Changes In Mean Fluorescence Intensity Research Articles

Monocyte response to SARS-CoV-2 protein ORF8 is associated with severe COVID-19 infection in patients with chronic lymphocytic leukemia.

The open reading frame 8 (ORF8) protein, encoded by the SARS-CoV-2 virus after infection, stimulates monocytes/macrophages to produce pro-inflammatory cytokines. We hypothesized that a positive ex vivo monocyte response to ORF8 protein pre-COVID-19 would be associated with subsequent severe Coronavirus disease 2019 (COVID-19). We tested ORF8 ex vivo on peripheral blood mononuclear cells from 26 anonymous healthy blood donors and measured intracellular cytokine/ chemokine levels in monocytes by flow cytometry. The percentage of positive monocyte staining in the sample and change in mean fluorescence intensity (ΔMFI) after ORF8 were used to calculate the adjusted MFI for each cytokine. We then tested pre-COVID-19 peripheral blood mononuclear cell samples from 60 chronic lymphocytic leukemia (CLL) patients who subsequently developed COVID-19 infection. Severe COVID-19 was defined as hospitalization due to COVID-19. In the 26 normal donor samples, the adjusted MFI for interleukin (IL)-1β, IL-6, IL-8, and CCL-2 were significantly different with ORF8 stimulation versus controls. We next analyzed monocytes from pre-COVID-19 PBMC samples from 60 CLL patients. The adjusted MFI to ORF8 stimulation of monocyte intracellular IL-1β was associated with severe COVID-19 and a reactive ORF8 monocyte response was defined as an IL-1β adjusted MFI ≥0.18 (sensitivity 67%, specificity 75%). The median time to hospitalization after infection in CLL patients with a reactive ORF8 response was 12 days versus not reached for patients with a non-reactive ORF8 response with a hazard ratio of 7.7 (95% confidence interval: 2.4-132; P=0.005). These results provide new insight on the monocyte inflammatory response to virus with implications in a broad range of disorders involving monocytes.

The natural history of de novo donor-specific HLA antibodies after kidney transplantation.

BackgroundDe novo donor-specific HLA antibodies (dnDSA) are key factors in the diagnosis of antibody-mediated rejection (ABMR) and related to graft loss.MethodsThis retrospective study was designed to evaluate the natural course of dnDSA in graft function and kidney allograft survival and to assess the impact of mean fluorescence intensity (MFI) evolution as detected by annual Luminex® screening. All 400 kidney transplant recipients with 731 dnDSA against the last graft (01/03/2000-31/05/2021) were included.ResultsDuring 8.3 years of follow-up, ABMR occurred in 24.8% and graft loss in 33.3% of the cases, especially in patients with class I and II dnDSA, and those with multiple dnDSA. We observed frequent changes in MFI with 5-year allograft survivals post-dnDSA of 74.0% in patients with MFI reduction ≥ 50%, 62.4% with fluctuating MFI (MFI reduction ≥ 50% and doubling), and 52.7% with doubling MFI (log-rank p < 0.001). Interestingly, dnDSA in 168 (24.3%) cases became negative at some point during follow-up, and 38/400 (9.5%) patients became stable negative, which was associated with better graft survival. Multivariable analysis revealed the importance of MFI evolution and rejection, while class and number of dnDSA were not contributors in this model.ConclusionIn summary, we provide an in-depth analysis of the natural course of dnDSA after kidney transplantation, first evidence for the impact of MFI evolution on graft outcomes, and describe a relevant number of patients with a stable disappearance of dnDSA, related to better allograft survival.

The different predictive effects of the intensity and proportion of CD20 expression on the prognosis of B-lineage acute lymphocyte leukemia.

The prognostic effects of the CD20 positivity have been studied extensively in B‐lineage acute lymphocyte leukemia (B‐ALL) patients, but the results remain controversial. The aim of this study is to investigate the different predictive effects of the intensity and proportion of CD20 expression on the prognosis for B‐ALL patients by retrospective analysis. The mean fluorescence intensity (MFI) and percentage of CD20 on B‐ALL cells from 206 patients with B‐ALL were dynamically measured by flow cytometry, and their optimal cut‐off values were determined using the receiver operating characteristic curve. Changes in MFI and percentage of CD20 at various time points and their relationship with prognosis were analyzed. We found that a low baseline CD20 MFI or high CD20 proportion was significantly associated with shorter 5‐year overall survival and progression‐free survival, and the combination of these two factors could more accurately predict worse survival for B‐ALL patients. Furthermore, low CD20 MFI or a high CD20 proportion had different predictive effects for ALL patients with different clinical characteristics and could serve as an independent risk factor for adverse prognosis. There were significant decreases in both the intensity and proportion of CD20 after recurrence in the absence of rituximab treatment, particularly with CD20 intensity. Notably, the decrease of CD20 intensity after recurrence indicated a more shortened survival time. Finally, we conclude that a low intensity or high proportion of CD20 expression may be used as an indicator for inferior prognosis for B‐ALL patients. CD20 intensity is more likely to be a more universal biomarker for worse prognosis.

