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1.
Genes Immun ; 5(7): 521-9, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15334114

ABSTRACT

Rheumatoid arthritis (RA) is associated with autoantibodies, the best known of which is rheumatoid factor (RF). RF/IgG complexes interact with FcgammaR on the surface of neutrophils, NK cells and monocyte/macrophages. We have analyzed the expression pattern and allelic polymorphisms of three FcgammaR genes (FcgammaRIIA, FcgammaRIIC and FcgammaRIIIA) in a large sample of RA patients and normal donors. We have found that the level of FcgammaR (CD16 and CD32) expression on NK cells is lower in RA patients than in normal individuals. Genotypic analysis demonstrated that the CD32 isoform expressed by the majority of RA patients was not the activating FcgammaRIIc1 isoform, commonly seen in normal individuals, but rather the inhibitory FcgammaRIIb isoform. The combination of the FcgammaRIIIA-176F allele with a lack of CD32 expression in NK cells appeared to be characteristic of RA subjects with aggressive disease. Since FcgammaRII and FcgammaRIIIA are predominantly expressed by NK cells, these data further suggest that FcgammaR-mediated activation of NK cells could be a disease-determining factor in RA patients.


Subject(s)
Antigens, CD/biosynthesis , Arthritis, Rheumatoid/epidemiology , Arthritis, Rheumatoid/metabolism , Killer Cells, Natural/metabolism , Receptors, IgG/biosynthesis , Antigens, CD/genetics , Arthritis, Rheumatoid/genetics , Female , Gene Expression Regulation/physiology , Gene Frequency/genetics , Genotype , Humans , Killer Cells, Natural/immunology , Male , Receptors, IgG/genetics , Severity of Illness Index
2.
J Immunol Methods ; 258(1-2): 85-95, 2001 Dec 01.
Article in English | MEDLINE | ID: mdl-11684126

ABSTRACT

We have recently reported that in addition to FcgammaRIIIa (CD16), approximately 45% of normal individuals also express FcgammaRIIc (CD32) on their natural killer (NK) cells. We found this expression to be regulated by an allelic polymorphism localized in the first extracellular exon (EC1) of the FcgammaRIIC gene, corresponding to aa 13. This is determined by a single nucleotide substitution, which results in either a functional open reading frame (glutamine-Q) or a premature stop codon (STP). Identification of this polymorphism provided a good explanation for the lack of CD32 expression previously observed with NK cells in some normal individuals. Here, we describe a new method for detection of FcgammaRIIc allelism based on RT-PCR amplification followed by an allele-specific restriction enzyme digestion. This method is rapid, reliable and time saving, as compared to the currently available allele-specific oligo-nucleotide probe-based Southern Blotting.


Subject(s)
Polymerase Chain Reaction/methods , Polymorphism, Genetic , Receptors, IgG/genetics , Alleles , Base Sequence , Blotting, Southern , Codon, Terminator , DNA Primers/genetics , DNA Restriction Enzymes , Exons , Flow Cytometry , Genotype , Humans , Immunophenotyping , Killer Cells, Natural/immunology , Open Reading Frames , Polymorphism, Restriction Fragment Length , Polymorphism, Single Nucleotide
3.
Am J Reprod Immunol ; 44(1): 16-21, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10976808

ABSTRACT

PROBLEM: Although leukocytes do not possess significant numbers of ovarian steroid hormone receptors, their numbers in the endometrium vary consistently, relative to the menstrual cycle. The possibility that cell types within the endometrium express leukocyte-attracting genes in response to ovarian hormones was investigated. METHOD OF STUDY: Endometrial biopsies were collected 10 days post-leutinizing hormone surge; the cell types were separated and cultured individually for 5 days in the presence of increasing amounts of estrogen or progesterone. Following culture, RNA was collected from cells and reverse-transcription-polymerase chain reaction was used to determine relative levels of gene expression of monocyte chemotactic proteins (MCP)-1, -2, and -3, and interleukin (IL)-12 p35 and p40. RESULTS: Although both endometrial stroma and glands were able to make MCP mRNA, steady-state levels of gene expression did not vary significantly relative to hormone treatment. The same was found for the p35 molecule of the IL-12 gene; however, differences were observed for the p40 subunit. CONCLUSIONS: Within the human endometrium, chemokines other than MCP and IL-12 are most likely responsible for cycle-related leukocyte recruitment.


