Mean Fluorescence Intensity Levels Research Articles

The association of endothelial progenitor cell markers with arteriovenous fistula maturation in hemodialysis patients.

Arteriovenous fistula (AVF) failure is one of the most important clinical problems in end-stage renal disease. Endothelial progenitor cells (EPCs) have a role on vascular angiogenesis and endothelialization. We aimed to investigate the association markers of EPCs on AVF maturation by measuring the surface expressions of CD34, CD309 and CD133 on the monocytes. This prospective observational study was conducted in 54 voluntary patients with end-stage renal disease who were admitted for their first renal replacement therapy and were available for AVF creation. Venography was performed in all patients before AVF creation. Six patients were excluded due to inadequate veins after venographic imaging, and also seven patients were excluded due to postoperative thrombosis. The blood samples were analyzed a day before the fistula operation, and the expressions of CD34, CD133 and CD309 on the surface of monocytes were measured. Patients were divided into two groups after the evaluation of AVF maturation, as the mature group and the failure group. The CD309 expression level on the monocytes was 338.00 (35.00-479.00) in the mature group; however, it was 36.00 (5.50-237.00) (p 0.031) in the failure group. Multiple logistic regression analyses showed that both BMI and the mean fluorescence intensity level of CD309 expression on monocytes independently predicted AVF maturation. The presence of DM and increased BMI negatively correlated with AVF maturation. High intensity of CD309 expression on monocytes was observed in patients with successful AVF maturation.

Early and Sustained Reduction in Donor-Specific Antibodies in Desensitized Living Donor Kidney Transplant Recipients: A 3-Year Prospective Study.

BackgroundDesensitization with IVIG and rituximab allows acceptable graft survival in sensitized kidney transplant recipients with preexisting donor-specific antibodies (DSAs) and a positive crossmatch. There is little published data reporting the durability of DSA removal in kidney transplant recipients treated with IVIG and rituximab.MethodsWe conducted a 3-year prospective DSA monitoring study in living donor kidney recipients with preexisting DSA to assess the durability of DSA removal after a perioperative protocol of IVIG and rituximab. All recipients had flow crossmatch titers less than 1:32. Data were analyzed using linear mixed effects models and Kaplan-Meier survival methods.ResultsThe longitudinal database comprised 210 mean fluorescence intensity (MFI) determinations. Forty-two DSAs were identified in 29 patients. Pretreatment MFI averaged 4715 ± 3962 (range, 947-20 129). At 1 month posttransplant, 18 patients (62%) had a complete response (MFI < 1000) and an additional 9 patients (31%) had a partial response (MFI reduced but >1000). There was a 46% reduction (P < 0.001) in DSA MFI at 1 month posttransplant that was sustained throughout the 3-year follow-up period and was observed for both class I and II DSAs regardless of pretreatment MFI levels. With a mean posttransplant follow-up of 1048 ± 574 days, 3-year patient and graft survivals were 95% and 90%. Four patients (14%) had acute rejection between days 125 and 560.ConclusionsDesensitization with IVIG and rituximab results in early and sustained DSA removal over a 3-year posttransplant period in living donor kidney transplant recipients with pretransplant DSA and a positive crossmatch, excellent patient and graft survivals and a low incidence of acute rejection.

PD-1 expression on classical monocytes (CM) as an independent predictor of cancer specific survival in clear cell renal carcinoma (ccRCC).

562 Background: We have previously shown that PD-1 expression in peripheral blood lymphocytes is elevated in RCC patients at diagnosis and decreases subsequent to surgery. We theorize that classical monocyte (CM) inhibition through PD-1 interferes with antigen presentation necessary to stimulate adaptive immune responses, and, thus, preop CM PD-1 expression is prognostic of poor outcomes. Methods: Blood samples were obtained from RCC patients (n = 90) preop and age-matched healthy donors (n = 25). Flow cytometric (FC) data were quantified as mean fluorescence intensity (MFI) or % of cells that express a biomarker. Patients were excluded if they had comorbid CLL, had prior surgeries for RCC, had non-ccRCC histology, or if the FC data was missing. We have defined elevated CM PD-1 expression at the third quartile level of CM PD-1 expression (784 MFI) in the healthy donors. CM PD-1 level was correlated with patient clinico-pathologic data using non-parametric tests. Cancer specific survival (CSS) curves were estimated with Kaplan-Meier methods and a multivariate analysis was performed. Results: The average age of the ccRCC cohort (n = 68) was 60 (range 25-88) years. 35 patients were Fuhrman Grade III/IV high-grade (HG) histology. With median follow-up of 41 months, 10 patients died with ccRCC. No patients with low-grade disease died. Median CM (%) was lower in those who died of disease compare to censored (64% vs 52%, p = 0.029). Patients with CM PD-1 expression &gt; 784 MFI had lower survival compared to patients with CM PD-1 &lt; 784 MFI (p = 0.02). Among HG patients, CSS at 24 months for CM PD-1 MFI &gt; 784 was 62.1% (95% CI 34.2-81.0) vs 93.3% (95% CI 61.3-99.0) for CM PD-1 MFI &lt; 784. On multivariable analysis, controlling for stage related covariates, CM PD-1 MFI &gt; 784 is associated with inferior CSS (hazard ratio = 5.1 [95% CI: 1.1-24.4], p = 0.04) among HG disease patients. Conclusions: In patients with ccRCC, an elevated preoperative CM PD-1 MFI level was associated with lower survival independent of pathological T stage and localized disease status. Preoperative CM PD-1 MFI may be an important biomarker to stratify patients at risk for poor outcome who may benefit from immunotherapy.

