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1.
Pediatr Transplant ; 18(2): E57-63, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24438382

ABSTRACT

The most common identifiable causes of acute liver failure in pediatric patients are infection, drug toxicity, metabolic disease, and autoimmune processes. In many cases, the etiology of acute liver failure cannot be determined. Acute leukemia is an extremely rare cause of acute liver failure, and liver transplantation has traditionally been contraindicated in this setting. We report a case of acute liver failure in a previously healthy 15-yr-old male from pre-B-cell acute lymphoblastic leukemia. He underwent liver transplantation before the diagnosis was established, and has subsequently received chemotherapy for pre-B-cell acute lymphoblastic leukemia. He is currently alive 31 months post-transplantation. The published literature describing acute lymphoblastic leukemia as a cause of acute liver failure is reviewed.


Subject(s)
Leukemia, B-Cell/complications , Leukemia, B-Cell/therapy , Liver Failure, Acute/complications , Liver Failure, Acute/surgery , Liver Transplantation , Precursor Cell Lymphoblastic Leukemia-Lymphoma/complications , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Adolescent , Biopsy , Humans , Immunosuppressive Agents/therapeutic use , Liver/pathology , Liver Function Tests , Male , Tissue Donors , Treatment Outcome
2.
Am J Clin Pathol ; 116(6): 893-7, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11764079

ABSTRACT

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) share many morphologic and immunophenotypic features. In addition to histomorphologic examination, it is customary to use the absence of CD23 to differentiate MCL from CLL/SLL, based primarily on reported comparisons of immunohistochemical staining of tissue sections. These findings are widely extrapolated to flow cytometric analysis, although available data are contradictory and not sufficiently detailed. We compared expression of CD23 by flow cytometry in 22 cases of MCL and 25 cases of CLL/SLL. Lymphoma cells in 12 of 22 MCLs were negative for CD23, and 10 showed dim expression. In contrast, none of 25 CLL/SLLs were negative for CD23, 4 were dimly positive, and 21 were moderately or brightly positive. Thus, a significant proportion of MCL exhibited overlap of CD23 expression in the low-intensity range with CLL/SLL. Clinically, there was no correlation between the intensity of CD23 expression and clinical stage at diagnosis or survival. These findings emphasize that by flow cytometry, MCL can be differentiated reliably from CLL/SLL using CD23 if negative expression is observed. However, with dimly positive expression, interpretation should be cautious.


Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/diagnosis , Lymphoma, Mantle-Cell/diagnosis , Receptors, IgE/analysis , Adult , Aged , Biomarkers, Tumor/analysis , Diagnosis, Differential , Female , Flow Cytometry/methods , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/chemistry , Lymphoma, Mantle-Cell/chemistry , Male , Middle Aged
3.
Transplantation ; 70(6): 877-86, 2000 Sep 27.
Article in English | MEDLINE | ID: mdl-11014641

ABSTRACT

BACKGROUND: In our previously described primate renal allograft model, T cell ablation leads to long-term graft survival. The role of endothelial cell alteration in chronic rejection was examined in our model. METHODS: Renal transplants were performed in rhesus monkeys using a T cell- depleting immunotoxin, FN18-CRM9. Sections from 10 rejected kidneys (5 acute and 7 chronic rejection) were examined after immunohistochemical staining for expression of endothelium-related proteins [von Willebrand factor (vWF), CD62P, and CD31], fibrinogen, and a macrophage marker (CD68). Glomerular staining for each antigen was graded on a semiquantitative scale. RESULTS: Intense staining for vWF was consistently observed in glomerular endothelium, subendothelium, and mesangium in all kidneys removed due to chronic rejection. vWF staining was weak in kidneys showing acute rejection. The difference in glomerular staining was statistically significant. Staining for vWF in extraglomerular vessels was nearly identical in kidneys showing acute and chronic rejection. Expression of CD62P was increased in extraglomerular vessels in allografts with chronic rejection, but the glomeruli showed little or no staining. There was no significant difference in the glomerular staining for CD62P or CD31 in organs showing acute and chronic rejection. Fibrinogen staining of glomerular mesangium was seen in kidneys with chronic rejection. Macrophages (CD68+) infiltrating glomeruli were more numerous in kidneys showing chronic rejection. CONCLUSION: Increased glomerular deposition of vWF in renal allografts showing chronic rejection, without increased staining for CD62P or CD31, suggests increased constitutive secretion of vWF from endothelial cells as a component of the mechanism of chronic rejection in our model.


