Evaluation of Temporal Changes in Urine-based Metabolomic and Kidney Injury Markers to Detect Compound Induced Acute Kidney Tubular Toxicity in Beagle Dogs.

Urinary protein biomarkers and metabolomic markers have been leveraged to detect acute Drug Induced Kidney Injury (DIKI) in rats; however, the utility of these indicators to enable early detection of DIKI in canine models has not been well documented. Therefore, we evaluated temporal changes in biomarkers and metabolites in urine from male and female beagle dogs. Gentamicin- induced kidney lesions in male dogs were characterized by moderate to severe tubular epithelial cell degeneration/necrosis, epithelial cell regeneration and dilation; and a unique urinebased metabolomic fingerprint. These metabolite changes included time and treatment-dependent increases in lactate, taurine, glucose, lactate, alanine, and citrate as well as 9 other known metabolites. As early as 3 days post dose, gentamicin induced increases in urinary albumin, clusterin, neutrophil gelatinase associated protein (NGAL) and total protein concentrations. Urinary albumin, clusterin, and NGAL showed earlier and more robust elevations than traditional kidney safety biomarkers, blood urea nitrogen and serum creatinine. Elevations in urinary kidney injury molecule 1 (KIM-1) were less reliable for detection of gentamicin nephrotoxicity in dogs based on values generated utilizing multiple first-generation, canine-specific KIM-1 immunoassays. The metabolic fingerprint was further evaluated in male and female dogs that received Compound A which induced slightly reversible renal tubular alterations characterized as degeneration/necrosis and concurrent significant increases in urinary taurine amongst other markers. These data support further investigations to demonstrate the value of urinary metabolites, albumin, clusterin, NGAL and taurine as promising markers to enable early detection of DIKI in dogs.

A prospective multicentre phase III validation study of AZGP1 as a biomarker in localized prostate cancer.

BACKGROUND: Prostate cancers (PCs) with similar characteristics at the time of diagnosis can have very different disease outcomes. Conventional biomarkers of PC still lack precision in identifying individuals at high risk of PC recurrence. While many candidate biomarkers are proposed in the literature, few are in clinical practice as they lack rigorous validation. This study prospectively enrolled an independent phase III cohort to evaluate the clinical utility of zinc-alpha 2-glycoprotein (AZGP1) as a prognostic biomarker in localized PC. PATIENTS AND METHODS: In our multicentre, prospective phase III study, AZGP1 status in 347 radical prostatectomy specimens was assayed by immunohistochemistry in a NATA-accredited laboratory. The AZGP1 score was assessed in a multivariable model incorporating established prognostic factors. We also report extended outcomes from our previous phase II study. The primary endpoint was biochemical relapse-free survival (BRFS). Secondary endpoints were metastasis-free survival (MFS) and PC-specific survival (PCSS). RESULTS: In the phase II cohort, with a median follow-up of 15.8 years, low/absent AZGP1 expression was an independent predictor of poor BRFS (HR, 1.4; 95% CI, 1.1-1.9; P = 0.03), MFS (HR, 2.8; 95% CI, 1.2-6.6; P = 0.02) and PCSS (HR, 3.8; 95% CI, 1.5-9.5; P = 0.005). These results were validated in our prospective phase III cohort. Low/absent AZGP1 expression independently predicted for BRFS (HR, 1.9; 95% CI, 1.1-3.3; P = 0.02), with shorter MFS (HR, 2.0; 95% CI, 1.1-3.4; P = 0.02). AZGP1 improved the discriminatory value when incorporated into existing prognostic risk models. CONCLUSION: Our study provides prospective phase III validation that absent/low AZGP1 expression provides independent prognostic value in PC. This study provides robust evidence for the incorporation of this biomarker into clinical practice.

Clinical algorithms for the diagnosis and management of urological leaks following pelvic exenteration.