Prospective De Novo Donor-Specific Antibody Monitoring in Renal Transplant Patients

De novo donor-specific antibodies (dnDSA) are associated with antibody-mediated rejection leading to kidney transplant failure. However, many transplant patients are stable with no signs of graft dysfunction at the time of dnDSA detection at screening test. We prospectively studied 26 kidney transplant patients for 3 years, with dnDSA detected using the Luminex single-antigen bead assay in a routine test. Proteinuria and estimated glomerular filtration rate were evaluated along with dnDSA monitoring at least annually. Graft loss associated with dnDSA occurred in 8 (31%) patients, 14 ± 11 months after the initial detection of dnDSA. These patients had more frequently multiple dnDSA (P=.004) and remarkable proteinuria, more than 1 g daily (P < .001). The remaining 18 patients were followed for 38 ± 15 months and considered stable as there was no deterioration regarding estimated glomerular filtration rate and proteinuria. In 7 (39%) of these patients, dnDSA waned and were not detected anymore at follow-up. Antibodies against HLA class I had a significantly (P < .001) lower mean fluorescence intensity (MFI) (2603 ± 1098) compared with those against HLA class II (11,109 ± 6414). In regression analysis, they were predictive of dnDSA wane (P=.043; odds ratio, 0.18; 95% confidence interval, 0.04-0.94). Despite fluctuations during follow-up, there was no significant change in MFI for those who retained the dnDSA. The presence of dnDSA is transient in a significant proportion of stable renal transplant patients, especially in those with antibodies against HLA class I and a low MFI. Multiple dnDSA and severe proteinuria adversely affect renal graft survival.

Comprehensive Longitudinal Surveillance of the IgG Autoantibody Repertoire in Established Systemic Lupus Erythematosus.

To investigate the role of epitope spreading in established systemic lupus erythematosus (SLE). IgG autoantibody reactivity with 398 distinct recombinant proteins was measured over a period of 6 years in 69 SLE patients and compared to that in 45 controls. Changes in mean fluorescence intensity (MFI), number of autoantibodies to distinct antigens, and reactivity with distinct clones of established antigenic targets (e.g., U1 RNP, Sm, and ribosomal P) representing epitope fine mapping were assessed. Linear mixed modeling, adjusted with Bonferroni correction for age and sex, was applied. The total number of autoantibodies, mean MFI, and number of autoantibodies in epitope fine mapping were higher in SLE patients compared to controls (P < 0.0001). The total number of antibodies to distinct autoantigens remained stable over time, while the mean MFI decreased over time in SLE (P < 0.021). SLE patients showed variable recognition of epitopes in fine mapping over time. In particular, in SLE patients, more clones of the U1 RNP complex were recognized at the time of new organ involvement (+0.65) (P = 0.007). Mean MFI was higher in patients with lupus nephritis (P = 0.047). The time-averaged MFIs of 22 individual autoantibodies (including double-stranded DNA [dsDNA]) were higher, after Bonferroni correction, in SLE (P < 0.0001). The MFIs of dsDNA and histone cluster 2 H3c were associated with scores on the Systemic Lupus Activity Measure (P < 0.0001). Longitudinal surveillance of the IgG autoantibody repertoire in established SLE reveals evidence of sustained breadth of autoantibody repertoire without significant expansion. Associations of disease activity with dsDNA and with histone H3 autoantibodies were confirmed.

Signaling Responses to Stroma-Derived Soluble Factors Are Associated with Outcome and with Expression of Microenvironment-Related Genes in Pediatric AML

Pediatric AML has a relapse rate approaching 40%, indicating that resistance to chemotherapy remains a critical problem. We are focused on identifying and overcoming environment-mediated mechanisms of resistance. We previously reported that the sensitivity of G-CSF- and IL-6-induced STAT3 signaling was significantly associated with outcome in pediatric AML patients. In this follow-up study, we used conditioned medium (CM) from HS5 stromal cells as a more physiological stimulus to evaluate the capacity of primary AML cells to activate signaling pathways.We studied 111 diagnostic bone marrow samples from patients who enrolled on Children's Oncology Group (COG) AML treatment studies, AAML03P1 or AAML0531, and provided informed consent to bank bone marrow. The same chemotherapy backbone was used in both trials. All samples had at least 70% viability after thawing. Thawed cells were divided into aliquots for stimulation with 50% CM, 10 ng/ml G-CSF, or 5 ng/ml IL-6. Unstimulated cells were used for isotype controls and for determination of basal signaling levels. Following 15 min stimulation, cells were fixed and processed for FACS analysis of CD45, pY-STAT3, pY-STAT5, pERK1/2, and pAKT. Responses were expressed as the fold change in mean fluorescence intensity for stimulated cells over unstimulated cells (ΔMFI).The ΔMFIs for pY-STAT3 and pY-STAT5 varied between 0.1 and 31.8. The ΔMFIs for pERK1/2 and pAKT rarely exceeded 2. Samples with a robust response to one stimulus generally responded robustly to the others. The Pearson correlation coefficient (r) for CM-induced pY-STAT3 v. G-CSF-induced pY-STAT3 was 0.8511 (p<0.00001). The CM-induced pY-STAT5 and G-CSF-induced pY-STAT5 responses were significantly but less strongly correlated (r=0.2765; p=0.0043). Cut point analyses identified response thresholds that distinguished patients with higher EFS from those with lower EFS. We found that higher CM-induced pY-STAT5 was significantly associated with an inferior EFS (Figure 1), with HR 2.06 for those with ΔMFI > 1.96 (p=0.034). Previously we found that higher inducible pY-STAT3 was associated with significantly better EFS (Redell, et al, Blood, 2013; Long, et al, Oncotarget, 2017). In this study, no cut point for CM-induced pY-STAT3 ΔMFI distinguished patients with good or poor EFS. Cytogenetics and FLT3/ITD were not significantly different for groups above and below the ΔMFI cut points. The finding that robust STAT5 signaling is associated with inferior EFS suggests that a factor that signals primarily through STAT5 and not STAT3 (e.g. GM-CSF, IL-3, FL) could contribute to treatment resistance and relapse.Interestingly, most of the samples that failed to activate STAT3/5 pathways in response to G-CSF also failed to respond to the cocktail of factors in CM, suggesting a generalized signaling dysfunction. To investigate intrinsic gene expression differences, we leveraged existing RNA-seq data for the samples for which we generated signaling responses. We obtained RNA-seq data for 27 samples from the NCI Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. RNA-seq data for an additional 45 samples were provided through collaboration with the University of Washington. Of these 72 samples, 29 samples were resistant to CM-induced pY-STAT3 (ΔMFI <2, CM-R) and 43 samples were sensitive (ΔMFI > 2, CM-S). Raw counts were normalized and analyzed by EdgeR with GLM algorithm and blocking to control for batch effects from the two datasets. We found that 219 features were significantly differentially expressed (DE; FDR <0.05), with 192 being expressed more highly in the CM-R group. Gene set enrichment analysis identified the “matrisome” gene set, including genes encoding growth factors and matrix proteins, as being significantly enriched in the CM-R group. For example, genes encoding G-CSF, GM-CSF, WNT7B, and integrin B3 were upregulated in CM-R samples. Additionally, a number of non-HOX homeobox genes, including DLX2, DLX3 and MSX2, were increased in CM-R samples. There was no difference in the mean %blasts for the samples in the CM-R and CM-S groups, arguing against the increased expression of matrix-related genes being due to a higher proportion of non-blast cells in the CM-R samples. Our integration of gene expression with inducible STAT3/5 responses will yield novel insights into extrinsic survival signaling and mechanisms of dysfunction. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prognostic tools to assess candidacy for and efficacy of antibody-removal therapy.

