Analysis of HLA-G expression in renal tissue in lupus nephritis: a pilot study.

BACKGROUND: The study aimed to investigate whether HLA-G antigen is expressed in the kidneys of patients affected by lupus nephritis (LN) and whether its detection in renal biopsies could be adopted as a marker of treatment response and prognosis. METHODS: Thirty renal biopsies from patients with LN were selected and analyzed through immunohistochemistry. Laboratory and clinical data were retrospectively collected at baseline, 6 and 12 months and at the latest clinical appointment. A number of patients (63.3%) were treated with rituximab (RTX) +/- methylprednisolone in the induction phase. The expression of HLA-G in glomeruli, tubules and infiltrating cells was examined and compared between lupus patients who achieved either complete or partial renal response and those who did not respond to treatment. RESULTS: HLA-G staining was observed in the glomeruli of 20 of 30 samples from patients with LN. The expression of the antigen was detected in podocytes, along glomerular capillary walls, on parietal glomerular epithelial cells and within the juxtaglomerular apparatus. Seventy per cent of patients whose glomeruli expressed HLA-G achieved partial or complete response at 6 months and 75% at the latest available follow up compared with 30% and 40%, respectively, of those who did not show any expression. The pattern of staining in tubules and infiltrating cells was highly variable precluding any clinical correlation. CONCLUSION: This study demonstrates that HLA-G is expressed in renal tissue in LN. Our retrospective data suggest that its expression could correlate with response to treatment.

IgG4-related disease in a multi-ethnic community: clinical characteristics and association with malignancy.

BACKGROUND: Immunoglobulin-G4-related disease (IgG4-RD) is a recently recognized fibro-inflammatory condition that can affect multiple organs. Despite growing interest in this condition, the natural history and management of IgG4-RD remain poorly understood. AIM: To describe the clinical characteristics, treatment and outcomes of IgG4-RD in a multi-ethnic UK cohort, and investigate its possible association with malignancy. DESIGN: Retrospective analysis of case-note and electronic data. METHODS: Cases were identified from sub-specialty cohorts and a systematic search of an NHS trust histopathology database using 'IgG4' or 'inflammatory pseudotumour' as search terms. Electronic records, imaging and histopathology reports were reviewed. RESULTS: In total, 66 identified cases of IgG4-RD showed a similar multi-ethnic spread to the local population of North West London. The median age was 59 years and 71% of patients were male. Presenting symptoms relating to mass effect of a lesion were present in 48% of cases and the mean number of organs involved was 2.4. Total of 10 patients had reported malignancies with 6 of these being haematological. 83% of those treated with steroids had good initial response; however, 50% had relapsing-remitting disease. Rituximab was administered in 11 cases and all achieved an initial serological response. Despite this, seven patients subsequently relapsed after a mean duration of 11 months and four progressed despite treatment. CONCLUSIONS: We report a large UK-based cohort of IgG4-RD that shows no clear ethnic predisposition and a wide range of affected organs. We discuss the use of serum IgG4 concentrations as a disease marker in IgG4-RD, the association with malignant disease and outcomes according to differing treatment regimens.

Updating genome annotation for the microbial cell factory Aspergillus niger using gene co-expression networks.

A significant challenge in our understanding of biological systems is the high number of genes with unknown function in many genomes. The fungal genus Aspergillus contains important pathogens of humans, model organisms, and microbial cell factories. Aspergillus niger is used to produce organic acids, proteins, and is a promising source of new bioactive secondary metabolites. Out of the 14,165 open reading frames predicted in the A. niger genome only 2% have been experimentally verified and over 6,000 are hypothetical. Here, we show that gene co-expression network analysis can be used to overcome this limitation. A meta-analysis of 155 transcriptomics experiments generated co-expression networks for 9,579 genes (∼65%) of the A. niger genome. By populating this dataset with over 1,200 gene functional experiments from the genus Aspergillus and performing gene ontology enrichment, we could infer biological processes for 9,263 of A. niger genes, including 2,970 hypothetical genes. Experimental validation of selected co-expression sub-networks uncovered four transcription factors involved in secondary metabolite synthesis, which were used to activate production of multiple natural products. This study constitutes a significant step towards systems-level understanding of A. niger, and the datasets can be used to fuel discoveries of model systems, fungal pathogens, and biotechnology.

Exploitation of sulfonylurea resistance marker and non-homologous end joining mutants for functional analysis in Zymoseptoria tritici.