BACKGROUND: Urine leak following pelvic exenteration for locally advanced pelvic malignancy is a major complication leading to increased mortality, morbidity and length of stay. We reviewed our experience and developed a diagnostic and management algorithm for urine leaks in this patient population. METHODS: Consecutive patients who underwent en bloc cystectomy and conduit formation as part of pelvic exenteration at a single quaternary referral centre from 1995 to 2012 were reviewed. Patients with urine leak were identified. Medical records were reviewed to extract data on diagnosis and management and a suggested clinical algorithm was developed. RESULTS: Of 325 exenterations, there were 102 conduits, of which 15 patients (15%) developed a conduit related urine leak. Most (14/15) patients were symptomatic. Diagnosis was made by drain creatinine studies (12/15) and/or imaging (15/15). Management comprised of conservative management, radiologic urinary diversion, early surgical revision and late surgical revision in 3, 11, 2 and 1 patients respectively. Important lessons from our 17 year experience include a high index of suspicion in a patient who is persistently septic despite appropriate treatment, the importance of regular drain creatinine studies, CT (computer tomography) with delayed images (CT intravenous pyelogram) when performing a CT for investigation of sepsis and early aggressive management with radiologic urinary diversion to facilitate early healing. CONCLUSION: Urine leak after pelvic exenteration is a complex problem. Conservative management usually fails and early diagnosis and intervention is the key. It is hoped that our algorithms will facilitate diagnosis and subsequent management of this group of patients.

Urological leaks after pelvic exenterations comparing formation of colonic and ileal conduits.

BACKGROUND: The aim of this study was to assess possible risk factors for urinary leakage of a newly formed urinary conduit after a partial or total pelvic exenteration. METHODS: An analysis was conducted from prospectively collected data of patients who underwent a pelvic exenteration with conduit formation for advanced and recurrent pelvic cancer. RESULTS: Of 232 patients undergoing a pelvic exenteration, 74 (32%) had a conduit formed. Of these, 47 (64%) had an ileal conduit compared with 27 (36%) a colonic conduit. Twelve (16%) patients developed a leak, of which nine occurred within the first month. Factors associated with a conduit leak included involvement of R2 surgical margins (43%), the magnitude of the exenteration and a current cardiovascular medical history (27%). Leaks were not found to be associated with either radiotherapy or chemotherapy. The 30-day leak rate for ileal conduits was 17% (8/47) and 4% (1/27) for colonic conduits with enterocutaneous fistula only occurring in the ileal conduit group (2/47). Fistula, drained collections and sepsis occurred in 40% of ileal and 19% of colonic conduits (p < 0.01). Patients with a conduit leak had a longer length of stay (59 versus 23 days, p < 0.001). CONCLUSIONS: Urine leaks after conduit formation in association with exenterations are relatively common with a prolonged length of hospital stay. Positive surgical margins and exenterations involving all four quadrants of the pelvis were associated with higher leak rates. There was no evidence of a difference between ileal and colonic conduits and number of leaks. However colonic conduits had less total complications including sepsis, leak and pelvic collections with comparatively no complications of a small bowel fistula.

Prostate-regenerating capacity of cultured human adult prostate epithelial cells.

Experimentation with the progenitor/stem cells in adult prostate epithelium can be inconvenient due to a tight time line from tissue acquisition to cell isolation and to downstream experiments. To circumvent this inconvenience, we developed a simple technical procedure for culturing epithelial cells derived from human prostate tissue. In this study, benign prostate tissue was enzymatically digested and fractionated into epithelium and stroma, which were then cultured in the medium designed for prostate epithelial and stromal cells, respectively. The cultured cells were analyzed by immunocytochemical staining and flow cytometry. Prostate tissue-regenerating capacity of cultured cells in vitro was determined by co-culturing epithelial and stromal cells in dihydrotestosterone-containing RPMI. Cell lineages in formed acini-like structures were determined by immunohistochemistry. The culture of epithelial cells mainly consisted of basal cells. A minor population was negative for known lineage markers and positive for CD133. The culture also contained cells with high activity of aldehyde dehydrogenase. After co-culturing with stromal cells, the epithelial cells were able to form acini-like structures containing multiple cell lineages. Thus, the established culture of prostate epithelial cells provides an alternative source for studying progenitor/stem cells of prostate epithelium.

