Assessment of salivary pro inflammatory cytokines profile level in patients treated with labial and lingual fixed orthodontic appliances.

The secretions of certain cytokines, chemokines and growth factors are triggered by orthodontic appliances, which often affect the remodelling of periodontal tissues. Critical cumulative forces are applied by various types of orthodontic appliances to the periodontium. The secretion of such molecules is probably responsible, through molecular and cellular communications, for the optimal resorption of hard tissues in the periodontal setting, which therefore enables the coordination of multiple movements of tooth. This study assessed and compared a wide range of cytokines, cellular marker analysis and defensins present in the saliva samples of human subjected to orthodontic treatment with two different treatment modalities, i.e., conventional lingual and labial fixed orthodontic appliances. A total 40 samples of saliva were obtained, of which 20 were treated with traditional lingual appliances and 20 were treated with labial fixed appliances. After 21 days of treatment, all salivary samples were collected from the subjects. In order to analyse a broad range of soluble cytokine levels in saliva by flow cytometry, a bead-based immunoassay was performed. Cell surface markers were analysed by flow cytometry. Protein levels of saliva for defensins were quantified by ELISA. Non-significant differences were observed in the cytokine levels in the saliva except for the significant effects for CCL2, IL-17A and IL-6. Cellular markers CD45 and CD326 showed high percentage in conventional lingual samples. Defensin levels were found to be lower in conventional lingual patients. Subjects with conventional lingual appliances had significantly higher salivary protein levels of IL-1ß, CCL2, IL17A, and IL-6, higher CD45+ and CD326+ cells and lower defensin levels than subjects with fixed labial appliances. The current study provided a clear basis for the development of innovative methods to aid in the improvement of various procedural treatments and orthodontic equipment of next generation.

Evaluation of a 10color protocol as part of a 2tube screening panel for flow cytometric assessment of peripheral blood leukocytic subsets.

Peripheral blood (PB) immunophenotyping is commonly required for initial evaluation of various suspected disease entities. Several approaches have been proposed. The objective of this work is to explore the value of a 10color protocol developed in our laboratory for flow cytometric assessment of PB leukocytic subsets, as part of a 2tube screening panel. A combination of CD16/CD56/CD34/CD33/CD19/CD4/CD8/CD3/CD20/CD45 antibodies in 1 tube was applied routinely during flow cytometric analysis of PB samples for diagnostic purposes. The protocol was systematically complemented by a 2nd tube with anti-kappa, anti-lambda, CD5, CD19, and CD45 antibodies for adults and selected pediatric patients, and specifically oriented panels when necessary. 25 samples with no detectable neoplastic PB involvement and 31 samples with a hematolymphoid disorder were investigated retrospectively. The contribution of CD33 in the separation of leukocytic populations, as well as the benefits from the simultaneous assessment of CD20/CD19/CD45, CD16/CD56 and the detection of CD34+ cells were examined. The gating strategy with the use of CD33 provided additional information in certain cases. The protocol enabled recognition of differential expression of CD20 and CD45 in CD19+ cells with chronic lymphocytic leukemia phenotype, overall evaluation of NK and NK like T cells, estimation of CD16- granulocytes and CD56/CD16 expression in monocytes, as well as identification of minor cell subsets, such as CD34+ cells. The proposed 10color combination of antibodies analyzed in a standardized manner can offer significant information in the initial evaluation of PB samples, thus, guiding subsequent investigation if needed.

Functional assessment of hematopoietic niche cells derived from human embryonic stem cells.

To evaluate hematopoietic niche cell populations isolated from human embryonic stem cells (hESCs), we tested the ability of hESC-derived stromal lines to support CD34(+) umbilical cord blood (UCB)- and hESC-derived CD34(+)45(+) cells in long-term culture initiating cell (LTC-IC) assays. Specifically, these hematopoietic populations were cocultured with hESC-derived mesenchymal stromal cells (hESC-MSCs) and hESC-derived endothelial cells (hESC-ECs), and then assessed for their LTC-IC potential in comparison to coculture with bone marrow (BM)-derived MSCs and the mouse stromal line M2-10B4. We found that the hESC-derived stromal lines supported LTC-ICs from UCB similar to M2-10B4 cells and better than BM-MSCs. However, none of the stromal populations supported LTC-IC from hESC-derived CD34(+)45(+) cells. Engraftment data using the output from LTC-IC assays showed long-term repopulation (12 weeks) of NSG mice to correlate with LTC-IC support on a given stromal layer. Therefore, hESC-derived stromal lines can be used to efficiently evaluate putative hematopoietic stem/progenitor cells derived from hESCs or other cell sources.

Histologic assessment of tumor-associated CD45(+) cell numbers is an independent predictor of prognosis in small cell lung cancer.

