A CCL24-dependent pathway augments eosinophilic airway inflammation in house dust mite-challenged Cd163(-/-) mice.

CD163 is a macrophage scavenger receptor with anti-inflammatory and pro-inflammatory functions. Here, we report that alveolar macrophages (AMΦs) from asthmatic subjects had reduced cell-surface expression of CD163, which suggested that CD163 might modulate the pathogenesis of asthma. Consistent with this, house dust mite (HDM)-challenged Cd163(-/-) mice displayed increases in airway eosinophils and mucous cell metaplasia (MCM). The increased airway eosinophils and MCM in HDM-challenged Cd163(-/-) mice were mediated by augmented CCL24 production and could be reversed by administration of a neutralizing anti-CCL24 antibody. A proteomic analysis identified the calcium-dependent binding of CD163 to Dermatophagoides pteronyssinus peptidase 1 (Der p1). Der p1-challenged Cd163(-/-) mice had the same phenotype as HDM-challenged Cd163(-/-) mice with increases in airway eosinophils, MCM and CCL24 production, while Der p1 induced CCL24 secretion by bone marrow-derived macrophages (BMMΦs) from Cd163(-/-) mice, but not BMMΦs from wild-type (WT) mice. Finally, airway eosinophils and bronchoalveolar lavage fluid CCL24 levels were increased in Der p1-challenged WT mice that received adoptively transferred AMΦ's from Cd163(-/-) mice. Thus, we have identified CD163 as a macrophage receptor that binds Der p1. Furthermore, we have shown that HDM-challenged Cd163(-/-) mice have increased eosinophilic airway inflammation and MCM that are mediated by a CCL24-dependent mechanism.

Decreased plasma cytokines are associated with low platelet counts in aplastic anemia and immune thrombocytopenic purpura.

BACKGROUND: We previously found plasma levels of CD40 ligand (CD40L), chemokine (C-X-C motif) ligand 5 (CXCL5), chemokine (C-C motif) ligand 5 (CCL5) and epidermal growth factor (EGF) to be low in aplastic anemia (AA) patients and to be correlated with platelet count. OBJECTIVES: To study the association of CD40L, CXCL5, CCL5 and EGF with platelets. METHODS: We measured cytokines in the plasma of immune thrombocytopenic purpura (ITP) and AA patients using the Luminex assay and confirmed the results in a mouse model and in vitro experiments. RESULTS: Both ITP and AA showed similarly low levels of CD40L, CXCL5, CCL5 and EGF, compared with healthy controls. In ITP, levels of these proteins were significantly greater in patients with higher platelet counts than in those with lower platelet counts. In a murine thrombocytopenia model, levels of CD40L, CXCL5, CCL5 and EGF decreased with platelet count after immune-mediated destruction, while the cytokine levels increased when the platelet count recovered. In vitro, concentrations of these cytokines in the supernatants of platelet suspensions were proportional to platelet numbers, and levels in sera prepared by simple blood coagulation were equivalent to those in platelet-rich plasma-converted sera. mRNA expression for CXCL5, CCL5 and EGF was higher in platelets than in megakaryocytes, peripheral blood mononuclear cells, granulocytes and non-megakaryocytic bone marrow cells. CONCLUSIONS: Plasma CD40L, CXCL5, CCL5 and EGF are mainly platelet-derived, suggesting a role of platelets in immune responses and inflammation. Measurement of CD40L, CXCL5, CCL5 and EGF in human blood allowed testable inferences concerning physiology and pathophysiology in quantitative platelet disorders.

Short-term induction and long-term suppression of HPV16 oncogene silencing by RNA interference in cervical cancer cells.

