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F I Preffer

Publications and source records attributed to F I Preffer.

At least 19 recordsLinked to original sources
PubMed2001

Fine-needle aspiration biopsy in the diagnosis and classification of primary and recurrent lymphoma: a retrospective analysis of the utility of cytomorphology and flow cytometry.

We retrospectively reviewed our experience with the fine-needle aspiration biopsy (FNAB) diagnosis of primary and recurrent lymphoma to assess the ability of cytomorphology with and without ancillary flow cytometry (FCM) analysis to diagnose and subclassify these tumors according to the Revised European-American Lymphoma/World Health Organization classifications. We reviewed 139 consecutive FNABS of 84 primary and 55 recurrent lymphomas. FCM was successful in 105 (75%) cases. The overall results, including cases without FCM, included 93/139 (67%) true positive, 7 (5%) false negative, and 39 indeterminate (27 [19%] suspicious and 12 [9%] atypical) diagnoses of lymphoma. In cases with FCM, there were 80/105 (77%) true positive, no false negative, and 25 indeterminate diagnoses (15 [14%] suspicious and 10 [9%] atypical). The overall results of the 84 primary lymphomas were 55 (67%) true positive, 5 (5%) false negative, and 24 indeterminate (14[16%] suspicious and 10 [12%] atypical) diagnoses for lymphoma. Of the 68 primary lymphomas analyzed with FCM, 50 [74%] were true positives, and 28 were indeterminate (11 [16%] suspicious and 7 [10%] atypical). There were no false negatives. Diagnostic accuracy varied among lymphoma subtypes. Subclassification of the positive cases were initially conclusive in only 55/93 cases (59%). However, a retrospective review of the morphologic together with FCM data in 15 of the 23 unclassified cases improved the overall subclassification of positive cases to 77%. Subclassification was best in small lymphocytic lymphoma/chronic lymphocytic leukemia, lymphoplasmacytic lymphoma, Burkitt's lymphoma, mantle cell lymphoma, and plasmacytoma (all 100%). Subclassification was poor in marginal-zone lymphoma (33%), and initially as well in diffuse large B-cell lymphoma (62%), but it improved on review (95%), as did subclassification of follicular lymphoma (77 to 100% on review). Hodgkin's disease was recognized as malignant in only 44% of the cases (7/16) and was classified as such based on morphology alone. This review of our early efforts to diagnose and subclassify lymphoma with FNAB and FCM indicates that although a diagnosis and proper subclassification of lymphoma can be made with certainty in the majority of cases, recurrent or primary, it requires close coordination of cytomorphology and immunophenotyping data, which often comes with close cooperation of cytopathologists and hematopathologists. A mere cytological diagnosis of positive for lymphoma is no longer acceptable if FNAB is to become an independent diagnostic tool for lymphoma.

Biopsy, Needle↗
PubMed2000

In vitro priming with adenovirus/gp100 antigen-transduced dendritic cells reveals the epitope specificity of HLA-A*0201-restricted CD8+ T cells in patients with melanoma.

Replication-deficient recombinant adenovirus (Ad) encoding human gp100 or MART-1 melanoma Ag was used to transduce human dendritic cells (DC) ex vivo as a model system for cancer vaccine therapy. A second generation E1/E4 region deleted Ad which harbors the CMV immediate-early promoter/enhancer and a unique E4-ORF6/pIX chimeric gene was employed as the backbone vector. We demonstrate that human monocyte-derived DC are permissive to Ad infection at multiplicity of infection between 100 and 500 and occurs independent of the coxsackie Ad receptor. Fluorescent-labeled Ad was used to assess the kinetics and distribution of viral vector within DC. Ad-transduced DC show peak transgene expression at 24-48 h and expression remains detectable for at least 7 days. DC transduced with replication-deficient Ad do not exhibit any unusual phenotypic characteristics or cytopathic effects. DC transduced with Ad2/gp100v2 can elicit tumor-specific CTL in vitro from patients bearing gp100+ metastatic melanoma. Using a panel of gp100-derived synthetic peptides, we show that Ad2/gp100v2-transduced DC elicit Ag-specific CTL that recognize only the G209 and G280 epitopes, both of which display relatively short half-lives ( approximately 7-8 h) on the surface of HLA-A*0201+ cells. Thus, patients with metastatic melanoma are not tolerant to gp100 Ag based on the detection of CD8+ T cells specific for multiple HLA-A*0201-restricted, gp100-derived epitopes.