Subject(s)
Cytokines , Endometrium/metabolism , Estrogens/pharmacology , Interleukin-12/metabolism , Monocyte Chemoattractant Proteins/metabolism , Progesterone/pharmacology , Cells, Cultured , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Chemokine CCL7 , Chemokine CCL8 , Endometrium/cytology , Endometrium/drug effects , Female , Gene Expression Regulation , Humans , Interleukin-12/genetics , Monocyte Chemoattractant Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction
4.
Am J Reprod Immunol ; 43(4): 187-96, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10836246

ABSTRACT

PROBLEM: Preeclampsia is the leading cause of maternal morbidity and premature fetal delivery in the United States, most likely involving the immune system in disease genesis. In this report, we tested the hypothesis that a superantigen phenomenon is an important factor in the pathogenesis of the disease. METHOD OF STUDY: A semi-quantitative polymerase chain reaction (PCR) was used to assess T-cell receptor (TCR) beta chain variable (Vbeta) regions as an indicator of T-cell expansion in both peripheral blood and basal plate of preeclamptic patients. All the subjects were also molecularly typed to identify their HLA-class II alleles. RESULTS: In peripheral blood of the majority of the patients, there was a high abundance of the Vbeta4 gene family, which was not observed in the control group. Polyclonality of this Vbeta gene family was confirmed by analysis of the Valpha chain and the complementary determining region 3 (CDR3). The majority of patients carried the Human Leukocyte Antigens (HLA)-DRB1*13 allele. CONCLUSION: We present evidence for the existence of a superantigen-like effect in at least a subset of patients with preeclampsia.


Subject(s)
Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor , HLA-DR Antigens/genetics , Pre-Eclampsia/immunology , Superantigens/immunology , T-Lymphocyte Subsets/immunology , Alleles , Endothelium, Vascular/immunology , Female , Genes, MHC Class II , Genetic Predisposition to Disease , Genotype , HLA-DRB1 Chains , Humans , Lymphocyte Activation , Placenta/immunology , Pre-Eclampsia/genetics , Pregnancy , Reverse Transcriptase Polymerase Chain Reaction
6.
Am J Obstet Gynecol ; 179(3 Pt 1): 604-9, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9757959

ABSTRACT

OBJECTIVE: Our purpose was to determine whether methotrexate affects the trophoblast or corpus luteum when administered for abortion. STUDY DESIGN: A randomized controlled trial was performed in women requesting an abortion up to 49 days' gestation. Twenty patients were treated with intramuscular methotrexate 50 mg/m2 (10 women) or 60 mg/m2 (10 women). Serum beta-human chorionic gonadotropin, progesterone, and 17-hydroxyprogesterone levels were determined at baseline and then serially after methotrexate administration for the first 24 hours, then every 24 hours for 7 days. On the seventh day misoprostol 800 microg was administered vaginally. RESULTS: Serum beta-human chorionic gonadotropin increased at a lower rate than occurs in normal pregnancy. Progesterone levels averaged 56.9 +/- 19.8 nmol/L at baseline and 45.5 +/- 20.5 nmol/L (P = .01) 1 week after methotrexate. Progesterone decreased in 16 women over the 7 days and increased in the other 4; these latter women all aborted after a single dose of misoprostol. Levels of 17-hydroxyprogesterone plateaued during the first day after methotrexate administration; both progesterone and 17-hydroxyprogesterone declined simultaneously between the third and fourth day after methotrexate. CONCLUSIONS: Methotrexate most likely primarily affects trophoblast production of human chorionic gonadotropin, as evidenced by a blunting of the expected increase in serum beta-human chorionic gonadotropin resulting in less support for the production of progesterone by the corpus luteum. However, changes in progesterone levels after methotrexate administration were inconsistent and are unlikely to represent the ultimate effect of methotrexate in abortion. The less-than-normal increase in serum beta-human chorionic gonadotropin levels after methotrexate administration is most likely a result of disruption of cytotrophoblast syncytialization. This disruption may be the true effect of methotrexate in destabilizing the implantation site of an early pregnancy.


Subject(s)
Abortifacient Agents/pharmacology , Corpus Luteum/drug effects , Methotrexate/pharmacology , Pregnancy/physiology , Trophoblasts/drug effects , 17-alpha-Hydroxyprogesterone/blood , Adult , Chorionic Gonadotropin, beta Subunit, Human/blood , Female , Humans , Injections, Intramuscular , Misoprostol/pharmacology , Pregnancy Trimester, First , Progesterone/blood , Prospective Studies
7.
Hum Reprod ; 13(4): 1063-9, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9619571