Subject(s)
Kidney Glomerulus/chemistry , Kidney Transplantation/immunology , von Willebrand Factor/metabolism , Acute Disease , Animals , Antigens, CD , Antigens, Differentiation, Myelomonocytic , Chronic Disease , Coloring Agents , Fibrinogen , Graft Rejection/diagnosis , Graft Rejection/metabolism , Immune Tolerance , Immunotoxins/administration & dosage , Macaca mulatta , Macrophages/immunology , Male
4.
Eur J Immunol ; 28(8): 2300-10, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9710208

ABSTRACT

We recently demonstrated that different CD45 monoclonal antibodies (mAb) are able to induce cellular aggregation in human peripheral blood mononuclear cells (PBMC) through LFA-1/ICAM-1 interactions. Such interactions could be down-modulated by protein kinase (PK) A/G inhibitors, but were unaffected by inhibitors of PKC, suggesting the involvement of PKA or PKG in CD45 mAb-induced adhesion. In this study we show that after incubation of PBMC with several (but not all) mAb to CD45, CD45RO and CD45RA, intracellular cAMP, but not cGMP concentrations readily increase, reaching a maximum 30 min after start of activation. As evidenced by several lines of investigation cAMP accumulation was independent of Fc receptor-associated signaling as well as tyrosine phosphatase activity of CD45. In highly pure T lymphocytes, CD45 mAb were unable to induce cAMP synthesis, but readily did so after addition of autologous monocytes. After paraformaldehyde fixation of both quiescent or IFN-gamma/TNF-alpha-preactivated monocytes, cAMP production was no longer detectable, suggesting monocytes as the cell of origin for the increased cAMP synthesis. Further, cAMP accumulation in monocytes occurred after reconstitution to T lymphocytes preincubated with CD45 mAb and extensively washed. Importantly, pretreatment of T lymphocyte/monocyte mixtures with LFA-1 mAb and/or ICAM-1 mAb down-regulated CD45 mAb-induced cAMP synthesis. Finally, we demonstrate that CD45 mAb are not only capable of inducing cAMP production, but also of directly stimulating PKA enzyme activity. Based on the data presented, we propose that CD45 signaling in T lymphocytes subsequently activates cAMP accumulation and PKA activation in monocytes via LFA-1/ICAM-1-dependent cellular interactions.


Subject(s)
Cyclic AMP/biosynthesis , Leukocyte Common Antigens/metabolism , Monocytes/metabolism , T-Lymphocytes/immunology , Antibodies, Monoclonal/pharmacology , Cell Communication , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic GMP/biosynthesis , Enzyme Activation , Epitopes , Humans , In Vitro Techniques , Intercellular Adhesion Molecule-1/metabolism , Lymphocyte Function-Associated Antigen-1/metabolism , Monocytes/immunology , Signal Transduction , T-Lymphocytes/metabolism
5.
J Heart Lung Transplant ; 15(2): 206-17, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8672525