RNA interference-mediated gene silencing has the potential to block gene expression. A synthetic double-stranded small interfering RNA (siRNA) based on a sequence motif of 21 nucleotides from human papillomavirus 16 (HPV16) E6E7 bicistronic RNA was found to be a potent siRNA that suppresses expression of both the E6 and E7 oncogenes in HPV16+ CaSki and SiHa cells. When stably expressed as a short hairpin RNA in these cells, however, siRNA silencing of E6 and E7 expression was efficient only at early cell passages, but became inefficient with increased cell passages despite the continued expression of the siRNA at the same level. The loss of the siRNA function was duplicable in stable p53 siRNA cells, but not in stable lamin A/C siRNA cells, suggesting that it is gene selective. The cells resistant to siRNA function retained normal siRNA processing, duplex unwinding and degradation of the unwound sense strand and RNA-induced silencing complex formation, suggesting that loss of the siRNA function occurred at a later step. Surprisingly, the siRNA-resistant cells were found to express notably a cytoplasmic protein of approximately 50 kDa that specifically and characteristically interacted with the unwound, antisense strand E7 siRNA. Altogether, our data indicate that a potent siRNA targeting to an essential or regulatory gene might induce a cell to develop siRNA-suppressive function.

Persistence of recipient plasma cells and anti-donor isohaemagglutinins in patients with delayed donor erythropoiesis after major ABO incompatible non-myeloablative haematopoietic cell transplantation.

Delayed donor erythropoiesis and pure red-cell aplasia (PRCA) complicate major-ABO mismatched non-myeloablative allogeneic stem-cell transplantation. To characterize these events, we analysed red-cell serology and chimaerism in lymphohaematopoietic lineages, including plasma cells and B cells, in 12 consecutive major-ABO incompatible transplants following cyclophosphamide/fludarabine-based conditioning. Donor erythropoiesis was delayed to more than 100 days in nine (75%) patients including six (50%) who developed PRCA. During PRCA, all patients had persistent anti-donor isohaemagglutinins and recipient plasma cells (5-42%), while myeloid and T cells were completely donor in origin. In contrast, B-cell chimaerism was frequently full-donor when significant anti-donor isohaemagglutinins persisted. Four patients with early mixed haematopoietic chimaerism and the prolonged presence of anti-donor isohaemagglutinins and recipient plasma cells developed delayed-onset (>100 days post-transplant) red cell transfusion dependence and PRCA after myeloid chimaerism converted from mixed to full donor. These findings confirm that donor-erythropoiesis is impacted by temporal disparities in donor immune-mediated eradication of recipient lymphohaematopoietic cells during major-ABO incompatibility and suggest that plasma cells are relatively resistant to graft-versus-host haematopoietic effects.

Phenotype and function of a CD56+ peripheral blood monocyte.

G-CSF primed CD34 cells cultured for 2-3 weeks in IL-2 and stem cell factor generate CD56(high) cells with phenotypic and morphologic features of NK cells, and a novel adherent CD56(low) CD16- population expressing myeloid markers (CD33 and HLA-DR). We hypothesized that similar cells might also occur in peripheral blood. In 13/13 normal individuals, we found a circulating population of CD56(low), CD33+, FcgammaRI+, FcgammaRII+, HLA-DR+, CD11b(high), CD14+ monocytes closely resembling the cultured CD56(low)CD33+ cells. They may represent a normal counterpart of the CD56+ CD33+ hybrid myeloid/natural killer cell leukemia. Their mean frequency was 1.3+/-1% (standard deviation), range 0.16-3.5%, of total mononuclear cells. CD56(low)CD33+ cells, primed with cytomegalovirus antigen, induced autologous T-lymphocyte proliferation comparably to CD56-, CD14+ peripheral blood monocytes (PBM). Conversely, CD56(low) cells induced greater T-cell proliferation than CD56- PBM when lymphocyte responders were HLA mismatched. Unstimulated CD56(low)CD33+ cells showed a low antiproliferative effect on K562, which was increased upon LPS stimulation. The pattern of cytokine production by CD56(low)CD33+ cells and PBM largely overlapped; however, they produced detectable levels of IL-6 and IL-1beta. These results define a minor monocyte population with distinct phenotypic and functional features.

Medicaid and Medicare reimbursement for flow cytometry.