ABSTRACT

Methotrexate is a folic acid analogue that has been used successfully for the treatment of ectopic pregnancy and, in conjunction with misoprostol, for medical abortions of early intrauterine pregnancies. To administer the most efficacious treatment requires knowledge of the mechanism underlying the induction of methotrexate-induced abortion. This study was designed to ascertain trophoblast integrity, proliferation and differentiation following administration of methotrexate. In addition, to determine if methotrexate affects the local uterine immune response, we ascertained the numbers and identities of decidual leukocytes following treatment. Ten women with undesired intrauterine pregnancies of 42-49 days gestation were recruited to receive methotrexte 50 mg/m2 i.m. A suction aspiration was performed 7 days later. Tissues from gestational age-matched elective surgical abortions were used as controls. Additionally, specimens from women who received methotrexate and misoprostol for abortion in a clinical trial of oral methotrexate in combination with misoprostol, who had a suction abortion because of continued embryonic cardiac activity 14 days after the methotrexate, were evaluated. Immunoreactivity to proliferating cell nuclear antigen and cyclin D3 antibodies was used to demonstrate a marked reduction in the proliferation index of cytotrophoblasts from methotrexate-treated abortions. Methotrexate treatment failures and non-treated pregnancies had a much higher proliferation index. There was no direct destruction of the syncytiotrophoblast, as indicated by the continued presence of human placental lactogen and beta-human chorionic gonadotrophin proteins. A decrease in the total number of leukocyte cells was observed in the decidua of methotrexate-treated samples, with the large granular lymphocyte (LGL) cells showing the greatest decline in numbers. Our conclusions from this study are that methotrexate acts primarily to derail the normal developmental programme of the trophoblast stem cell population, as well as to decrease LGL cell numbers in the decidua.


Subject(s)
Immune System/drug effects , Immunosuppressive Agents/pharmacology , Methotrexate/pharmacology , Nucleic Acid Synthesis Inhibitors/pharmacology , Trophoblasts/drug effects , Adult , Antibody Formation/drug effects , Cell Differentiation/drug effects , Cell Division , Cell Movement/drug effects , Cyclin D3 , Cyclins/metabolism , Decidua/cytology , Decidua/drug effects , Female , Humans , Lymphocytes/drug effects , Lymphocytes/physiology , Placenta/drug effects , Placenta/metabolism , Pregnancy , Proliferating Cell Nuclear Antigen/metabolism , Trophoblasts/cytology
8.
Am J Reprod Immunol ; 39(1): 1-11, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9458927

ABSTRACT

PROBLEM: A significant cohort of women with autoimmune thyroid disease (ATD) also suffer from reduced fertility. The finding that neither exogenous hormones nor donor eggs correct the infertility suggests that the problem involves an inherent endometrial defect. METHOD OF STUDY: Endometrial leukocyte populations in women with ATD were quantitated by immunohistochemistry. A semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique was used to examine the expression of transforming growth factor (TGF)-beta 1, interleukin (IL)-4, IL-10, and interferon (IFN)-gamma in the endometrial samples. RESULTS: A significant increase in the endometrial T-cell population was observed in women with ATD compared to controls. The relative abundance of IL-4 and IL-10 was decreased in women with ATD compared to controls, whereas IFN-gamma was increased. No difference was noted in the abundance of TGF-beta 1. The source of cytokine production for IL-4, IL-10, and IFN-gamma was the endometrial leukocytes. CONCLUSIONS: Both the leukocyte numbers and cytokine expression profile were altered significantly in a well-defined group of women with implantation defects.


Subject(s)
Autoimmune Diseases/complications , Embryo Implantation , Endometrium/cytology , Hyperthyroidism/complications , Hypothyroidism/complications , Infertility, Female/etiology , Leukocytes/physiology , Thyroid Diseases/complications , Autoimmune Diseases/pathology , Cytokines/biosynthesis , Endometrium/metabolism , Female , Histocompatibility Antigens Class II/biosynthesis , Humans , Hyperthyroidism/pathology , Hypothyroidism/pathology , Immunohistochemistry , Leukocytes/cytology , Leukocytes/metabolism , Polymerase Chain Reaction , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , T-Lymphocytes/physiology , Thyroid Diseases/immunology , Thyroid Diseases/pathology , Transcription, Genetic
9.
Fertil Steril ; 68(6): 1103-7, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9418705

ABSTRACT

OBJECTIVE: To determine whether the recruitment of decidual leukocytes during pregnancy is the same in intrauterine pregnancies (IUPs) versus ectopic pregnancies (EPs). DESIGN: Intrauterine decidual samples from both EPs and IUPs were obtained for the evaluation of leukocyte populations. SETTING: In vitro experiment. PATIENT(S): Women with EPs and women with IUPs. MAIN OUTCOME MEASURE(S): Immunohistochemical identification of decidual leukocyte populations. RESULT(S): We have analyzed the decidual leukocyte populations from three women with EPs by immunohistochemical analysis. The data demonstrate a leukocyte infiltration similar to that found in decidua from normal pregnancies. CONCLUSIONS(S): These data support the hypothesis that decidual leukocyte recruitment and/or increases during pregnancy is primarily hormonally regulated.