Adenoviruses, Human↗
PubMed2000

Pulsed electric fields for selection of hematopoietic cells and depletion of tumor cell contaminants.

Purging of tumor cells and selection of stem cells are key technologies for enabling stem cell transplantation and stem cell gene therapy. Here we report a strategy for cell selection based on physical properties of the cells. Exposing cells to an external pulsed electric field (PEF) increases the natural potential difference across the cell membrane until a critical threshold is reached and pore formation occurs, resulting in fatal perturbation of cell physiology. Attaining this threshold is a function of the applied field intensity and cell size, with larger cells porated at lower field intensities than smaller cells. Since hematopoietic stem cells are smaller than other hematopoietic cells and tumor cells, we found that exposure of peripheral blood mononuclear cells (PBMCs) to PEFs caused stepwise elimination of monocytes without affecting the function of smaller lymphocyte populations. Mobilized peripheral blood exposed to PEFs was enriched for CD34+/CD38- cells and stem cell function was preserved. Furthermore, PEF treatment was able to selectively purge blood preparations of tumor cells and eradicate transplantable tumor.

ADP-ribosyl Cyclase↗
PubMed2000

Regulatory role of mature B cells in a murine model of inflammatory bowel disease.

The spontaneous chronic colitis in TCR alpha mutant (TCRalpha(-/-)) mice mediated by CD4(+) TCRalpha(-)beta(+) T cells is more severe in the absence of mature B cells, suggesting a suppressive role of B cells and Ig in the development of chronic colitis. To investigate the direct role of B cells in the suppression of this colitis, cell transfer studies were performed in TCRalpha(-/-) x Igmu(-/-) (alphamu(-/-)) double-knockout mice. The chronic colitis was markedly attenuated in alphamu(-/-) mice after the adoptive transfer of peripheral B cells from TCRalpha(-/-) mice into 3- to 4-week-old alphamu(-/-) mice prior to the development of colitis. Furthermore, transfer of mature B cells from TCRalpha(-/-) mice markedly decreased the number of pathogenic colonic CD4(+) TCRalpha(-)beta(+) T cells in alphamu(-/-) mice with established colitis. This B cell effect required the presence of functional co-stimulatory molecules CD40 and B7-2 (CD86) but not B7-1 (CD80). These results indicate that mature B cells play an important role in the development of chronic colitis in TCRalpha(-/-) mice by directly regulating the pathogenic T cells (CD4(+) TCRalpha(-)beta(+) T cells).

Adoptive Transfer↗
PubMed2000

Accumulation of macrophages with dendritic cell characteristics in the pulmonary response to Listeria.

Pulmonary immunity reflects a balance between proinflammatory and immunosuppressive factors in the lung. To determine the immune activities of exudate macrophages in the pulmonary immune response, Lewis rats were injected intratracheally with heat-killed Listeria (HKL), labeled ex vivo with the lipophilic dye PKH-26. At 24 h, macrophages from bronchoalveolar lavage fluid were purified on the basis of their surface membrane expression of RMA, a macrophage-specific antigen, which is brightly expressed by resident alveolar macrophages but dimly expressed by monocytes. Pulmonary macrophages were analyzed for uptake of PKH-26-HKL, and RMA(bright/dim) macrophages sorted by FACS were compared for cytokine expression, nitric oxide (NO) release, and APC activities. RMA(bright) macrophages were OX-62(-), B7(-), and factor XIIIa(-); they were the dominant mediators of phagocytosis when low doses of HKL were administered intratracheally but did not support the proliferation of T lymphocytes. RMA(dim) exudate macrophages were OX-62(+), B7(+), and factor XIIIa(+). They expressed more IL-1 and TNF, but less nitric oxide, than did RMA(bright) macrophages; they were excellent APCs for T cell responses. We conclude that a subset of RMA(dim) exudate macrophages shows phenotypic and functional evidence of dendritic cell differentiation.

Animals↗
PubMed2000

Treatment of multiple myeloma by antibody mediated immunotherapy and induction of myeloma selective antigens.

