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Clusterin, ubiquitously distributed in mammalians, was cloned and identified as the most potently induced gene during rat prostate involution following androgen deprivation. Also found to be involved in many other patho-physiological processes, its biological significance is still controversial, particularly with regard to apoptosis. We previously showed that transient over-expression of clusterin blocked cell cycle progression of simian-virus-40-immortalized human prostate epithelial cell lines PNT1A and PNT2. We show in the present study that the accumulation of an intracellular 45 kDa clusterin isoform was an early event closely associated with death of PNT1A cells caused by cell detachment followed by apoptosis induction (anoikis). Cell morphological changes, decreased proliferation rate and cell cycle arrest at G0/G1-S-phase checkpoint were all strictly associated with the production and early translocation to the nucleus of a 45 kDa clusterin isoform. Later, nuclear clusterin was found accumulated in detached cells and apoptotic bodies. These results suggest that a 45 kDa isoform of clusterin, when targeted to the nucleus, can decrease cell proliferation and promotes cell-detachment-induced apoptosis, suggesting a possible major role for clusterin as an anti-proliferative gene in human prostate epithelial cells.

The impact of subclinical inflammation (SCI) noted on early kidney allograft biopsies remains unclear. This study evaluated the outcome of SCI noted on 3-month biopsy. A total of 273/363 (75%) kidney transplant recipients with a functioning kidney underwent allograft biopsies 3-months posttransplant. Among those with stable allograft function at 3 months, 200 biopsies that did not meet the Banff criteria for acute rejection were identified. These were Group I: No Inflammation (NI, n = 71) and Group II: Subclinical Inflammation (SCI, n = 129). We evaluated differences in kidney function at 24-months and allograft histology score at 12-month biopsy. SCI patients had a higher serum creatinine (1.6 ± 0.7 vs 1.38 ± 0.45; P = .02) at 24-months posttransplant, and at last follow-up at a mean of 42.5 months (1.69 ± 0.9 vs 1.46 ± 0.5 mg/dL; P = .027). The allograft chronicity score (ci + ct + cg + cv) at 12-months posttransplant was higher in the SCI group (2.4 ± 1.35 vs 1.9 ± 1.2; P = .02). The incidence of subsequent rejections within the first year in SCI and NI groups was 24% vs 10%, respectively (P = .015). De novo donor-specific antibody within 12 months was more prevalent in the SCI group (12/129 vs 1/71, P = .03). SCI is likely not a benign finding and may have long-term implications for kidney allograft function.

IntroductionPatients with chronic lymphocytic leukemia (CLL) who received intravenous infusions of autologous leukemia cells transfected with an adenovirus vector encoding the CD40 ligand (Ad-CD154) experienced acute reductions in leukemia cell counts and lymph node size. 1 This rapid cytoreduction was unexpected and suggested the possible contribution of innate immune-effector mechanisms to the early clearance of CLL cells following CD154 gene therapy.Following intravenous infusion of Ad-CD154-transduced CLL cells, we observed that bystander, nontransduced CLL cells were induced to express Fas (CD95) and DR5, 1,2 a receptor for the tumor necrosis factor (TNF)-receptor apoptosis-inducing ligand (TRAIL). Furthermore, activated CD4 T cells of patients treated with CD154 gene therapy were noted to express the ligands for such death receptors, namely Fas ligand (CD178) and TRAIL. 2 In vitro studies demonstrated that cells that expressed both CD178 and TRAIL could kill CLL cells within 1 day after CD40 ligation in a CD95-dependent fashion through coligation of both CD95 and DR5. 2 Moreover, CLL cells became increasingly sensitive to apoptosis induced by cells bearing CD178 and/or TRAIL over 3 to 5 days following CD40 activation. 2,3 CLL cells also can be induced to express high levels of CD95 and DR5 following coculture with CD154-bearing cells in vitro.Although initially resistant to CD95-or DR5-mediated apoptosis 1 day after such coculture, CD40-activated CLL cells become increasingly sensitive to apoptosis triggered by ligation of such extrinsic death receptors over the ensuing 3 to 5 days, an phenomenon termed "latent sensitivity to Fas-mediated apoptosis". 2,3 The initial resistance of CLL cells to CD95-mediated apoptosis following CD40 ligation may be secondary to CLL cell expression of inhibitors of apoptosis proteins (IAPs), such as the X-linked IAP (XIAP). IAPs negatively regulate apoptosis by inhibiting caspase activity directly. 4 Moreover, IAPs can block the execution phase of apoptosis through direct inhibition of the effector caspase-3 and/or caspase-7. 5 In addition, IAPs can prevent initiation of the intrinsic caspase activation cascade by directly inhibiting the apical caspase-9. Finally, high-level expression of XIAP, such as that found in CLL, 6-8 can inhibit CD95-mediated apoptosis of cells that express CD95. 9 Conversely, the latent sensitivity of CLL cells to CD95-mediated apoptosis following CD40 ligation may be due to release of intrinsic inhibitors to the IAPs that are sequestered within the 2 In different cell lines, it has been shown that Bid is degraded following ligation of extrinsic death receptors, such as CD95 or DR5, thereby generating a small truncated Bid (tBid) that rapidly trafficks to the mitochondria where it can trigger the release of the second mitochondria-derived activator of caspases (Smac), a potent natural IAP inhibitor that also is referred to as the direct IAP-binding protein with low isoelectric point (pI) (DIABLO). [11][12][13][14] Conceivably, inhibition of IAPs by ...