Medicaid, a program administered by individual states but involving federal funding, is the source of medical coverage for many low-income patients. This method of reimbursement is crucial for many flow cytometry laboratories, but is not well understood by many laboratory professionals. Conversely, flow cytometry often is not well understood by administrators in Medicaid offices. The potential exists for great variation in Medicaid reimbursement for flow cytometry services from state to state. As a first step toward elucidating the extent of this variation and bringing more information about Medicaid to laboratory professionals, state Medicaid offices were asked to provide the fee-for-service reimbursement for flow cytometry services. These services included Current Procedural Terminology (CPT) codes 85045 (reticulocyte counts), 86359 (total T-cell count), 86360 (absolute CD4 and CD8 counts, with ratio), 86361 (absolute CD4 count), 86812 (HLA typing, single antigen [B27]), 88180 (immunophenotyping, per surface marker), and 88182 (DNA, cell cycle analysis). Data were collected on technical and professional components and on global reimbursement. Wide variation exists in reimbursement amounts for these tests. Variation for CPT code 88180 was markedly pronounced.

Handling, storage, and preparation of human blood cells.

Human peripheral blood samples are probably the most common specimens submitted for flow cytometric analysis. Handling and preparation will vary depending on the cell lineage to be examined and the type of assay. This unit presents protocols for separating white cells by lysing erythrocytes, isolating mononuclear cells by trilineage separation, and assorted methods for isolating or enriching specific cell populations, including monocytes, neutrophils, T lymphocytes, B lymphocytes, NK cells, stem cells, and platelets.

Preparation of cells from blood.

Peripheral blood is a bountiful source of numerous cell types that are easily analyzed by flow cytometry for a variety of properties. The monodisperse nature of blood makes preparation of these cells relatively easy if the coagulation cascade is inhibited. For some studies the presence of serum can be a confounding factor, although this is often overcome by merely pelleting the cells from the serum and washing the remaining cells several times. Blood, particularly from humans and primates, should be considered highly infectious whether or not from a healthy donor. Therefore universal precautions should be followed at all times. Techniques and route of collection may have a profound influence on the condition and nature of the blood being obtained. Performing venipuncture with an extremely small gauge needle may disrupt cells and prevent satisfactory analysis. Venous blood and arterial blood may yield differing data. Even blood collection at different times of day may yield diurnal variations in some assays. The demands of each cell type and of each assay dictate specific preparation, fixation, storage, and staining protocols. However, the overall goal for preparation of cells from blood, for all of the assays, remains the same--collect and prepare the cells in such a manner that they accurately represent the in vivo state.

Current practices in clinical flow cytometry. A practice survey by the American Society of Clinical Pathologists.

The American Society of Clinical Pathologists surveyed 136 laboratories actively engaged in performing clinical flow cytometric testing to determine the demographics of these laboratories, the credentials of the personnel involved with testing, the volume and types of tests performed, and how data are analyzed and interpreted. These results are reported with commentary based on previous surveys and recommended practice guidelines.

Complete replication of human sperm genome in egg extracts from Xenopus laevis.

To examine the ability of Xenopus egg extracts to support a complete replication cycle of human sperm genome, demembranated human spermatozoa were incubated with the extract from activated Xenopus laevis eggs. Most sperm heads were decondensed within 15 min. The heads became round within 30 min with diameters of 10-30 microns. The process of DNA replication in the pronuclei was monitored by two methods, bromodeoxyuridine incorporation and flow cytometry. The results indicate that DNA replication was initiated approximately 1.5 h after membrane structure formation and that it lasted up to 9 h. The amounts of DNA in most pronuclei were doubled by 4-9 h, depending on which donor toad was the source of the egg extract. Inclusion of the protein synthesis inhibitor, cycloheximide (100 micrograms/ml), had no obvious effect on human sperm DNA replication but appeared to prevent the pronuclei from degradation after a prolonged period (> 6 h) of incubation. After storage in liquid nitrogen for > 1.5 mo, the efficiency of the egg extracts in supporting sperm head decondensation and DNA replication was reduced for human sperm but not for Xenopus sperm. Possible applications of the use of Xenopus egg extract for human sperm activation and DNA replication are discussed.

A survey of current practices in clinical flow cytometry.

In mid-1995, a 40-question survey was distributed to determine the current practices of clinical flow cytometry laboratories. Forty-seven responses were received. Included in the survey were questions regarding the affiliation and size of the laboratory, the qualifications of the staff, the nature and number of assays performed, whether laboratory resources and specimen loads were changing, and how data were analyzed and interpreted. The results indicate considerable variability in many aspects of clinical cytometry, such as number of markers used for immunophenotyping leukemias and lymphomas, types of specimens analyzed for DNA-ploidy, and charges for specific tests.

