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Abstract: The present investigation was designed to evaluate the renal microvascular endothelial cell responses following exposure to preformed antibodies against human leukocyte antigens (HLA) in the recipient. We hypothesize that activation of endothelial cell genes has a pivotal role in renal allograft survival. In this study, we used cultured human umbilical cord vein endothelial cells (HUVEC), human microvascular glomerular endothelial cells (HMGEC), activated with and without IFN-γ and TNF-α, and pre-transplant blood group O patient sera containing multispecific HLA class I and class II antibodies. Molecular HLA typing revealed the HMGEC haplotype to be HLA-A*01, HLA-A*68, HLA-B*14, HLA-B*35, HLA-C*04, HLA-C*08, HLA-DRβ1*13, and HLA-DRβ1*15. Flow cytometry was used for phenotypic characterization of both inactivated and activated HUVECs and HMGECs with IFN-γ and TNF-α. HUVECs were positive for HLA-ABC, HLA-DR/DQ, von Willebrand factor, endoglin, PECAM, ICAM, MCAM, integrin beta-3, thrombomodulin, E-selectin, VCAM-1, and tissue factor, and negative for alpha smooth muscle actin and P-selectin antibodies. HMGECs were positive for HLA-ABC, HLA-DR/DQ, von Willebrand factor, endoglin, ICAM, MCAM, integrin beta-3, thrombomodulin, VCAM-1, and tissue factor; and negative for PECAM, E-selectin, P-selectin, and for blood group antigens A and B. 42 samples were analyzed by real time PCR and categorized into the following groups: the control group (HMGEC only, n=12), group 1 (HMGECs incubated with patient sera, n=15), and group 2 (HMGECs activated by TNF-α and IFN-γ and incubated with patient sera, n=15). Expression levels of the vasoconstriction genes endothelin 1 (EDN1), endothelin 2 (EDN2), and endothelin receptor type A (EDNRA) were up-regulated in both groups 1 and 2 compared to the control group. The thrombomodulin (THBD) gene was also up-regulated in both groups 1 and 2 compared to the control. Chemokine genes CCL5 and CX3CL1 were up-regulated in both groups 1 and 2 compared to the controls; whereas, CCL2 was up-regulated only in group 2. Cytokine activity genes colony stimulating factor 2 (CSF2), tumor necrosis factor (TNF), tumor necrosis factor (ligand) superfamilymember 10 (TNFSF10), interleukin 1 beta (IL1B), and interleukin 6 (IL6) were up-regulated in both groups 1 and 2 compared to the control; whereas, IL11 was up-regulated only in group 1 and IFNB1 in group 2. Adhesion molecule genes intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), and integrin beta 3 (ITGB3) were up-regulated in both groups 1 and 2 compared to the control; whereas, CDH5 and COL18A1 were up-regulated only in group 2. Anti-apoptosis genes BCL2A1, CFLAR, and SPHK1 were up-regulated only in group 2 compared to controls. Apoptosis and caspase-activation genes CASP, RIPK1, and FAS were up-regulated only in group 2 compared to the control. Angiopoietin 1 (ANGPT1) and prostaglandin I2 (prostacyclin) synthase (PTGIS) were down-regulated in both groups 1 and 2 compared to the control group. Our results indicate that expression of the endothelin gene in endothelial cells may contribute to vasoconstriction of blood vessels in post-renal allograft transplantation. In addition, thrombomodulin, by reducing thrombogenic activity, and interleukin 11, through its cytoprotective effects, may have a role in transplant accommodation in the presence of pre-formed HLA antibodies. This study showed that activation of the vasoconstriction genes, thrombomodulin gene, chemokine genes, cytokine activity genes, adhesion genes, anti-apoptosis genes, and apoptosis and caspase-activation genes could have consequential effects on renal allograft survival. [Copyright &y& Elsevier]

Abstract: Acute myelogenous leukemia (AML) is divided into 8 FAB subgroups based on differentiation and maturation properties of the neoplastic cells. Acute promyelocytic leukemia (APL), or M3 AML, is associated with disseminated intravascular coagulation (DIC). Flow cytometric immunophenotyping differentiates among the AML subtypes. Key markers in this classification include the myeloid antigens CD13 and CD33 and the hematopoietic precursor markers CD34 and HLA-DR. The present study analyzes and compares differences in the expression of these markers in 27 M0–M2 cases and 8 M3 cases. The M0–M2 cases generally expressed all four antigens. CD13 and CD33 were positively expressed in 23 (85.2%) and 21 (77.8%) of the 27 cases, respectively. CD34 and HLA-DR were present in 25 (92.6%) and 26 (96.3%) of the 27 cases, respectively. Analysis of the M3 cases revealed a different immunophenotype as CD13 and CD33 were each positive in all 8 (100%) M3 AML cases while CD34 and HLA-DR were negative in 6 (75%) and 8 (100%) of the 8 M3 cases, respectively. In contrast to expression of the early markers CD34 and HLA-DR in the M0–M2 group, these were negative in the M3 cases which were characterized by heterogeneous CD13 and generally homogeneous and bright CD33 expression. [Copyright &y& Elsevier]

