Encapsulation of packaging cell line results in successful retroviral-mediated transfer of a suicide gene in vivo in an experimental model of glioblastoma.

AIMS: Retroviral-mediated gene therapy has been proposed as a primary or adjuvant treatment for advanced cancer, because retroviruses selectively infect dividing cells. Efficacy of retroviral-mediated gene transfer, however, is limited in vivo. Although packaging cell lines can produce viral vectors continuously, such allo- or xenogeneic cells are normally rejected when used in vivo. Encapsulation using microporous membranes can protect the packaging cells from rejection. In this study, we used an encapsulated murine packaging cell line to test the effects of in situ delivery of a retrovirus bearing the herpes simplex virus thymidine kinase suicide gene in a rat model of orthotopic glioblastoma. MATERIALS AND METHODS: To test gene transfer in vitro, encapsulated murine psi2-VIK packaging cells were co-cultured with baby hamster kidney (BHK) cells, and the percentage of transfected BHK cells was determined. For in vivo experiments, orthotopic C6 glioblastomas were established in Wistar rats. Capsules containing psi2-VIK cells were stereotaxically implanted into these tumours and the animals were treated with ganciclovir (GCV). Tumours were harvested 14 days after initiation of GCV therapy for morphometric analysis. RESULTS: Encapsulation of psi2-VIK cells increased transfection rates of BHK target cells significantly in vitro compared to psi2-VIK conditioned medium (3 x 10(6) vs 2.3 x 10(4) cells; P<0.001). In vivo treatment with encapsulated packaging cells resulted in 3% to 5% of C6 tumour cells transduced and 45% of tumour volume replaced by necrosis after GCV (P<0.01 compared to controls). CONCLUSION: In this experimental model of glioblastoma, encapsulation of a xenogeneic packaging cell line increased half-life and transduction efficacy of retrovirus-mediated gene transfer and caused significant tumour necrosis.

The distribution of congenital anomalies within the VACTERL association among tumor necrosis factor antagonist-exposed pregnancies is similar to the general population.

OBJECTIVE: To compare the distribution of congenital anomalies within the VACTERL association (vertebral defects, anal atresia, cardiac, tracheoesophageal, renal, and limb abnormalities) between patients exposed to tumor necrosis factor-α (TNF-α) antagonist and the general population. METHODS: Analysis for comparison of proportional differences to a previous publication between anomaly subgroups, according to subgroup definitions of the European Surveillance of Congenital Anomalies (EUROCAT), a population-based database. RESULTS: Most EUROCAT subgroups belonging to the VACTERL association contained only one or 2 records of TNF-α antagonist exposure, so comparison of proportions was imprecise. Only the category "limb abnormalities" showed a significantly higher proportion in the general population. CONCLUSION: The high number of congenital anomalies belonging to the VACTERL association from a report of pregnancies exposed to TNF-α antagonists could not be confirmed using a population-based congenital anomaly database.

Not all inflammatory markers are linked to kidney function: results from a population-based study.

BACKGROUND: Several studies have reported increased levels of inflammatory biomarkers in chronic kidney disease (CKD), but data from the general population are sparse. In this study, we assessed levels of the inflammatory markers C-reactive protein (hsCRP), tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 across all ranges of renal function. METHODS: We conducted a cross-sectional study in a random sample of 6,184 Caucasian subjects aged 35-75 years in Lausanne, Switzerland. Serum levels of hsCRP, TNF-α, IL-6, and IL-1β were measured in 6,067 participants (98.1%); serum creatinine-based estimated glomerular filtration rate (eGFR(creat), CKD-EPI formula) was used to assess renal function, and albumin/creatinine ratio on spot morning urine to assess microalbuminuria (MAU). RESULTS: Higher serum levels of IL-6, TNF-α and hsCRP and lower levels of IL-1β were associated with a lower renal function, CKD (eGFR(creat) <60 ml/min/1.73 m(2); n = 283), and MAU (n = 583). In multivariate linear regression analysis adjusted for age, sex, hypertension, smoking, diabetes, body mass index, lipids, antihypertensive and hypolipemic therapy, only log-transformed TNF-α remained independently associated with lower renal function (β -0.54 ±0.19). In multivariate logistic regression analysis, higher TNF-α levels were associated with CKD (OR 1.17; 95% CI 1.01-1.35), whereas higher levels of IL-6 (OR 1.09; 95% CI 1.02-1.16) and hsCRP (OR 1.21; 95% CI 1.10-1.32) were associated with MAU. CONCLUSION: We did not confirm a significant association between renal function and IL-6, IL-1β and hsCRP in the general population. However, our results demonstrate a significant association between TNF-α and renal function, suggesting a potential link between inflammation and the development of CKD. These data also confirm the association between MAU and inflammation.