The lack of techniques for rapid assembly of gene deletion vectors, paucity of selectable marker genes available for genetic manipulation and low frequency of homologous recombination are major constraints in construction of gene deletion mutants in Zymoseptoria tritici. To address these issues, we have constructed ternary vectors for Agrobacterium tumefaciens mediated transformation of Z. tritici, which enable the single step assembly of multiple fragments via yeast recombinational cloning. The sulfonylurea resistance gene, which is a mutated allele of the Magnaporthe oryzae ILV2 gene, was established as a new dominant selectable marker for Z. tritici. To increase the frequency of homologous recombination, we have constructed Z. tritici strains deficient in the non-homologous end joining pathway of DNA double stranded break repair by inactivating the KU70 and KU80 genes. Targeted gene deletion frequency increased to more than 85% in both Z. tritici ku70 and ku80 null strains, compared to ⩽10% seen in the wild type parental strain IPO323. The in vitro growth and in planta pathogenicity of the Z. tritici ku70 and ku80 null strains were comparable to strain IPO323. Together these molecular tools add significantly to the platform available for genomic analysis through targeted gene deletion or promoter replacements and will facilitate large-scale functional characterization projects in Z. tritici.

Construction and high-throughput phenotypic screening ofZymoseptoria tritici over-expression strains.

Targeted gene deletion has been instrumental in elucidating many aspects of Zymoseptoria tritici pathogenicity. Gene over-expression is a complementary approach that is amenable to rapid strain construction and high-throughput screening, which has not been exploited to analyze Z. tritici, largely due to a lack of available techniques. Here we exploit the Gateway® cloning technology for rapid construction of over-expression vectors and improved homologous integration efficiency of a Z. tritici Δku70 strain to build a pilot over-expression library encompassing 32 genes encoding putative DNA binding proteins, GTPases or kinases. We developed a protocol using a Rotor-HDA robot for rapid and reproducible cell pinning for high-throughput in vitro screening. This screen identified an over-expression strain that demonstrated a marked reduction in hyphal production relative to the isogenic progenitor. This study provides a protocol for rapid generation of Z. tritici over-expression libraries and a technique for functional genomic screening in this important pathogen.

A suite of Gateway® compatible ternary expression vectors for functional analysis in Zymoseptoria tritici.

Gene overexpression is a widely used functional genomics approach in fungal biology. However, to date it has not been established in Zymoseptoria tritici which is an important pathogen of wheat (Triticum species). Here we report a suite of Gateway® recombination compatible ternary expression vectors for Agrobacterium tumefaciens mediated transformation of Z. tritici. The suite of 32 vectors is based on a combination of four resistance markers for positive selection against glufosinate ammonium, geneticin, hygromycin and sulfonylurea; three constitutive Z. tritici promoters (pZtATUB, pZtGAPDH and pZtTEF) and a nitrogen responsive promoter (pZtNIA1) for controlled expression of the open reading frames. Half of the vectors facilitate expression of proteins tagged with C-terminal EGFP. All 32 vectors allow high frequency targeting of the overexpression cassette into the Ku70 locus and complement the Ku70 gene when transformed into a Z. tritici ku70 null strain, thus circumventing additional phenotypes that can arise from random integration. This suite of ternary expression vectors will be a useful tool for functional analysis through gene overexpression in Z. tritici.

In vitro investigation of a terbinafine impregnated subcutaneous implant for veterinary use.

A terbinafine impregnated subcutaneous implant was evaluated to determine if drug was released into isotonic saline over the course of 6 months at two different temperatures, 37°C and 4°C. These temperatures were chosen to simulate the nonhibernating (37°C) and hibernating body (4°C) temperatures of little brown bats (Myotis lucifugus). Insectivorous bats of North America, including little brown bats, have been devastated by white nose syndrome, a fungal infection caused by Geomyces destructans. No treatments exist for bats infected with G. destructans. Implants were placed into isotonic saline; samples were collected once per week and analyzed with HPLC to determine terbinafine concentrations. The mean amount of terbinafine released weekly across the 28 weeks was approximately 1.7 µg at 4°C and 4.3 µg at 37°C. Although significant differences in the amount released did occur at some time points, these differences were not consistently greater or less at either of the temperatures. This study showed that terbinafine was released from an impregnated implant over the course of 6 months at concentrations ranging from 0.02 to 0.06 µg/mL depending on temperature, which may be appropriate for little brown bats (Myotis lucifugus) infected with Geomyces destructans, the etiologic agent of white nose syndrome.

Outcome of patients with preformed donor-specific antibodies following alemtuzumab induction and tacrolimus monotherapy.