Abstract: Flow cytometric analysis of cluster of differentiation (CD) markers in abnormal lymphocyte populations is crucial in the diagnosis of precursor T cell acute lymphoblastic leukemia (T-ALL)/lymphoblastic lymphoma (LBL). The World Health Organization (WHO) suggested immunophenotype for pre-T ALL/LBL typically includes the expression of TdT, cCD3, and CD7, while CD2, CD3, CD4, CD5, CD8, and CD10 are variably expressed. The myeloid antigens CD13 and CD33 are usually positive, whereas CD117 and cCD79a are infrequently expressed. Furthermore, there is frequent dual expression of CD4 and CD8. In the present investigation, 19 cases of pre-TALL/LBL were analyzed for selected CD marker expression. Fifteen of 19 cases studied were evaluated for TdT, cCD3, and cCD79a expression. Eleven (73.3%) positively expressed TdT, 15 (100%) positively expressed cCD3, and 9 (60%) positively expressed cCD79a. Of the 17 cases analyzed for CD7, CD5, and CD10 expression, CD7 and CD5 were positive in all 17 (100%) cases, whereas CD10 was positive in 8 (47.1%) cases. Of the 18 cases evaluated for CD2, CD3, CD4, CD8, and dual expression of CD4 and CD8, CD2 was expressed in 14 (77.8%), while CD3 was expressed in 7 (38.9%) cases. CD4 was positive in 11 (61.1%), and CD8 was expressed in 9 (50%). Dual expression of CD4 and CD8 occurred in only 4 (22.2%) of the cases. Of the 16 analyzed for CD13, CD33, and CD117, only 1 case (6.3%) expressed CD13, while 2 (12.5%) cases expressed CD33 and CD117. Thus, these data point to the need for a more extensive study to reevaluate the current WHO defined immunophenotype used in the diagnosis of pre-TALL/LBL. [Copyright &y& Elsevier]

Abstract: The WHO immunophenotype for plasma cell myeloma is deletion of CD19 and CD20, usual expression of CD38, CD138, and CD56, and occasional expression of CD10. Of the 39 cases of plasma cell dyscrasia in our study, the mean fluorescence intensities (MFI) of CD38, CD138, CD56, and CD19 were quantified in 30 cases. CD19 was absent in 38 of the cases (97.4%), whereas CD138 and CD38 were expressed in all 39 cases (100%). Most cases expressed CD38 and CD138 brightly with MFI values greater than 501, whereas all other marker expression was moderate with MFI greater than 301. Whereas CD38/CD56 dual expression was observed in 25 cases (64.1%), 14 failed to express CD56 (35.9%). CD56 expression was bright in 16 cases (53.3%), moderate in 2 cases (6.7%), and negative in the remaining 12 cases (40%) with MFI values of 200 or less. CD117 expression was positive in 9 of 24 cases (37.5%). In 32 of 39 cases, 27 were negative for CD20 (84.4%) and 28 were negative for CD10 (87.5%). Our results point to the value of quantitative fluorescence intensity in the flow cytometric evaluation of CD molecular expression or deletion in the diagnosis of hematopathologic disorders, such as plasma cell dyscrasia. [Copyright &y& Elsevier]

Abstract: Flow cytometric analysis of abnormal lymphocyte populations in chronic lymphocytic leukemia (CLL) has been widely reported to show weak expression of surface immunoglobulin (sIg). The international scoring system to help discriminate between CLL and other B-cell lymphoproliferative disorders lists this as the first of 5 criteria worth 1 point each. In the present study, 30 cases of CLL were studied for surface and cytoplasmic Ig expression. 23 of these 30 (76.7%) cases were positive for sIg. Of these 23, 14 cases (60.9%) showed moderate to bright sIg expression. All of these 23 cases were positive for CD5 and CD23; all were negative for CD10 and only 6 (26.1%) were positive for FMC7. 27 of 30 (90.0%) cases expressed cytoplasmic immunoglobulin (cIg) compared to 5% reported by others. This shows that cytoplasmic Ig occurs in a much greater percentage of cases than reported previously. 23 of 30 (76.7%) cases showed positivity for both surface and cytoplasmic Ig, with 15 showing kappa light chain restriction and 8 showing lambda light chain restriction. Six expressed cytoplasmic Ig only, with 4 showing kappa light chain restriction and 2 showing lambda light chain restriction. One case expressed neither cytoplasmic nor surface Ig. CD38 positivity portends a worse prognosis. Of the 29 cases tested for CD38, 13 (44.8%) were positive. Of these 13 cases, 12 were in the surface Ig/cytoplasmic Ig+ group and 1 in the cytoplasmic Ig+ group only. Also, of the 23 cases tested for CD22 expression, 16 (69.6%) were positive. These data question the use of both “weak” surface Ig expression and lack of CD22 expression as valid scoring criteria for CLL. [Copyright &y& Elsevier]