BACKGROUND: In view of the successful use of serotherapy in many B-cell malignancies, we and others have sought to identify tumor selective antigens for the serotherapy of plasma cell dyscrasias (PCD) including multiple myeloma (MM), and Waldenstrom's macroglobulinemia (WM). We recently identified Muc-1 core protein as a MM selective antigen. Though Muc-1 core protein is abundantly expressed on most MM plasma cells, expression of this antigen can be absent, or weak on some plasma cells which could potentially result in the selection of Muc-1 core protein negative clones following serotherapy of PCD. In addition to Muc-1 core protein, we have also been examining the use of CD20 directed serotherapy for PCD. DESIGN: As part of these efforts, we recently initiated a phase II clinical trial examining the use of Rituximab (Rituxan, MabThera) as a single agent in MM patients; as well several WM patients have been treated with Rituximab at our Institutions. RESULTS: In previous studies, we have shown that CD20 is abundantly expressed on the plasma cells of most WM patients; in contrast, CD20 is expressed on plasma cells from a minority of MM patients, and in these patients expression of CD20 can be weak or heterogeneous with both CD20+ and CD20- plasma cells present. As such, we have sought out clinically useful inducers of Muc-1 core protein, and of CD20 on malignant plasma cells. CONCLUSIONS: These efforts resulted in the identification of dexamethasone (Dex) as a potent inducer of Muc-1 core protein on MM plasma cells, and interferon-gamma (IFN-gamma) as a potent inducer of CD20 on MM plasma cells and B-cells. Importantly, these agents induced their respective antigens at pharmacologically achievable doses.

Animals↗
PubMed1999

Intrinsic human immunodeficiency virus type 1 resistance of hematopoietic stem cells despite coreceptor expression.

Interactions of human immunodeficiency virus type 1 (HIV-1) with hematopoietic stem cells may define restrictions on immune reconstitution following effective antiretroviral therapy and affect stem cell gene therapy strategies for AIDS. In the present study, we demonstrated mRNA and cell surface expression of HIV-1 receptors CD4 and the chemokine receptors CCR-5 and CXCR-4 in fractionated cells representing multiple stages of hematopoietic development. Chemokine receptor function was documented in subsets of cells by calcium flux in response to a cognate ligand. Productive infection by HIV-1 via these receptors was observed with the notable exception of stem cells, in which case the presence of CD4, CXCR-4, and CCR-5, as documented by single-cell analysis for expression and function, was insufficient for infection. Neither productive infection, transgene expression, nor virus entry was detectable following exposure of stem cells to either wild-type HIV-1 or lentivirus constructs pseudotyped in HIV-1 envelopes of macrophage-tropic, T-cell-tropic, or dualtropic specificity. Successful entry into stem cells of a vesicular stomatitis virus G protein-pseudotyped HIV-1 construct demonstrated that the resistance to HIV-1 was mediated at the level of virus-cell membrane fusion and entry. These data define the hematopoietic stem cell as a sanctuary cell which is resistant to HIV-1 infection by a mechanism independent of receptor and coreceptor expression that suggests a novel means of cellular protection from HIV-1.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Müllerian-inhibiting substance type II receptor expression and function in purified rat Leydig cells.

Müllerian-inhibiting substance (MIS), a gonadal hormone in the transforming growth factor-beta superfamily, induces Müllerian duct involution during male sexual differentiation. Mice with null mutations of the MIS ligand or receptor develop Leydig cell hyperplasia and neoplasia in addition to retained Müllerian ducts, whereas MIS-overexpressing transgenic mice have decreased testosterone concentrations and Leydig cell numbers. We hypothesized that MIS directly modulates Leydig cell proliferation and differentiated function in the maturing testis. Therefore, highly purified rat Leydig and Sertoli cells were isolated to examine cell-specific expression, binding, and function of the MIS type II receptor. These studies revealed that this receptor is expressed abundantly in progenitor (21-day) and immature (35-day) Leydig cells as well as in Sertoli cells. Prepubertal progenitor Leydig cells exhibit high affinity (Kd = 15 nM), saturable binding of MIS. No binding, however, is detected with either peripubertal immature Leydig cells or Sertoli cells at either age. Moreover, progenitor, but not immature Leydig cells, respond to MIS by decreasing DNA synthesis. These data demonstrate that functional MIS type II receptors are expressed in progenitor Leydig cells and support the hypothesis that MIS has a direct role in the regulation of postnatal testicular development.

Animals↗
PubMed1999

Treatment of plasma cell dyscrasias by antibody-mediated immunotherapy.