BACKGROUND: Small cell lung carcinoma (SCLC) continues to have a poor prognosis, with a 2-year survival of < 20%. Studies have suggested that SCLC may affect the immune system to allow it to evade immunologic responses. We hypothesized that any such effect would be characterized by a decrease in the lymphoid cells associated with the tumor in biopsy specimens and that this might relate to patient outcome. METHODS: Sixty-four SCLC biopsy specimens were immunohistochemically stained with anti-CD45 antibody to identify immune cells associated with the tumor. A mean CD45 count per high-power field for each case was obtained, and the results were correlated with age, sex, stage, performance status (PS), treatment with chemotherapy/radiotherapy, and overall survival. RESULTS: The median CD45 count for all cases was taken as 40 (CD45(40)). Kaplan-Meier plots demonstrated better survival for patients with a CD45(40) > 40 ( P < .009). No relationship between CD45 40 and age, sex, stage, or treatment by chemotherapy or radiotherapy was identified. Although PS was a significant predictor of survival ( P = .014), it did not correlate with CD45 40. In patients with better Eastern Cooperative Oncology Group PS (≤ 2), the CD45(40) demonstrated a highly significant survival advantage for those with CD45(40) > 40 ( P < .0001). CONCLUSIONS: The data indicate that (1) simple immunohistochemical assessment of immune cell infiltrates in routinely processed and stained biopsy specimens of primary tumors can provide prognostic information in SCLC and (2) tumor-associated CD45(+) cells in SCLC biopsy specimens may be a good clinical marker to identify patients with poor prognosis despite good PS.

Optimizing antibody panels for efficient and cost-effective flow cytometric diagnosis of acute leukemia.

BACKGROUND: Differentiating acute myeloid leukemia (AML) from acute lymphoblastic leukemia (ALL) determines effective patient management and often depends on flow cytometry. Antibodies used in flow cytometry are costly, and the expenses are not always reimbursed. Having observed that AML and ALL have distinct patterns in the CD45/SSC panel, we set to analyze more leukemia cases and establish an algorithm for the efficient diagnosis of acute leukemia. METHODS: We retrospectively analyzed 127 consecutive cases of acute leukemia within the last 2 years and correlated the blast distribution patterns in the CD45/SSC panel, with the morphology and the detailed immunophenotype. RESULTS: Our results show that all the acute leukemias can be initially triaged into AML, ALL, and Indeterminate provisional groups based on the blast distribution patterns in the CD45/SSC panel and morphology. Each group was then further analyzed with tailored AML, ALL, and Indeterminate flow panels. Using this approach, we have efficiently and correctly diagnosed almost all the acute leukemias. Our analysis also determined the minimal numbers of immunological markers needed for the lineage assignment of acute leukemia. CONCLUSION: The algorithmic approach with tailored subsequent antibody selection could maintain diagnostic accuracy while significantly reducing reagent use, labor, and time. With a shrinking reimbursement for flow cytometric studies, an increase in laboratory efficiency without compromising diagnostic accuracy or turnaround time will contribute to preserving revenue and optimizing clinical service.

Dynamic regulation of CD45 lateral mobility by the spectrin-ankyrin cytoskeleton of T cells.

The leukocyte common antigen, CD45, is a critical immune regulator whose activity is modulated by cytoskeletal interactions. Components of the spectrin-ankyrin cytoskeleton have been implicated in the trafficking and signaling of CD45. We have examined the lateral mobility of CD45 in resting and activated T lymphocytes using single-particle tracking and found that the receptor has decreased mobility caused by increased cytoskeletal contacts in activated cells. Experiments with cells that have disrupted betaI spectrin interactions show decreased cytoskeletal contacts in resting cells and attenuation of receptor immobilization in activated cells. Applying two types of population analyses to single-particle tracking trajectories, we find good agreement between the diffusion coefficients obtained using either a mean squared displacement analysis or a hidden Markov model analysis. Hidden Markov model analysis also reveals the rate of association and dissociation of CD45-cytoskeleton contacts, demonstrating the importance of this analysis for measuring cytoskeleton binding events in live cells. Our findings are consistent with a model in which multiple cytoskeletal contacts, including those with spectrin and ankyrin, participate in the regulation of CD45 lateral mobility. These interactions are a major factor in CD45 immobilization in activated cells. Furthermore, cellular activation leads to CD45 immobilization by reduction of the CD45-cytoskeleton dissociation rate. Short peptides that mimic spectrin repeat domains alter the association rate of CD45 to the cytoskeleton and cause an apparent decrease in dissociation rates. We propose a model for CD45-cytoskeleton interactions and conclude that the spectrin-ankyrin-actin network is an essential determinant of immunoreceptor mobility.