Abstract: Zeta-chain (TCR)-associated protein kinase 70 kDa (Zap-70) and CD38 expression may be of prognostic significance in chronic lymphocytic leukemia (CLL). Previous studies indicate that Zap-70 and CD38 are usually positive in cases of CLL with unmutated immunoglobulin variable region genes (IgVH) and may be used to predict IgVH mutation status and prognosis. Usually cases of CLL positive for Zap-70 or CD38 indicate a worse prognosis. In the present investigation, 47 cases of CLL were evaluated for CD38 expression, and 17 cases were evaluated for both Zap-70 and CD38 expression. Of the 47 cases, 19 (40.4%) positively expressed CD38. Of the 17 cases evaluated for Zap-70, 11 (64.7%) were positive for Zap-70, while only 6 (35.3%) were positive for CD38 expression; the remaining cases were negative for CD38. The results of this study show that Zap-70 expression may be a better indicator of the mutational status of IgVH and prognosis of CLL than CD38 expression. In addition, CD38 negativity does not necessarily indicate that IgVH mutation has occurred. These data point to the need for a more extensive study to evaluate the significance of Zap-70 and CD38 expression as indicators of IgVH mutation status and prognosis of CLL patients. [Copyright &y& Elsevier]

Abstract: Flow cytometric analysis of cluster of differentiation (CD) markers of myeloid cells has been used in conjunction with cell morphology to diagnose chronic myelogenous leukemia (CML). In the present study, 16 cases of CML were studied for levels of expression of myeloid markers CD15, CD13, CD33, and CALLA, i.e., CD10 which is also expressed on mature granulocytes. In 11 (68.8%) of 16 cases, a differentiated granulocyte population was detected that showed decreased expression of both CD10 and CD13. CD10 was found to be negative in 1 (6.3%) case and showed decreased expression in 10 (62.5%) of the cases. CD13 showed decreased expression in 11 (68.8%) of the 16 cases. Of the 15 cases analyzed for CD15, 2 (13.3%) were negative and 6 (40%) showed decreased expression. Of the 11 cases which showed simultaneous diminished expression of CD10 and CD13, 8 (72.7%) also showed decreased expression of CD15. Of the antigens studied, CD33 was the only one to be consistently expressed at normal levels, i.e., 13 (81.3%) cases demonstrated normal expression. Therefore, these results point to frequently decreased expression levels of CD10, CD13, and CD15 and rarely decreased expression levels of CD33 in association with CML. [Copyright &y& Elsevier]

Abstract: The prognostic significance of selected markers of leukemic cells is well known. CD7 and CD56 expression at diagnosis has been associated with low remission rates and biological aggressiveness in a significant proportion of acute leukemias. Among 46 patients with acute myeloid leukemia, we found CD7 expression in 15 cases (32.6%) and CD56 positivity in 10 patients (21.7%). Six of these myeloid leukemia cases (13%) showed expression of both CD7 and CD56. Four of 46 (8.7%) patients expressed CD79a. Among the 10 that were acute myeloblastic leukemia, 8 expressed CD7, 4 expressed CD56, and 4 were positive for CD79a. Thus, these markers were expressed early in hemopoietic ontogeny in the lesser-differentiated acute myeloid leukemia subtypes, including FAB M0, M1, and M2. Whereas CD7 and CD56 were each positive in 4 cases of acute myelomonocytic leukemia (FAB M4 subtype), there was no CD79a expression in the M4 cases. CD7 is expressed by mature T cells, NK cells, and an immature myeloid cell subset. NK cells and a T cell subset express CD56. By contrast, CD79a is a B cell marker that is assigned a high score of 2.0 in the differentiation of acute leukemias of ambiguous lineage in the WHO classification. The aberrant expression of CD7, CD56, and CD79a, representing the capacity of these leukemias for trilineal expression of leukocyte differentiation antigens, portends a poor prognosis. [Copyright &y& Elsevier]

Abstract: According to WHO, several T-cell immunophenotypic markers may be aberrantly expressed in acute myeloid leukemia (AML). TdT may be expressed in greater than one-third of cases, and CD2 and CD7 may be expressed frequently at low intensity; however, the T-cell lineage specific antigen CD3 is usually absent. In this investigation, 30 cases of AML were evaluated for CD2, CD3, CD5, CD7, CD8 and TdT expression, and mean fluorescence intensities (MFI). Of the 3 (10%) cases positive for CD3 and CD8, 1 was bright (MFI>501), and 2 cases were moderate. TdT was moderately expressed in 4 (13.3%) cases with MFI values between 301 and 500. CD2 and CD5 were positive in 5 (16.7%) cases. While CD2 was moderate in all 5 cases, CD5 was bright in 3, moderate in 1 and dim in 1. Of the 15 (50%) cases positive for CD7, 9 were bright, 5 were moderate, and 1 was dim with a MFI value between 201 and 300. These data indicate that CD2 and CD7 may be frequently expressed at greater intensities than WHO specified. These results point to the need for a more extensive study to evaluate the potential prognostic significance of aberrant T-cell marker expression in AML. [Copyright &y& Elsevier]

