T Cook

Nephropathy caused by Chinese herbs in the UK

The use of Chinese herbal remedies is increasing in the UK. We report the presence of a nephrotoxic compound in herb samples, which led to end-stage renal failure in two patients. We suggest that use of these products is regulated more tightly.

Urothelial malignant disease and chinese herbal nephropathy

We have previously reported occurrence of a specific type of nephropathy due to ingestion of Chinese herbs (Chinese herbal nephropathy [CHN]) in two patients in the UK. These cases highlighted the role of aristolochic acid in causing this nephropathy, which was first described in a Belgian cohort. We now report development of invasive transitional cell carcinoma of the urinary tract associated with the presence of aristolochic acid-DNA adducts in one of these patients. This work clearly shows the carcinogenic potential of aristolochic acid in this new type of nephropathy.

Foetal gene delivery in mice by intra-amniotic administration of retroviral producer cells and adenovirus

With the aim of developing foetal gene therapy for cystic fibrosis, we have investigated the possibility of gene targeting to the mouse foetus with two different viral vector systems and at different times of gestation. We report here that recombinant retrovirus producing cells administered into the intra-amniotic cavity of mid- to late-gestation mouse MF1 foetuses survive in the amniotic fluid and are able to engraft to a certain extent in foetal tissues. By production of infectious virus they mediate transduction and β-galactosidase transgene expression in neighbouring foetal tissues 24 to 72 h following injection. Retrovirus producer cells could, therefore, become a means to overcome the limitations of low retroviral titre, for in vivo foetal gene transfer. To investigate the developmental stage at which transduction of the airways and enteral systems can be obtained we also administered a highly infective first generation adenoviral vector (AdRSVβgal) into the amniotic cavity of foetal mice between 13 to 16 days post coitus, β-galactosidase activity was detected between 24 to 120 h after injection. The highest levels of transgene expression were generally observed between 48 to 72 h following injection of the adenoviral vector. We demonstrate that infection of the pulmonary airways is dependent on the developmental stage of the foetus and can be achieved on the 15th day of gestation.

Successful expression of β-galactosidase and factor IX transgenes in fetal and neonatal sheep after ultrasound-guided percutaneous adenovirus vector administration into the umbilical vein

In utero somatic gene therapy in the later stages of pregnancy may allow targeting of organ systems which are difficult to reach later in life and to prevent the development of tissue damage otherwise caused by the early onset of inherited diseases. We report here on the percutaneous delivery of two adenoviral vectors, containing the β-galactosidase reporter gene and the human Factor IX gene respectively, to the fetal liver and circulation by ultrasound-guided umbilical vein puncture similar to procedures used in human pregnancy. Vector spread, as detected by PCR analysis for the β-galactosidase encoding vector, was found in almost all fetal and neonatal organs and in the maternal liver. Expression of the β-galactosidase transgene was detected in many fetal tissues by RT-PCR. High β-galactosidase production was shown by immuno-histochemistry predominantly in the liver, where about 30% of the hepatocytes stained positive, and in the adrenal cortex. Production of factor IX was determined by ELISA in the plasma of treated fetuses and newborn lambs and reached at birth up to 80% of the normal human plasma concentration. This demonstrates a very hopeful proof of principle for the development of prenatal treatment of many genetic diseases but also requires more detailed investigations with respect to the observed systemic spread of the vector.

Local delivery of VEGF adenovirus to the uterine artery increases vasorelaxation and uterine blood flow in the pregnant sheep

Impaired materno-placental perfusion causes two important obstetric complications, fetal growth restriction and preeclampsia. This study investigated whether adenoviral vector-mediated overexpression of vascular endothelial growth factor (VEGF) in the uterine arteries (UtAs) increases uterine artery blood flow (UBF). First-generation adenovirus vectors (5 × 1011 particles) containing the VEGF gene (Ad.VEGF-A or -D) or the β-galactosidase reporter gene (Ad.lacZ) were injected into the UtAs of pregnant sheep (n=6) at 88–102 days of gestation (term=145 days). UBF was measured using Doppler sonography before, and 4–7 days after injection. Mean UBF increased significantly from 233±156 (s.d.) ml min−1 to 753±415 ml min−1 following Ad.VEGF-A injection (P=0.005, n=5); Ad.lacZ infection had no significant effect. Organ bath experiments on uterine arterial sections 4–7 days after injection showed that, compared with Ad.lacZ vessels, Ad.VEGF-A-transduced vessels had a reduced contractile response to phenylephrine (Emax 148±10.9 vs Emax 228.2±27.5, P<0.05) but increased relaxation with bradykinin (pD2 (−log EC50) values 9.11±0.01 vs 8.65±0.11, P<0.05). Injection of Ad.VEGF-A into the UtAs increases UBF by enhancing vasodilatation. This may provide the basis for therapy in pregnancies complicated by uteroplacental insufficiency.

Ultrasound-guided percutaneous delivery of adenoviral vectors encoding the beta-galactosidase and human factor IX genes to early gestation fetal sheep in utero.

