Comprehensive assessment and standardization of solid phase multiplex-bead arrays for the detection of antibodies to HLA.

Solid phase multiplex-bead arrays for the detection and characterization of HLA antibodies provide increased sensitivity and specificity compared to conventional lymphocyte-based assays. Assay variability due to inconsistencies in commercial kits and differences in standard operating procedures (SOP) hamper comparison of results between laboratories. The Clinical Trials in Organ Transplantation Antibody Core Laboratories investigated sources of assay variation and determined if reproducibility improved through utilization of SOP, common reagents and normalization algorithms. Ten commercial kits from two manufacturers were assessed in each of seven laboratories using 20 HLA reference sera. Implementation of a standardized (vs. a nonstandardized) operating procedure greatly reduced MFI variation from 62% to 25%. Although laboratory agreements exceeded 90% (R(2) ), small systematic differences were observed suggesting center specific factors still contribute to variation. MFI varied according to manufacturer, kit, bead type and lot. ROC analyses showed excellent consistency in antibody assignments between manufacturers (AUC > 0.9) and suggested optimal cutoffs from 1000 to 1500 MFI. Global normalization further reduced MFI variation to levels near 20%. Standardization and normalization of solid phase HLA antibody tests will enable comparison of data across laboratories for clinical trials and diagnostic testing.

Preformed and de novo donor specific antibodies in visceral transplantation: long-term outcome with special reference to the liver.

Despite improvement in early outcome, rejection particularly chronic allograft enteropathy continues to be a major barrier to long-term visceral engraftment. The potential role of donor specific antibodies (DSA) was examined in 194 primary adult recipients. All underwent complement-dependent lymphocytotoxic crossmatch (CDC-XM) with pre- and posttransplant solid phase HLA-DSA assay in 156 (80%). Grafts were ABO-identical with random HLA-match. Liver was included in 71 (37%) allografts. Immunosuppression was tacrolimus-based with antilymphocyte recipient pretreatment in 150 (77%). CDC-XM was positive in 55 (28%). HLA-DSA was detectable before transplant in 49 (31%) recipients with 19 continuing to have circulating antibodies. Another 19 (18%) developed de novo DSA. Ninety percent of patients with preformed DSA harbored HLA Class-I whereas 74% of recipients with de novo antibodies had Class-II. Gender, age, ABO blood-type, cold ischemia, splenectomy and allograft type were significant DSA predictors. Preformed DSA significantly (p < 0.05) increased risk of acute rejection. Persistent and de novo HLA-DSA significantly (p < 0.001) increased risk of chronic rejection and associated graft loss. Inclusion of the liver was a significant predictor of better outcome (p = 0.004, HR = 0.347) with significant clearance of preformed antibodies (p = 0.04, OR = 56) and lower induction of de novo DSA (p = 0.07, OR = 24). Innovative multifaceted anti-DSA strategies are required to further improve long-term survival particularly of liver-free allografts.

Re-examination of the lymphocytotoxic crossmatch in liver transplantation: can C4d stains help in monitoring?

C4d-assisted recognition of antibody-mediated rejection (AMR) in formalin-fixed paraffin-embedded tissues (FFPE) from donor-specific antibody-positive (DSA+) renal allograft recipients prompted study of DSA+ liver allograft recipients as measured by lymphocytotoxic crossmatch (XM) and/or Luminex. XM results did not influence patient or allograft survival, or cellular rejection rates, but XM+ recipients received significantly more prophylactic steroids. Endothelial C4d staining strongly correlates with XM+ (<3 weeks posttransplantation) and DSA+ status and cellular rejection, but not with worse Banff grading or treatment response. Diffuse C4d staining, XM+, DSA+ and ABO- incompatibility status, histopathology and clinical-serologic profile helped establish an isolated AMR diagnosis in 5 of 100 (5%) XM+ and one ABO-incompatible, recipients. C4d staining later after transplantation was associated with rejection and nonrejection-related causes of allograft dysfunction in DSA- and DSA+ recipients, some of whom had good outcomes without additional therapy. Liver allograft FFPE C4d staining: (a) can help classify liver allograft dysfunction; (b) substantiates antibody contribution to rejection; (c) probably represents nonalloantibody insults and/or complete absorption in DSA- recipients and (d) alone, is an imperfect AMR marker needing correlation with routine histopathology, clinical and serologic profiles. Further study in late biopsies and other tissue markers of liver AMR with simultaneous DSA measurements are needed.

A critical appraisal of methods to grade transplant glomerulitis in renal allograft biopsies.