Abstract: We aimed to examine the use of circulating tumor cells (CTCs) as an effective measure of treatment efficacy and immune system function in metastatic breast cancer patients. CTCs are believed to be indicators of residual disease and thus pose an increased risk of metastasis and poorer outcomes to those patients who are CTC-positive. We obtained peripheral blood samples from 45 patients previously diagnosed with metastatic disease originating in the breast. Using TLR agonists that bind TLR ligands and upregulate immune effects versus unstimulated cells, we calculated a percent specific lysis using chromium-51 assay to illustrate the functional abilities of patient natural killer (NK) cells. We found those with greater than 5 CTCs per 7.5mL blood had significantly decreased responses by their immune cells when compared with those patients who had 5 CTCs or less. We furthermore found a correlation between disease progression and CTC-positive patients, indicating that those who have a positive test should be closely monitored by their clinician. CTCs represent an exciting new clinical opportunity that will ideally utilize their low invasiveness and quick turnaround time to best benefit clinical scenarios. [Copyright &y& Elsevier]

Abstract: Systemic lupus erythematosus (SLE) is a chronic, relapsing, and remitting disease affecting primarily African American females of child bearing age. Familial aggregation of this disease suggests that at least part of the susceptibility for this disease is genetic, although environmental and hormonal influences are also likely to play a role. Early studies of genetic susceptibility to SLE revealed several of the major histocompatibility complex molecules, namely HLA DR, to be linked to SLE. Meta-analysis of genome scans has yielded loci significant for lupus patients, one of which includes the MHC region. Regulatory T cells are immunoregulatory cells that modulate activated immune cells. These cells play a large role in homeostasis of the immune responses and maintenance of immunologic tolerance, i.e., prevention of autoimmunity. Decreased numbers of regulatory T cells have been described in many autoimmune diseases, including systemic lupus erythematosus. Autoantibody production in systemic lupus erythematosus and the resulting immune complex formation and complex deposition into tissues are arguably the central core of immune dysregulation leading to disease manifestations and symptoms. Inability of the immune system to recognize and inhibit autoreactive immune cells in this particular autoimmune disease may be the result of inappropriate numbers and function of regulatory T cells. This study aims to characterize the immune cell population in patients from our community suffering from systemic lupus erythematosus and to prove that these patients exhibit a unique cellular profile compared to healthy age, race and gender matched control subjects. Surprisingly, our findings demonstrate that patients from the local Mississippi area exhibit increased proportions of CD25+ FoxP3+ regulatory T cells and CD25+ FoxP3− T cells (of CD45+ CD3+ CD4+ helper T cells) as compared to healthy controls. HLA tissue-typing of these lupus patients revealed a prominent subgroup (~30%) of patients possessing the HLA DRB1*1503 allele. The investigation of this subgroup demonstrated regulatory T cell composition similar to that of the total lupus group and to that of the non-HLA DRB1*1503 subgroup. Genetic analysis for molecular gene expression levels of various lupus-associated genes by real-time PCR demonstrated a unique profile as compared to healthy controls. Increased gene expression of FoxP3 together with decreased gene expression levels of GATA3, TNFAIP3, and TNFSF4 suggest that variations in gene products compared to healthy controls may be playing a role in the immune cell dysregulation and disproportionate CD25+ FoxP3+ regulatory T cells. [Copyright &y& Elsevier]

Abstract: In the present investigation, flow cytometric techniques were utilized to evaluate 100 cases of HIV and 20 normal controls for CXCR4, CCR5, and TLR4 expression in CD4-positive T cells, CD8-positive T cells, regulatory T cells, and Th17 cells, and fluorescence intensity values were determined. TLR4 was expressed by CD4+ T cells and CD8+ T cells in 97 cases, by regulatory T cells in 88 of 95 cases, and by Th17 cells in 93 of 95 cases, while it remained negative in all 20 normal controls. These data indicate that TLR4 upregulation is not limited to gram-negative bacterial infection nor is expression limited to myeloid cells. Upregulation of TLR4 in HIV patients may either be directly or indirectly related to the presence of the virus. CXCR4 was positively expressed by CD4+ T cells in 96 HIV cases, CD8+ T cells in 95, regulatory T cells in 89 of 95 cases evaluated, and Th17 cells in all 92 cases evaluated, while expression remained negative in the majority of normal controls. CCR5 was positively expressed by CD4+ and CD8+ T cells in all 100 HIV cases and by regulatory T cells in 89 of 95 cases evaluated, while expression was negative in most CD4+ and regulatory T cells of normal controls. Statistically significant differences were detected when TLR4 expression by CD4+ and CD8+ T cells was compared to stage of disease. TLR4 expression decreased as infection progressed from acute phase to AIDS. In addition, expression of TLR4 by all T cell subsets was slightly decreased in patients receiving HAART therapy. Results also reveal a positive correlation between CXCR4, CCR5, and TLR4 expression indicating that TLR4 expression and chemokine expression pathways are linked. [Copyright &y& Elsevier]

Abstract: The present investigation examined variations in cytokine and Toll-like receptor expression by T-lymphocytes in HIV infection. 50 HIV cases and 10 normal controls were evaluated for TLR3, TLR4, TLR8, TLR9, IL-2, IL-4, IL-6, IL-10, IL-12, TNF-α, and IFN-γ expression and staining intensities. TLR3 was expressed in 37 HIV (74%) cases, and TLR4 was brightly expressed in all (100%) HIV cases. However, TLR3 and TLR4 remained negative in all normal controls. Results reveal that TLR4 up-regulation is not limited to gram-negative bacterial infection. No statistical difference was detected when TLR8 and TLR9 expression were compared between the two test populations. A statistically significant difference was detected when IL-2, IL-4, IL-6, IL-10, IL-12, and IFN-γ expression were analyzed in HIV cases and normal controls. When cytokine expression was compared with stage of disease, results indicated that a Th1 to Th2 cytokine switch did not occur. Only TNF-α expression decreased as infection progressed from the chronic phase into AIDS. When TLR expression was compared to cytokine expression, no statistical differences were detected. These data point to the need for a more in-depth study to determine whether or not the up-regulation of Toll-like receptor expression increases cytokine expression via the NFκB pathway. [Copyright &y& Elsevier]