Comprehensive assessment of T-cell receptor beta-chain diversity in alphabeta T cells.

The adaptive immune system uses several strategies to generate a repertoire of T- and B-cell antigen receptors with sufficient diversity to recognize the universe of potential pathogens. In alphabeta T cells, which primarily recognize peptide antigens presented by major histocompatibility complex molecules, most of this receptor diversity is contained within the third complementarity-determining region (CDR3) of the T-cell receptor (TCR) alpha and beta chains. Although it has been estimated that the adaptive immune system can generate up to 10(16) distinct alphabeta pairs, direct assessment of TCR CDR3 diversity has not proved amenable to standard capillary electrophoresis-based DNA sequencing. We developed a novel experimental and computational approach to measure TCR CDR3 diversity based on single-molecule DNA sequencing, and used this approach to determine the CDR3 sequence in millions of rearranged TCRbeta genes from T cells of 2 adults. We find that total TCRbeta receptor diversity is at least 4-fold higher than previous estimates, and the diversity in the subset of CD45RO(+) antigen-experienced alphabeta T cells is at least 10-fold higher than previous estimates. These methods should prove valuable for assessment of alphabeta T-cell repertoire diversity after hematopoietic cell transplantation, in states of congenital or acquired immunodeficiency, and during normal aging.

Recovery from chronic demyelination by thyroid hormone therapy: myelinogenesis induction and assessment by diffusion tensor magnetic resonance imaging.

The failure of the remyelination processes in multiple sclerosis contributes to the formation of chronic demyelinated plaques that lead to severe neurological deficits. Long-term cuprizone treatment of C57BL/6 mice resulted in pronounced white matter pathology characterized by oligodendrocyte depletion, irreversible demyelination and persistent functional deficits after cuprizone withdrawal. The use of a combination of in vivo diffusion tensor magnetic resonance imaging (DT-MRI) and histological analyses allowed for an accurate longitudinal assessment of demyelination. Injection of triiodothyronine (T(3)) hormone over a 3 week interval after cuprizone withdrawal progressively restored the normal DT-MRI phenotype accompanied by an improvement of clinical signs and remyelination. The effects of T(3) were not restricted to the later stages of remyelination but increased the expression of sonic hedgehog and the numbers of Olig2(+) and PSA-NCAM(+) precursors and proliferative cells. Our findings establish a role for T(3) as an inducer of oligodendrocyte progenitor cells in adult mouse brain following chronic demyelination.

Oxidative burst assessment and neutrophil-platelet complexes in unlysed whole blood.

BACKGROUND: Methods currently employed for measuring reactive oxygen species production can lead to both cellular depletion and in artifactual activation. The objective of this study was to design a methodology allowing the measurement of oxidative burst (OB) with minimal sample manipulation. METHODS: To that purpose a flow cytometry technique developed in our laboratory, based on nucleic acid staining to discriminate erythrocytes and debris, was employed. DRAQ5 dye and PECy5-CD45 monoclonal antibody (MoAb) were simultaneously used in FL3 to identify the leukocyte population and the PE-CD14 MoAb emission was detected in FL2 for monocytes. OB was measured by using the fluorogenic probe dihydrorhodamine 123, a marker of hydrogen peroxide production. Phorbol myristate acetate (PMA), Opsonized Zymosan (OZ), fMLP and calcium ionophore A23187 activators were also used in this study. For OB assays, dose-response curves were performed for each activator. In addition, the effect of activator concentration on annexin V binding, as a measure of phosphatidylserine translocation, was evaluated. RESULTS: With this method no-lysis and no-wash steps are required, thus avoiding an unwanted damage to leukocytes. PMA and Zymosan produced an increase in annexin V binding, while fMLP and calcium ionophore did not. CONCLUSIONS: This study reports a feasible and reproducible new flow cytometry assay for assessing OB of neutrophils and monocytes with minimal sample manipulation. In addition, under PMA and OZ conditions, the number of neutrophils showing annexin V binding was strikingly increased. This effect is not related with a phagocytic overstimulation, but with an increased neutrophil-platelet complexes formation.

Assessment of lymphocyte development in radiation bone marrow chimeras.

Transplantation of marrow between mouse strains congenic for CD45 after lethal irradiation establishes hematopoiesis driven by genetically marked cells in recipient animals. After several weeks, peripheral blood or primary and secondary lymphoid organs of transplant recipients can be evaluated for the presence of donor-derived cells. Two- or three-color flow cytometry can be used to identify the progeny of transplanted cells, to document their cell-surface phenotypes, and to follow development of T, B, and myeloid lineages in vivo.