Currently, the ability to predict or monitor the efficacy of HLA antibody-removal therapies is deficient. We previously reported that titration studies are a consistent and accurate means of assessing antibody strength. To test whether titration studies can also predict which patients are better candidates for desensitization, we studied 38 patients from 3 centers (29 receiving plasmapheresis/low-dose intravenous immunoglobulin [IVIg]; 9 patients receiving high-dose IVIg). For patients undergoing plasmapheresis/low-dose IVIg, antibody titer reduction correlated with number of treatment cycles for both class I and II antibodies but only up to approximately 4 cycles. Reduction in titer slowed with additional cycles, suggesting a limit to the efficacy of this approach. Furthermore, initial titer (predesensitization) can guide the selection of candidates for successful antibody-removal treatment. In our experience, patients with antibodies at an initial titer>1:512 could not be reduced to the goal of a negative lymphocyte crossmatch, corresponding to a 1:16 titer, despite a significant increase in the number of treatment cycles. Change in mean fluorescence intensity (MFI) value did not correlate with success of treatment if initial MFI values were>10 000, likely due to single antigen bead saturation. Overall, we present a potential prognostic tool to predict candidacy and a monitoring tool to assess efficacy of desensitization treatment.

P241 A study of immunocomplex capture fluorescence analysis using lymphocyte and monocyte fraction by freezing isolation

Aim Immunocomplex capture fluorescence analysis (ICFA) is a novel method to predict rejection/acceptance responses in organ transplantation. The method is less susceptible to the effect of drug compared with traditional methods. The method uses fluorescent beads to sensitively detect antibodies directed against HLA I/II antigens on blood cells.In this study, the changes in mean fluorescence intensity (MFI) control values of lymphocyte and monocyte fraction from donor cells before cryopreservation and donor cells after one month of cryopreservation were monitored using ICFA for a lymphocyte crossmatch. Methods The subjects were patients who were going to undergo living-donor kidney transplantation. ICFA method for a lymphocyte crossmatch was performed. The stability of MFI control values was monitored for donor cells before cryopreservation and those after one month of cryopreservation. Results The monitoring results showed that the MFI control value of lymphocyte and monocyte fraction from donor cells after one month of cryopreservation was 9.4% higher than that of lymphocyte and monocyte fraction from donor cells before cryopreservation. On the other hand, the MIF control value of leukocytes after one month of cryopreservation using the conventional method was 100%. Conclusions More reliable donor-specific HLA antibody test results can be obtained by lymphocyte crossmatch test using lymphocyte and monocyte fraction by freezing isolation from cryopreserved donor cells.

Quantitative Evaluation of the Impact of Ethylenediaminetetraacetic Acid Pretreatment on Single-Antigen Bead Assay.

BackgroundEthylenediaminetetraacetic acid (EDTA) pretreatment has been shown to overcome complement interference in the single-antigen bead (SAB) assay. However, a quantitative evaluation of its impact on the assay for preemptive application to diverse clinical samples is still lacking.MethodsSerum samples from 95 renal transplant candidates were tested with and without EDTA-pretreatment in parallel. Changes in mean fluorescence intensity (MFI) values were analyzed to determine the impact of EDTA-pretreatment and the characteristics of complement interference.ResultsMFI values from EDTA-treated and untreated sera showed good correlations (r = 0.99) and were linear after excluding outliers (slopes, 1; intercepts, −63.7 and −24.2 for class I and II, respectively). Using an assay cutoff of 2000 MFI, positive/negative assignments were concordant for 99% of the 9215 class I beads and 9025 class II beads tested. As defined by an MFI increment above 4000 after EDTA pretreatment, complement interference affected 172 class I beads in 12 samples (12.6%) and 60 class II beads in 7 samples (7.4%), and the findings were supported in 83% and 86% of these samples by dilution studies. In a case study, EDTA pretreatment prevented falsely low MFI values and facilitated the interpretation of titration curves. Finally, EDTA pretreatment reduced the coefficient of variance (CV) by 2.1% and 2.4% for class I and II beads respectively (P < 0.0001).ConclusionsIt is safe to preemptively treat all clinical samples with EDTA before SAB assay to prevent false negative results or falsely low MFI values. EDTA pretreatment has the added benefit of improved assay precision.

