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The p53 oncosuppressor protein is a critical mediator of the response to injury in mammalian cells and is mutationally inactivated in the majority of lung malignancies. In this analysis, the effects of p53-deficiency were investigated in short-term primary cultures of murine bronchiolar Clara cells. Clara cells, isolated from gene-targeted p53-deficient mice, were compared to cells derived from wild type littermates. p53 null cultures displayed abnormal morphology; specifically, a high incidence of multinucleation, which increased with time in culture. Multinucleated cells were proficient in S phase DNA synthesis, as determined by BrdU incorporation. However, multinucleation did not reflect altered rates of S phase synthesis, which were similar between wild type and p53-/- cultures. Nucleation defects in p53-/- Clara cells associated with increased centrosome number, as determined by confocal microscopy of pericentrin-stained cultures, and may highlight a novel role of p53 in preserving genomic integrity in lung epithelial cells. Effects of p53-deficiency were also studied following exposure to DNA damage. A p53-dependent reduction in the BrdU index was observed in Clara cells following ionizing radiation. The reduction in BrdU index in wild type cells displayed serum-dependency, and occurred only in the absence of serum. Taken together, these findings demonstrate that in murine primary Clara cell culture, cell cycle arrest is a p53-mediated response to DNA damage, and that extracellular factors, such as serum, influence this response. These findings highlight functions of wild type p53 protein in bipolar spindle formation, centrosome regulation, and growth control in bronchiolar Clara cells.

Halothane hepatitis can be life-threatening and this severe adverse reaction may arise via an immune process. We have detected autoantibodies to purified human liver microsomal carboxylesterase in sera of 17 out of 20 patients with halothane hepatitis (85%) but not in 9 halothane-exposed controls and in only 2 (at low levels) of 33 patients with liver disease due to other causes. Immunohistochemical studies localised the carboxylesterase predominantly to the cnetrilobular region of liver sections, which is consistent with the area affected by halothane hepatitis. Human hepatic micromosal carboxylesterase is a target antgen in halothane hepatitis, and an immune response to this protein may be involved in the liver damage observed. Lancet 1993; 342: 963–64