Immunophenotypic analysis of the T cell receptor V beta repertoire in CD4+ and CD8+ lymphocytes from normal peripheral blood.

The antigenic specificity of the majority of T lymphocytes in the peripheral blood is determined by the combination of alpha and beta variable region chains present in the T cell receptor complex. Currently, the V beta chains are grouped into 25 families. Historically, determination of V beta usage has relied on detection of gene rearrangement on the nucleic acid level; however, with the increased availability of monoclonal antibodies to the product of these genomic rearrangements, immunophenotypic methods are rapidly becoming a reliable alternative method for studying the usage of V beta regions by T cells and T cell subsets. In the present study, multiparametric flow cytometry was used to determine the use of 10 V beta chains on CD4+ and CD8+ T cells in the peripheral blood of 28 normal donors. By obtaining absolute lymphocyte counts at the time blood was drawn, the absolute number of both CD4+ and CD8+ cells using particular V beta regions could be determined. Additionally, the intradonor consistency of V beta usage was examined by obtaining blood from 5 of the volunteers at an interval of approximately 1 year. The results of this study suggest a fairly homogeneous pattern of use for these V beta regions. The most striking longitudinal differences were observed in one individual who underwent a tonsillectomy midway between the T cell receptor V beta determinations.

Method to make paraffin-embedded breast and lymph tissue mimic fresh tissue in DNA analysis.

Determination of DNA ploidy has been found to be of diagnostic and prognostic value with regard to many solid tumors. Flow cytometric analysis of DNA ploidy is dependent on the binding of fluorescent dyes to DNA. Preserving cell morphology by fixing the tissue in formalin interferes with the binding of propidium iodide (PI) and other fluorescent dyes to DNA. This distortion of DNA content measurement can cause inaccuracies in DNA-ploidy determinations of formalin-fixed tissue specimens and precludes the use of appropriate DNA standards. Therefore, it has been impossible to determine accurately the DNA ploidy of formalin-fixed, paraffin-embedded (FFPE) tissues. Using formalin-fixed cells as a model for FFPE cells, we developed a thermal treatment method to reverse the effect of formalin on the binding of propidium iodide to DNA. Applying this approach to the preparation of FFPE lymph node and breast tissue for DNA analysis, we have developed a method that makes the binding of PI to the DNA of FFPE tissue mimic that of fresh tissue. Following dewaxing, rehydration, and trypsin treatment, the FFPE tissue, resuspended in PBS, was heated to 75 degrees C for 90 min to restore the PI binding to that of fresh cells. This method makes it possible to use fresh, DNA-diploid cells as an internal control and, thus, determine more accurately the DNA ploidy of tumors preserved in formalin and paraffin.

Detection and monitoring of a concomitant atypical myeloproliferative disorder and chronic lymphocytic leukemia by flow-cytometric immunophenotyping.

OBJECTIVE: To illustrate the utility of a broad panel of monoclonal antibodies to detect secondary processes or unexpected characteristics of the primary blood dyscrasia. DESIGN: Case report and discussion. SETTING: Regional academic medical center. PATIENT: A 64-year-old man presenting with an apparent acute myeloid leukemia. INTERVENTIONS: Sequential immunophenotyping with a broad panel of monoclonal antibodies to monitor progression of disease and response to therapy. MAIN OUTCOME MEASURE: Identification and monitoring of the two atypical populations in this patient with correlation to the clinical status of the patient. RESULTS: Identification of an unsuspected mature lymphoid clone and characterization of the evolution of the myelomonocytic clone. CONCLUSION: The evolving mature lymphoid clone may have been overlooked in the context of a predominant atypical myeloproliferative process, particularly if a limited panel of monoclonal antibodies had been used for immunophenotyping. Sequential immunophenotyping was useful in monitoring the progression of each atypical process.

Immunophenotyping of congenital leukemia.