Abstract: Toll-like receptors (TLRs) and cytokines function in immune regulation and thus may be dysregulated in disease. In this study, 25 leukemia cases and 10 normal controls were analyzed by flow cytometry for differential cytokine and TLR expression. The percentages of CD3+ cells producing TNF-α, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, and IFN-γ were determined. Statistically significant differences between lymphocytic leukemia cases and normals were observed for all cytokines except IL-12. Differences in expression of all cytokines other than IL-6 and IFN-γ proved to be statistically significant between myeloid leukemic and normal cases. IFN-γ and IL-3 dual staining was observed to be most prominent in leukemic samples. Additionally, the staining intensities of TLR3, TLR4, TLR8, and TLR9 in regard to CD3+ cells were evaluated and compared among the three groups. The increased staining intensity of TLR9 in leukemic cases compared to levels in normal controls was statistically significant. No differences of statistical significance were observed between the two leukemic groups for cytokine or TLR expression. These results warrant further study of the mechanism and potential therapeutic value targeting these leukemic patterns. [Copyright &y& Elsevier]

Dendritic cells (DCs) consist of a heterogeneous population of hematopoietic cells characterized by their unique dendritic morphology, their efficient antigen-presenting capability to activate nai¨ve CD4+ and CD8+ T cells, as well as their lack of lineage-specific markers. Functional properties comparing umbilical cord blood monocyte-derived and umbilical cord blood stem cell-derived DCs have not yet been investigated. Human umbilical cord blood CD14+ monocytes and CD34+ stem cells were induced to differentiate into dendritic cells using 100 ng/mL granulocyte–macrophage colony-stimulating factor (GM-CSF), 25 ng/mL interleukin (II)-4, 2.5 ng/mL tumor necrosis factor α (TNF-α) and 100 ng/mL GM-CSF, 25 ng/mL stem cell factor, and 2.5 ng/mL TNF-α, respectively. Differentiated dendritic cells were CD80+, CD86+, CD83+, CD54+, CD1a+, CD11b+, CD11c+, HLA-DR+, CD34−, CD3−, CD19−, CD14−, and CD16−. Reverse transcription polymerase chain reaction revealed that differentiating monocytes initially expressed CD86 mRNA while CD80 mRNA appeared on Day 2. Differentiating stem cells expressed both CD80 and CD86 mRNA on Day 2 of culture. Mixed lymphocyte reaction was employed to evaluate the two types of lineage-derived DCs. Monoclonal antibodies (mabs) to CD80 and CD86 were employed to assess their costimulatory roles. CD14 and CD34 derived DCs prior to the functional assay were stimulated for 18 h with 0.1 and 1.0 mg/mL Escherichia coli lipopolyssacharide, respectively. A decrease in stimulation as depicted by decreased T-cell activation was significant with mabs to both CD80 and CD86 on monocyte-derived DCs while only mabs to CD86 induced decreased T-cell activation by stem cell-derived DCs. The varied functional role of CD80 and CD86 costimulatory molecules is associated with DC differentiation from distinct cord blood-isolated hematopoietic lineages. These studies demonstrate that DC association with distinct hematopoietic lineages is of relevance in transplantation and vaccine therapies. [Copyright &y& Elsevier]

Dendritic cells (DCs) consist of a heterogeneous population of hematopoietic cells characterized by their unique dendritic morphology, their efficient antigen-presenting capability to activate naive CD4+ and CD8+ T cells, and their lack of lineage specific markers. Functional properties comparing umbilical cord blood monocyte-derived and umbilical cord blood stem cell-derived DCs have not yet been investigated. CD14+ monocytes and CD34+ stem cells were isolated from human umbilical cord blood and were induced to differentiate into dendritic cells using 100 ng/mL granulocyte–macrophage colony stimulating factor (GM-CSF), 25 ng/mL IL-4, 2.5 ng/mL tumor necrosis factor-α (TNF-α), 100 ng/mL GM-CSF, 25 ng/mL stem cell factor, and 2.5 ng/mL TNF-α, respectively. Flow cytometric analysis revealed that the 14-day-old dendritic cells were CD80+, CD86+, CD83+, CD54+, CD1a+, CD11b+, CD11c+, HLA-DR+, CD34−, CD3−, CD19−, CD14−, and CD16−. Reverse transcription polymerase chain reaction was employed to detect expression of mRNA for CD80 and CD86. Differentiating monocytes initially expressed CD86 while CD80 appeared on day 2. Differentiating stem cells expressed CD80 and CD86 on day 2 of culture. The surface expression of CD80 and CD86 was studied over the course of differentiation. Mixed lymphocyte reaction was employed to evaluate the two types of lineage-derived DCs. Prior to the functional assay, CD14+ and CD34+ derived DCs were stimulated for 18 h with 0.1 mg/mL and 1.0 mg/mL E. coli lipopolyssacharide, respectively. Monoclonal antibodies (mabs) to CD80 and CD86 were employed to assess their costimulatory roles. A decrease of stimulation as depicted by decreased T cell activation was significant with mabs to both CD80 and CD86 on monocyte-derived DCs while only mabs to CD86 induced decreased T cell activation by stem cell-derived DCs. The varied functional role of CD80 and CD86 costimulatory molecules is associated with DC differentiation from distinct cord blood isolated hematopoietic lineages. These studies demonstrate that DC association with distinct hematopoietic lineages is of relevance in transplantation and vaccine therapies. [Copyright &y& Elsevier]