Protein A-Based Immunoadsorption Is More Efficient Than Conventional Plasma Exchange to Remove Circulating Anti-HLA Antibodies

We retrospectively evaluated the ability of protein-A immunoadsorption (IA) as compared to that of conventional plasma exchanges (PE) in reducing the mean fluorescence intensity (MFI) of anti-HLA antibodies assessed by the single antigen assay in sensitized renal transplant recipients. Change in MFI of 441 anti-HLA antibodies was measured after 1 single session of IA or after 3 consecutive daily PE sessions. While both strategies were able to significantly lower the amount of anti-HLA antibodies, the relative reduction in MFI was higher after IA as compared to PE (-69 vs. -58%, respectively, p = 0.003). This better efficacy of IA was observed despite a lower total volume of treated plasma (105 ± 6 vs. 160 ± 16 ml/kg after IA and after PE, respectively). Our data suggest a higher efficiency of IA over conventional PE sessions to remove anti-HLA antibodies and call for a larger evaluation of IA to confirm its potential added value in desensitization protocols.

Abstract 4206: Increased responsiveness to ligand stimulation of the STAT pathway at relapse in acute myelogenous leukemia

Abstract Background: We have shown that Stat pathway sensitivity to G-CSF and IL-6, measured by the increase in tyrosine phosphorylated Stat3 (pY-Stat3), is associated with clinical outcome in pediatric AML patients. These results support a possible relationship between chemoresponsiveness and ligand-induced Stat3 response. We hypothesized that consistently altered changes in Stat3 signaling between diagnosis and relapse would represent advantageous adaptations that promote chemotherapy resistance in pediatric AML. Methods: 25 sample pairs from initial diagnosis and relapse from pediatric AML patients treated on the COG frontline trial AAML0531 were analyzed. After thawing, ≥80% viability was confirmed by Trypan exclusion. Constitutively phosphorylated Stats (pY-Stat3, pS-Stat3, and pY-Stat5), total Stat3 (TStat3), G-CSF receptor, and gp130 were measured in unstimulated cells. Additionally, cells were stimulated for 15 minutes with 10 or 100 ng/ml G-CSF, or 5 or 50 ng/ml IL-6 with 10 or 100 ng/ml soluble IL-6 receptor, respectively, for measurement of ligand-induced pStats. Data were collected on the LSR II (BD) and analyzed with FCS Express 4 (DeNovo). For the ligand-induced pStats, data are expressed as the fold change in mean fluorescence intensity (ΔMFI) of the stimulated sample compared to the corresponding unstimulated sample. Constitutive pStats, receptors, and TStat3 data are expressed as percent in the positive region, as defined by the upper limit of the signal in the isotype control. The Wilcoxon Signed Rank test was used to test for significant differences in parameters between diagnosis and relapse. Results: 24/25 sample pairs had adequate cell numbers and viability for analysis. At both doses of G-CSF, 21/24 pairs demonstrated an increase in pY-Stat3 ΔMFI. At the lower G-CSF dose, the mean ±SEM ΔMFI increased from 1.41±0.13 to 2.36±0.28 (p=0.0001). At the higher dose, ΔMFI increased from 1.65±0.20 to 2.71±0.33 (p=0.0002). Similarly, at both doses of IL-6, 17/24 pairs demonstrated an increase in pY-Stat3 ΔMFI. At the lower IL-6 dose, the ΔMFI of pY-Stat3 increased from 1.25±0.09 to 1.56±0.16 (p=0.0168). At the higher dose, ΔMFI increased from 1.49±0.17 to 2.01±0.24 (p=0.0051). Only 3/24 pairs showed &amp;gt;15% increase in G-CSF receptor expression, while 10/24 had a &amp;gt;15% increase in gp130 expression. No significant changes were seen in constitutive activity of pStats, or TStat3 expression. Conclusions: Our data demonstrate that ligand-induced Stat3 signaling pathways evolve to become stronger at relapse in pediatric AML. More robust signaling was not due to increased expression of total Stat3 and was rarely associated with increased receptor expression. Our data suggest that ligand-induced Stat pathway activation may be promoting survival in relapsed AML. Our results provide support for further development and evaluation of targeted agents against this pathway in AML. Citation Format: Alexandra M. Stevens, Marcos Ruiz, Michele S. Redell. Increased responsiveness to ligand stimulation of the STAT pathway at relapse in acute myelogenous leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4206. doi:10.1158/1538-7445.AM2014-4206

Bone Marrow Stromal Cells Enhance DNA Damage Signaling Independent Of Stat3 Activation In Pediatric AML