ABSTRACT

BACKGROUND: Cytokines play a central role in inflammatory responses and in specific immune responses directed toward alloantigens. The pattern and quantity of cytokines produced in graft rejection can yield valuable information regarding the cellular and molecular mechanisms of the antigraft response. METHODS: We used the polymerase chain reaction to semiquantitatively measure changes in the amount of messenger RNA from the interleukin-1 beta, interleukin-2, interleukin-4, interleukin-6, interleukin-10, tumor necrosis factor-alpha, interferon-gamma, interleukin-1 receptor antagonist, and the interleukin-2 receptor genes in the peripheral blood and endomyocardium of cardiac allograft recipients during the first 8 weeks after transplantation. A total of 328 samples of resting (n = 251) and stimulated (n = 77, stimulated with phytohemagglutinin and lipopolysaccharide for 18 hours) peripheral blood mononuclear cells collected from 16 patients were measured. To measure intragraft cytokine levels, we analyzed 150 endomyocardial biopsy specimens from 19 patients. RESULTS: No elevation in expression was seen before injection, but, after the onset of rejection and concomitant with treatment, there was a decrease in detectable mRNA (p < 0.05) for the pro-inflammatory monokines interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha in resting peripheral blood mononuclear cells, and a decrease for the T-cell derived cytokines interleukin-4 and interleukin-10 in stimulated peripheral blood mononuclear cells. These changes in mRNA expression occurred coincidentally with decreases in the percentage of lymphocytes and monocytes in the peripheral blood after administration of rejection therapy. In endomyocardial biopsy specimens, there were no detectable changes in the quantities of cytokine mRNA specimens for the interferon-gamma, interleukin-6, interleukin-10, interleukin-1ra, and interleukin-1 beta genes before rejection. In general, the levels of these cytokines were near the lower limits of detection by our assay in endomyocardial biopsies, mRNA from the interleukin-2, interleukin-4, tumor necrosis factor-alpha, and interleukin-2R genes were undetectable. CONCLUSIONS: We conclude that changes in the expression of cytokine mRNA in both peripheral blood mononuclear cells and endomyocardial biopsy specimens as measured by the semiquantitative polymerase chain reaction method used in this study does not effectively predict rejection. The decline in peripheral blood mononuclear cell cytokine mRNA after rejection treatment is likely due to changes in the proportion of lymphocytes and monocytes in the peripheral blood in concert with a steroid-induced downregulation by cytokine gene transcription.


Subject(s)
Cytokines/genetics , Endocardium/immunology , Graft Rejection/diagnosis , Heart Transplantation/immunology , Monocytes/immunology , Myocardium/immunology , Polymerase Chain Reaction , RNA, Messenger/genetics , Biopsy , Diagnosis, Differential , Endocardium/pathology , Gene Expression/physiology , Graft Rejection/immunology , Graft Rejection/pathology , Heart Transplantation/pathology , Humans , Myocardium/pathology , Predictive Value of Tests , Receptors, Cytokine/genetics
7.
J Immunol ; 154(11): 5611-9, 1995 Jun 01.
Article in English | MEDLINE | ID: mdl-7751614

ABSTRACT

Intraepithelial lymphocytes (IELs) have been extensively studied in the murine small intestine. However, to date no studies have assessed IEL in the large intestine, despite the marked differences in function and lumenal environment. In the present study, we isolated IEL from both small and large intestine of three mouse strains (BALB/c, C3H/HeN, C57BL/6) and determined the frequency of CD2, CD4, and CD8 expression on CD3+ IEL, as well as the frequency of alpha beta and gamma delta TCR usage and V beta distribution. Higher numbers of IEL/unit length were always isolated from the small intestine (20-30 x 10(6)/5 mice) compared with large intestine (1.1-2.5 x 10(6)/5 mice). Interestingly, IEL from the large intestine of all strains were predominantly alpha beta TCR+ whereas gamma delta TCR+ IELs predominated in small intestine. Large intestinal IELs were mainly CD4+, in both BALB/c and C3H/HeN mouse strains. IELs from large intestine of C57BL/6 mice were mainly CD8+; however, the CD4+ subset was fourfold higher when compared with small intestine IEL. Potential functional differences between IEL subsets was assessed by determining the relative levels of mRNA for IL-1, 2, 4, 5, 10, IFN-gamma, TGF-beta, and TNF-gamma. Similar patterns of IL-1, IFN-gamma and TNF-alpha were seen while more IL-2, IL-4, IL-5, and IL-10 mRNA was noted in large intestinal IEL. Stimulation of C3H/HeJ IEL with anti-CD3 also resulted in higher levels of IL-3/GM-CSF, IL-4, and IL-6 by IEL from large intestine. These results show that marked differences occur among the T cell subsets present in IELs from mouse small and large intestine.