Transplant glomerulitis is an increasingly recognized lesion in renal transplant biopsies. To develop a refined grading system, we defined glomerulitis by the presence of ≥5 leukocytes/glomerulus and evaluated 111 biopsies using three different grading systems: (i) percentage of glomerular involvement, (ii) peak inflammation in the most severely affected glomerulus and (iii) presence/absence of endocapillary occlusion by inflammatory cells. Endocapillary occlusion had no impact on graft survival, but was associated with increased serum creatinine, proteinuria and subsequent transplant glomerulopathy. Grading based on either percent or peak glomerular involvement correlated with graft failure and peritubular capillaritis. However, the percent glomerular involvement method had the additional advantage of displaying associations with: concurrent proteinuria, focal or diffuse immunoperoxidase peritubular capillary C4d staining, 1-year postbiopsy serum creatinine, subsequent detection of donor-specific antibody and development of transplant glomerulopathy. Patients with >75% glomerular involvement also revealed persistent high-grade glomerulitis on follow-up biopsies despite antirejection treatment. In conclusion, grading of glomerulitis is a meaningful exercise, and a quantification system based on percentage of glomerular involvement shows the most robust associations with clinical parameters and prognosis.

1.7 year follow-up after bortezomib therapy for refractory antibody mediated rejection.

In summary, 1.7 years after transplantation, bortezomib rescue has been durably effective in salvaging our patient with refractory antibody mediated rejection. The only price has been persistently high levels of BK viruria. The presence of ongoing and even recurrent donor specific antibody has made it difficult to reduce immunosuppression further, and the concern that the high levels of BK viruria will eventually progress to viremia and nephropathy necessitates continued therapy with very low dose cidofovir and leflunomide. The absence of C1q binding DSA with stable renal function may provide some reserved optimism that the DSA that is detectable by convention Luminex assay may have reduced pathological implications. However, more data and prolonged follow-up are needed to determine whether or not non-complement binding DSA has an adverse pathological role.

Serum and prostatic tissue concentrations of moxifloxacin in patients undergoing transurethral resection of the prostate.

The spectrum of pathogens causing chronic bacterial prostatitis comprises Gram-negative, Gram-positive and atypical microorganisms. Because of its broad spectrum of activity, the group 4 fluoroquinolone moxifloxacin might be a suitable antibiotic for treatment of bacterial prostatitis. The aim of this prospective study was to investigate the penetration of moxifloxacin into prostatic tissue in patients with benign prostatic hyperplasia. Patients received a single dose of moxifloxacin 400 mg in an 1 hour lasting infusion (250 ml) for perioperative prophylaxis before undergoing transurethral resection of the prostate (TURP). Serum concentrations were determined in all patients before infusion, at the end of infusion (time point 0), 0.5, 1 and 2 h after the end of infusion. Patients were randomized for tissue sampling either 0, 0.5, 1 or 2 h after the end of infusion. At beginning of TURP approximately 1 g of tissue was sampled for analysis. Concentrations of moxifloxacin in serum and tissue were determined by HPLC. 39 patients were evaluated. Median serum and prostatic tissue concentrations peaked at 0 h (4.94 mg/ L and 8.50 mg/ kg, respectively). The lowest concentrations were quantified at 2 h after the end of infusion (2.46 mg/ L and 3.88 mg/ kg, respectively). The prostatic tissue concentrations of moxifloxacin were approximately twice as high as in corresponding serum. At the end of infusion the tissue and serum concentrations seemed to be already equilibrated, as their ratios did not differ significantly during the time of investigation. After an intravenous infusion of 400 mg the serum and prostatic tissue concentrations of moxifloxacin were well above the MIC values of most important prostatic pathogens. The high tissue/ serum ratio and the extended antibacterial spectrum suggests active concentration in the prostate which may translate into increased efficacy compared to group 2 and 3 fluoroquinolones in the treatment of chronic bacterial prostatitis.

Replicative senescence of biliary epithelial cells precedes bile duct loss in chronic liver allograft rejection: increased expression of p21(WAF1/Cip1) as a disease marker and the influence of immunosuppressive drugs.