IgG Donor-Specific Anti-Human HLA Antibody Subclasses and Kidney Allograft Antibody-Mediated Injury.

Antibodies may have different pathogenicities according to IgG subclass. We investigated the association between IgG subclasses of circulating anti-human HLA antibodies and antibody-mediated kidney allograft injury. Among 635 consecutive kidney transplantations performed between 2008 and 2010, we enrolled 125 patients with donor-specific anti-human HLA antibodies (DSA) detected in the first year post-transplant. We assessed DSA characteristics, including specificity, HLA class specificity, mean fluorescence intensity (MFI), C1q-binding, and IgG subclass, and graft injury phenotype at the time of sera evaluation. Overall, 51 (40.8%) patients had acute antibody-mediated rejection (aABMR), 36 (28.8%) patients had subclinical ABMR (sABMR), and 38 (30.4%) patients were ABMR-free. The MFI of the immunodominant DSA (iDSA, the DSA with the highest MFI level) was 6724±464, and 41.6% of patients had iDSA showing C1q positivity. The distribution of iDSA IgG1-4 subclasses among the population was 75.2%, 44.0%, 28.0%, and 26.4%, respectively. An unsupervised principal component analysis integrating iDSA IgG subclasses revealed aABMR was mainly driven by IgG3 iDSA, whereas sABMR was driven by IgG4 iDSA. IgG3 iDSA was associated with a shorter time to rejection (P<0.001), increased microcirculation injury (P=0.002), and C4d capillary deposition (P<0.001). IgG4 iDSA was associated with later allograft injury with increased allograft glomerulopathy and interstitial fibrosis/tubular atrophy lesions (P<0.001 for all comparisons). Integrating iDSA HLA class specificity, MFI level, C1q-binding status, and IgG subclasses in a Cox survival model revealed IgG3 iDSA and C1q-binding iDSA were strongly and independently associated with allograft failure. These results suggest IgG iDSA subclasses identify distinct phenotypes of kidney allograft antibody-mediated injury.

C1q Binding Activity of De Novo Donor-specific HLA Antibodies in Renal Transplant Recipients With and Without Antibody-mediated Rejection.

Complement fixation by donor-specific HLA antibodies (DSA) is a primary mechanism for antibody-mediated damage of organ allografts. Using a recently developed kit that measures C1q binding to distinguish complement fixing and nonfixing antibodies, studies showed that C1q + DSAs have a higher risk of rejection and graft loss compared to C1q-DSA. The objective of this study was to assess the ability of the C1q-binding assay to identify clinically significant de novo DSA in renal transplant recipients and to define the properties of DSA that confer C1q binding ability. The DSA-positive sera from 34 kidney recipients, 19 with biopsy-proven antibody-mediated rejection (AMR) + and 15 who were AMR-, were assayed in C1q-binding assays (C1q Screen; One Lambda, Inc. Canoga Park, CA). The correlation between C1q-binding activity, presence of AMR, DSA mean fluorescence intensity (MFI) values, and immunoglobulin G isotype was determined. Fifty-three percent (10/19) of sera from AMR+ patients had C1q + DSA, whereas only 13% (2/15) of sera from AMR- patients contained C1q + DSA. C1q + DSA exhibited significantly higher MFI values regardless of whether they were from AMR+ or AMR- patients (16,118 ± 6698 vs 6429 ± 4003; P < 0.0001). C1q + DSA converted to C1q - when diluted to a comparable MFI level as the C1q - DSA from AMR- patients, and some C1q - antibodies converted to C1q + when concentrated to MFI levels comparable to those observed for AMR+/C1q + sera. The C1q binding activity by de novo DSA in patients with AMR largely reflects differences in antibody strength. The C1q assay does not appear to distinguish functionally distinct DSA with clinical significance.

De novo donor-specific human leukocyte antigen antibodies early after kidney transplantation.

Our aim was to determine the incidence of de novo donor-specific human leukocyte antigen (HLA) antibody (dnDSA) during the first year after kidney transplantation and the impact of early dnDSA on acute rejection and protocol biopsy findings. We selected all patients who received a kidney transplant at our center between July 2010 and March 2012. Single antigen bead assay was performed at 1, 4 and 12 months after transplantation. Only DSAs with a mean fluorescence intensity (MFI) of greater 999 were included. We included 245 kidney transplant recipients who did not have a DSA before transplantation. At 12 months, 8.2% of the patients developed dnDSA; 2.4% of them were to HLA class I and 6.5% to HLA class II. Of the 32 patients with a dnDSA at 1 or 4 months, only 8 (25%) persisted at 12 months. The risk of antibody-mediated rejection (AMR) was higher in the dnDSA group. For the dnDSA group with MFI of 3,000 or greater (compared with the group with MFI<3,000), the hazard ratio for AMR was 10.6 (95% confidence interval, 2.27-49.5). The cumulative incidence of AMR or mixed rejection at 1 year was 30% in the group with dnDSA MFI level of 3,000 or greater but only 4% for the group with dnDSA with MFI less than 3,000. On 1-year protocol biopsies, the dnDSA group showed more interstitial inflammation, tubulitis, and glomerulitis. We conclude that dnDSA occurring during the first posttransplantation year may be transient, and the risk of AMR is higher in patients with a dnDSA MFI level that is greater than 3,000.

