression, but the suppression was positively correlated with IFN-gamma secretion. Addition of neutralizing Abs to IFN-gamma to cultures of Con A-stimulated spleen cells from infected BALB/c mice effectively reversed this suppression. Furthermore, administration of anti-IFN-gamma Abs to BALB/c mice early during infection dramatically shifted the phenotype of these susceptible mice to a more resistant-like phenotype, as expressed by a low and undulating parasitemia and a >300% increase in survival period. These results strongly suggest that the enhanced induction and secretion of IFN-gamma during T. congolense infections contribute to the relative susceptibility of BALB/c mice to the disease.

Animals↗