In utero gene therapy may provide treatment of genetic diseases before significant organ damage, allow permanent genetic correction by reaching stem cell populations, and provide immune tolerance against the therapeutic transgenes and vectors. We have used percutaneous ultrasound-guided injection as a minimally invasive fetal procedure. First-generation adenoviruses encoding the nuclear localizing beta-galactosidase reporter gene or the human factor IX (hFIX) gene, or colloidal carbon were delivered via the umbilical vein (UV, n = 4), heart (intracardiac [IC], n = 2), liver parenchyma (intrahepatic [HE], n = 11), peritoneal cavity (intraperitoneal [IP], n = 14), skeletal musculature ([intramuscular [IM], n = 11), or the amniotic cavity (intraamniotic [IA], n = 14) to early-gestation fetal sheep (0.3 gestation = day 33-61). Postmortem analysis was performed at 2, 9, or 28 days after injection. Although fetal survival was between 77% and 91% for IP, HE, IA, and IM routes, no fetuses survived UV or IC procedures. The hFIX levels reaching 1900 and 401 ng/ml (IP), 30 ng/ml (HE), 66.5 and 39 ng/ml (IA), and 83 and 65.5 ng/ml (IM), respectively, were determined 2 days after injection and decreased at birth to 16.5 ng/ml (IP), 7 ng/ml (HE), 4.5 ng/ml (IA), and 4 and 0 ng/ml (IM). Polymerase chain reaction (PCR) and immunohistochemistry showed broadest hFIX transgene spread and highest localised beta-galactosidase expression, respectively, after IP administration. Antibodies were observed against vector but not against hFIX.

Therapeutic plasma concentrations of human factor IX in mice after gene delivery into the amniotic cavity: a model for the prenatal treatment of haemophilia B

Several groups including our own have reported gene delivery to fetal organs by vector administration into the amniotic cavity. Based on these studies we hypothesised that the large surface of the fetal skin may be exploitable for high level production of systemically required gene products to be released into the fetal circulation.

Preformed complement-activating low-level donor-specific antibody predicts early antibody-mediated rejection in renal allografts.

Background. Donor-specific anti-HLA antibodies (DSA) are a major cause of alloimmune injury. Transplant recipients with negative complement-dependent cytotoxic crossmatch (CDC-XM) and donor cell-based flow cytometric crossmatch (flow-XM) but low level DSA (i.e., by Luminex) have worse outcomes compared with nonsensitized patients. The aim of this study was to establish whether complement-activating ability in this low-level DSA, present before transplantation, as determined by this technique is important in dictating pathogenicity. Methods. We retrospectively studied 52 patients with preformed DSA detected by single-antigen flow cytometric fluorescent beads (SAFBs). Patients were transplanted using a steroid-sparing regimen consisting of alemtuzumab induction, 1 week of corticosteroids and tacrolimus monotherapy.Fifteen (29%) of 52 patients experienced antibody-mediated rejection (AMR), whereas 37 (71%) patients did not. There were no demographic differences between patients with AMR and those without. Pretransplant sera were retested using a modified (SAFB) assay, which detects the presence of the complement fragment C4d as a result of DSA-induced complement activation. Results. C4d+DSA were detected in 10 (19%) of 52 patients. Biopsy-proven AMR occurred in 7 (70%) of the 10 patients with C4d+DSA and in 8 (19%) of 42 patients with C4d-DSA. AMR-free survival was worse in patients with C4d+DSA (P<0.001). Conclusions. The ability of preformed, low-level, DSA to trigger C4d fixation in vitro in patients with negative conventional crossmatch tests is predictive for AMR. C4d SAFB is potentially a powerful tool for risk stratification prior to transplantation and may allow identification of unacceptable donor antigens, or patients who may require enhanced immunosuppression.

Antibody-mediated rejection after alemtuzumab induction: incidence, risk factors, and predictors of poor outcome.

Background. Antibody-mediated rejection (AMR) is associated with allograft loss. Identification of factors associated with poor outcome has not been extensively studied. Methods. We retrospectively studied 469 patients who received a negative crossmatch renal transplant with alemtuzumab induction. Forty-eight of 469 (10.2%) patients were treated for AMR. Thirty of 48 (62.5%) of the cases fulfilled the Banff criteria for definite AMR, whereas 18 of 48 (37.5%) were categorized as suspicious for AMR (tissue injury with C4d staining or donor-specific antibodies [DSAbs]). Sensitization, high human leukocyte antigen, and -DR mismatch were risk factors for the development of AMR (P=0.0016, 0.001, and 0.012, respectively). Results. Allograft survival was inferior in the AMR group (70.2%) compared with the nonrejector group (97.0%) (P<0.001). Forty-two of 48 (87.5%) of patients with acute AMR had DSAbs. Patients with CII DSAbs at the time of AMR, whether alone or in combination with CI DSAbs had the worst allograft survival (P=0.014). Both the mean cumulative and immunodominant mean fluorescence index were higher in those patients who subsequently lost their grafts (P<0.001). Patients with diffuse C4d staining had inferior allograft survival than those with focal C4d or no staining (P=0.02). There was no significant difference in survival by histological grade but a trend to inferior outcomes in those with vascular involvement (P=0.06). Those patients who met the full Banff criteria had worse survival than those with suspicion for AMR only (P=0.04). Conclusion. This study identifies patients at risk of graft failure from AMR. These patients may benefit from newer therapeutic strategies including the use of eculizumab or bortezomib.

Lesson of the week: cholesterol emboli syndrome.

Cholesterol embolism is a common but underrecognised complication arising from a variety of vascular insults