P005 : LOW LEVEL ANTI-HLA ANTIBODIES: DETERMINING RELEVANT UNACCEPTABLE ANTIGENS BY SINGLE ANTIGEN MICROARRAY AND SPECIFIC/SURROGATE FLOW CROSSMATCHES

A challenge to any HLA laboratory is to determine the cutoff or level of anti-HLA antibody that ultimately determines the unacceptable antigens (UA) of a potential transplant patient. These UA’s are placed In UNET for virtual crossmatching. Once accomplished, this level is accepted for all HLA antigens as demonstrated by the single antigen bead arrays. The microarray assay is not quantitative and may give false positive reactions at low levels. The challenge is to determine if these antibodies would cause a positive flow T and B cell Flow crossmatch (FLXM). The object of this study is to verify that a low or intermediate Mean Fluorescence Intensity (MFI) anti HLA antibody will or will not cause a positive FLXM with extra deceased donor or normal individual’s lymphocytes. We performed 159 FLXM on 31 patient sera. Only one antibody specificity that could cause a positive FLXM was demonstrated on each of these cells by molecular typing. Our MFI cutoff was 2000 MFI (+DTT). The pronase FLXM Molecules of Equivalent Soluble Fluorochrome (MESF) cutoff was 1500 (+500) for T cell and 2500 (+500) for B cell. Twenty-three patients had negative crossmatches when cross matched by at least five different cells and their serum. The false positive UA as demonstrated by a negative FLXM were removed from UNET. The MFI values for the UA’s varied from 1000 to 5000. There was no correlation of MFI levels to outcome of FLXM. One patient received a renal transplant and two were removed from the wait list. In conclusion: the use of FLXM to demonstrate relevant levels of anti-HLA antibody for each individual patient and allowed us to remove false positive UA’s. This type of analysis gives a potential kidney recipient a greater chance to receive a renal transplant.

The Role of Donor-Specific Antibodies in Acute Cardiac Allograft Dysfunction in the Absence of Cellular Rejection

Acute allograft dysfunction (AAD) is an important cause of morbidity among heart transplant recipients. The role of donor-specific antibodies (DSAs) in AAD, with the increasing use of single antigen bead (SAB) assays that have improved the ability to detect DSA, remains unclear. We retrospectively reviewed 329 heart transplant recipients followed up at our institution. AAD was defined as an acute decline in left ventricular ejection fraction to less than 50% and a decrement of 10% or higher compared to baseline in the absence of cellular rejection. Patients with AAD were compared with matched 30 heart transplant controls. There were 10 (3%) patients with AAD, 4 (40%) had DSA detectable by SAB assay compared to 16 (53%) controls (P=0.43). Peak DSA mean fluorescent intensity (MFI) levels were significantly higher at baseline (class I and class II) in AAD compared to controls. DSA MFI values increased at the time of AAD and returned to baseline values during follow-up for these patients with AAD (P<0.05) but remained unchanged over time for controls. Six (60%) patients with AAD and 1 (3%) control had antibody-mediated rejection (AMR) by endomyocardial biopsy (P<0.01). There were 4 (40%) patients with AAD with no DSA or AMR. AAD after heart transplant is a heterogeneous process characterized by 1) AMR and DSA, 2) AMR but no DSA, and 3) no AMR or DSA. The presence of DSA is not associated with AAD, but the quantity assessed by MFI levels may play a role.

Reduced tumour necrosis factor receptor superfamily 13C inversely correlated with tumour necrosis factor superfamily 13B in patients with immune thrombocytopenia.

To investigate the expression of tumour necrosis factor superfamily 13B (TNFSF13B) receptors in immune thrombocytopenia (ITP) and their correlation with disease activity, we investigated the protein and mRNA levels of TNFSF13B, tumour necrosis factor receptor superfamily 13C (TNFRSF13C), TNFRSF13B and TNFRSF17 by flow cytometry, enzyme-linked immunosorbent assay and real time quantitative polymerase chain reaction. All CD19(+) B lymphocytes expressed TNFRSF13C by flow cytometry, but the mean fluorescence intensity (MFI) was decreased in patients with active disease compared to patients in remission and healthy controls, while no significant difference of TNFRSF13C mRNA was found between ITP patients and controls. The mRNA and plasma TNFSF13B were elevated in active ITP patients, and TNFRSF13C MFI level was inversely correlated with plasma TNFSF13B in active patients. In vitro assays showed that TNFRSF13C MFI was decreased after long exposure to TNFSF13B. No significant difference for TNFRSF13B or TNFRSF17 was found between ITP patients and controls. In conclusion, TNFRSF13C expression is reduced on CD19(+) cells in active ITP patients. This down-regulation occurs through a post-transcriptional mechanism and could be a consequence of chronic increase of TNFSF13B.