Subject(s)
Intestinal Mucosa/immunology , Intestine, Large/immunology , Intestine, Small/immunology , T-Lymphocyte Subsets/immunology , Animals , Antibodies, Monoclonal/immunology , CD2 Antigens/biosynthesis , CD3 Complex/immunology , CD4-Positive T-Lymphocytes/immunology , Cytokines/biosynthesis , Flow Cytometry , Intestinal Mucosa/cytology , Intestine, Large/cytology , Intestine, Small/cytology , Lymphocyte Activation/immunology , Mice , Mice, Inbred C3H , Polymerase Chain Reaction , RNA, Messenger/biosynthesis
8.
Transpl Immunol ; 3(2): 124-34, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7582903

ABSTRACT

Previous studies have shown that post-transplantation infusion of donor specific bone marrow following a non-specific potent immunosuppressive agent such as antilymphocyte globulin (ALG) can significantly enhance graft survival compared to ALG alone. This enhancement remains variable and is thought to occur through the induction of specific partial tolerance to the renal allograft, but the underlying cellular mechanisms have not been clearly identified. In order to improve the efficacy of this specific immunosuppressive treatment and to study the events leading to enhanced allograft survival, we sought to establish a simple in vitro model based on a mixed lymphocyte reaction (MLR). We show that cellular proliferation seen in a normal MLR can be suppressed by addition of donor specific bone marrow cells (BMC). Significantly, this suppression is not observed with either third party BMC or donor specific peripheral blood mononuclear cells (PBMC). We have defined the optimum conditions of bone marrow infusion regarding number of BMC, their handling and culture, and simple enrichment procedures. Using a semiquantitative polymerase chain reaction assay, we have studied the cytokine gene expression in MLR modulated by donor specific BMC. In an unmodified allogeneic response, the responder cells show increased expression of interleukin-2 (IL-2) gamma-interferon IFN-gamma and receptor (IL-2R) mRNA, and no IL-10 mRNA. When responder cells are cultured with BMC of the stimulator, there is a 256-fold decrease in IL-2 mRNA, and a 64-fold decrease in IFN-gamma and IL-2R mRNA. There is also a 64-fold increase in IL-10 mRNA. This effect is even more marked when the BMC are depleted of CD3+ cells. The kinetics of addition of donor specific BMC to the normal allogeneic MLR culture and specificity of the action of BMC are also elucidated. Our data suggest that the enhancement of graft survival observed with donor BMC may operate through decreased proliferation of reactive T cell clones (due to decreased IL-2/IL-2R) and suppressed monocyte functions (due to decreased IFN-gamma and increased IL-10 gene expression).


Subject(s)
Bone Marrow Transplantation/immunology , Cytokines/genetics , HLA Antigens/immunology , Immune Tolerance , Lymphocyte Culture Test, Mixed , Th1 Cells/metabolism , Th2 Cells/metabolism , Bone Marrow/immunology , Bone Marrow Cells , Bone Marrow Transplantation/pathology , CD3 Complex/analysis , Cell Division/drug effects , Cells, Cultured , Cryopreservation , Cytokines/metabolism , Fibroblast Growth Factor 1/pharmacology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , HLA Antigens/genetics , Haplotypes , Humans , Immune Tolerance/genetics , Kinetics , Lymphocyte Depletion , Phenotype , Th1 Cells/immunology , Th2 Cells/immunology , Tissue Donors
9.
Arthritis Rheum ; 38(6): 750-4, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7540009