It has been shown that low-level preformed donor-specific antibodies (DSAbs) detected by luminex beads in the setting of a negative CDC and flow cytometry crossmatch (CDC/FCXM) are associated with inferior allograft outcomes. The relevance of preformed DSAbs in patients receiving alemtuzumab induction and tacrolimus monotherapy has not been studied. Four hundred and eighty renal transplant recipients with a negative CDC/FCXM had their pretransplant sera retrospectively screened for DSAbs. 45/480 (9.4%) of patients were found to have preformed DSAbs. Females and patients receiving regrafts were more likely to have a DSAb (p = 0.008 and p < 0.0001, respectively). Patients with DSAbs had inferior allograft survival (p = 0.047), increased incidence of antibody-mediated rejection (p < 0.0001) and inferior allograft function at 6 months posttransplant (p = 0.017). Patients with HLA class I DSAb (alone or in combination with a Class II DSAb) with high mean fluorescence intensities (MFIs) were at highest risk. We conclude that patients with preformed DSAb are at high risk of adverse outcomes when receiving a minimal immunosuppressive regime incorporating alemtuzumab induction. Patients found to have a preformed DSAb despite a negative crossmatch might benefit from augmented immunosuppression.

Mycophenolic acid 12-h trough level monitoring in renal transplantation: association with acute rejection and toxicity.

Studies of renal transplantation utilizing trough plasma level monitoring of mycophenolic acid (MPA) have shown inconsistent associations with toxicity and rejection. In this study, 5600 12-h trough MPA samples from 121 renal transplant recipients immunosuppressed with mycophenolate mofetil (MMF) and tacrolimus in a steroid sparing protocol (steroids for 7 days only) were sequentially analyzed. Higher MPA levels were associated with lower hemoglobin concentrations and anemia (hemoglobin <10 g/dL). Similarly, higher MPA levels were associated with lower total white cell counts and an increased incidence of leucopenia (total white cell count <4.0 x 10(9)/L). Hypoalbuminemia and renal impairment were also associated with hemotoxicity. MMF-associated diarrhea and viral infection were associated with higher MPA levels. Conversely, biopsy-proven acute rejection within the first month post-transplantation was associated with lower MPA levels. Anti-CD25 antibody induction was also associated with reduced rejection rates. No association was seen between MPA levels and platelet count, thrombocytopenia or bacterial infection. An MPA level of 1.60 mg/L early post-transplantation best discriminated patients with and without rejection, and an MPA level of 2.75 mg/L best discriminated patients with and without toxicity later post-transplantation.

Tacrolimus monitoring in renal transplantation: a comparison between high-performance liquid chromatography and immunoassay.

It is recommended that specific methods of tacrolimus monitoring rather than immunoassays, which overestimate tacrolimus levels, should be used in transplant recipients. Direct comparison of these techniques, however, has not been conducted in renal transplantation. In this study, 40 renal transplant recipients with tacrolimus monitoring by microparticle enzyme immunoassay (MEIA; target trough level 10 to 15 ng/mL) were compared with 40 patients monitored by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS; target trough level 8 to 13 ng/mL). All patients received anti CD25 antibody induction and mycophenolate mofetil in a steroid-sparing protocol. No differences were seen between MEIA and HPLC-MS groups in patient demographics. All patients were followed for 6 months. Patient survival was 100% in both groups; graft survival was 100% in the MEIA group and 97.5% in the HPLC-MS group. The groups did not differ in the number of dose changes required in the first 6 months or in the number of patients displaying tacrolimus levels within target range at 3 and 6 months. Delayed graft function occurred in 14 patients in the MEIA group and 12 patients in the HPLC-MS group (P = NS). Biopsy-proven acute rejection occurred in four patients in the MEIA group and one patient in the HPLC-MS group (P < .2). No differences were seen for the following parameters at 3 or 6 months: biopsy-proven tacrolimus nephrotoxicity, serum creatinine or estimated creatinine clearance, systolic or diastolic blood pressure, cholesterol, cytomegalovirus disease, posttransplant diabetes, or tremor. This study suggests that renal transplantation with HPLC-MS monitoring of tacrolimus is safe and effective.

Cytokine polymorphisms do not influence acute rejection in renal transplantation under tacrolimus-based immunosuppression.