The use of serotherapy to treat patients with plasma cell dyscrasias (PCDs) has been sought by us and others. Candidate antigens that have been targeted or proposed for targeting in PCDs include the immunoglobulin idiotype, CD19, CD38, CD54, CD126, HM1.24, and Muc-1 core protein. Unfortunately, many of these antigens are not ideal for use in serotherapy since they are not selectively expressed, are either shed or secreted, or have not been fully characterized. Serotherapy with an anti-CD19 monoclonal antibody (B4) conjugated to a blocked ricin toxin had no significant activity in patients with multiple myeloma (MM). Circulating CD20+ clonotypic B cells have been detected in the circulation of most MM and Waldenstrom's macroglobulinemia (WM) patients. Plasma cells from most WM patients express CD20, but most MM patient plasma cells either lack CD20 or express it weakly. In view of recent successes with anti-CD20-directed serotherapy in other B-cell malignancies, we initiated a phase II trial to study the anti-CD20 monoclonal antibody rituximab (Rituxan; IDEC Pharmaceuticals, San Diego, CA, and Genentech, Inc, San Francisco, CA) in patients with MM. We describe two PCD patients (one with WM and one with MM) who responded to therapy. By flow cytometric analysis, CD20+ plasma cells and B cells present in the bone marrow and peripheral blood of a patient with MM disappeared with response to rituximab therapy. However, residual CD20- tumor cells remained in the bone marrow following rituximab therapy, and after 6 months this patient progressed with CD20- myeloma cells. As a potential strategy to overcome this limitation, we demonstrated that interferon-gamma at pharmacologically achievable levels induced CD20 expression on these CD20- plasma cells, consistent with our recent findings that interferon-gamma is a potent inducer of CD20 expression on MM patient plasma cells and B cells. We also characterize a response to rituximab with a decrease in paraprotein and resolution of anemia in a patient with WM whose response to rituximab is ongoing after 19+ months. This preliminary experience supports the potential use of serotherapy targeting CD20 in PCDs. Our studies further suggest that interferon-gamma may enhance CD20 expression on MM plasma cells, thereby increasing their susceptibility to anti-CD20 monoclonal antibody therapies.

Aged↗
PubMed1999

Human ovarian cancer, cell lines, and primary ascites cells express the human Mullerian inhibiting substance (MIS) type II receptor, bind, and are responsive to MIS.

Six human ovarian cancer cell lines and samples of ascites cells isolated from 27 patients with stage III or IV ovarian papillary serous cystadenocarcinoma were studied individually to test whether recombinant human Mullerian inhibiting substance (rhMIS) acts via its receptor. To do these experiments, we scaled up production of rhMIS and labeled it successfully with biotin for binding studies, cloned the human MIS type II receptor for mRNA detection, and raised antibodies to an extracellular domain peptide for protein detection. These probes were first tested on the human ovarian cancer cell lines and then applied to primary ovarian ascites cells. rhMIS inhibited colony growth of five of six cell lines that expressed the human MIS type II receptor mRNA by Northern analysis while not inhibiting receptor-negative COS cells. Flow cytometry performed on MIS-sensitive ovarian cancer cell lines demonstrated specific and saturable binding of rhMIS (Kd = 10.2 nM). Ascites cells from 15 of 27 or 56% of patients tested bound biotinylated MIS (MIS-biotin) and, of the 11 that grew in soft agarose, 9 of 11 or 82% showed statistically significant inhibition of colony formation. Of the 15 patients who bound biotinylated MIS, mRNA was available for analysis from 9, and 8 of 9 expressed MIS type II receptor mRNA by reverse transcription-PCR, showing a statistically significant correlation, compared with binding, by chi2 analysis (P = 0.025). Solid ovarian cancers were positive for the MIS type II receptor protein by immunohistochemical staining, which colocalized with staining for antibody to CA-125 (OC-125). Thus, the detection of the MIS type I receptor by flow cytometry may be a useful predictor of therapeutic response to MIS and may be a modality to rapidly choose patients with late-stage ovarian cancer for treatment with MIS.

Adult↗
PubMed1998

Humanized IgG1 and IgG4 anti-CD4 monoclonal antibodies: effects on lymphocytes in the blood, lymph nodes, and renal allografts in cynomolgus monkeys.