ABSTRACT

OBJECTIVE: To examine the de novo synthesis and cellular distribution of the E-selectin adhesion molecule in synovial tissues obtained from patients with rheumatoid arthritis (RA). METHODS: Immunohistochemistry techniques combined with in situ hybridization were used to examine RA synovium. RESULTS: There were numerous endothelial cells positive for E-selectin and E-selectin messenger RNA in the RA synovial membranes. Moreover, E-selectin expression appeared to correlate with inflammatory activity. CONCLUSION: The strong vascular expression of E-selectin indicates an activation of endothelial cells in the recruitment of cells associated with the chronic inflammation of RA.


Subject(s)
Arthritis, Rheumatoid/metabolism , Cell Adhesion Molecules/analysis , Cell Adhesion Molecules/genetics , RNA, Messenger/analysis , Adult , Aged , Arthritis, Rheumatoid/genetics , Base Sequence , Cell Adhesion Molecules/biosynthesis , DNA Primers/analysis , DNA Primers/chemistry , DNA Primers/genetics , E-Selectin , Female , Humans , Immunohistochemistry , In Situ Hybridization , Male , Middle Aged , Molecular Sequence Data , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Synovial Membrane/chemistry , Synovial Membrane/metabolism , Up-Regulation
10.
Eur J Immunol ; 24(12): 3087-92, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7805737

ABSTRACT

Analysis of cytokine gene expression by reverse transcription-polymerase chain reaction (RT-PCR) demonstrated high spontaneous levels of transcripts for multiple cytokines in murine Peyer's patches (PP) compared to spleen and peripheral lymph nodes. This is consistent with the presence of active germinal centers in PP and their continuous exposure to lumenal antigen including bacterial endotoxin. RT-PCR analysis of cytokine transcripts in purified PP T cell populations revealed the presence of transcripts for interleukin-4 (IL-4), IL-5 and IL-10 in addition to interferon-gamma (IFN-gamma) in CD8+ cells purified by flow cytometry. The majority of PP CD8+ T cells were also CD45RBlo (MB23G2-), suggesting that these cells were activated/memory cells. CD8+ cells in spleen and mesenteric lymph nodes (MLN) were predominantly CD45RBhi (MB23G2+) consistent with a resting/naive phenotype. PP and MLN CD8+ T cells also secreted IL-5 and IL-10 when stimulated with anti-CD3 monoclonal antibody and when co-cultured with PP B cells enhanced secretion of both IgG and IgA. These studies suggest that CD8+ T cells at mucosal sites secrete T helper type 2 cytokines and can provide functional help for B cells in these tissues.


Subject(s)
Antibody Formation , CD8-Positive T-Lymphocytes/immunology , Cytokines/metabolism , Immunologic Memory , Peyer's Patches/immunology , T-Lymphocyte Subsets/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Animals , Cytokines/genetics , Gene Expression , Immunoglobulin A, Secretory/metabolism , Immunoglobulin G/metabolism , Interleukin-10/metabolism , Interleukin-5/metabolism , Mice , Mice, Inbred C3H , RNA, Messenger/genetics
11.
Blood ; 83(7): 1862-70, 1994 Apr 01.
Article in English | MEDLINE | ID: mdl-7908233