Scenario-analysis evaluating semergency strategies after rabies re-introduction.

Now that the elimination of rabies in Western Europe is nearly complete, thanks to the oral mass vaccination of foxes (ORV) which took place over the last 25 years, it is necessary to prepare for emergency situations due to the re-introduction of rabies from still infected areas. Such emergency strategies should aim at minimizing the risk of falling back to large-scale vaccination, in a cost efficient manner. An approved spatially-explicit simulation model of spread and control of rabies was adapted to the new problem of re-introduction of rabies into free areas. The logic of the model and options for local emergency vaccination (for example ring-vaccination vs. compact area treatment or heavily concentrated vs. thin extended control areas) were determined. Based on systematic simulation experiments the performance of strategic options was assessed. Key issues such as public health risk (i.e. number of rabies cases), failure risk (i.e. disease breakout from the control area), and budgetary risk (i.e. duration of the emergency program) were simultaneously considered. The results obtained reveal efficiency relations that contradict a priori derived management suggestions.

What is the future of wildlife rabies control in Europe?

Over the last fifteen years or so, classical rabies in terrestrial wildlife has been eliminated from large areas of Western Europe. Over the next few years, terrestrial rabies is likely to occur only east of a line from the Baltic Sea to the Black Sea; the overall aim is to eliminate terrestrial rabies from the whole European Union. Elimination of rabies from the less rich countries of Eastern Europe, and the protection of Europe against a resurgence of rabies in the longer term requires modifications to existing OIE and WHO strategies. Here we discuss the options available to eliminate rabies in wildlife while taking account of financial cost, and how to maintain a 'cordon sanitaire' along the eastern boundary of the EU in order to protect the rabies-free areas from rabies incursion. Minimising financial costs at the national level is obviously essential, considering the competing priorities for development and health. This could be achieved either by increasing external funding (for example by the EU) and/or by changing the currently agreed vaccination strategy to reduce costs; any such change must not substantially reduce the chances of rabies elimination. A cordon sanitaire might be placed outside the economic area of the EU, to protect the whole of the EU, or it might be placed within the easternmost countries to ensure logistical consistency of vaccination. Policy must also anticipate an emergency due to rabies breaking out in a previously freed region. Strategic planning may be complicated by the increasing range and abundance of the raccoon dog, an introduced species that is increasingly important as a host for fox rabies. It is argued here that models help to evaluate altemative strategies, exploring options for optimising costs by minimising bait density and frequency or by reducing the vaccination area.

The strength of 70%: revision of a standard threshold of rabies control.

The success of oral vaccination of foxes (ORV) conceptually is linked to the immunisation of host individuals beyond the herd immunity threshold. However, field evidence and theoretical analysis suggests that mathematically derived values of herd immunity might be rather conservative and, moreover, restrict the adjustment of standard ORV protocols in the case of limited resources. Here, the relationship between baiting effort, duration of ORV programmes and rabies elimination is analysed. An individual-based, spatially explicit model for the control of rabies in foxes that incorporates the important peculiarities of the vaccination process, i.e. the spatial distribution of infected hosts, irregular home-range use, heterogeneous bait coverage etc., is applied. Using multiple repetitions of simulated ORV programmes, the control outcome is analysed in a chance-like fashion overriding the yes-or-no prediction inherent in the herd immunity concept. It is shown why control planning must not only aim at particular immunisation levels but, simultaneously, has to specify the allowed time horizon of control success. It is demonstrated that planning a higher chance of elimination increases necessary effort non-linearly. It was found that low immunisation results (i.e. 50%) still provide a reasonable chance of control success. The potential changes in ORV planning and evaluation allowing for the integration of risk concepts in strategies are discussed.

The peptide recognized by HLA-A68.2-restricted, squamous cell carcinoma of the lung-specific cytotoxic T lymphocytes is derived from a mutated elongation factor 2 gene.