INTRODUCTION: Acute rejection remains an important cause of graft loss after renal transplantation. It has been suggested that cytokine genotyping may play a predictive role in identifying individuals who are at higher risk of acute rejection with a view to individualizing their immunosuppression. The aim of this study was to investigate any possible associations between acute rejection and certain cytokine polymorphisms. METHODS: We genotyped 91 cadaveric renal transplant recipients on tacrolimus-based immunosuppression and 84 of their donors. The cytokine polymorphisms studied were the following: tumor necrosis factor (TNF)-alpha-1032 T/C, TNF-alpha-865 C/A, TNF-alpha-859 G/A, interleukin (IL)1-R1-970 C/T, IL-10 haplotype [-1082, -819, -592], and IL-6-174 C/G. RESULTS: We found no association between any polymorphism and the incidence of acute rejection. This was true for both the recipient and donor population. CONCLUSION: Cytokine polymorphisms did not influence acute rejection in our study. We conclude that in the modern era of immunosuppression cytokine genotyping is not a significant predictor of acute rejection in renal transplantation.

Steroid sparing in renal transplantation with tacrolimus and mycophenolate mofetil: three-year results.

Although renal transplantation with a 7-day steroid-sparing regimen, tacrolimus and mycophenolate, is associated with good short-term outcomes, late allograft dysfunction and failure remain concerns. In this study 101 consecutive patients underwent renal transplantation using this immunosuppressive regimen. In addition, anti-CD25 monoclonal antibody was used in 25 high-risk patients (regrafts, two-antigen human leukocyte antigen (HLA)-DR mismatch or sensitized with anti-HLA panel reactivity >30%). After a median follow-up of 39 months (range 29 to 49), overall patient survival is 98%, with two cardiac deaths. Three other graft losses occurred, one each to early venous thrombosis, polyoma viral nephropathy, and late rejection due to noncompliance. Therefore, overall graft survival is 95%. The acute rejection rate at 6 and 12 months was 19% (no rejection occurred between months 6 and 12). Late rejection was uncommon, with only two further episodes beyond 12 months. Mean creatinine at 12 months was 144 micromol/L and mean estimated glomerular filtration rate (GFR) of 55 mL/min. Graft function was stable at 3 years with a mean creatinine of 142 micromol/L and mean estimated GFR 56 mL/min. During the study, five patients developed posttransplant diabetes mellitus (two cases beyond 12 months). Tissue-invasive cytomegalovirus disease and BK viral nephropathy each occurred in three patients, with all episodes in the first 12 months. Mean weight gain is 3.3 kg and mean blood pressure is 135/81 on an average of 1.5 antihypertensive agents. This steroid-avoidance regimen is associated with excellent medium-term patient and graft outcomes and a low incidence of side effects.

Late steroid withdrawal for renal transplant recipients on tacrolimus and MMF is safe.

INTRODUCTION: We conducted a study to assess the safety of staged, late steroid withdrawal in kidney or kidney/pancreas transplant recipients on steroids, tacrolimus, and mycophenolate mofetil (MMF). MATERIALS AND METHODS: We studied 50 patients including 33 recipients of cadaveric kidneys, eight living donor kidneys, and nine kidney-pancreas transplants. The mean time posttransplantation was 5.1 years (range 2.1 to 7.9 years). All patients were induced on prednisolone, tacrolimus, and MMF; steroids were withdrawn over 5 to 6 months. The rate of steroid reduction was altered in the face of typical steroid withdrawal symptoms (limb-girdle arthralgia/myalgia). RESULTS: No rejection episodes occurred during steroid withdrawal. No patient required transplant biopsy for graft dysfunction. Six patients failed steroid withdrawal: five due to arthralgia/myalgia and one due to recurrent pulmonary sarcoidosis. The unexplained rise in serum creatinine following steroid withdrawal described in several other steroid withdrawal studies was not observed in this patient cohort. The mean serum creatinine was 137 micromol/L with deltacreatinine -6.8 micromol/y per year prior to steroid cessation versus 132 micromol/L with deltacreatinine -5.9 micromol/y in the year post-steroid cessation. There were 14 patients with posttransplant diabetes mellitus in this cohort: eight on gliclazide and six on insulin. We observed a reduction in their daily insulin/gliclazide requirements from 52 units to 41 units, and 73 mg to 65 mg, respectively. Two patients became gliclazide-independent at the time of steroid cessation. CONCLUSIONS: Careful steroid withdrawal from a platform of tacrolimus and MMF is safe and not associated with a significant risk of rejection or graft dysfunction.

Five-year results of kidney transplantation under tacrolimus-based regimes: the persisting significance of vascular rejection.