Pre-transplant donor HLA-specific antibodies: Characteristics causing detrimental effects on survival after lung transplantation

The impact of Luminex-detected HLA antibodies on outcomes after lung transplantation is unclear. Herein we have undertaken a retrospective study of pre-transplant sera from 425 lung transplants performed between 1991 and 2003. Pre-transplant sera, originally screened by complement-dependent cytotoxicity (CDC) assays, were retrospectively tested for the presence of HLA-specific antibodies using HLA-coated Luminex beads and C4d deposition on Luminex beads. The results were correlated with graft survival at 1 year. Twenty-seven patients were retrospectively identified as having been transplanted against donor-specific HLA antibodies (DSA) and 36 patients against non-donor-specific HLA antibodies (NDSA). DSA-positive patients had 1-year survival of 51.9% compared with 77.8% for NDSA and 71.8% for antibody-negative patients (p = 0.029). One-year survival of patients with complement-fixing DSA was 12.5% compared with 62.5% for non-complement-fixing DSA, 75.8% for non-complement-fixing NDSA and 71.8% for antibody-negative patients (p < 0.0001). DSA-positive patients with mean fluorescence intensity (MFI) >5,000 had 1-year survival of 33.3% compared with 71.4% for MFI 2,000 to 5000 and 62.5% for MFI <2,000 (p = 0.0046). Multivariable analysis revealed DSA to be an independent predictor of poor patient survival within 1 year (p = 0.0010, hazard ratio [HR] = 3.569) as well as complement-fixing DSA (p < 0.0001, HR = 11.083) and DSA with MFI >5,000 (p = 0.0001, HR = 5.512). Pre-formed DSA, particularly complement-fixing DSA, and high MFI are associated with poor survival within the first year after lung transplantation. Risk stratification according to complement fixation or MFI levels may allow for increased transplantation in sensitized patients.

The value of flow cytometry in the measurement of platelet activation and aggregation in human immunodeficiency virus infection

Human immunodeficiency deficiency virus (HIV) infection is associated with chronic inflammation and an increased risk of thrombotic events. Activated platelets (PLTs) play an important role in both thrombosis and inflammation, and HIV has been shown to induce PLT activation by both direct and indirect mechanisms. P-selectin (CD62P) is a well-described marker of PLT activation, and PLT glycoprotein (GP) IV (CD36) has been identified as a marker of PLT aggregation. Data on PLT function in the context of HIV infection remain inconclusive. Laboratory techniques, such as flow cytometry, enable the assessment of PLTs in their physiological state and environment, with minimal artifactual in vitro activation and aggregation. In this study, we describe a novel flow cytometry PLT assay, which enabled the measurement of PLT function in HIV infection. Forty-one antiretroviral-naïve HIV-positive individuals and 41 HIV-negative controls were recruited from a clinic in the Western Cape. Platelet function was evaluated by assessing the response of platelets to adenosine diphosphate (ADP) at two concentrations (0.04 mM, 0.2 mM). The percentage expression and mean fluorescence intensity (MFI) of CD62P and CD36 was used to evaluate platelet function. These were then correlated with platelet (PLT) count; CD4 count; % CD38/8; viral load and D-dimers. The % CD62P levels were higher in HIV-positive patients (HIV % CD62P 11.33[5.96–29.36] vs. control 2.48[1.56–6.04]; p < 0.0001). In addition, the HIV group showed higher CD62P MFI levels (HIV CD62P MFI 3.25 ± 7.23 vs. control 2.35 ± 1.31, p = 0.0292). Baseline levels of %CD36 expression were significantly higher in HIV-positive patients (%CD36 12.41[6.31–21.83] vs. control 6.04[1.34–13.15]; p = 0.0091). However, the baseline CD36MFI showed no significant difference between the two groups (HIV CD36 MFI 3.09 ± 0.64 vs. control 2.44 ± 0.11, p = 0.4591). The HIV group showed higher levels of % CD36 expression post stimulation with 0.04 mM ADP 43.32 ± 27.41 vs. control 27.47 ± 12.95; p < 0.0214) and no significant difference at 0.2 mM ADP (HIV % CD36 39.06 ± 17.91 vs. control 44.61 ± 18.76; p = 0.3277). Furthermore, the HIV group showed a single phase response to ADP as compared to the control group, which showed a normal biphasic response. We concluded that PLT flow cytometry is valuable in the assessment of levels of PLT activation, and further, that the addition of an endogenous agonist, such as ADP, enabled the measurement of PLT function in HIV infection. We were able to show that, although PLTs are significantly activated in HIV compared to uninfected controls, they retain their functional capacity.

Early response roles for prolactin cortisol and circulating and cellular levels of heat shock proteins 72 and 90α in severe sepsis and SIRS.