The identification of naturally processed tumor peptides that can stimulate a tumor-specific, CTL response is crucial to the development of a vaccine-based, immunotherapeutic approach to cancer treatment. One type of cancer in which a tumor-specific, CTL response has been observed is squamous cell carcinoma of the lung. In the system investigated here, the tumor-specific CTLs are HLA-A68.2 restricted. Immunoaffinity chromatography was used to isolate the HLA-A68.2 molecules from the tumor cell line, and peptide was eluted with acid from the HLA-A68.2 molecules and subjected to three rounds of separation by reversed phase-high performance liquid chromatography (RP-HPLC). To determine which fractions contained the peptide recognized by the tumor-specific CTLs, an aliquot of each RP-HPLC fraction was added to the autologous, B-lymphoblastoid cell line, and the cells were then tested as targets for tumor-specific CTLs. After the third round of RP-HPLC, mass spectrometry was used to sequence individual peptide candidates, and a peptide with a m/z of 497 was identified as the active peptide. Collision-activated dissociation of m/z 497 allowed identification of the peptide sequence as ETVSEQSNV. With the exception of a single amino acid difference (glutamic acid versus glutamine as the sixth position in the peptide), this peptide is identical to residues 581 to 589 of elongation factor 2. The PCR was used to amplify the elongation factor 2 gene in both the tumor cells and the autologous B cell line, and DNA sequencing of the products revealed the presence of a heterozygous mutation in the tumor cells that accounts for the difference between the two peptide sequences. Although a similar analysis did not reveal the presence of the mutation in three additional lung cell carcinomas, this does not rule out the possibility that a survey of a larger population of tumor cells would reveal the presence of the mutation at a low frequency. These results demonstrate the utility of this approach for identifying tumor-specific antigens that are the targets of a CTL response.

Nephrogenic adenoma of the bladder in renal transplant and non-renal transplant patients: a review of 22 cases.

OBJECTIVES: To review diagnoses of nephrogenic adenoma and in particular to evaluate its association with transitional cell carcinoma (TCC) of the bladder and its relationship to renal transplantation. METHODS: A retrospective review of 22 cases of nephrogenic adenoma (NA) diagnosed between 1989 and 1996 was conducted, 7 of which were in renal transplant patients. Data collected in each case included demographic details, predisposing factors, associated urologic pathology, mode of presentation, cystoscopic finding, management, and follow-up. RESULTS: There was a 3:1 predominance of men. Mean follow-up was 21.4 months (range 3 to 50). Six patients (27%) had one or more recurrences. All 22 patients had some form of previous bladder insult or surgery, including recurrent urine infections, urinary tract instrumentation, placement of ureteric stents, cystodiathermy, and open bladder surgery. Six cases were associated with TCC of the bladder, of which 4 had NA lesions directly over or close to the site of previous fulguration. In 4 patients, there was a temporal relationship between the administration of intravesical doxorubicin hydrochloride or bacille Calmette-Guérin (BCG) and the onset of NA lesions. One case was associated with an inverted papilloma that had not been described before. In 7 renal transplant cases, 3 lesions were found contralateral to the side of the ureterovesical anastomosis. All 22 cases were benign histologically, but one NA was found within a low-grade baldder TCC. Nineteen cases were followed up regularly with no malignant transformation. Three patients were lost to follow-up. CONCLUSIONS: This study has demonstrated an association between NA and bladder cancer. Patients with NA, especially those treated with intravesical chemotherapy or BCG, should have regular cystoscopies. Fulguration or transurethral resection appear to be sufficient treatment. No renal transplant patients had vesical TCC and NA simultaneously. Neither immunosuppression nor ureterovesical anastomosis appeared to be a significant predisposing factor in the transplant patients.

Qsr1p, a 60S ribosomal subunit protein, is required for joining of 40S and 60S subunits.