Subject(s)
Antigens, CD/analysis , Decidua/pathology , Leukocytes , Pregnancy, Ectopic/pathology , Pregnancy , CD3 Complex/analysis , CD56 Antigen/analysis , Decidua/cytology , Female , Flow Cytometry , Humans , Immunohistochemistry , Leukocyte Common Antigens/analysis , Leukocyte Count , Leukocytes/immunology , Lipopolysaccharide Receptors/analysis
10.
Blood ; 83(3): 713-23, 1994 Feb 01.
Article in English | MEDLINE | ID: mdl-8298134

ABSTRACT

The effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) are not confined to cells of the myeloid lineage. GM-CSF has been shown to have effects on mature T cells and both mature and immature T-cell lines. We therefore examined the GM-CSF responsiveness of murine thymocytes to investigate whether GM-CSF also affected normal immature T lymphocytes. The studies presented here indicate that GM-CSF augments accessory cell (AC)-dependent T-cell receptor (TCR)-mediated proliferation of unseparated thymocyte populations. To identify the GM-CSF responsive cell type, thymic AC and T cells were examined for GM-CSF responsiveness. We found that GM-CSF augmentation of TCR-induced thymocyte proliferation appears to be mediated via augmentation of AC function, and not via direct effects on mature single-positive (SP) thymocytes. Enriched double-negative (DN) thymocytes were also tested for GM-CSF responsiveness. GM-CSF induced the proliferation of adult and fetal DN thymocytes in an AC-independent and TCR-independent single-cell assay. Thus, in contrast to the SP thymocytes, a DN thymocyte population was directly responsive to GM-CSF. GM-CSF therefore may play a direct role in the expansion of DN thymocytes and an indirect role in the expansion of SP thymocytes.


Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Lymphocyte Activation/drug effects , Receptors, Antigen, T-Cell/physiology , T-Lymphocytes/immunology , Animals , Antigen-Presenting Cells/drug effects , Female , Male , Mice , Mice, Inbred C57BL , Receptors, Interleukin-2/analysis
11.
Blood ; 81(10): 2671-8, 1993 May 15.
Article in English | MEDLINE | ID: mdl-8490177

ABSTRACT

The treatment of cancer with lymphokine-activated killer (LAK) cells in conjunction with high-dose interleukin-2 (IL-2) has been limited by the toxicity of IL-2 and the narrow range of tumors that respond to therapy. Cytokines that are capable of augmenting lower doses of IL-2 are, therefore, a major focus of research. We report here that granulocyte-macrophage colony-stimulating factor (GM-CSF) can augment low-dose IL-2 LAK induction from murine splenocytes. Anti-tumor necrosis factor alpha (anti-TNF alpha) or anti-interferon gamma (anti-IFN gamma) monoclonal antibodies did not inhibit (IL-2 + GM-CSF)-induced LAK generation, indicating that GM-CSF augmentation does not require TNF alpha or IFN gamma activity. Depletion of natural killer cells before culture did not inhibit low-dose IL-2-induced LAK generation or the ability of GM-CSF to augment LAK generation. In contrast, depletion of both CD4+ and CD8+ T cells before culture inhibited the generation of LAK activity. However, depletion of only CD4+ T cells, or only CD8+ T cells, did not inhibit the generation of IL-2 or (IL-2 + GM-CSF) LAK activity. These results suggest that LAK precursors are present in both the CD4+ and CD8+ T-cell populations and suggest that the addition of GM-CSF to low-dose IL-2 may result in the generation of T-derived LAK cells.


Subject(s)
Cytotoxicity, Immunologic/drug effects , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Interleukin-2/pharmacology , Killer Cells, Lymphokine-Activated/immunology , Animals , Antibodies, Monoclonal/pharmacology , CD4 Antigens/analysis , CD8 Antigens/analysis , Cell Line , Cells, Cultured , Dose-Response Relationship, Drug , Drug Synergism , Female , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Humans , Interferon-gamma/immunology , Killer Cells, Lymphokine-Activated/drug effects , Kinetics , Lymphocyte Depletion , Male , Mice , Mice, Inbred C57BL , Recombinant Proteins/immunology , Recombinant Proteins/pharmacology , Spleen/immunology , T-Lymphocyte Subsets/immunology , Tumor Necrosis Factor-alpha/immunology
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