Complementary DNA clones encoding human aflatoxin B1 aldehyde reductase (AKR7A2), aldehyde reductase (AKR1A1), aldose reductase (AKR1B1), dihydrodiol dehydrogenase 1 (AKR1C1) and chlordecone reductase (AKR1C4) have been expressed in Escherichia coli. These members of the aldo-keto reductase (AKR) superfamily have been purified from E. coli as recombinant proteins. The recently identified AKR7A2 was shown to differ from the AKR1 isoenzymes in being able to catalyse the reduction of 2-carboxybenzaldehyde. Also, AKR7A2 was found to exhibit a narrow substrate specificity, with activity being restricted to succinic semialdehyde (SSA), 2-nitrobenzaldehyde, pyridine-2-aldehyde, isatin, 1,2-naphthoquinone (1,2-NQ) and 9,10-phenanthrenequinone. In contrast, AKR1A1 reduces a broad spectrum of carbonyl-containing compounds, displaying highest specific activity for SSA, 4-carboxybenzaldehyde, 4-nitrobenzaldehyde, pyridine-3-aldehyde, pyridine-4-aldehyde, 4-hydroxynonenal, phenylglyoxal, methylglyoxal, 2,3-hexanedione, 1,2-NQ, 16-ketoestrone and D-glucuronic acid. Comparison between the kinetic properties of AKR7A2 and AKR1A1 showed that both recombinant enzymes exhibited roughly similar kcat/Km values for SSA, 1,2-NQ and 16-ketoestrone. Many of the compounds which are substrates for AKR1A1 also serve as substrates for AKR1B1, though the latter enzyme was shown to display a specific activity significantly less than that of AKR1A1 for most of the aromatic and aliphatic aldehydes studied. Neither AKR1C1 nor AKR1C4 was found to possess high reductase activity towards aliphatic aldehydes, aromatic aldehydes, aldoses or dicarbonyls. However, unlike AKR1A1 and AKR1B1, both AKR1C1 and AKR1C4 were able to catalyse the oxidation of 1-acenaphthenol and, in addition, AKR1C4 could oxidize di- and tri-hydroxylated bile acids. Specific antibodies raised against AKR7A2, AKR1A1, AKR1B1, AKR1C1 and AKR1C4 have been used to show the presence of all of the reductases in human hepatic cytosol; the levels of AKR1B1 and AKR1C1 were markedly elevated in livers with alcohol-associated injury, and indeed AKR1B1 was only detectable in livers with evidence of alcoholic liver disease. Western blotting of extracts from brain, heart, kidney, liver, lung, prostate, skeletal muscle, small intestine, spleen and testis showed that AKR7A2 is present in all of the organs examined, and AKR1B1 is similarly widely distributed in human tissues. These experiments revealed however, that the expression of AKR1A1 is restricted primarily to brain, kidney, liver and small intestine. The AKR1C family members proved not to be as widely expressed as the other reductases, with AKR1C1 being observed in only kidney, liver and testis, and AKR1C4 being found in liver alone. As human kidney is a rich source of AKR, the isoenzymes in this organ have been studied further. Anion-exchange chromatography of human renal cytosol on Q-Sepharose allowed resolution of AKR1A1, AKR1B1, AKR1C1 and AKR7A2, as identified by substrate specificity and Western blotting. Immunohistochemistry of human kidney demonstrated that AKR7A2 is expressed in a similar fashion to the AKR1 family members in proximal and distal convoluted renal tubules. Furthermore, both AKR7A2 and AKR1 members were expressed in renal carcinoma cells, suggesting that these groups of isoenzymes may be engaged in related physiological functions.

Glutathione S-transferase (GST) in man comprise at least four gene families. Three of these families give rise to cytosolic isoenzymes (alpha, mu and pi classes), whilst the remainder is membrane bound and has been called microsomal GST. These enzymes are implicated in tumourogenesis and both pi class GST and alpha class GST have been described in four cases of human renal cell carcinoma. Using specific polyclonal rabbit antisera we have demonstrated by immunohistochemistry that all 12 renal carcinomas studied contained GST pi. Most tumours also contained GST alpha, GST mu and microsomal GST isoenzymes but their distribution was heterogeneous and sometimes very focal. This heterogeneity of GST isoenzyme distribution within tumours has not been well documented previously, but is relevant to our understanding of the functions of GST, and to the interpretation of biochemical quantification experiments using tissue extracts.

Seventeen patients with Wegener's granuloma (WG), all of whom had renal biopsies taken at presentation, are reviewed. In conjunction with nasal or transbronchial biopsies renal biopsies aided diagnosis and also yielded prognostic information. The detection of antineutrophil cytoplasm antibodies in the serum of patients with WG is a useful supplementary diagnostic tool.

Glutathione S-transferases are a group of drug metabolising and detoxification enzymes. We have studied the distribution of four isoenzymes, acidic, basic, neutral, and microsomal GST in human liver, gall bladder, and small and large intestinal epithelium by immunohistochemistry. Antibodies were raised in rabbits to purified GST subunits and several formalin fixed paraffin sections of each human tissue studied using the peroxidase-antiperoxidase method. Staining density was graded from very strong (+++) to negative (-). All four enzymes were identified within the liver, the acidic GST being found almost exclusively within the biliary epithelium. The gall bladder epithelium stained strongly for acidic and basic GST. In the small intestinal epithelium the acidic and neutral GST were readily identified in villi and crypts, whilst basic GST was found only in the villi and microsomal only in the crypts. In the colonic mucosa only acidic GST could consistently be identified. This histological heterogeneity may have functional implications for these enzymes in human hepatobiliary and intestinal tissue.