The uterus contains all the components of a tertiary lymphoid compartment. We hypothesize that specific leukocyte recruitment to the endometrium during the secretory phase of the menstrual cycle and early pregnancy limits the type of immunocyte that gains access. The present study utilized flow cytometry to define and quantify adhesion molecules possibly used by decidual infiltrating lymphocytes (DIL) as homing receptors, uterine microvascular myometrial endothelial cells (UtMVE-Myo) as addressins, and secretory endometrial stroma cells (STO) as retainment factors. Human umbilical cord vein endothelial cells and peripheral blood lymphocytes were used as control cells for comparison studies. DIL were composed of predominantly lymphocyte function-associated antigen (LFA)-1+, intercellular adhesion molecule (ICAM)-1+, LFA-2+, LFA-3+, gp150,95+, α1β1+, Hermes cell adhesion molecule (H-CAM)+, and neural cell adhesion molecule (N-CAM)+ (CD56bright) memory/effector natural killer cells. A significant number of UtMVEC-Myo expressed platelet endothelial cell adhesion molecule (PECAM)-1, a percentage were uniquely LFA-3+, and α4 integrin expression was uniquely high. An increased number of STO uniquely expressed α3, β3, and LFA-3, whereas α2, α4, αVβ3, and H-CAM were significantly increased. Possible unique adhesions of DIL:UtMVEC-Myo included SLe×:PECAM, vascular cell adhesion molecule-1:α4, and LFA-2:LFA-3, whereas DIL:STO included LFA-2:LFA-3 and N-CAM:N-CAM. Unique molecules on DIL may also associate with extracellular matrix (ECM) or complement on UtMVEC-Myo or STO to form gp150,95:fibrinogen/iC3b/C3dg, α1β1:laminin (LM)/collagen (CO), and ICAM-1:fibronectin (FN) interactions. Bridges of ECM may also form between DIL and UtMVEC-Myo adhesion molecules including ICAM-1:FN:ICAM-1 and α4β1:FN:α4β1. DIL:ECM:STO interactions may involve α2β1:CO:α2β1, α3β1:LM/CO/FN:α3β1, αVβ3:VN:αVβ3, and H-CAM:hyaluronate:H-CAM. It is likely that many adhesion molecules play a role in the recruitment and retainment of specialized lymphocytes within the uterine microenvironment. (Mackay et al., 1990). [Copyright &y& Elsevier]

Abstract: The present investigation was designed to show the effect of human leukocyte antigen (HLA) class II molecular allelic specificities in the recipient on the induction of humoral antibody rejection, identified by C4d peritubular capillary staining, as well as specific antibody identified by Luminex technology. Major histocompatibility complex (MHC) class II molecules are expressed on dendritic cells, macrophages, and B lymphocytes and they present antigenic peptides to CD4 positive T lymphocytes. Human renal peritubular and glomerular capillaries express class II MHC molecules upon activation. Expression of class II molecules on renal microvascular endothelial cells exposes them to possible interaction with specific circulating antibodies. We hypothesize that HLA-DQβ1*0202 expression in recipients decreases the likelihood of antibody-mediated renal allograft rejection. We found that 80% (=25) of DQ2 positive haplotype recipients failed to induce humoral antibody renal allograft rejection and 20% (n =25) of DQ2 positive haplotype recipients induced humoral antibody renal allograft rejection (p =0.008). By contrast, 48% (n =46) of DQ2 negative haplotype recipients failed to induce a humoral antibody component of renal allograft rejection and 52% (n =46) of DQ2 negative haplotype recipients induced humoral antibody-mediated renal allograft rejection. Our results suggest that recipients who express the DQβ1*0202 allele are less likely to induce a humoral antibody component of acute renal allograft rejection than are those expressing DQ1, DQ3, or DQ4 alleles. DQβ1*0202 allele expression in recipients could possibly be protective against acute humoral allograft rejection and might serve as a future criterion in recipient selection and in appropriate therapy for acute renal rejection episodes. [Copyright &y& Elsevier]