Early chronic liver allograft rejection (CR) is characterized by distinctive cytological changes in biliary epithelial cells (BECs) that resemble cellular senescence, in vitro, and precede bile duct loss. If patients suffering from early CR are treated aggressively, the clinical and histopathological manifestations of CR can be completely reversed and bile duct loss can be prevented. We first tested whether the senescence-related p21(WAF1/Cip1) protein is increased in BECs during early CR, and whether treatment reversed the expression. The percentage of p21+ BECs and the number of p21+ BECs per portal tract is significantly increased in early CR (26 +/- 17% and 3.6 +/- 3.1) compared to BECs in normal liver allograft biopsies or those with nonspecific changes (1 +/- 1% and 0.1 +/- 0.3; P: < 0.0001 and P: < 0.02), chronic hepatitis C (2 +/- 3% and 0.7 +/- 1; P: < 0.0001 and P: < 0.04) or obstructive cholangiopathy (7 +/- 7% and 0.7 +/- 0.6; P: < 0.006 and P: = 0.04). Successful treatment of early CR is associated with a decrease in the percentage of p21+ BECs and the number of p21+ BECs per portal tract. In vitro, nuclear p21(WAF1/Cip1) expression is increased in large and multinucleated BECs, and is induced by transforming growth factor (TGF)-beta. TGF-beta1 also increases expression of TGF-beta receptor II, causes phosphorylation of SMAD-2 and nuclear translocation of p21(WAF1/Cip1), which inhibits BEC growth. Because conversion from cyclosporine to tacrolimus is an effective treatment for early CR, we next tested whether these two immunosuppressive drugs directly influenced BEC growth in vitro. The results show that cyclosporine, but not tacrolimus, stimulates BEC TGF-beta1 production, which in turn, causes BEC mito-inhibition and up-regulation of nuclear p21(WAF1/Cip1). In conclusion, expression of the senescence-related p21(WAF1/Cip1) protein is increased in BECs during early CR and decreases with successful recovery. Replicative senescence accounts for the characteristic BEC cytological alterations used for the diagnosis of early CR and lack of a proliferative response to injury. The ability of cyclosporine to inhibit the growth of damaged BECs likely accounts for the relative duct sparing properties of tacrolimus.

Environmental impacts of PAH and oil release as a NAPL or as contaminated pore water from the construction of a 90-cm in situ isolation cap.

The placement of a sediment cap was the remedial alternative selected in the Record of Decision for the containment of PAH-contaminated sediments near the Wyckoff/Eagle Harbor Superfund site shoreline, a former log rafting area at this closed wood treatment site. Soft sediments with substantial quantities of nonaqueous phase liquid (NAPL) occurred in this area, which raised a concern that there would be environmental releases or potential cap failure in this area of the site. As part of the investigations to guide cap design, a laboratory bench study was devised to evaluate consolidation-driven NAPL and dissolved phase PAH permeation of the cap. Sediment cores collected from the site were extruded side-by-side into 20 cm diameter, 120 cm high acrylic columns to maintain sediment stratification. Synthetic seawater was added until approximately 60 cm of water covered the site sediment. The simulated cap material was added to each column in such a manner as to fall through the overlying water at a uniform rate to simulate settling velocities expected during a barge wash-off placement event. Vertical loads were applied incrementally to the cap/sediment columns until the total consolidation stress was equivalent to a 90-cm cap. Each column was extruded, inspected visually for the migration of NAPL, and sectioned into three layers with each analyzed for total petroleum hydrocarbons and PAHs. In all three test cylinders, there was no indication of impact to the top 10 cm of the cap (the biologically active zone). The results suggest that the chemicals detected at high concentrations in the native sediments would stay in place and not migrate through a overlying cap via consolidation-induced advection.

Concanavalin A simultaneously primes liver hematopoietic and epithelial progenitor cells for parallel expansion during liver regeneration after partial hepatectomy in mice.

Liver hematopoietic progenitor cells (LHPC) and liver epithelial progenitor cells (LEPC) share a remarkable number of growth and differentiation-controlling receptor-ligand signaling systems. These likely account for the ability of the liver to support hematopoiesis in fetal life, and possibly for suggestions that LHPC can differentiate into hepatocytes. In these experiments, the kinetics and magnitude of LHPC and LEPC activation and expansion were studied by using a concanavalin A (Con A) liver injury model followed by partial hepatectomy (PH). Studies were performed in interleukin 6-deficient (IL-6(-/-)) mice and wild-type (IL-6(+/+)) controls, which show equal susceptibility to Con A- induced injury, because IL-6/gp130 signaling has been implicated in both LHPC and LEPC expansion. Con A pretreatment primed LHPC and LEPC for a rapid and parallel expansion after PH in IL-6(+/+) mice, which was significantly blunted and delayed in the IL-6(-/-) mice. Exogenous IL-6 given immediately before PH after Con A, augmented both LHPC and LEPC expansion in the IL-6(-/-) mice. Thus, the proinflammatory cytokine IL-6, commonly produced in liver injury and inflammatory disease, is an important growth factor involved in the expansion of LHPC and LEPC. This observation has implications for both hepatic carcinogenesis and transplantation.

Growth control of human biliary epithelial cells by interleukin 6, hepatocyte growth factor, transforming growth factor beta1, and activin A: comparison of a cholangiocarcinoma cell line with primary cultures of non-neoplastic biliary epithelial cells.