ABSTRACT

We recently reported that cross-linking the leukocyte common antigen (CD45) can rapidly induce aggregation of human peripheral blood mononuclear cells via lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) interactions. Herein, we have examined both T-cell--monocyte cellular interactions and the molecular signaling that are involved in this phenomenon. Experiments using highly purified T lymphocytes showed that CD45-induced aggregation requires the presence of both T cells and monocytes. Cross-linking CD45 only on T lymphocytes, but not on monocytes, initiated cellular clustering after reconstituting to the respective untreated cell type. By several criteria, CD45-induced clustering of T cells to autologous monocytes was shown to be Fc-receptor--independent. When comparing intracellular signaling in leukocyte aggregation induced by CD45 cross-linking versus phorbol myristate-12-13-acetate (PMA) treatment, the former was found to be fivefold to 10-fold more sensitive to H-8, a reagent that effectively blocks cAMP- and cGMP-dependent protein kinases. On the other hand, reagents that increase intracellular cAMP levels (eg, dbcAMP, forskolin, and IBMX), protein kinase C (PKC) inhibitors (eg, staurosporine), and tyrosine kinase inhibitors (eg, herbimycin A and genistein) all readily inhibited PMA-induced, but not CD45 monoclonal antibody-induced, aggregation. We conclude that cross-linking the leukocyte common antigen on T cells induces LFA-1--/ICAM-1--dependent T-cell--monocyte aggregation through a unique signaling pathway independent of PKC, which involves instead cAMP-/cGMP-dependent protein kinases.


Subject(s)
Cell Adhesion Molecules/physiology , Leukocyte Common Antigens/physiology , Leukocytes/physiology , Lymphocyte Function-Associated Antigen-1/physiology , Alkaloids/pharmacology , Antigens, CD/metabolism , CD18 Antigens , Cell Adhesion , Cell Aggregation , Cyclic AMP-Dependent Protein Kinases/physiology , Humans , Intercellular Adhesion Molecule-1 , Phosphorylation , Protein Kinase C/physiology , Protein-Tyrosine Kinases/physiology , Staurosporine
12.
J Immunol ; 152(4): 1641-52, 1994 Feb 15.
Article in English | MEDLINE | ID: mdl-8120374

ABSTRACT

In the complete absence of APCs staphylococcal superantigens induced IL-2, IL-4, IL-5, IFN-gamma, and IL-2R gene transcripts in both highly purified human T cells and FACs sorted CD4+ memory (CD45RA-) T cells. Secretion of IL-2, IL-4, and IFN-gamma, as well as DNA synthesis, on the other hand, required the presence of monocytes. At cytokine gene transcript level, three patterns of expression were noted after superantigen activation of T cells in the presence vs the absence of APC. mRNA levels for IL-2 were markedly up-regulated in the presence of monocytes, IL-4 and IFN-gamma transcripts increased only modestly, and IL-5 and IL-2R mRNA levels were unaffected. Blocking mAbs against LFA-1 and LFA-3 added to staphylococcal enterotoxin B (SEB)-activated cultures of T cells and autologous monocytes, reproducibly decreased both T cell proliferation and genetic expression of IL-2, IL-4, IL-5, and IL-2R, although having little or no effect on IFN-gamma transcripts. Further, under those conditions of blocking, secretion of IL-2 and IL-4 was dramatically decreased, whereas IFN-gamma secretion remained essentially unchanged. In contrast, LFA-1 and LFA-3 mAbs completely abrogated IFN-gamma secretion from PHA-activated T cell-monocyte mixtures, although having no inhibitory effect on T cell proliferation. These results indicate a characteristic and differential involvement of adhesion molecule-mediated signals in superantigen-induced T cell proliferation, differential cytokine gene expression, and cytokine secretion.


Subject(s)
Cell Adhesion Molecules/physiology , Cytokines/genetics , Lymphocyte Activation , Superantigens/immunology , T-Lymphocytes/metabolism , Adult , Antibodies, Monoclonal/immunology , Antigen-Presenting Cells/physiology , Cytokines/metabolism , Gene Expression , Humans , Interferon-gamma/genetics , Interferon-gamma/metabolism , Interleukin-2/genetics , Interleukin-2/metabolism , Interleukin-4/genetics , Interleukin-4/metabolism , Monocytes/metabolism , T-Lymphocytes/immunology
13.
Eur J Immunol ; 23(8): 2049-52, 1993 Aug.
Article in English | MEDLINE | ID: mdl-8344373