Despite aggressive treatments, death from chemoresistant disease still occurs for almost half of children with AML. It is clear that the bone marrow microenvironment protects a subset of cells from chemotherapy, but the mechanisms of resistance remain unknown. We recently reported that patients whose AML blasts activated Stat3 in response to two bone marrow-derived ligands, G-CSF and IL-6, had a significantly superior survival rate, compared to patients whose blasts failed to activate Stat3 (Redell, et al, 2013, Blood121:1083). In this followup study of a subset of these patients, we further investigated the basis for the failure of the Stat3 response, as well as mechanisms of environment-mediated chemotherapy resistance. Our hypotheses were A) Stat3 responsiveness to G-CSF/IL-6 reflects the cell's general ability to respond to ligand stimulation, rather than a specific defect in a Stat3 pathway; and B) Stat3 responsiveness correlates with other functional responses to stromal exposure, including survival, proliferation, and chemotherapy resistance. Second samples from 27 patients were studied by FACS analysis of intracellular markers. To study stroma-induced changes, AML cells were co-cultured on stromal cells overnight, or briefly stimulated with stroma-conditioned medium (CM). We used HS5 cells, which secrete high levels of many soluble factors, and HS27A cells, which secrete very few factors. The stromal cell lines express mOrange for exclusion by FACS.First, primary AML cells were stimulated with G-CSF (10 ng/ml) or IL-6 + soluble IL-6R (5 + 10 ng/ml) for 15 min, then pY-Stat3 (PE) and pERK1/2 (AlexaFluor488) were measured by FACS. Data were acquired on the LSRII (BD) and analyzed with FCSExpress4 (DeNovo). Ligand responses were expressed as the fold change in mean fluorescence intensity over unstimulated cells (ΔMFI). We found a strong correlation between G-CSF-induced pY-Stat3 ΔMFI and pERK1/2 ΔMFI (R=0.858, p<0.001), suggesting that failure to activate Stat3 in unresponsive samples was not due to a Stat3-specific block. As expected, HS5 CM strongly activated Stat3 in samples that also responded to G-CSF and IL-6, while HS27A CM did not activate Stat3 in any samples. We compared responses to G-CSF v. HS5 CM, and found a strong correlation for pY-Stat3 ΔMFI (R=0.886, p<0.001) and for pERK1/2 ΔMFI (R=0.907, p<0.001). Samples that failed to respond to G-CSF also failed to respond to a combination of multiple soluble factors, supporting the idea that signaling function or dysfunction is a generalized property of AML cells.To further investigate functional changes induced in AML cells by stromal cells, we co-cultured blasts from 17 samples overnight with HS5 or HS27A cells. We labeled cleaved PARP (AlexaFluor647) to measure viable cells (%cPARP-) in each condition. Within the viable AML cell population, we measured the proliferative fraction (Ki67-FITC+ or Ki67-AlexaFluor700+) and the fraction with active DNA damage signaling (γH2AX-AlexaFluor488+). We found a small increase in the %viable for co-cultured cells v. cultured alone (p<0.05 for HS27A v. alone; Wilcoxon Signed Rank Test) and in the %Ki67+ (p<0.005 for HS27A v. alone). Moreover, increases were seen in γH2AX, with 5.6 ±6%+ in cells cultured alone, v. 7.9 ±6% in HS5 co-cultures (p<0.05) and v. 11.5 ±9% in HS27A co-cultures (p<0.001). For 10 samples, cell number was sufficient for additional overnight cultures with etoposide (up to 10 mM). Both stromal cell lines enhanced DNA damage signaling, reflected by an increase in γH2AX from 7 ±10% in etoposide-treated cells cultured alone, to 18 ±19% in HS5 co-cultures (p<0.05) and 19 ±16% in HS27A co-cultures (p<0.005). To test whether HS5 CM-induced pY-Stat3 or pERK1/2 were related to these parameters, we examined bivariate correlations. We found no correlation in most cases, but an inverse relationship between both ΔMFIs and %viable after etoposide (pY-Stat3: R=-0.733, p<0.05 and pERK1/2: R=-0.683, p<0.05). In other words, samples characterized by signaling failure tended to have better survival after etoposide treatment. This result is consistent with our prior study showing that patients whose blasts failed to activate Stat3 had poorer outcome than those whose blasts activated Stat3 normally. In summary, we found that environment-induced Stat3 signaling did not promote chemoresistance, and that stromal cells may instead confer chemoresistance by enhancing DNA damage signaling. Disclosures:No relevant conflicts of interest to declare.

Measurement of donor‐specific HLA antibodies following plasma exchange therapy predicts clinical outcome in pediatric heart and lung transplant recipients with antibody‐mediated rejection

Therapeutic plasma exchange (TPE) is an increasingly utilized immunosuppressive adjunct for treatment of antibody-mediated rejection (AMR) following organ transplantation. TPE works through removal of donor-specific HLA antibodies (DSAs) in the recipient's plasma. However, there is no clear laboratory measure evaluating efficacy of removal of DSAs or predicting clinical outcome. We hypothesized that semi-quantitative DSA measurement by multiplex HLA antibody immunoassay may provide qualitative and quantitative data for DSA clearance and predict treatment efficacy. To evaluate this, we retrospectively investigated DSA concentrations and clinical outcome for 21 pediatric patients who received 31 cycles of TPE peri-operatively as an adjunct treatment for transplantation in the setting of a positive cytotoxic crossmatch (CXM) and in recipients with AMR following heart or lung transplantation. Immunoassay measurement of DSAs during 15/20 cycles correlated significantly with clinical outcome in the AMR treatment group (P = 0.02), demonstrating the utility of DSA measurement in predicting clinical outcome. In contrast, immunoassay correlated with clinical outcome in only 7/11 patients treated peri-operatively with TPE for CXM-positive transplantations (P = 0.58). Changes in mean fluorescence intensity (MFI) for the DSAs correlated better with clinical response than surrogate CXM titers in a subset of patients. We conclude that semi-quantitative measurement of DSAs by immunoassay can predict clinical response to TPE for treatment of AMR is more reliable than surrogate CXM titer, and should be used to guide TPE treatment of AMR.