Urinalysis and pre-renal acute kidney injury: time to move on.

Urinary indices are classically believed to allow differentiation of transient (or pre-renal) acute kidney injury (AKI) from persistent (or acute tubular necrosis) AKI. However, the data validating urinalysis in critically ill patients are weak. In the previous issue of Critical Care, Pons and colleagues demonstrate in a multicenter observational study that sodium and urea excretion fractions as well as urinary over plasma ratios performed poorly as diagnostic tests to separate such entities. This study confirms the limited diagnostic and prognostic ability of urine testing. Together with other studies, this study raises more fundamental questions about the value, meaning and pathophysiologic validity of the pre-renal AKI paradigm and suggests that AKI (like all other forms of organ injury) is a continuum of injury that cannot be neatly divided into functional (pre-renal or transient) or structural (acute tubular necrosis or persistent).

MAP/ERK kinase kinase 1 (MEKK1) mediates transcriptional repression by interacting with polycystic kidney disease-1 (PKD1) promoter-bound p53 tumor suppressor protein.

Mitogen-activated protein kinase (MAPK) cascades regulate a wide variety of cellular processes that ultimately depend on changes in gene expression. We have found a novel mechanism whereby one of the key MAP3 kinases, Mekk1, regulates transcriptional activity through an interaction with p53. The tumor suppressor protein p53 down-regulates a number of genes, including the gene most frequently mutated in autosomal dominant polycystic kidney disease (PKD1). We have discovered that Mekk1 translocates to the nucleus and acts as a co-repressor with p53 to down-regulate PKD1 transcriptional activity. This repression does not require Mekk1 kinase activity, excluding the need for an Mekk1 phosphorylation cascade. However, this PKD1 repression can also be induced by the stress-pathway stimuli, including TNFα, suggesting that Mekk1 activation induces both JNK-dependent and JNK-independent pathways that target the PKD1 gene. An Mekk1-p53 interaction at the PKD1 promoter suggests a new mechanism by which abnormally elevated stress-pathway stimuli might directly down-regulate the PKD1 gene, possibly causing haploinsufficiency and cyst formation.

Specific delineation of BK polyomavirus in kidney tissue with a digoxigenin-labeled DNA probe.

The detection of BK polyomavirus (BK virus, BKV) in kidney tissue is hampered by nonspecificity of antibodies suited to immunohistochemistry, and nonspecific background with in situ hybridization. The biotin-labeled DNA probe that is commercially available (Enzo Life Sciences, Inc.) shows good signal, but the intrinsic background in kidney tissue is high. We determined that the intrinsic background is due to endogenous biotin or biotin-binding activity in the renal tubular epithelium. Neither antibody blocking procedures nor an avidin/biotin block were entirely satisfactory for eliminating this background staining. We developed a digoxigenin-labeled DNA probe, and protocol, for detecting BK virus in formalin-fixed, paraffin embedded, kidney tissue obtained at autopsy. The hybridization signal is strong and there is no perceptible background staining. Eleven negative control kidneys all failed to hybridize. Conditions for low stringency hybridization may be employed, detecting both the related JC polyomavirus and BKV. Alternatively, high stringency hybridization conditions may be utilized, detecting BKV only. BK associated tubular necrosis is clearly demonstrated in two cases of BK nephritis.

Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4.

In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI.

New measurements of energy expenditure and physical activity in chronic kidney disease.

The accurate estimation of total daily energy expenditure (TEE) in chronic kidney patients is essential to allow the provision of nutritional requirements; however, it remains a challenge to collect actual physical activity and resting energy expenditure in maintenance dialysis patients. The direct measurement of TEE by direct calorimetry or doubly labeled water cannot be used easily so that, in clinical practice, TEE is usually estimated from resting energy expenditure and physical activity. Prediction equations may also be used to estimate resting energy expenditure; however, their use has been poorly documented in dialysis patients. Recently, a new system called SenseWear Armband (BodyMedia, Pittsburgh, PA) was developed to assess TEE, but so far no data have been published in chronic kidney disease patients. The aim of this review is to describe new measurements of energy expenditure and physical activity in chronic kidney disease patients.

Giant warts in a kidney transplant patient: regression with sirolimus.