A well characterized human cholangiocarcinoma (CC) cell line, SG231, was compared with primary cultures of normal human biliary epithelial cells (BECs) for alterations in interleukin 6 (IL-6) and hepatocyte growth factor (HGF)-mediated stimulation and transforming growth factor beta1 (TGF-beta1) and activin A-mediated inhibition of growth. Results were compared with immunolabeling of the original tumor and after injection of SG231 into the liver of BALB/cByJ-scid mice. In vitro, both BECs and CCs expressed met, gp80, and gp130 messenger RNA (mRNA) and protein, but the levels of expression were higher in the CCs than in the BECs. In both the CCs and BECs, exogenous HGF or IL-6 induced phosphorylation of met or gp130, respectively, and a concentration-dependent increase in DNA synthesis. However, the CCs but not BECs, continued to grow in basal serum-free medium (SFM) and spontaneously produced both IL-6 and HGF under these conditions, which resulted in auto-phosphorylation of gp130 and met, respectively; and neutralizing anti-HGF or anti-IL-6 alone inhibited CC growth, indicative of autocrine growth control circuits. Conversely, activin A inhibits the growth of both BECs and CCs, but does not significantly increase apoptosis. Activin-A-induced growth inhibition of both CCs and BECs can be reversed by 100 ng/mL exogenous IL-6, but not by 10 to 100 ng/mL HGF. TGF-beta1 inhibited the growth of BECs but had no mitoinhibitory or proapoptotic effects on CCs. Immunolabeling of the original tumor and after inoculation into scid mice showed positive staining for met, gp130, gp80, and IL-6. This study contributes to a further understanding of BEC growth control and derangements that can occur during cholangiocarcinogenesis.

The effect of interleukin-6 (IL-6)/gp130 signalling on biliary epithelial cell growth, in vitro.

The effect of IL-6 on the growth of mouse biliary epithelial cells (BEC), in vitro, was tested by comparing BEC obtained IL-6-deficient mice (IL-6(-/-)) to wild-type littermate controls (IL-6(+/+)), in two different media: simple serum-free media (S-SFM), and complete serum-free media (C-SFM) containing forskolin, which stimulates BEC IL-6 production. In S-SFM, neither IL-6(+/+)nor IL-6(-/-)BEC constitutively produced IL-6 mRNA or protein, and there was no difference between IL-6(+/+)and IL-6(-/-)BEC growth. In contrast, when the BEC were maintained in C-SFM, over 48 h, the growth of IL-6(+/+)BEC was 40% greater than IL-6(-/-)BEC (P<0.006). Enhanced IL-6(+/+)BEC growth in C-SFM was associated with induced expression of IL-6 mRNA and IL-6 protein secretion into the medium, upregulation of the IL-6Ralpha (gp80) and phosphorylation of the signal transducing molecule gp130. In C-SFM, anti-IL-6 neutralizing antibodies blocked enhanced IL-6(+/+)BEC growth, whereas exogenous rhIL-6 stimulated retarded growth of IL-6(-/-)BEC. Thus, under conditions that mimic an inflammatory or stressful microenvironment in vivo, BEC produce, secrete and respond to IL-6, via upregulation and activation of the IL-6Ralpha (gp80)/gp130 signaling system in an autocrine/paracrine manner.

The development and compensation of biliary cirrhosis in interleukin-6-deficient mice.

In an effort to understand the role of IL-6/gp130 signaling in chronic liver injury, IL-6 deficient (IL-6(-/-)) and wild-type control (IL-6(+/+)) mice were subjected to bile duct ligation (BDL) for 12 weeks. This maneuver causes chronic biomechanical stress and liver injury, fueling sustained biliary epithelial and hepatocyte proliferation. By 12 weeks after BDL, IL-6(-/-) mice develop significantly higher total serum bilirubin levels (23.2 +/- 2.3 versus 14.9 +/- 2.1 mg/dl, P < 0.0001; delta bilirubin subfraction 16.7 +/- 4.0% versus 9.2 +/- 1.8%; P < 0.002), and the majority (15/18) show "black" gallbladder bile, compared to IL-6(+/+) mice (5/16; P < 0.003). The IL-6(-/-) mice also cannot sustain the compensatory liver mass increase commonly seen with chronic obstructive cholangiopathy, because of less hepatocyte proliferation, despite a rate of hepatocyte apoptosis similar to that of IL-6(+/+) mice. Moreover, IL-6(-/-) mice show a more advanced stage of biliary fibrosis and a higher mortality rate than the IL-6(+/+) controls (51% versus 23%; P < 0.02). These phenotypic changes in the IL-6(-/-) mice are associated with decreased expression and phosphorylation of gp130 and the transcription factor STAT3, compared to IL-6(+/+) mice. Daily treatment with exogenous recombinant IL-6 for 3-6 weeks starting at 6 weeks after BDL significantly lowers the serum total bilirubin in both groups. In the IL-6(-/-) mice, exogenous IL-6 treatment also increases the level of gp130 protein expression and completely reverses the loss of liver mass by increasing the hepatocyte proliferation. In conclusion, IL-6 appears to contribute to biliary tree integrity and maintenance of hepatocyte mass during chronic injury.