ABSTRACT

Murine intraepithelial lymphocytes (IEL) respond poorly to T cell mitogens and to monoclonal antibody stimulation of T cell receptor (TCR)- and CD3- associated molecules. In contrast, we found that a soluble extract of Mycobacterium tuberculosis (Mtb), but not purified protein derivative of tuberculin, induced significant proliferative responses in IEL cultures. The active component was apparently a heat shock protein (HSP), since recombinant 71-kDa HSP from Mtb induced IEL to proliferate, while 65-kDa HSP from M. bovis and M. leprae did not. Both alpha/beta and gamma/delta TCR-enriched IEL gave proliferative responses to 71-kDa HSP. Further, culture supernatants from IEL stimulated with 71-kDa HSP contained elevated levels of interleukin-(IL)-3/granulocyte-macrophage colony-stimulating factor, interferon-gamma and IL-6, but not IL-2, IL-4, IL-5 or transforming growth factor-beta. Finally, several IEL T cell clones have been maintained for up to 6 weeks, when stimulated with 71-kDa HSP, IL-2 and feeder cells. Our results show that the 71-kDa HSP of Mtb induces IEL T cells to divide and to secrete cytokines and this may offer a model for cloning and study of IEL T cells in vitro.


Subject(s)
Bacterial Proteins/pharmacology , Cytokines/biosynthesis , Heat-Shock Proteins/pharmacology , Intestines/immunology , Lymphocyte Activation/drug effects , Mycobacterium tuberculosis/immunology , Animals , Cells, Cultured , Interleukin-2/pharmacology , Intestines/cytology , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL
14.
Lymphokine Cytokine Res ; 12(2): 59-67, 1993 Apr.
Article in English | MEDLINE | ID: mdl-8324078

ABSTRACT

A simple, rapid, reproducible, and nonradioisotopic method for semiquantitative analysis of cytokine mRNAs based on polymerase chain reaction (PCR) is described. RNA isolated from 2.5 million cells has proven sufficient to perform semiquantitative analysis of mRNA for 10 different cytokines. By this approach accurate assessment of mRNA levels for multiple cytokines can be made from as little as 2 ml of blood or about 3 mg of biopsy material. Total cellular RNA is quantitatively recovered by guanidinium isothiocyanate-acid-phenol extraction of a constant number of cells. Further quantitation of RNA is unnecessary. Highly reproducible PCR product formation occurs after specific amplification of aliquots of reverse transcribed test RNA. The photographic image of the ethidium bromide-stained gel accurately reflects the amount of PCR product loaded, both densitometrically and visually. PCR product generation is not affected by the presence of carrier RNA. Thus quantitative recovery of total RNA is possible even from a very small number of cells. Similarly, presence of a large excess of nonspecific RNA from nonexpressing cells does not affect amplification of the specific mRNA under study. A linear relationship between mRNA frequency and PCR product formation is observed over a 256- to 512-fold range. The actual mRNA concentration for each cytokine varies depending on the relative abundance of mRNA for that cytokine relative to total RNA. By performing two amplification cycles (28 and 35) on undiluted and 10-fold diluted RNA samples, the range of detection linearity is extended over a 5000-fold difference in input RNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Cytokines/analysis , Polymerase Chain Reaction/methods , RNA, Messenger/analysis , Base Sequence , Cell Count , Ethidium , Gels , Humans , Molecular Sequence Data , Sepharose , Transcription, Genetic
15.
Cell Immunol ; 147(1): 110-28, 1993 Mar.
Article in English | MEDLINE | ID: mdl-8096435