Objective. To evaluate the early heat shock protein (HSP) and hormonal stress response of intensive care unit (ICU) patients with severe sepsis/septic shock (SS) or systemic inflammatory response syndrome (SIRS) compared to healthy subjects (H). Methods. Patients with early (first 48 hrs) SS (n = 29) or SIRS (n = 29) admitted to a university ICU and 16 H were enrolled in the study. Serum prolactin, cortisol, and plasma ACTH were determined using immunoassay analyzers. ELISA was used to evaluate extracellular HSPs (eHSP90α, eHSP72) and interleukins. Mean fluorescence intensity (MFI) values for intracellular HSPs (iHSP72, iHSP90α) were measured using 4-colour flow-cytometry. Results. Prolactin, cortisol, and eHSP90α levels were significantly increased in SS patients compared to SIRS and H (P < 0.003). ACTH and eHSP72 were significantly higher in SS and SIRS compared to H (P < 0.005). SS monocytes expressed lower iHSP72 MFI levels compared to H (P = 0.03). Prolactin was related with SAPS III and APACHE II scores and cortisol with eHSP90α, IL-6, and lactate (P < 0.05). In SS and SIRS eHSP90α was related with eHSP72, IL-6, and IL-10. Conclusion. Prolactin, apart from cortisol, may have a role in the acute stress response in severe sepsis. In this early-onset inflammatory process, cortisol relates to eHSP90α, monocytes suppress iHSP72, and plasma eHSP72 increases.

Liver transplantation with a strongly positive crossmatch: Case study and literature review

A positive crossmatch has been associated with increased risk in liver transplantation. To study the clinical significance of preformed donor-specific human leukocyte antigen antibodies (DSAs) in liver transplantation, we reviewed patients who underwent liver transplantation with a strongly positive flow cytometry crossmatch. DSAs were evaluated with a Luminex solid phase assay. The complement-fixing ability of DSAs was tested with a complement component 1q (C1q) assay. Using an assay correlation between complement-dependent cytotoxicity crossmatch, flow cytometry crossmatch, and DSA results, we reviewed the effects of DSAs on the outcomes of our patients as well as reported cases in the literature. Five of 69 liver recipients had a strongly positive crossmatch: 4 had a positive T cell crossmatch [median channel shift (MCS) = 383.5 ± 38.9], and 5 had a positive B cell crossmatch (MCS = 408.8 ± 52.3). The DSAs were class I only in 1 patient, class I and II in 3 patients, and class II only in 1 patient. Cholestasis, acute rejection, or both were observed in 3 of the 4 patients with a positive T cell crossmatch with an MCS approximately greater than 300. The C1q assay was positive for 3 patients. Two had either persistent cholestasis or early acute rejection. One patient who was treated with preemptive intravenous immunoglobulin had an unremarkable outcome despite a positive C1q result. One of the 2 patients with a negative C1q assay experienced persistent cholestasis and early and recurrent acute rejection; the other had an unremarkable outcome. None of the patients died or lost a graft within the first year of transplantation. Our study suggests that human leukocyte antigen antibody screening, flow cytometry crossmatch MCS levels, DSA mean fluorescent intensity levels, and C1q assays may be useful in assessing the risk of antibody-mediated rejection and timely interventions in liver transplantation.

Tetramethylpyrazine upregulates the aquaporin 8 expression of hepatocellular mitochondria in septic rats

BackgroundThe sepsis-induced acute liver injury majorly depends on the dysfunction of mitochondria and the loss of cellular energy. Aquaporin 8 (AQP8) can modulate water transport and osmotic swelling of mitochondria in the inner mitochondrial membrane of the liver. In this study, we explore the effects of tetramethylpyrazine (TMP) on protecting the structure of hepatocyte mitochondria and modulating the expression of AQP8. Materials and methodsForty-eight rats were randomly allocated to four groups: control group receiving sham procedure, septic group receiving cecal ligation and puncture (CLP), therapeutic group receiving 60 mg/kg of ligustrazine (TMP) intravenously from caudal vein immediately after CLP, and preventive group receiving 60 mg/kg/d of ligustrazine intravenously from caudal vein for 7 d before CLP. The mitochondrial ultrastructure of rat liver was observed. The protein expression of AQP8 was assayed by Western blot. Analysis of AQP8 messenger RNA (mRNA) expression level was performed by the reverse transcription–polymerase chain reaction. The mean fluorescence intensity (MFI) of rhodamine 123 (Rh 123) was measured by flow cytometry. The serum tumor necrosis factor alpha (TNF-α) level was determined by the enzyme-linked immunosorbent assay. ResultsThe mitochondrial ultrastructure was markedly damaged in the septic group, whereas it was lightly damaged in the therapeutic and preventive groups. Compared with the control group, the AQP8 protein expression and MFI were significantly reduced, and the steady-state AQP8 mRNA and serum TNF-α levels were increased in the septic, therapeutic, and preventive groups. Compared with the septic group, the AQP8 protein expression and MFI were increased, and the steady-state AQP8 mRNA and serum TNF-α levels were decreased significantly in the therapeutic and preventive groups. There was no significant difference in morphologic characteristics, AQP8 protein level, AQP8 mRNA level, MFI, and serum TNF-α level between the therapeutic and the preventive groups. Linear positive correlation was observed between the AQP8 protein level and the MFI of Rh 123. Linear negative correlation was observed between the AQP8 protein level or the MFI of Rh 123 and serum TNF-α level. ConclusionsTMP has protective effect on hepatocellular mitochondria from damage in sepsis by ameliorating the expression of AQP8 protein in liver mitochondria. The protective effect of TMP on the liver mitochondria might not have a difference between using TMP before or after the occurrence of sepsis.