Abstract: The World Health Organization (WHO) characterization of the immunophenotype of precursor B-cell acute lymphoblastic leukemia (pre-B ALL) includes the possible expression of myeloid cluster of differentiation (CD) markers CD13 and CD33. In precursor T-cell acute lymphoblastic leukemia (pre-T ALL), myeloid markers CD13 and CD33 are frequent while CD117 is rare. In the present investigation, 71 cases of confirmed pre-B ALL were evaluated for the presence of CD13 and CD33. Of the 19 (27%) cases that positively expressed myeloid markers, 10 (53%) expressed CD13, 17 (89%) expressed CD33, and 1 (5%) expressed CD117. Eight (42%) expressed both CD13 and CD33, and 1 (5%) expressed CD13, CD33, and CD117. Twenty-one cases of confirmed pre-T ALL were analyzed for myeloid markers CD13, CD33, CD117, and MPO. Of the 6 (29%) expressing myeloid markers, 4 (67%) were positive for CD13, 4 (67%) for CD33, 3 50(%) for CD117, and 1 (17%) for MPO. One (17%) was positive for both CD13 and CD117; one (17%) for CD13 and CD33; one (17%) for CD13, CD33 and CD117; and one (17%) for CD13, CD33 and MPO. These markers portend a poor prognosis compared to ALL cases without myeloid antigens, and a poor response to drug therapies targeting conventional ALL. Future studies will be directed to correlation of these markers with prognosis and therapeutic response, as well as whether drug therapies targeting myeloid antigens could be of use in treatment. [Copyright &y& Elsevier]

Breast cancer (BC) is a major health problem for women around the world. Although advances in the field of molecular therapy have been achieved, the successful therapeutic management of BC, particularly metastatic disease, remains a challenge for patients and clinicians. One of the areas of current investigation is the circulating tumor cells (CTCs), which have a determinant role in the development of distant metastasis. At the present, many of the available treatment strategies for metastatic disease are of limited benefit. However, the elucidation of the mechanisms of tumor progression and metastasis may help to identify key molecules/components that may function as therapeutic targets in the future. In the present study, the functional analysis of CTCs revealed their ability to grow and proliferate to form colonies. Immunofluorescence staining of the CTCs' colonies exhibits elevated expression of cell growth and survival associated proteins such as, survivin, ERK and Akt1. More importantly, the functional screening of the chemokine profile in BC patients' sera revealed an HR-independent elevation of the chemokine CXCL10 when compared to healthy controls. The analysis of chemokines CXCL9 and CXCL11 demonstrated an HR-dependent production pattern. The levels of both CXCL9 and CXCL11 were markedly high in HR + patients' sera when compared to HR − patients and healthy controls. The functional analysis of HR + and HR − BC derived cell lines when cultivated in media supplemented with patients' sera demonstrated the alteration of tumor progression and metastasis related proteins. We noted the induction of survivin, β-catenin, MKP-1, pERK, CXCR4 and MMP-1 both at the protein and mRNA levels. The induction of those proteins was in keeping with patients' sera induced cell proliferation as measured by the MTT assay. In conclusion, our data emphasizes the role of chemokines, especially CXCL10, in BC progression and metastasis via the induction of signaling pathways, which mainly involve survivin, β-catenin, MKP-1 and MMP-1. [ABSTRACT FROM AUTHOR]

The risk posed by breast cancer represents a complex interaction among factors affecting tumor immunity of the host. Toll-like receptors (TLRs) are members of the innate immune system and generally function to attract host immune cells upon activation. However, the good intentions of TLRs are sometimes not transferred to positive long-term effects, due to their involvement in exacerbating inflammatory effects and even contributing to continued inflammation. Chronic inflammatory states are considered to favor an increased predisposition to cancer, with continuous activation of inflammatory cytokines and other hallmarks of inflammation exerting a deleterious effect. Circulating tumor cells (CTCs) are neoplastic cells present in the peripheral blood circulation that have been found to be an indicator of disease progression and long-term survival. In the present study, we examined the expression of TLRs on dendritic cells, which play a major role in eliciting anti-tumor immunity, in metastatic breast cancer patients with CTCs. Flow cytometric data showed significant differences between circulating tumor cell (CTC) positive patients and CTC negative patients in their expression of TLR2 by CD8 positive cytotoxic T cells and TLR2, TLR4, TLR3, and TLR8 by CD11c positive dendritic cells (p < 0.05). Expression of TLR2, TLR4, and TLR8 was increased in CTC positive patients, whereas TLR3 expression was decreased in the dendritic cell population. [ABSTRACT FROM AUTHOR]