Low cost CD4 enumeration using generic monoclonal antibody reagents and a two-color user-defined MultiSET protocol.

BACKGROUND: The standard three-tube, three-color flow cytometric method utilizing the TriTEST reagents in conjunction with the MultiSET software commonly used in most laboratories in Thailand for CD4 enumeration is expensive and thus unavailable to most HIV-infected patients. A more affordable method, i.e., the PanLeucogating protocol using only two monoclonal antibody reagents, has been described but requires the use of the CellQUEST software that does not have automatic gating and reporting facilities. We describe a simple protocol that utilizes a two-color user-defined protocol with the automated MultiSET software for the acquisition, analysis, and reporting of CD4 results. METHODS: A two-color user-defined protocol was set up following instructions in the Becton Dickinson Biosciences MultiSET manual, adhering strictly to the information regarding the Gate and Attractor Hierarchy for analyzing various reagent combinations. This simple two-color user-defined MultiSET software was evaluated using generic monoclonal reagents in comparison with the standard TriTEST/MultiSET protocol. RESULTS: The two-color user-defined MultiSET software is easy to use. It requires only modification of the original MultiSET program and the results obtained are comparable with those derived from the standard TriTEST/MultiSET protocol. CONCLUSION: The use of this easy and reliable two-color user-defined MultiSET protocol represents an affordable alternative to CD4 testing in resource-poor settings.

Assessment of thymic output in common variable immunodeficiency patients by evaluation of T cell receptor excision circles.

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by repeated infections and hypogammaglobulinaemia. Additionally, T-cell abnormalities including lymphopenia, decreased proliferation to mitogens and antigens, and the reduced production and expression of cytokines, have also been observed. In this study we have investigated the expression of naive, memory and activation markers in T-cell subpopulations in 17 CVID patients in comparison to age-matched normal controls. The numbers of CD4+ T cells, including CD45RA+CD62L+ and, to a lesser extent, CD45RA-CD62L+/RA+CD62L- were significantly reduced in patients, whereas CD8+ T cells were within normal range. In contrast, HLA-DR+ cells were increased both in CD4+ and CD8+ T cells. To assess the thymic output, we analysed the presence of T-cell receptor excision circles (TRECs) in CD4+ and CD8+ T cells by quantitative PCR. TRECs were decreased significantly in patients and the rate of TREC loss was higher with increasing age. TRECs correlated with naive CD4+ T cells, whereas there was an inverse relationship between TRECs and CD8+HLA-DR+ and CD8+CD45RA-CD62L+/RA+CD62L- T cells. Our results suggest the presence of a defect in the naive T cell compartment with origin at the thymic level in CVID, and indicate that TREC may be a useful marker to monitor thymic function in this primary immunodeficiency.

Functional assessment of precursors from murine bone marrow suggests a sequence of early B lineage differentiation events.

Most lineage marker-negative (Lin-)TdT+ cells from murine marrow lack CD34 but display c-kit at low density as well as IL-7Ralpha and Flk-2/Flt-3 receptors. Single cells with these characteristics generated CD45RA+CD19- as well as CD19+ lymphocytes in culture. CD45RA+CD19- marrow cells were resolved into three nonoverlapping subsets. One subset, lacking DX5 and Ly-6C antigens, yielded CD19+ cells in culture. Further analysis demonstrated CD24 on most Lin-TdT+ cells and all CD45R+CD19-DX5-Ly-6C- cells. Mac-1/CD11b was absent from these two subsets of B lineage precursors, while IL-7Ralpha was retained during subsequent differentiation to a CD19+ and stromal cell-independent stage. These findings contrast with previous descriptions of B lymphocyte precursors and suggest a sequence of early differentiation events.

Mechanical anchoring strength of L-selectin, beta2 integrins, and CD45 to neutrophil cytoskeleton and membrane.

The strength of anchoring of transmembrane receptors to cytoskeleton and membrane is important in cell adhesion and cell migration. With micropipette suction, we applied pulling forces to human neutrophils adhering to latex beads that were coated with antibodies to CD62L (L-selectin), CD18 (beta2 integrins), or CD45. In each case, the adhesion frequency between the neutrophil and bead was low, and our Monte Carlo simulation indicates that only a single bond was probably involved in every adhesion event. When the adhesion between the neutrophil and bead was ruptured, it was very likely that receptors were extracted from neutrophil surfaces. We found that it took 1-2 s to extract an L-selectin at a force range of 25-45 pN, 1-4 s to extract a beta2 integrin at a force range of 60-130 pN, and 1-11 s to extract a CD45 at a force range of 35-85 pN. Our results strongly support the conclusion that, during neutrophil rolling, L-selectin is unbound from its ligand when the adhesion between neutrophils and endothelium is ruptured.