Expression of selected regulatory molecules on the CD83+ monocyte-derived dendritic cells generated from patients with laryngeal cancer and their clinical significance

B7H1 and B7H4 overexpression is associated with inhibition of the immune system in many solid tumors, and altogether with CD200 molecule plays an important role in tumor invasion by promoting malignant transformation. However, there is no report about impact of these molecules on laryngeal squamous cell carcinoma. The objective of the present study was to assess by means of flow cytometry the expression of B7H1, B7H4, CD200, and CD200R on CD83+ monocyte-derived dendritic cells (Mo-DC), pulsed with autologous tumor cell lysates (aTCL) in patients who suffer from G1, G2, or G3 laryngeal carcinoma (LC, n = 60) in comparison to healthy donors (HD, n = 15). It has been demonstrated that median value of the percentages of CD83+ B7H1+, CD83+ B7H4+, and CD83+ CD200+ cells were higher in LC patients than HD (p = 0.041, p ≤ 0.0001, and p = 0.02, respectively). Mean fluorescence intensity (MFI) of CD200, CD200R, B7H1, and B7H4 on the Mo-DC pulsed with aTCL of the patients was also higher than on the Mo-DC of HD (p ≤ 0.0001, p ≤ 0.0001, p = 0.002, and p ≤ 0.0001, respectively). The highest MFI levels of all molecules were noted in grade 3 LC. The aforementioned results prove that there is a relation between the presence of laryngeal cancer and the expression of B7H1, B7H4, CD200, and CD200R regulatory molecules on the CD83+ Mo-DC pulsed with autologous cancer cell lysates. Strong association of LC grade and the tested antigens expression suggests a critical role for these proteins in LC biology.

Detection of Antibodies Against Major Histocompatibility Complex Class I-Related Chain A in Long-term Renal Graft Recipients

To determine the prevalence, allele specificity, and intensity of anti-MICA antibodies in long-term renal graft recipients and to investigate their association with impaired renal function. Sixty-eight long-term (> 10 y) renal graft recipients were divided into 2 groups: (1) patients with impaired renal function (serum creatinine ≥ 2 mg/dL, n=6); (2) patients with normal renal function (serum creatinine < 176.8 μmol/L, n=62). Anti-MICA antibodies were tested using Luminex single antigen beads assays and the frequency, specificity, and intensity of these antibodies were compared between 2 patient groups. MICA antibodies were detected in 33% of impaired renal function patients and 15% of normal renal function patients (P > .05). Anti-MICA*027 antibodies were found in 11.76% of patients, whereas antibody to MICA*012 was found in 2.94% of patients. Interestingly, among all antibody specificities, MICA*001,*004, *007, *009, *012, and *018 were found more frequently in impaired renal function patients than in normal renal function patients. The peak mean fluorescence intensity levels of MICA antibodies in impaired renal function patients were significantly higher than those in normal renal function patients (P < .05). Our data suggest that increased prevalence and intensity of anti-MICA antibodies are associated with impaired renal graft function in long-term renal graft recipients and some MICA antibodies might be more important than others in mediating graft rejection.

Monocytes/Macrophages Are the Major Targets of the CCL3 Chemokine Produced by CD38+CD49d+ Chronic Lymphocytic Leukemia Cells.