Abstract: In recent years, circulating tumor cells (CTCs) in metastatic cancer patients have been found to be a promising biomarker to predict overall survival and tumor progression in these patients. A relatively high number of CTCs has been correlated with disease progression and poorer prognosis. This study was designed to assess innate immune system function, known to be responsible for the immune defense against developing neoplasms, in metastatic cancer patients with CTCs. Our aim is to provide a link between indication of poorer prognosis, represented by the number of CTCs to the cytotoxic activity of natural killer cells, an important component of the innate immune system, and to represent a promising expanded approach to management of metastatic cancer patients with CTCs. Seventy-four patients, with metastatic breast, colorectal, or prostate cancer, were recruited for this study. Using a flow cytometric assay, we measured natural killer (NK) cell cytotoxicity against K562 target cells; and CTCs were enumerated using the CellSearch System. Toll-like receptors 2 and 4 expression was also determined by flow cytometry. We found that within each of our three metastatic cancer patient groups, NK cell cytotoxic activity was decreased in patients with a relatively high number of CTCs in peripheral blood compared to patients with a relatively low number of CTCs. In the breast and prostate cancer group, patients with CTCs greater than 5 had decreased NK cell cytotoxicity when compared to patients with less than 5 CTCs. In the colorectal cancer group, we found that 3 or more CTCs in the blood was the level at which NK cell cytotoxicity is diminished. Additionally, we found that the toll-like receptors 2 and 4 expression was decreased in intensity in all the metastatic cancer patients when compared to the healthy controls. Furthermore, within each cancer group, the expression of both toll-like receptors was decreased in the patients with relatively high number of CTCs, i.e. greater than 5 for the breast and prostate cancer group and greater than 3 for the colorectal cancer group, compared to the patients with relatively low number, i.e. less than 5 or 3, respectively. Treatment options to increase NK cell cytotoxic activity should be considered in patients with relatively high numbers of CTCs. [Copyright &y& Elsevier]

Abstract: The present investigation was designed to show the effect of molecular HLA class II DR and DQ allelic differences between donor and recipient on humoral antibody rejection identified by C4d peritubular capillary staining. The hypothesis is that expression of the DRβ1*1501, DQβ1*0602 allele in the donor kidney increases the likelihood of humoral antibody rejection. We found that 67% (n=18) of DR15 and/or DQ6 haplotype donor kidneys induced humoral antibody renal allograft rejection; 35% (n=40) of DR15 and/or DQ6 haplotype donor kidneys failed to induce humoral antibody renal allograft rejection (p=0.02). 42% (n=31) of C4d+ recipients had donors with DR15; 17% (n=42) of C4d recipients had donors with HLA-DR15 (p=0.01).We compared donor haplotype alleles of 4 C4d+ with 6 C4d− recipients by high resolution molecular typing; 3 of 4 C4d+ recipients had a donor with the DRβ1*1501/DQβ1*0602 allele. This allele was absent in all C4d− donors. 35% of C4d+ recipients had 2 DR mismatches when compared to 36% of C4d− recipients. Our results, suggest that the DRβ1*1501, DQβ1*0602 allele in the donor kidney increases the risk of humoral antibody episodes of acute rejection, and signals the need for C4d staining of renal biopsies. Future analysis of additional donor and recipient haplotypes will establish whether or not this is a useful predictor of humoral rejection episodes. [ABSTRACT FROM AUTHOR]

Abstract: C4d “staining” of interstitial capillaries of endomyocardial biopsies serves as an indicator of a humoral component of cardiac allograft rejection. In the present study, two cardiac allograft recipients were monitored serially for both cellular and humoral rejection. Cellular rejection, evaluated by light microscopy, and humoral rejection, judged by C4d immunofluorescent “staining”, were treated with appropriate immunosuppressants. Weekly serial biopsies of the first patient revealed maximal humoral rejection (3+) after 1 week but diminished to 2+ thereafter. Cellular rejection was graded as 3A after 3 weeks but declined steadily to negligible cellular rejection through week 15. Whereas, cellular rejection peaked at 14–21 days, humoral rejection was greatest in the early post-transplant period. Biopsies of the second patient for 12 weeks revealed grade 1A to 1B moderate cellular rejection. Humoral rejection peaked at 3+ “staining” for C4d after 1 week transplant, and then wavered between 2+ (moderate “staining”) and 1+ (weak “staining”). Results revealed the significance not only of traditional light microscopy in evaluating the severity of cellular rejection in endomyocardial biopsies of cardiac allotransplants, but also the value of C4d immunofluorescent “staining” of interstitial capillaries as an indicator of humoral rejection episodes, which may require modified therapy. [Copyright &y& Elsevier]

Dendritic cells (DCs) are a diverse group of hematopoietic-derived cells that play a prominent role in initiating the body''s immune response. Tumor necrosis factor-α (TNFα) aids CD34+ hematopoietic stem cells in the development of DCs. In this study, we aimed to further define the relationship between TNFα and DC maturation. CD34+ stem cells were isolated from umbilical cord blood and cultured using granulocyte–macrophage colony stimulating factor, stem cell factor, and varying concentrations of TNFα. An anti-TNF receptor 1 (anti-TNFR1) antibody was used to show the specificity of TNFα. Flow cytometry and light microscopy analyses were performed at days 0, 7, and 14 of culture, revealing mature DCs at all concentrations of TNFα by day 14, excluding those with anti-TNFR1 bound to the cell''s TNF receptor 1. DCs possessed a characteristic veiled appearance and were consistent with a DC panel of surface markers. TNFα was essential to the development of DCs, as those with bound anti-TNFR1 were virtually unable to develop into DCs. Increasing TNFα enhanced the survival of culturing stem cells and resulted in a parallel increase in day 14 DCs. Although increases in TNFα produced more DCs, these cells were not as phenotypically mature, expressing less CD80 than those receiving only a single initial dosage of TNFα. These studies support the prevalence of large numbers of DCs under inflammatory conditions, such as the rheumatoid joint, where local concentrations of TNFα are high. [Copyright &y& Elsevier]