Purpose: Recent reports have suggested that intraabdominal postoperative infection is associated with higher rates of overall and local recurrence and cancer-specific mortality. However, the mechanisms responsible for this association are unknown. We hypothesized that the greater inflammatory response in patients with postoperative intraabdominal infection is associated to an increase in local and systemic angiogenesis. Methods: We designed a prospective cohorts study with matched controls. Patients with postoperative intra-abdominal infection (abscess and/or anastomotic leakage) (group 1; n=17) after elective colorectal cancer resection operated on for cure were compared to patients with an uncomplicated postoperative course (group 2; n=17). IL-6 and VEGF levels were determined by ELISA in serum and peritoneal fluid at baseline, 48 hours and postoperative day 4 or at the time the peritoneal infection occurred. Results: No differences were observed in age, gender, preoperative CEA, tumor stage and location and type of procedure performed. Although there were no differences in serum IL-6 levels at 48 hours, this pro-inflammatory cytokine was higher in group 1 on postoperative day 4 (group 1: 21533 + 27900 vs. group 2: 1130 + 3563 pg/ml; p < 0.001). Serum VEGF levels were higher in group 1 on postoperative day 4 (group 1: 1212 + 1025 vs. group 2: 408 + 407 pg/ml; p < 0.01). Peritoneal fluid VEGF levels were also higher in group 1 at 48 hours (group 1: 4857 + 4384 vs. group 2: 630 + 461 pg/ml; p < 0.001) and postoperative day 4 (group 1: 32807 + 98486 vs. group 2: 1002 + 1229 pg/ml; p < 0.001). A positive correlation between serum IL-6 and VEGF serum levels was observed on postoperative day 4 (r=0.7; p<0.01). Conclusions: These results suggest that not only the inflammatory response but also the angiogenic pathways are stimulated in patients with intra-abdominal infection after surgery for colorectal cancer. The implications of this finding on long-term follow-up need to be evaluated.

La lithiase rénale: un marqueur du risque cardiovasculaire [Kidney stone as a cardiovascular risk marker].

Si le passage d'un calcul rénal est souvent considéré comme un événement médical mineur, quoique très douloureux, de plus en plus d'études indiquent qu'il doit être pris au sérieux puisqu'il peut indiquer un risque cardiovasculaire augmenté. Nous revoyons ici les études qui associent risque cardiovasculaire et calcul rénal et les liens physiopathologiques qui les unissent. Nous montrons que la lithiase est un événement intervenant tôt dans la vie d'un individu à risque de développer des complications cardiovasculaires. Ainsi, la lithiase ne doit pas être banalisée, mais doit être considérée comme une première alerte devant inciter le médecin traitant à recenser précocement les facteurs de risque cardiovasculaires et à mettre en place une stratégie de prévention. Cette approche pourrait permettre de diminuer l'incidence d'événements cardiovasculaires chez les patients formeurs de lithiases. Most of the time, kidney stones are considered as minor, but painful events. However, several studies have recently shown an association between kidney stone and an increased cardio-vascular risk. We review here these studies and explore the underlying pathophysiological hypotheses. At the end, we propose that lithiasis should be considered as a red flag intervening early during life-time and allowing a check of cardiovascular risk factors and early preventive intervention. Such approach may be successful in reducing the incidence of cardio-vascular events in stone formers.

Treatment-dependent loss of polyfunctional CD8+ T-cell responses in HIV-infected kidney transplant recipients is associated with herpesvirus reactivation.

Antiretroviral-therapy has dramatically changed the course of HIV infection and HIV-infected (HIV(+)) individuals are becoming more frequently eligible for solid-organ transplantation. However, only scarce data are available on how immunosuppressive (IS) strategies relate to transplantation outcome and immune function. We determined the impact of transplantation and immune-depleting treatment on CD4+ T-cell counts, HIV-, EBV-, and Cytomegalovirus (CMV)-viral loads and virus-specific T-cell immunity in a 1-year prospective cohort of 27 HIV(+) kidney transplant recipients. While the results show an increasing breadth and magnitude of the herpesvirus-specific cytotoxic T-cell (CTL) response over-time, they also revealed a significant depletion of polyfunctional virus-specific CTL in individuals receiving thymoglobulin as a lymphocyte-depleting treatment. The disappearance of polyfunctional CTL was accompanied by virologic EBV-reactivation events, directly linking the absence of specific polyfunctional CTL to viral reactivation. The data provide first insights into the immune-reserve in HIV+ infected transplant recipients and highlight new immunological effects of thymoglobulin treatment. Long-term studies will be needed to assess the clinical risk associated with thymoglobulin treatment, in particular with regards to EBV-associated lymphoproliferative diseases.