Abstract 2350Poster Board II-327 Introduction:CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Recent gene expression profiling studies comparing CLL cases expressing low versus high levels of CD38 and CD49d, identified CCL3 as a gene upregulated by CD38+CD49d+ CLL. The release of CCL3 by cultured CLL cells was also demonstrated upon CD38 triggering, and CCL3 protein was found in CLL cells from bone marrow biopsies (BMB) of CD38+ cases (Zucchetto et al., Cancer Res, 2009; 69:4001-9). Given the role of CCL3 as potent chemoattractant for different cell types, we aimed at identifying the major targets of CCL3, as produced by CD38+CD49d+ CLL cells. Methods:CLL infiltrates of BMB were characterized by immunohistochemistry (IHC). Expression of the CCL3 receptors CCR1 and CCR5 by PB CLL subpopulations was evaluated by flow cytometry. T lymphocyte and monocyte migrations were performed by in-vitro transwell chemotaxis assays. Results:IHC analysis of BMB from 16 CLL cases revealed a higher number of infiltrating CD68+ cells in the context of CLL-involved areas of BMB from CD38+CD49d+CCL3+, compared to CD38−CD49d−CCL3− cases (p=0.01). CD3+ lymphocytes were interspersed in the CLL aggregates, but with no significant difference between the two subgroups. Evaluation of CCR1 and CCR5 in PB cell subpopulations from 40 CLL cases expressing or not surface CD38 and CD49d, showed the highest mean fluorescence intensity (MFI) levels for both CCR1 (624±60) and CCR5 (64±9) in the monocytic component, irrespective of CD38 and CD49d expression by CLL cells. Conversely, both CLL cells and residual T lymphocytes showed low MFI levels for CCR1 (19±4 and 14±3) and CCR5 (21±2 and 20±2). High CCR1 and CCR5 expression levels were detected in in-vitro differentiated monocytes from purified PB cells of four CD38+CD49d+ CLL. Accordingly, CCR1 expression was documented in macrophage-like cells in BMB from CD38+CD49d+ CLL. Next, we evaluated the capability of purified monocytes and T lymphocytes from 10 CLL cases to migrate in response to CCL3. In keeping with the strong expression of CCR1, monocytes migrated toward CCL3 at a concentration of 3 ng/mL (migration index, MI= 8.8±0.9, p=0.03), whereas T lymphocytes required a higher CCL3 concentration (100 ng/mL) to display slight migration capability (MI= 1.6±0.2, p=ns). The increased infiltration of macrophages in BMB from CCL3-producing CD38+CD49d+ CLL, prompted us to verify the capability of CCL3-stimulated macrophages to induce the expression by endothelial cells (EC) of the CD49d specific ligand VCAM-1. By using two different EC models (HUVEC and ADMEC), we documented a significant up-regulation of VCAM-1 by EC exposed to conditioned media (CM) collected from cultures of macrophages challenged in-vitro with CCL3 (p=0.002). Notably, increased levels of the pro-inflammatory cytokine TNF-α were detected in CCL3-CM (p=0.006), and neutralization of TNF-α by specific antibodies reverted the capability of CCL3-CM to induce VCAM-1 by EC models. In agreement with these in-vitro data, we found a more prominent meshwork of VCAM-1+ stromal/endothelial cells in lymphoid infiltrates from CD38+CD49d+ CLL compared to CD38−CD49d− cases (p=0.002), and engagement of CD49d by VCAM-1 was able to significantly delay the spontaneous apoptosis observed in cultured CLL cells. Conclusions:CD68+ monocytes/macrophages are likely the main targets for the CLL3 chemokine produced by CD38+CD49d+ CLL cells, and are active in determining, through the release of TNF-α and other yet unidentified cytokines, the overexpression of VCAM-1 by endothelial cells. Experiments aimed at investigating further roles of CD68+ monocytes/macrophage in CLL are currently matter of study. Disclosures:No relevant conflicts of interest to declare.

Ventilation during Cardiopulmonary Bypass: Impact on Neutrophil Activation and Pulmonary Sequestration

Background: Cardiopulmonary bypass (CPB) is associated with neutrophil activation, pulmonary sequestration, and release of inflammatory mediators leading to pulmonary dysfunction. We investigate the effect of continuous ventilation during cardiopulmonary bypass on neutrophil activation and pulmonary sequestration. Methods: Forty-six patients undergoing coronary artery bypass grafting with cardiopulmonary bypass were prospectively randomized to continuous ventilation and nonventilation groups. Blood samples were collected, and bronchoalveolar lavage (BAL) was performed following induction of anesthesia and at 4 hr after aortic declamping. Differential white cell count was measured, and flow cytometry to determine cell count numbers and quantify CD45 and CD11b leukocyte cell surface adhesion molecule expression was performed on the blood and BAL samples. Results: Twenty-three patients were randomized to standard nonventilated CPB and 23 patients to ventilation throughout CPB. Significant increases in blood and BAL neutrophil numbers were detected at 4 hr following aortic declamping in both groups (Blood: NV p <. 0001, V p <. 0001; BAL: NV p =. 017, V p =. 0007). No significant inter-group differences in BAL and blood neutrophil numbers were found. Significantly higher blood neutrophil CD11b mean fluorescent intensity levels were present 4 hr following declamping compared with baseline in both groups (NV Blood, p =. 021; V Blood p <. 0001). No significant inter- or intragroup differences in BAL neutrophil CD11b mean fluorescent intensity levels were found. There was no death or major complication. Conclusions: Cardiopulmonary bypass during coronary artery bypass grafting is associated with increased neutrophil pulmonary sequestration, and blood neutrophil CD11b activation. Continuous ventilation during cardiopulmonary bypass does not significantly reduce neutrophil pulmonary sequestration or activation.