BACKGROUND: Optimizing therapeutic monoclonal antibody (mAb) depends on the incorporation of the necessary effector functions and the development of hypoantigenic "humanized" antibodies by genetic engineering, which then need to be tested in appropriate preclinical trials. METHODS: Constructs of humanized OKT4A containing the complementarity-determining region (CDR) of murine OKT4A and the framework and constant regions of human light (kappa) and heavy chains (IgG1 and IgG4) were prepared and tested in cynomolgus monkeys who received a renal allograft. A prophylactic course of CDR-OKT4A/human (h) IgG1 or CDR-OKT4A/ hIgG4, either as high-dose single bolus (10 mg/kg) or as low-dose multiple infusion (1 mg/kg for 12 days) was given, and the effects on graft survival, immunohistology, circulating cells, and lymph node cells were assessed. RESULTS: The IgG1 isotype induced coating of T cells, modulation of surface CD4 molecules, and profound depletion of CD4+ lymphocytes in peripheral blood, which persisted as long as the animals were followed (up to 7 weeks). The IgG4 isotype induced only cell coating without cell clearance or modulation. In lymph nodes, coating of lymphocytes (approximately 60%) was seen with both isotypes in the earliest sample (6 hr). After 2 days, significant depletion of lymph node CD4 cells was evident, with a decrease in the CD4 to CD8 ratio in the IgG1-treated group; no depletion occurred in the IgG4 group. The emigration of CD4+ cells into the allograft was significantly delayed in the CDR-OKT4A/hIgG1-treated animals when compared with the CDR-OKT4A/hIgG4 group as judged by immunocytochemistry (23.8+/-13.2 days vs. 7.4+/-1.5 days, P<0.001) or interleukin-2-promoted T-cell outgrowth from allograft biopsies (22.2+/-11.0 days vs. 6.3+/-0.5 days, P<0.01). CONCLUSIONS: This study demonstrates that the in vivo effects of CDR-grafted OKT4A are dependent on its isotype. The depleting mAb CDR-OKT4A/hIgG1 significantly delays the entry of CD4+ cells into the graft, inhibiting the early phase of rejection. However, graft rejection occurs when CD4+ cells eventually infiltrate the graft, even in the presence of depressed levels of circulating CD4+ cells. Both isotypes demonstrated therapeutic efficacy: graft survival was prolonged over controls. In the case of CDR-OKT4A/hIgG4, neither lymphocyte depletion, antigenic modulation, nor prevention of infiltration is necessary for a beneficial effect, which indicates that this mAb blocks CD4 function or renders the CD4+ cell less responsive. The lack of depletion is a feature of potential clinical advantage in minimizing the risk of excessive immunosuppression.

Animals↗
PubMed1998

Myeloperoxidase detection by three-color flow cytometry and by enzyme cytochemistry in the classification of acute leukemia.

In the classification of acute leukemia, the presence of myeloperoxidase (MPO) within the leukemic blasts indicates myeloid leukemia. Previous studies compared enzyme cytochemistry (EC) or flow cytometry (FC) with immunocytochemistry, in detecting MPO. Our study is the first direct comparison of EC with 3-color FC in a large group of acute leukemias. We studied 26 cases of acute myeloid leukemia (AML) and 4 cases of B-precursor acute lymphoblastic leukemia (B-ALL). Classification was according to the French-American-British criteria. The cells were analyzed for MPO expression by 3-color FC after cell permeabilization followed by staining with anti-MPO antibody. For FC, the blasts were defined by a combination of light scatter characteristics and dim CD45 expression. Concordance between EC and FC was seen in 27 of 30 cases (23/26 AML and all B-ALL), including all AML cases of M1, M2, M3, and M4 subtypes. In 1 of 4 AML-MO and 2 of 5 AML-M5a cases, FC demonstrated the presence of MPO in 8%, 86%, and 94% blasts; EC detected none. Three-color FC may be more sensitive than routine EC in demonstrating the presence of MPO in AML cases and may offer the advantage of multiparametric analysis.

Acute Disease↗
PubMed1997

Suppressive role of B cells in chronic colitis of T cell receptor alpha mutant mice.