Tumor necrosis factor (TNF) signaling, but not TWEAK (TNF-like weak inducer of apoptosis)-triggered cIAP1 (cellular inhibitor of apoptosis protein 1) degradation, requires cIAP1 RING dimerization and E2 binding.

Cellular inhibitor of apoptosis (cIAP) proteins, cIAP1 and cIAP2, are important regulators of tumor necrosis factor (TNF) superfamily (SF) signaling and are amplified in a number of tumor types. They are targeted by IAP antagonist compounds that are undergoing clinical trials. IAP antagonist compounds trigger cIAP autoubiquitylation and degradation. The TNFSF member TWEAK induces lysosomal degradation of TRAF2 and cIAPs, leading to elevated NIK levels and activation of non-canonical NF-kappaB. To investigate the role of the ubiquitin ligase RING domain of cIAP1 in these pathways, we used cIAP-deleted cells reconstituted with cIAP1 point mutants designed to interfere with the ability of the RING to dimerize or to interact with E2 enzymes. We show that RING dimerization and E2 binding are required for IAP antagonists to induce cIAP1 degradation and protect cells from TNF-induced cell death. The RING functions of cIAP1 are required for full TNF-induced activation of NF-kappaB, however, delayed activation of NF-kappaB still occurs in cIAP1 and -2 double knock-out cells. The RING functions of cIAP1 are also required to prevent constitutive activation of non-canonical NF-kappaB by targeting NIK for proteasomal degradation. However, in cIAP double knock-out cells TWEAK was still able to increase NIK levels demonstrating that NIK can be regulated by cIAP-independent pathways. Finally we show that, unlike IAP antagonists, TWEAK was able to induce degradation of cIAP1 RING mutants. These results emphasize the critical importance of the RING of cIAP1 in many signaling scenarios, but also demonstrate that in some pathways RING functions are not required.

Angiotensinergic innervation of the kidney: present knowledge and its significance.

Intrarenal neurotransmission implies the co-release of neuropeptides at the neuro-effector junction with direct influence on parameters of kidney function. The presence of an angiotensin (Ang) II-containing phenotype in catecholaminergic postganglionic and sensory fibers of the kidney, based on immunocytological investigations, has only recently been reported. These angiotensinergic fibers display a distinct morphology and intrarenal distribution, suggesting anatomical and functional subspecialization linked to neuronal Ang II-expression. This review discusses the present knowledge concerning these fibers, and their significance for renal physiology and the pathogenesis of hypertension in light of established mechanisms. The data suggest a new role of Ang II as a co-transmitter stimulating renal target cells or modulating nerve traffic from or to the kidney. Neuronal Ang II is likely to be an independent source of intrarenal Ang II. Further physiological experimentation will have to explore the role of the angiotensinergic renal innervation and integrate it into existing concepts.

Which subgroup of patients with rheumatoid arthritis benefits from switching to rituximab versus alternative anti-tumour necrosis factor (TNF) agents after previous failure of an anti-TNF agent?

BACKGROUND: Patients with rheumatoid arthritis (RA) with an inadequate response to TNF antagonists (aTNFs) may switch to an alternative aTNF or start treatment from a different class of drugs, such as rituximab (RTX). It remains unclear in which clinical settings these therapeutic strategies offer most benefit. OBJECTIVE: To analyse the effectiveness of RTX versus alternative aTNFs on RA disease activity in different subgroups of patients. METHODS: A prospective cohort study of patients with RA who discontinued at least one aTNF and subsequently received either RTX or an alternative aTNF, nested within the Swiss RA registry (SCQM-RA) was carried out. The primary outcome, longitudinal improvement in 28-joint count Disease Activity Score (DAS28), was analysed using multivariate regression models for longitudinal data and adjusted for potential confounders. RESULTS: Of the 318 patients with RA included; 155 received RTX and 163 received an alternative aTNF. The relative benefit of RTX varied with the type of prior aTNF failure: when the motive for switching was ineffectiveness to previous aTNFs, the longitudinal improvement in DAS28 was significantly better with RTX than with an alternative aTNF (p = 0.03; at 6 months, -1.34 (95% CI -1.54 to -1.15) vs -0.93 (95% CI -1.28 to -0.59), respectively). When the motive for switching was other causes, the longitudinal improvement in DAS28 was similar for RTX and alternative aTNFs (p = 0.40). These results were not significantly modified by the number of previous aTNF failures, the type of aTNF switches, or the presence of co-treatment with a disease-modifying antirheumatic drug. CONCLUSION: This observational study suggests that in patients with RA who have stopped a previous aTNF treatment because of ineffectiveness changing to RTX is more effective than switching to an alternative aTNF.