Congenital leukemia is a rare but well-documented disease in which a leukemic process is detected at birth or very shortly thereafter. An estimated 175-200 reports of congenital leukemia have appeared in the literature. The majority of the cases reported have not undergone thorough immunophenotyping, but rather have been assigned lineage based on cytochemical and morphological studies. Historically, a large proportion of congenital leukemias have been thought to be of the myeloid lineage, in contrast to pediatric leukemias in general, which are primarily lymphoid. The precise proportions of the lineage assignments may be distorted by the inclusion of cases of transient myeloproliferative disorders (TMD) as congenital leukemia. The immunophenotyping data available to date suggest that congenital leukemias are phenotypically heterogeneous, lacking any common distinguishing markers. The prognosis for congenital leukemias is usually poor if leukemoid reactive processes, such as TMD, are carefully excluded.

Congenital leukemia: report of two cases.

Congenital leukemia is a rare disease in which a leukemic process is present at birth or immediately thereafter. The majority of cases presented in the literature were reported prior to the availability of contemporary immunophenotyping methods, and lineage assignment was often made on the basis of morphology alone. Congenital leukemias may be of various lineages, although, historically, monocytic and myelomonocytic congenital leukemias appear to be the most prevalent. We present two cases of congenital leukemia with detailed immunophenotypic and cytochemical characterization. One case is of the lymphoid lineage, and the second is of myelomonocytic lineage. Neither patient displayed trisomy 21.

Alterations of T-cell receptor variable region expression in human immunodeficiency virus disease.

Since only a small percentage of CD4+ lymphocytes is infected at any one time during the course of human immunodeficiency virus (HIV) disease, a question central to the pathogenesis of HIV is whether or not the depletion of CD4+ lymphocytes is a random or selective event. The majority of peripheral blood T lymphocytes use alpha and beta variable chains as components of their T-cell receptor (TCR) complex. Depletion of CD4+ T lymphocytes from the peripheral blood may be dependent on the V beta chain expressed by the CD4+ cell, based on the hypothesis that HIV may encode a superantigen. Peripheral blood from normal controls and HIV+ patients was studied for alterations in the expression of various V beta chains of the TCR. Three-color flow cytometry was used to determine the expression of V beta 2, V beta 3, V beta 8, V beta 13, and V beta 19 on all lymphocytes and on both CD4+ and CD8+ lymphocytes independently. Alteration of the V beta chains in HIV+ disease was analyzed as a function of absolute CD4 count and Centers for Disease Control (CDC) stage of the patient. These data suggest that the loss of T helper (CD4) lymphocytes during the course of HIV disease may be a selective event. These data are consistent with the hypothesis that selective depletion of CD4+, V beta 19+ lymphocytes may be due to the encoding of a superantigen by HIV. Furthermore, using multicolor flow cytometry and stratifying patients by absolute CD4 counts (or stage of disease) may reveal immunologic changes that might otherwise be overlooked.

Flow cytometric analysis of primary and metastatic squamous cell carcinoma of the oral cavity and oropharynx.

A retrospective analysis of formalin-fixed, paraffin-embedded tissue from patients with histologically confirmed metastatic squamous cell carcinoma was performed using flow cytometry. Ninety-eight sets of specimens from previously untreated patients with an oral cavity or oropharyngeal tumor and a simultaneous cervical metastatic deposit were analyzed. Normal mucosa and cervical lymph nodes were processed identically and run as controls. All patients underwent surgical resection at Wilford Hall USAF Medical Center or The Eye and Ear Hospital of Pittsburgh between 1980 and 1986. The specimens from 94 patients were technically adequate for interpretation. Diploid histograms in both the primary and metastatic tumors were present in 49 (52%) of 94 patients. Aneuploid histograms in either the primary and metastatic tumors were noted in 45 (47%) of 94 patients. In this group of 45 patients, the primary tumor and cervical metastasis were both aneuploid in 21 (46%), and aneuploid histograms occurred with equal incidence in either the primary or metastasis in the remaining 24 cases. No statistically significant prediction of survival could be made from any correlation with the histograms of either the primary or metastasis. The potential technical problems and limitations of flow cytometry in the determination of DNA content of formalin-fixed, paraffin-embedded tissue and the selection of patients with advanced disease warrant caution in the interpretation of results.