The role of antibodies (Abs) in the development of chronic colitis in T cell receptor (TCR)-alpha-/- mice was explored by creating double mutant mice (TCR-alpha-/- x immunoglobulin (Ig)mu-/-), which lack B cells. TCR-alpha-/- x Ig mu-/- mice spontaneously developed colitis at an earlier age, and the colitis was more severe than in TCR-alpha-/- mice. Colitis was induced in recombination-activating gene-1 (RAG-1-/-) mice by the transfer of mesenteric lymph node (MLN) cells from TCR-alpha-/- x Ig mu-/- mice. When purified B cells from TCR-alpha-/- mice were mixed with MLN cells before cell transfer, colitis did not develop in RAG-1-/- mice. Administration of the purified Ig from TCR-alpha-/- mice and a mixture of monoclonal autoAbs reactive with colonic epithelial cells led to attenuation of colitis in TCR-alpha-/- x Ig mu-/- mice. Apoptotic cells were increased in the colon, MLN, and spleen of TCR-alpha-/- x Ig mu-/- mice as compared to Ig mu-/- mice and TCR-alpha-/- mice. Administration of the purified Ig from TCR-alpha-/- mice into TCR-alpha-/- x Ig mu-/- mice led to decrease in the number of apoptotic cells. These findings suggest that although B cells are not required for the initiation of colitis, B cells and Igs (autoAbs) can suppress colitis, presumably by affecting the clearance of apoptotic cells.

Adoptive Transfer↗
PubMed1997

In vitro detection of Mycoplasma fermentans binding to B-lymphocytes in fresh peripheral blood using flow cytometry.

Previous reports have shown, using fluorescent probes conjugated to the organism, that Mycoplasma fermentans fuses with about 12% of peripheral blood lymphocytes. However, no lymphocyte subset was specified. To elucidate the specific subset of lymphocytes involved, we developed a three-color flow cytometric assay to detect M. fermentans binding to fresh peripheral blood cells. In our assay, two strains of M. fermentans were grown in SP4 glucose broth, mixed with fresh whole blood samples (n > 20), and incubated at 37 degrees C. The blood samples were then stained with a polyclonal antibody to M. fermentans, a monoclonal antibody to B-lymphocytes (CD19), and a monoclonal antibody to T-lymphocytes (CD3). Using three-color flow cytometry, we obtained data confirming binding of M. fermentans to 10%-15% of peripheral blood lymphocytes with minimal granulocyte or monocyte staining detected. Flow cytometric analysis showed that early binding appears predominantly directed towards B-lymphocytes (86.7 +/- 9.0%), and that this binding could not be blocked by antibodies directed towards common B lymphocyte cell surface antigens. M. fermentans binding to B-lymphocytes occurred within 5 min of in vitro inoculation, reached a maximum within 30-60 min (94-97%), and thereafter plateaued. The binding was concentration dependent over a three log dilution using 10(3) color changing units as standard. Binding to T-lymphocytes was minimal (<5% positive). B lineage tumor cells or peripheral blood B cells obtained from HIV infected individuals demonstrated reduced binding of M. fermentans. This assay provides a good method to study the cellular interactions of mycoplasma and may help to elucidate pathogenic mechanisms of mycoplasma infections.

Antibodies, Monoclonal↗
PubMed1997

Cytotoxicity and apoptosis in human renal allografts: identification, distribution, and quantitation of cells with a cytotoxic granule protein GMP-17 (TIA-1) and cells with fragmented nuclear DNA.