We present this observational study of lung transplant recipients (LTR) treated with carfilzomib (CFZ)-based therapy for antibody-mediated rejection (AMR) of the lung. Patients were considered responders to CFZ if complement-1q (C1q)-fixing ability of their immunodominant (ID) donor-specific anti-human leukocyte antibody (DSA) was suppressed after treatment. Treatment consisted of CFZ plus plasma exchange and immunoglobulins. Fourteen LTRs underwent CFZ for 20 ID DSA AMR. Ten (71.4%) of LTRs responded to CFZ. DSA IgG mean fluorescence intensity (MFI) fell from 7664 (IQR 3230-11 874) to 1878 (653-7791) after therapy (p = 0.001) and to 1400 (850-8287) 2 weeks later (p = 0.001). DSA C1q MFI fell from 3596 (IQR 714-14 405) to <30 after therapy (p = 0.01) and <30 2 weeks later (p = 0.02). Forced expiratory volume in 1s ( FEV ) fell from mean 2.11 L pre-AMR to 1.92 L at AMR (p = 0.04). FEV was unchanged after CFZ (1.91 L) and subsequently rose to a maximum of 2.13 L (p = 0.01). Mean forced expiratory flow during mid forced vital capacity (25-75) (FEF ) fell from mean 2.5 L pre-AMR to 1.95 L at AMR (p = 0.01). FEF rose after CFZ to 2.54 L and reached a maximum of 2.91 L (p = 0.01). Responders had less chronic lung allograft dysfunction or progression versus nonresponders (25% vs. 83%, p = 0.04). No deaths occurred within 120 days and 7 patients died post CFZ therapy of allograft failure. Larger prospective interventional studies are needed to further describe the benefit of CFZ-based therapy for pulmonary AMR.

Clusterin gene expression is potently induced in experimental models in which apoptosis is activated, such as rat prostate involution following castration. Nevertheless, its precise physiological role has not yet been established, and both anti-apoptotic and pro-apoptotic functions have been suggested for this gene. Clusterin expression level depends on cell proliferation state, and we recently showed that its over-expression inhibited cell cycle progression of SV40-immortalized human prostate epithelial cells PNT2 and PNT1a. Here we studied clusterin expression in PNT1a cells subjected to serum-starvation with the aim of defining clusterin early molecular changes following apoptosis induction. Under serum-starvation conditions, decreased growth rate, slow rounding-up of cells, cell detachment, and formation of apoptotic bodies indicative of anoikis (detachment-induced apoptosis) were preceded by significant downregulation of 70 kDa clusterin precursor and upregulation of 45-40 kDa isoforms. On the 8th day of serum-free culturing, only the higher molecular weight protein-band of about 45 kDa was clearly induced and accumulated in detached cells and apoptotic bodies in which PARP was activated. Anoikis was preceded by induction and transloction of a 45-kDa clusterin isoform to the nucleus. Thus, nuclear targeting of a specific 45-kDa isoform of clusterin appeared to be an early and specific molecular signal triggering anoikis-death. Considering also that clusterin is downregulated during prostate cancer onset and progression, and that its upregulation has inhibited DNA synthesis and cell cycle progression of immortalized human prostate epithelial cells, we suggest that clusterin might be a new anti-oncogene in the prostate.

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(i) End-stage renal disease was associated with low antipig antibody levels. (ii) Xenoreactivity decreased with increased genetic engineering of pig cells. (iii) High cPRA status had no significant effect on antibody binding or T-cell and B-cell response.