QSR1 is a recently discovered, essential Saccharomyces cerevisiae gene, which encodes a 60S ribosomal subunit protein. Thirty-one unique temperature-sensitive alleles of QSR1 were generated by regional codon randomization within a conserved 20-amino-acid sequence of the QSR1-encoded protein. The temperature-sensitive mutants arrest as viable, large, unbudded cells 24 to 48 h after a shift to 37 degrees C. Polysome and ribosomal subunit analysis by velocity gradient centrifugation of lysates from temperature-sensitive qsr1 mutants and from cells in which Qsr1p was depleted by down regulation of an inducible promoter revealed the presence of half-mer polysomes and a large pool of free 60S subunits that lack Qsr1p. In vitro subunit-joining assays and analysis of a mutant conditional for the synthesis of Qsr1p demonstrate that 60S subunits devoid of Qsr1p are unable to join with 40S subunits whereas 60S subunits that contain either wild-type or mutant forms of the protein are capable of subunit joining. The defective 60S subunits result from a reduced association of mutant Qsr1p with 60S subunits. These results indicate that Qsr1p is required for ribosomal subunit joining.

SQT1, which encodes an essential WD domain protein of Saccharomyces cerevisiae, suppresses dominant-negative mutations of the ribosomal protein gene QSR1.

QSR1 is an essential Saccharomyces cerevisiae gene, which encodes a 60S ribosomal subunit protein required for joining of 40S and 60S subunits. Truncations of QSR1 predicted to encode C-terminally truncated forms of Qsr1p do not substitute for QSR1 but do act as dominant negative mutations, inhibiting the growth of yeast when expressed from an inducible promoter. The dominant negative mutants exhibit a polysome profile characterized by 'half-mer' polysomes, indicative of a subunit joining defect like that seen in other qsr1 mutants (D. P. Eisinger, F. A. Dick, and B. L. Trumpower, Mol. Cell. Biol. 17:5136-5145, 1997.) By screening a high-copy yeast genomic library, we isolated several clones containing overlapping inserts of a novel gene that rescues the slow-growth phenotype of the dominant negative qsr1 truncations. The suppressor of qsr1 truncation mutants, SQT1, is an essential gene, which encodes a 47.1-kDa protein containing multiple WD repeats and which interacts strongly with Qsr1p in a yeast two-hybrid system. SQT1 restores growth and the "half-mer" polysome profile of the dominant negative qsr1 mutants to normal, but it does not rescue temperature-sensitive qsr1 mutants or the original qsr1-1 missense allele. In yeast cell lysates, Sqt1p fractionates as part of an oligomeric protein complex that is loosely associated with ribosomes but is distinct from known eukaryotic initiation factor complexes. Loss of SQT1 function by down regulation from an inducible promoter results in formation of half-mer polyribosomes and decreased Qsr1p levels on free 60S subunits. Sqt1p thus appears to be involved in a late step of 60S subunit assembly or modification in the cytoplasm.

Exchangeability of Qsr1p, a large ribosomal subunit protein required for subunit joining, suggests a novel translational regulatory mechanism.

Qsr1p is a 60S ribosomal subunit protein that is necessary for joining of large and small ribosomal subunits and is also one of the last proteins assembled onto the 60S ribosomal subunit in the cytoplasm. The finding that Qsr1p is identical to L7, a protein previously shown to cycle on and off large ribosomal subunits in the cytoplasm, suggests that the addition of Qsr1p onto the 60S ribosomal subunit could be utilized as a translational regulatory mechanism by limiting the supply of functional 60S subunits.

Effect of lumbar orthotics on trunk muscle strength.