Increased Responsiveness to Ligand Stimulation of the STAT Pathway At Relapse in Acute Myelogenous Leukemia

AbstractAbstract ▪3544▪This icon denotes a clinically relevant abstract Background:Acute myeloid leukemia (AML) is a very heterogeneous disease and changes in characteristics such as immunophenotype and mutation profiles are known to occur between diagnosis and relapse. Alterations in signaling pathways are likely to occur as well, but have not yet been reported. Our lab has recently shown that Stat3 signaling profiles are associated with outcome in pediatric AML patients. Specifically, patients whose blasts were sensitive to both G-CSF and IL-6, measured by increased tyrosine phosphorylation of Stat3 (pY-Stat3), had significantly improved event-free and overall survival compared to those whose blasts were resistant to one or both of the tested ligands. These results support the importance of ligand responsiveness within the STAT pathway in relation to chemoresponsiveness in AML. We hypothesized that comparison of STAT signaling pathways in paired diagnostic and relapse samples would identify changes that are likely to represent strengthening of pathways involved in chemoresistance. Methods:Twenty two sample pairs from initial diagnosis and relapse from pediatric AML patients treated on the Children’s Oncology Group protocol AAML0531 have been analyzed for this study. After thawing, ≥80% viability was confirmed by Trypan blue stain. Constitutively phosphorylated STATs (including pY-Stat3, pS-Stat3, and pY-Stat5), total Stat3 (TStat3), pY418-Src, G-CSF receptor, and gp130 were measured in unstimulated cells. Additionally, cells were stimulated for 15 minutes with 10 or 100ng/ul G-CSF, or 5 or 50ng/ul IL-6 with 10 or 100 ng/ul soluble IL-6 receptor, respectively, for measurement of ligand-induced pStats. Data were collected on the LSR II (BD) and analyzed with FCS Express 4 (DeNovo). For the ligand-induced pStats, data are expressed as the fold change in mean fluorescence intensity (DMFI) of the stimulated sample compared to the corresponding unstimulated sample. Constitutive pStats, receptors, TStat3, and pY418-Src data are expressed as percent positive compared to isotype control. The Wilcoxon Signed Rank test was used to test for significant differences in parameters between diagnosis and relapse. Results:Of the 22 sample pairs thawed, 21 had adequate cell numbers and viability for analysis. Increases in ΔMFI of pY-Stat3 between diagnosis and relapse were seen in response to both doses of G-CSF. At the lower dose, ΔMFI increased from 1.45±0.14 to 2.30±0.33 (p=0.0023), with increases demonstrated in 16/21 pairs. At the higher dose level, ΔMFI increased from 1.70±0.20 to 2.68±0.38 (p=0.0019) with increases in 16/21 pairs. Similarly, in response to the lower IL-6 dose, the ΔMFI of pY-Stat3 increased from 1.29±0.10 to 1.58±0.18 (p=0.0183), with increases in 14/21 pairs while at the higher dose, ΔMFI increased from 1.53±0.20 to 2.07±0.27 (p=0.0079) with increases in 12/21 pairs. Evaluated parameters that did not change significantly between diagnosis and relapse included G-CSF receptor expression (74.09% to 80.16; p=0.1526), gp130 expression (51.63% to 60.52%; p=0.0569), constitutive activity of pYStat3 (23.60% to 26.63%; p=0.0962), and TStat3 expression (35.32% to 43.96%; p=0.053). Though the changes were small, pY418-Src was found to be significantly increased between paired samples (0.90% to 2.58%; p=0.0011). Conclusions:STAT pathway signaling patterns evolve between diagnosis and relapse in pediatric AML. Our data demonstrate that ligand-induced Stat3 signaling pathways evolve to become stronger at relapse. This suggests that the resistance seen to chemotherapy in relapsed patients may be in part due to the increased activity of this anti-apoptotic pathway in response to growth factors and cytokines present in the bone marrow niche. The increases in activity within the STAT pathway at relapse provide support for further development and evaluation of targeted agents against this pathway in relapsed patients with AML. Disclosures:No relevant conflicts of interest to declare.

Protein Rebinding to a Surface‐Confined Imprint

AbstractA novel strategy to prepare a selective ultrathin molecularly imprinted polymer (MIP) film directly on the gold‐based transducer surface for the peptide and protein detection in aqueous solution is demonstrated using a combination of epitope‐ and electrochemical surface imprinting approach. The synthetic peptide derived from the surface‐exposed C‐terminus of cytochrome c (Cyt c, residues 96–104) is selected as the template for the imprinting. It is labeled with a fluorescent dye in order to quantitatively evaluate all stages of the imprinting process in terms of changes in mean fluorescence intensity. The labeled peptide template is first chemisorbed on the gold surface as an oriented submonolayer through an additional C‐terminal cysteine. After electropolymerization, the template is stripped off electrochemically. To allow the imprinted sites to be confined to the surface, the film thickness is controlled to be comparable to the thickness of the peptide layer. This is achieved by the electropolymerization of scopoletin. Recognition capabilities of the films are characterized and the resulting MIP film is able to selectively capture the template peptide and the corresponding target protein. In case of the peptide recognition, the MIP film can discriminate even the single amino acid mismatched sequences of the target peptide.

Cellular function of the mononuclear phagocyte system (MPS) as a phenotypic probe for pharmacokinetics (PK) and pharmacodynamics (PD) of PEGylated liposomal doxorubicin (PLD) in patients with recurrent ovarian cancer.