Expression and Prognostic Significance of the Inhibitor of Apoptosis Protein (IAPs) Family and Its Antagonists in Chronic Lymphocytic Leukemia

The apoptotic mode of cell death is a major regulatory process in all complex organisms. The slow accumulation of malignant cells in chronic lymphocytic leukemia (CLL) suggests that this disease is caused by a defect in apoptosis regulation. Apoptosis, defined as a programmed cell death, is executed through activity of caspases, cysteine proteases which are regulated by a number of pro- and anti-apoptotic proteins. One of such checkpoints is a control of caspase activation by the family of inhibitor of apoptosis proteins (IAPs). So far eight IAP proteins have been identified in human. They were XIAP, cIAP1, cIAP2, ILP-2, NAIP, survivin and BRUCE/Apollon. Moreover, an important role in the regulation of apoptosis play also three proteins which bind to IAPs and inhibit their activity, such as Smac/DIABLO, HtrA2/Omi and XAF1. Until recently IAPs and their antagonists were poorly researched in CLL. The majority of them were even not investigated in this disease yet. The primary aim of the study was to perform a complex analysis of expression of the IAPs family proteins and their antagonists in leukemic cells (ex vivo) in untreated CLL patients in comparison to the healthy controls. In this study we have also aimed to assess differences in expression of these proteins between patients with stable(SD) and progressive disease (PD). We tried to establish a potential prognostic significance of the IAPs expression and to investigate their correlation with the clinical course of the disease. The last aim was to analyze the expression of these proteins in the cell culture (in vitro settings) in response to the drugs routinely used in CLL. One hundred untreated patients with CLL were included in the study. Patients were divided into two groups: with SD (n=52) and PD (n=48). Twenty seven healthy donors served as a control group. Expression of IAPs and their inhibitors were investigated by flow cytometry, after careful comparative studies by Western blot and fluorescence microscopy. Protein expression was assessed on the basis of the mean fluorescence intensity (MFI). Results were compared with several disease-related parameters, such as clinical stage according to Rai, lymphocyte count, ZAP-70 and CD38 expression or serum beta-2-microglobulin concentration. The secondary endpoint of the study was to compare the results with the outcome of examined patients. Our study revealed significantly higher expression of cIAP1 (median MFI level: 81.1 vs 8.2; p=0.000001) and cIAP2 (median MFI level: 313.5 vs 146.8; p=0.014) and lower expression of IAP-antagonist - Smac/DIABLO (median MFI level: 147.9 vs 301.3; p=0.010) in the group of untreated CLL patients in comparison to the control group. Furthermore, patients with PD showed significantly higher expression of all analyzed IAPs compared with those with SD. Median MFI level for cIAP1 were: 131.2 vs 60.1; p=0.002, for cIAP2: 434.3 vs 301.5; p=0.026, for XIAP: 463.7 vs 225.4; p=0.002 and for survivin: 119.6 vs 38.6; p=0.00006. We also found a positive correlation between all examined protein's expression and clinical stage according to Rai and high lymphocytosis, but only XIAP correlated with beta-2-microglobulin concentration (p=0.017). Moreover the study showed that simultaneous expression of two or more IAPs is associated with shorter overall survival. Additionally, analyzing the expression of IAPs and their antagonists in the cell cultures (in vitro) in response to cytotoxic drugs (Cladribine, Fludarabine, Mafosfamide) we observed significantly lower level of cIAP1, cIAP2 and XIAP as well as a higher level of proapoptotic Smac/DLABLO in comparison to the control, cytostatic-free cultures. In conclusion, CLL cells are characterized by overexpression of cIAP1 and cIAP2 and downregulation of Smac/DIABLO. The anti-apoptotic pattern of these apoptosis-regulating proteins expression may be responsible for pathological accumulation of malignant cells in CLL. Moreover, expression of all IAP proteins is significantly elevated in patients with PD, what indicates a role of inhibited proclivity of CLL cells to undergo spontaneous apoptosis in the disease progression. Importantly, the co-expression of two or more IAP proteins in tumor cell seems to be unfavorable prognostic factor in CLL patients. Thus, all those data suggest that IAPs/IAP-antagonists system plays an important role in the biology of CLL and further studies to confirm their prognostic usefulness are warranted.

Identification of an Invasion Regulatory Domain within the Core Protein of Syndecan-1

Among the four members of the syndecan family there exists a high level of divergence in the ectodomain core protein sequence. This has led to speculation that these core proteins bear important functional domains. However, there is little information regarding these functions, and thus far, the biological activity of syndecans has been attributed largely to their heparan sulfate chains. We have previously demonstrated that cell surface syndecan-1 inhibits invasion of tumor cells into three-dimensional gels composed of type I collagen. Inhibition of invasion is dependent on the syndecan heparan sulfate chains, but a role for the syndecan-1 ectodomain core protein was also indicated. To more closely examine this possibility and to map the regions of the ectodomain essential for syndecan-1-mediated inhibition of invasion, a panel of syndecan-1 mutational constructs was generated, and each construct was transfected individually into myeloma tumor cells. The anti-invasive effect of syndecan-1 is dramatically reduced by deletion of an ectodomain region close to the plasma membrane. Further mutational analysis identified a stretch of 5 hydrophobic amino acids, AVAAV (amino acids 222-226), critical for syndecan-1-mediated inhibition of cell invasion. This invasion regulatory domain is 26 amino acids from the start of the transmembrane domain. Importantly, this domain is functionally specific because its mutation does not affect syndecan-1-mediated cell binding to collagen, syndecan-1-mediated cell spreading, or targeting of syndecan-1 to specific cell surface domains. This invasion regulatory domain may play an important role in inhibiting tumor cell invasion, thus explaining the observed loss of syndecan-1 in some highly invasive cancers.