Weakening of the trunk muscles is thought to be one disadvantage of prolonged lumbar orthotic use. This study examines weakness of the trunk flexor and extensor muscles in patients who are wearing lumbar orthotics for extended periods. Strength of the trunk flexor and trunk extensor muscles was tested in 24 individuals, using the Kinetic computer. Both concentric and eccentric forces were recorded. Four groups of patients were studied. Group 1 (n = 6) consisted of patients with low back pain who had used a lumbar orthotic for a prolonged period of time. Group 2 (n = 6) consisted of hospital employees with no history of low back pain, who wore lumbar orthotics prophylactically, for back protection. Group 1C (n = 6) consisted of healthy controls, with no history of either back pain or lumbar orthotic use, who were individually age- and gender-matched to each patient in Group 1. Group 2C (n = 6) consisted of healthy controls matched in the same fashion to each patient in Group 2. After consultation with a statistician, statistical analysis was performed using the Wilcoxon's test. Nonparametric statistics were chosen because of the lack of evidence of a normal distribution of the parameters being studied. This analysis revealed significant weakness in concentric flexion (P = 0.0464), concentric extension (P = 0.0277), and eccentric extension (P = 0.0464) in Group 1 compared with matched controls in Group 1C. The only significant weakness compared with controls in Group 2 was in eccentric flexion (P = 0.0277). Trends were toward weakness in the orthotic users for the other motions studied, with a P value of less than 0.1 for eccentric extension. Prolonged use of lumbar orthotics may be associated with trunk muscle weakness in the population studied. Prescribers should continue to limit duration of use when possible and to consider strengthening exercises when prolonged use is anticipated.

A novel mouse gene highly conserved throughout evolution: regulation in adipocyte differentiation and in tumorigenic cell lines.

A cDNA clone referred to as 168 was previously isolated from mouse 1246 adipocytes by differential hybridization on the basis of its down regulation in adipocytes when compared to preadipocytes. 5' RACE was used to obtain a full length clone of 761 bp encoding for a highly basic 25 kD polypeptide that is extremely conserved in several diverse species of eukaryotes. There is a single amino acid substitution at position 202 compared to the human homolog, QM, a putative tumor suppressor. Clone 168 mRNA decreases 80% in rat primary culture of adipocytes compared to preadipocytes and does not decrease when differentiation is blocked by PGF2 alpha or EGF, indicating that the decrease is correlated with expression of the differentiation phenotype. Finally, two 1246 cell line variants that exhibit altered growth and increased tumorigenicity have a similar level of 168 mRNA when compared to the non tumorigenic adipogenic parent cell line.

Structure of the gene encoding mouse adipose differentiation-related protein (ADRP).

Adipose differentiation-related protein (ADRP) is a novel 50-kDa membrane-associated protein whose message levels are induced rapidly and maximally after triggering adipocyte differentiation. The gene encoding mouse ADRP has been isolated and characterized from four overlapping lambda phage clones. The gene spans 14 kb and contains 8 exons and 7 introns. Exons range in size from 50 to 696 bp and intron sizes range from 87 bp to 4.3 kb. Major and minor transcription initiation sites were determined 76 and 78 bp, respectively, upstream of the initiator methionine. A TATTTTA sequence is centered 30 bp upstream of the major transcription start site, and within the 5'-flanking region there are several putative transcription factor binding sites. ADRP has been mapped to chromosome 4, specifically between the b and Ifa loci. A second ADRP-like gene was isolated and partially characterized. This second locus is not expressed in 12 different mouse tissues and shares 87% sequence similarity to ADRP over exon and intron regions analyzed. Finally, this is the first reported genomic structure of ADRP.

Nucleotide sequence of the C-terminal region of the mouse epidermal growth factor receptor and expression in teratoma-derived cell lines with increased tumorigenic properties.

The isolation of a cDNA corresponding to a portion (amino acid 943 to 1073) of the cytoplasmic domain of the mouse EGF receptor surrounding the auto phosphorylation sites was obtained by using the reverse transcriptase polymerase chain reaction (RT-PCR) approach. Deduced amino acid sequence of mouse EGF receptor (EGFr) shows a 92% and 76% homology to corresponding regions in the human and the chicken EGFr, respectively. This cDNA was used to develop a sensitive RNase protection assay to investigate EGF receptor mRNA expression in mouse C3H teratoma derived cell lines with increased tumorigenic properties which display a progressive decrease of EGF binding and response. The results show that increased tumorigenicity was not accompanied by a change in EGF receptor mRNA expression. Moreover, they indicate that the RNase protection assay developed using the probe described here is a sensitive approach to investigate EGF receptor expression in murine cells.