2591 Background: A phenotypic probe is a test that can be administered to a patient as a potential indicator of a drug’s PK/PD which can then be used to individualize therapy. Since nanoparticles are cleared via the MPS, including blood monocytes (MO), we hypothesize that circulating MO could potentially play a major role in, and be a surrogate marker of nanoparticle clearance (CL). Furthermore, we postulate that the ability to measure MO functional activity in blood samples can be used to predict CL and subsequently PD endpoints such as progression free survival (PFS) and hand-foot syndrome (HFS), in patients (pt). Methods: PLD 30/40 mg/m2 IV x 1 with or without carboplatin (AUC = 5 mg/mL/min) was administered to pts (n=10) with recurrent ovarian cancer. Serial PK samples were obtained at time 0 to day 28 post dose. Plasma was processed to measure encapsulated and released doxorubicin using solid phase separation and HPLC. Data was analyzed with WinNonlin to obtain PK parameters. MO and granulocyte phagocytic function (uptake of FITC-labeled opsonized E. Coli) and oxidative burst activity (intracellular oxidation of dihydrorhodamine 123) were assessed via flow cytometry at 0, 48, 96 h, and on day 28. For both assays, changes in mean fluorescence intensity of the gated MO population, following incubation of whole blood with the appropriate substrate, served as an index of activity. Pts were followed from the first dose of PLD until time of disease progression. Results: There was a direct linear relationship between encapsulated doxorubicin CL and both phagocytosis (R2 = 0.87) and oxidative burst activity (R2 = 0.64) in blood MO. Similarly, phagocytosis (R2 = 0.77) and oxidative burst probes correlated with PFS (R2 = 0.67) in the 4 pts who progressed while on PLD. Oxidative burst also correlated with degree of HFS (R2 = 0.56). There was no relationship between phagocytosis and oxidative burst in granulocytes and PK/PD of PLD. Conclusions: These findings indicate that probes of MPS function predict PLD CL, HFS and PFS and thus may be useful for individualizing PLD therapy in ovarian cancer and other malignancies.

Evaluation of Residual Promoter Activity in γ-Retroviral Self-inactivating (SIN) Vectors

Therapeutic gene delivery mediated by retroviral vectors has the advantage of stable integration into the host genome. A major safety concern for gene delivery achieved by murine leukemia virus (MLV)-based retroviral vectors is the activation of adjacent cellular genes including oncogenes following integration into the host genome. Self-inactivating (SIN) vectors lacking viral enhancers/promoters in their 3' long terminal repeat (LTR) have been proposed as a means of overcoming this safety concern. However the MLV-based SIN vectors currently used by laboratories to assess insertional mutagenesis, integration site selection, and the potency of transgene expression are not uniform in the composition of their 3' LTRs. We constructed a series of SIN vectors representative of the currently employed vectors, but lacking an internal promoter. Green fluorescent protein (GFP) was used as a reporter gene. Target cells exposed to these vectors were evaluated for number of integrants and GFP expression at the messenger RNA (mRNA) level and protein level. We found that viral promoter activity in the 3' LTR is not attenuated in many currently employed SIN vectors. These results suggest that the influence of strong residual promoter activity should be taken into consideration when interpreting experimental results obtained using SIN vectors in gene therapy research.

Flow cytometry for assessment of the efficacy of siRNA

Small interfering RNA (siRNA) has emerged as a powerful tool to study the loss-of-function phenotype by specifically silencing a target gene. The success of gene silencing depends on the choice of appropriate target sequence, and requires a rapid and sensitive assay for quantifying siRNA efficiency. Conventional assays include Western blotting and reverse transcription coupled with polymerase chain reaction. However, these methods are somewhat inaccurate owing to the variation in transfection efficiency. To avoid this, we have developed a flow cytometric method to provide a quantitative analysis of siRNA efficacy. We constructed a novel vector pHyper1G, which can express enhanced green fluorescent protein (EGFP). It contains a H1 promoter, which can drive expression of short hairpin RNA (shRNA) directed against a target gene. The target gene was cloned into pDsRed1-N1. The resulting construct can express a fusion protein between target protein and DsRed. These vectors were co-transfected into 293T cells. The transfected cells were analyzed by flow cytometry. The percentage of EGFP+, DsRed+ cells and the change in mean fluorescence intensity (MFI) of DsRed channel indicate changes in expression of target gene in a cell population, and hence the efficacy of the corresponding shRNA. In addition, the cells transfected with pHyper1G derivative were sorted, and analyzed for the activity of target gene. We designed an oligonucleotide duplex encoding shRNA against glucose-6-phosphate dehydrogenase (G6PD) gene, and cloned this into pHyper1G. The resulting vector pHyper1G101 effectively knocked down the expression of G6PD-DsRed from pDsRed301, as shown by significant reduction in both the percentage of EGFP+, DsRed+ cells, and MFI. Changes in these parameters were consistent with decreases in protein level and activity of G6PD. Moreover, albeit at low suboptimal transfection efficiency, cells transfected with pHyper1G101 alone were successfully sorted for those expressing shRNA, and their G6PD activity was found to be suppressed. Flow cytometric analysis provides a reliable assessment of the efficiency of siRNA in a cell population. This method can be easily automated and used for screening of appropriate shRNAs against certain genes. Moreover, using the present system, we can deliberately sort for and analyze those cells expressing shRNA. This can be applied to the hard-to-transfect cell types, and greatly facilitates the analyses of gene silencing in these cells.