In the present study, we analyzed human renal allografts using immunohistochemical techniques to determine the site, identity, and frequency of (a) cytotoxic and apoptotic cells, as identified by staining for GMP-17 (TIA-1), a component of cytotoxic granules; and (b) DNA fragmentation in situ, as detected by the TUNEL method. In acute cellular rejection (n = 15), GMP-17+ mononuclear cells accounted for 29% +/- 12% of the infiltrating cells in the interstitium (341 +/- 164/mm2) and were significantly more concentrated in tubulitis lesions, where they amounted to 65% +/- 14% of the mononuclear cells (96 +/- 61/mm2) (p < 0.01 versus interstitium). GMP-17+ mononuclear cells were also found in sites of endothelialitis. An estimated 80% of the GMP-17+ lymphocytes expressed CD8, and 10% to 20% expressed either CD4 or the macrophage marker CD14. The latter finding led us to analyze normal peripheral blood monocytes by flow cytometry, all of which were found to contain GMP-17. NK cells and neutrophils, which are known to express GMP-17, were detected only rarely in allografts. Specimens with cyclosporine A toxicity (n = 7) or acute tubular necrosis (n = 13) showed fewer GMP-17+ cells in the interstitium (22 +/- 46/mm2 and 62 +/- 50/mm2, respectively) and tubules (2 +/- 6/mm2 and 10 +/- 10/mm2, respectively) (all p < 0.01 versus rejection). These differences were due largely to less intense mononuclear cell infiltration. In cyclosporine A toxicity, however, the percentages of GMP-17+ mononuclear cells within tubules and the interstitium were significantly lower than in rejection (p = 0.02), whereas in acute tubular necrosis significantly lower percentages were found in the tubules (p = 0.04) but not in the interstitium. Native kidneys with end-stage diabetic nephropathy (n = 5) had very low proportions of GMP-17+ cells in interstitial infiltrates (7% +/- 6%) and in tubules (11% +/- 15%), although the infiltrates were focally intense (517 +/- 355/mm2). TUNEL+ cells were found in acute cellular rejection, predominantly in areas with intense mononuclear infiltrates and also within lesions of tubulitis and endothelialitis. Although some TUNEL+ cells were intrinsic renal cells, most appeared to be infiltrating mononuclear cells, and we were able to detect CD3 in some. In areas of intense cellular infiltration, the percentages of TUNEL+ cells (range, 0.5% to 4.2%) were comparable to those seen in the rat thymus, indicating a high level of apoptosis. Overall, in the allograft samples, the numbers of GMP-17+ cells and TUNEL+ cells were significantly correlated (r = 0.79; p < 0.01). These data provide new evidence that T cell (and possibly macrophage)-mediated cytotoxicity plays an important role in acute renal allograft rejection, particularly in the case of tubular injury, and furthermore suggest that apoptosis may be a mechanism not only for graft cell destruction, but also for elimination of activated T cells in the infiltrate.

Apoptosis↗
PubMed1996

Reduced fatty acid ethyl ester synthase activity in the white blood cells of alcoholics.

PURPOSE: Fatty acid ethyl esters (FAEEs), esterification products of ethanol and fatty acids, have been implicated as mediators of ethanol-induced organ damage. It has been shown that FAEE synthase, the enzyme responsible for the formation of FAEE, is present selectively in the organs commonly damaged by ethanol abuse. Recently, we have made the observation that FAEEs are also present in the serum after ethanol ingestion. The current study was performed to determine whether cellular elements of the blood and/or plasma are capable of synthesizing FAEEs from fatty acids and ethanol. MATERIALS AND METHODS: Heparinized blood samples were collected from 10 healthy volunteers, and the red blood cells, platelets, plasma, and several white blood cell populations were assayed for FAEE synthase activity. Blood samples from control subjects and individuals admitted to an alcoholic detoxification unit at a local hospital were also assayed for FAEE synthase activity. RESULTS: We observed that the FAEE synthase activity is present in whole blood, primarily within white blood cells. Fractionation of the white blood cells revealed that the lymphocyte-monocyte fraction isolated using Ficoll-hypaque contained approximately 3.5-fold higher activity than the granulocyte fraction. The cell type that contained the highest FAEE synthase activity (1220 pmol/hr/10(6) cells) was the natural killer (NK) cell population. B cells contained approximately 40% of the enzyme activity found in NK cells, and the B-cell activity was slightly greater than that found in CD4+ and CD8+ T cells. Having shown that FAEE synthase exists in a blood cell, we subsequently demonstrated that alcoholic individuals have approximately half the white blood cell FAEE synthase activity of that found in normal controls. We also demonstrated that white blood cell FAEE synthase could be induced nearly 2-fold upon ingestion of 2 oz of scotch whiskey for 6 days. The enzyme activity returned to baseline levels despite ingestion of 2 oz of scotch whiskey/day for 3 additional days. CONCLUSIONS: These data indicate that ethanol ingestion results in increased FAEE production, particularly by NK cells. FAEE synthesis after ethanol ingestion may explain the presence of FAEE in the serum. The lower enzyme activity observed in white blood cells of alcoholics from a detoxification center may be the result of years of ethanol abuse or it may be that alcoholics congenitally have low levels of FAEE synthase. If the latter is true, this finding may explain in part the genetic predisposition of many alcoholic individuals to ethanol abuse.

Acyltransferases↗