BACKGROUND: Acute rejection has been the major risk factor for medium-term kidney graft loss because of chronic allograft nephropathy. We investigated whether the use of improved immunosuppression has altered the relationship between acute and chronic rejection by analyzing data from 245 renal transplant patients receiving Tacrolimus-based immunosuppression. RESULTS: Five-year graft survival (censored for death with functioning graft) was 88.8% with no significant difference between living and cadaveric kidney transplants. The only significant predictor of medium-term graft loss was acute vascular rejection. CONCLUSION: Under Tacrolimus-based immunosuppression, the occurrence of acute interstitial rejection, even when occurring late, repeatedly, or with failure of graft function to return to baseline, was not associated with chronic allograft nephropathy or medium-term graft loss. Vascular rejection remains the major immunological obstacle to long-term transplant success. Five-year overall survival rates with a functioning graft of 80% with 90% graft survival censored for death with function seem to be realistic and achievable goals.

Urinary retinol binding protein in Indo-Asian patients with idiopathic interstitial nephritis.

BACKGROUND: Idiopathic interstitial nephritis (IIN) is common in the UK Indo-Asian population. Lack of systemic involvement and unremarkable urinalysis on stick testing suggest that it may underlie some cases of end-stage renal failure of undetermined cause. If IIN is diagnosed early, prompt initiation of treatment can improve long-term outcome. AIMS: To investigate whether urinary retinol binding protein (RBP) is elevated more commonly than urinary albumin in IIN, and might be useful in the early detection of renal disease in Indo-Asian patients. DESIGN: Preliminary observational study METHODS: We measured urinary RBP and urinary albumin in 19 Indo-Asian patients in whom a renal biopsy had shown IIN, 10 of whom had already been treated with corticosteroids at the time of specimen collection. A further 28 Indo-Asian patients with glomerular disease, and six with normal light-microscopic renal biopsy, were assessed in parallel. RESULTS: Urinary RBP/creatinine ratio (RCR) was elevated in all 19 cases of IIN, compared to 12/19 in whom the albumin/creatinine ratio (ACR) was elevated. Elevated urinary RBP was thus significantly more common than albuminuria in this group (p<0.01). Twelve of the 19 cases also satisfied the criteria for tubular proteinuria. RCR was elevated to >30 times the upper limit of normal in 7/9 who had not previously received corticosteroids, of whom four had normal ACR; none had ACR >5 times the upper limit of normal. DISCUSSION: These data suggest that measurement of urinary RBP should be explored as an adjunct to albuminuria, if screening for renal disease in the Indo-Asian population is contemplated.

Red cell traverse through thin glomerular basement membranes.

BACKGROUND: How red cells enter the urinary filtrate in most cases of hematuria of glomerular origin has remained a mystery despite the frequent ultrastructural examination of renal biopsy material. METHODS: Serial sections of glutaraldehyde-fixed, resin-embedded material from a case of sporadic microhematuria were examined by transmission electron microscopy when the site of a red cell traversing the glomerular capillary wall was fortuitously discovered on routine examination. RESULTS: The red cell assumed a dumbbell shape and traversed a localized gap 2.25 microm in diameter in the glomerular endothelium and basement membrane. Serial sections suggested a transcellular route. Apart from the thinning of the basement membrane (167 nm), there were no other generalized abnormalities. CONCLUSION: Red cells can traverse through gaps in the glomerular capillary walls to gain access to Bowman's space. This may be the origin of glomerular hematuria in common noninflammatory forms of glomerular disease, including thin basement membrane nephropathy.

Why is there so much end-stage renal failure of undetermined cause in UK Indo-Asians?

There is a high incidence of end-stage renal failure (ESRF) of undetermined cause in the Indo-Asian population of the UK. We studied patients presenting from the district of Brent and Harrow, which has a large Indo-Asian community, and whose renal services are largely provided by our centre. The diagnosis and ethnicity of patients starting renal replacement therapy and/or undergoing renal biopsy were collated. The incidences of ESRF, rates of renal biopsy and underlying diagnoses were calculated for Indo-Asians and Caucasians. Requirement for renal replacement therapy in Indo-Asians presenting to our centre from Brent and Harrow was 221/10(6)/year; no underlying diagnosis was identified in 77/10(6)/year. Renal biopsy rate in these patients was 456/10(6)/year, and the diagnostic categories significantly over-represented compared to Caucasians were: hypertension and ischaemia, focal segmental glomerulosclerosis (FSGS), idiopathic interstitial nephritis (IIN), diabetic nephropathy, minor glomerular abnormality, lupus nephritis and non-specific advanced chronic renal disease (p<0.001). The first three of these had a combined incidence of 135/10(6)/year in Indo-Asians and 31/10(6)/year in Caucasians. ESRF of undetermined cause is common in UK Indo-Asians, as is requirement for renal biopsy. Hypertension with ischaemia, FSGS and IIN are over-represented in the Indo-Asian population, and should be targeted for early diagnosis and treatment in this group.