ABSTRACT

A novel cell aggregation-inducing characteristic of the leukocyte common antigen, CD45, is described and its underlying molecular mechanisms investigated. Formation of strong cell clusters was consistently observed in human PBMCs after crosslinking CD45 molecules with antibodies, directed to epitopes common for all CD45 isoforms (e.g., mAb ROS220 or NIH45-2) or the CD45RA (e.g., mAb Alb11) or the CD45RO isoform (e.g., mAb UCHL1). This phenomenon was not seen after PBMC treatment with CD45RA mAb HB11 or CD2 mAb 39C1.5. Identical to phorbol 12-myristate 13-acetate (PMA)-induced clustering, CD45-mediated aggregation was also suppressed by EDTA, by cytochalasin B, and by incubation at 4 degrees C, all characteristics of adhesion mediated by integrins. The involvement of LFA-1 and ICAM-1 in CD45-mediated adhesion was supported by the observation that CD11a (LFA-1 alpha) mAb R7.1, CD18 (LFA-1 beta) mAb R3.3, and CD54 (ICAM-1) mAb R6.1 or RR/l all strongly inhibited CD45- and PMA-induced aggregation. Interestingly, highly pure T lymphocytes did not aggregate in response to CD45 mAb, but did after PMA treatment. These results indicate that triggering human PBMCs via CD45 can cause strong cell aggregation, largely through LFA-1/ICAM-1 interactions. Our findings support an important role of the CD45 antigen in signal transduction and intercellular interaction in human PBMCs.


Subject(s)
Cell Adhesion Molecules/immunology , Cell Adhesion/immunology , Leukocyte Common Antigens/immunology , Lymphocyte Function-Associated Antigen-1/immunology , Monocytes/immunology , Signal Transduction/immunology , Antibodies, Monoclonal/pharmacology , Cell Adhesion/drug effects , Cell Adhesion Molecules/biosynthesis , Cold Temperature , Cytochalasin B/pharmacology , Edetic Acid/pharmacology , Humans , Immunologic Capping , Integrins/immunology , Intercellular Adhesion Molecule-1 , Lymphocyte Function-Associated Antigen-1/biosynthesis , Monocytes/drug effects
16.
Lymphokine Cytokine Res ; 11(4): 207-13, 1992 Aug.
Article in English | MEDLINE | ID: mdl-1420599

ABSTRACT

Fc receptor-positive lymphocytes (FcR+) contain lymphokine-activated killer cell (LAK) precursors that in response to IL-2 develop potent antitumor cytotoxicity. These FcR+ cells are also capable of antibody-dependent cytotoxicity (ADCC), which can be detected using fresh human peripheral blood lymphocytes (PBL) directed to murine targets, however, PBL-mediated ADCC to human tumors usually is very low, requiring a stimulation of the PBL, which also can be accomplished with IL-2. Using human melanoma tumor target cells, with and without the 14G2a monoclonal antibody, we examined in parallel the role of p75 IL-2 receptor for regulation of the induction of both LAK and ADCC forms of antitumor cytotoxicity. Enrichment of FcR+ cells from fresh peripheral blood by elutriation and flow cytometry, followed by varying periods of IL-2 culture, revealed a differential kinetics of activation. ADCC was detectable after PBL exposure to IL-2 for as short as the 4 h cytotoxicity assay, while LAK activation required more than 24 h of exposure. Elimination of the FcR+ cells by magnetic bead depletion from large granular lymphocyte populations (LGL) resulted in a loss of both LAK and ADCC. Addition of antibody known to block the binding of IL-2 to the p75 molecule of the IL-2 receptor complex (Mik-beta 1) to activation cultures at zero time resulted in abrogation of both cytotoxicities. These results suggest that differentiation and maturation of the ADCC effectors occurs in response to IL-2 via the p75 molecule, as also does LAK activation.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Cytotoxicity, Immunologic/physiology , Killer Cells, Lymphokine-Activated/immunology , Receptors, Interleukin-2/physiology , Antibodies, Monoclonal , Antibody-Dependent Cell Cytotoxicity , Humans , Interleukin-2/physiology , Kinetics , Lymphocyte Depletion , Melanoma/immunology , Receptors, Fc/physiology , Receptors, Interleukin-2/antagonists & inhibitors , Tumor Cells, Cultured
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