Cellular Expression of Tissue Factor, CD40 Ligand and P-Selectin in Normal Healthy Subjects during Hyperglycemia and Hyperinsulinemia.

Type 2 diabetes mellitus (DM) patients have increased atherosclerotic and acute vascular complications. Both, high glucose, high insulin levels are independently associated risk factors for increased mortality. We have previously reported elevated circulating tissue factor procoagulant activity (TF-PCA) during hyperglycemia and hyperinsulinemia in normal subjects. To evaluate the effects of hyperglycemia and hyperinsulinemia on blood cells activation we measured monocyte tissue factor (TF) expression, monocyte-platelet aggregates, neutrophil-platelet aggregates, platelet P- selectin and CD40 ligand (CD40L) expression using flow cytometry. We studied 4 groups of healthy individuals at baseline and 24 hr after being subjected to following clamps. Hyperglycemia and Hyperinsulinemia Group, HG+HI: 10 subjects were infused with glucose to maintain levels at 200 mg/dl. Euglycemia and Hyperinsulinemia Group, EG+HI: 7 subjects were infused with insulin to maintain serum levels at 1000 pmol/l. Selective Hyperglycemia and Euinsulinemia Group, HG+EI: 6 subjects were infused with glucose (~200 mg/dl) and somatostatin to block endogenous insulin secretion. Placebo Group: 5 subjects received saline. TF expressing monocytes were increased from 20 to 32% (p < 0.002) and from 13 to 18% (p < 0.02) with HG+HI and EG+HI, respectively. Percent positive cells for monocyte-platelet aggregates increased in the HG+HI group, from 72 to 85% (p < 0.05). Platelet CD40L expression increased with HG+HI (33 to 46%, p < 0.001), EG+HI (31 to 38%, p = 0.01) and HG+EI (33 to 37 %, p = 0.05), whereas P-selectin and neutrophil-platelet aggregates did not increase in any group. There was no change in mean fluorescence intensity (MFI) for any of the measurements. No changes were noted in placebo group at 24 hrs.In addition, we studied the effect of activation of whole blood with ADP (1, 5 and 25μM) and thrombin peptide SFLLRN (25 μM). Upon stimulation with ADP and SFLLRN the percent positive cells and MFI for monocyte-platelet and neutrophil-platelet aggregates, and platelet P-selectin expression were elevated compared to untreated sample in all groups, both at baseline and 24 hr. Percent positive TF expressing monocytes and CD40L expressing platelets were also increased with stimulation.Conclusions: Combination of hyperglycemia with hyperinsulinemia increases circulating TF expression, monocyte-platelet aggregates, and platelet CD40L expression but not neutrophil-platelet aggregates or platelet P- selectin. Hyperinsulinemia and hyperglycemia alone also increased monocyte TF expression and platelets CD40L expression. Stimulation with ADP and SFLLRN increased TF, CD40L, P-selectin and monocyte-platelet aggregates compared to untreated cells. These studies provide evidence for platelet activation and enhanced monocyte TF expression by hyperglycemia- hyperinsulinemia. CD40L and P-selectin are recognized to increase monocyte TF production. The increase in platelet CD40L expression without increase in P-selectin suggests a role of CD40L in the enhanced TF expression. These studies provide evidence for a procoagulant and proinflammatory state that contributes to both the acute vascular events and atherogenesis in DM.

Cytokines modulate expression of cell-membrane complement inhibitory proteins in human lung cancer cell lines.

Human lung cancers overexpress several cell-membrane complement inhibitory proteins (CIP). These complement inhibitory proteins are membrane cofactor protein (CD46), decay-accelerating factor (DAF; CD55), and CD59 (protectin). These cell-membrane proteins have a wide normal tissue distribution, are known to protect normal host cells from homologous complement-mediated lysis, and are thought to facilitate tumor escape from immunosurveillance. To study whether proinflammatory cytokines that are involved in cancer growth can modulate cell-membrane CIP expression in lung cancer cells, we studied the effect of interleukin (IL)-1alpha, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma on two human lung cancer cell lines. ChaGo K-1 and NCI-H596 cell lines, undifferentiated carcinoma and lung adenosquamous carcinoma, respectively, were stimulated with different cytokines, and the effects of incubation time and cytokine concentration on cell-membrane CIP expression were studied. Cell-membrane CIP expression was evaluated using flow cytometry and cytokine effect was calculated as percent change in mean fluorescence intensity of each CIP molecule from its untreated control. We found that DAF was the lung cancer cell-membrane CIP molecule that was the most responsive to cytokine stimulation. Maximal stimulatory effect was usually noted 72 h after a cytokine was introduced. In ChaGo K-1 and NCI-H596 lung cancer cell lines, IL-1alpha and TNF-alpha increased DAF expression. IL-1alpha (100 U/ml/72 h) increased DAF expression up to a maximal mean of 45 and 48%, respectively, in comparison with untreated cells. TNF-alpha (1, 000 U/ml/72 h) increased DAF expression up to a mean of 131 and 46%, respectively. IFN-gamma (1 U/ml/72 h) increased DAF expression in NCI-H596 cells up to a mean of 100%, but had a slight inhibitory effect on DAF expression in ChaGo K-1 cells, decreasing expression by a mean of 17% in comparison with untreated cells. We conclude that cell-membrane DAF expression in the studied human lung cancer cell lines is modulated by IL-1alpha, TNF-alpha, and IFN-gamma, and speculate that cytokine-mediated modulation of cell-membrane DAF in human lung cancer cells might affect lung cancer cell biology.