Increased levels of glutathione (GSH) occur in the epithelial lining fluid (ELF) of chronic cigarette smokers. Therefore we investigated the effect of cigarette smoke condensate solution (CSC) on GSH synthesis and the regulation of gamma-glutamylcysteine synthetase (gammaGCS) in human type II alveolar epithelial cells (A549). CSC exposure increased GSH levels, gammaGCS activity and gammaGCS heavy subunit (HS) mRNA, as well as increasing DNA binding of the activator protein-1 (AP-1) and the human antioxidant response element (hARE). Transfection of deletion constructs of the gammaGCS-HS promoter in a chloramphenicol acetyl transferase (CAT) reporter system revealed that an hARE, present within promoter, is not required for the CSC mediated induction. We conclude that CSC induction of gammaGCS-HS expression is associated with AP-1/AP-1-like responsive elements.

Many patients with systemic vasculitis have antibodies to neutrophil cytoplasm antigens (ANCA) detectable by indirect immunoflourescencc. We sought to characterize further the nature of these antigens. Western blots of neutrophil protein extracts indicated that nine patients' sera, positive for ANCA by immunofluorescence, all reacted with a 45 kDa and a 27–31 kDa protein. Negative control sera, and sera taken in remission, did not react with either of these antigens. The results suggest that ANCA in vasculitis have the same target specificity and may therefore permit greater accuracy of diagnosis and increase our understanding of the pathogcncsis of the conditions.

In a case of focal and segmental glomerulonephritis unusual cell junctions were discovered between podocytes. These most closely resembled lesions described in aminonucleoside induced nephrosis in rats and were unlike anything previously seen in our experience. Shortly after renal transplantation nephrotic syndrome recurred and biopsy specimens showed recurrent focal and segmental glomerulonephritis, with the appearance of these unusual interpodocyte junctions in the graft kidney. This may be related to circulating factors in the blood of the patient.

A comparison was made between the renal biopsy appearances of Wegener's granuloma, microscopic polyarteritis and diffuse endocapillary proliferative glomerulonephritis, including the distribution of surface IgE-bearing mast cells. In microscopic polyarteritis and in other inflammatory conditions mast cells were usually associated with areas of interstitial inflammatory infiltrate, whereas in Wegener's granuloma mast cells were usually separate from areas of inflammation, suggesting a role for mast cells in the development of the renal lesion of Wegener's granulomatosis.

A series of renal biopsy specimens taken at the time of percutaneous nephrolithotomy were investigated for the presence and location of foci of microcalcification. Calcium was found in 18 of 25 (72%) of biopsy specimens from stone formers and in only seven of 30 (23%) of control biopsy specimens. This may indicate defective intrarenal handling of calcium as plasma calcium concentration was normal and 40% had a raised 24 hour urinary calcium excretion.

A single blind randomised trial of two different anti-emetic regimens (A and B) was performed in 26 patients with breast carcinoma undergoing emetic i.v. cytotoxic chemotherapy. They all received oral Motival (nortriptylene/fluphenazine) for 48 h after therapy and for regimen A received N-saline i.v. with their cytotoxics whilst for regimen B patients were given 16 mg dexamethasone i.v. Patients were given the alternative regimen at the subsequent course of treatment. They were asked to assess overall nausea and number of vomiting episodes in the 24 h following therapy. There was a statistically significant reduction in both for the regimen containing dexamethasone. This agent causes few side effects and is particularly suited for out-patient use.

The presence of apoptotic bodies in proliferative glomerulonephritis was investigated by light and electron microscopy. They were identified in small numbers in 35 of 68 biopsies studied, especially biopsies containing numerous neutrophils. Apoptosis may be a mechanism reducing acute glomerular damage and allowing a hypercellular glomerulus to return to normal.

We report novel renal histopathologic features in a patient with hemolytic uremic syndrome after allogeneic bone marrow transplantation who survived with plasma exchange therapy. This distribution of vascular lesions within the kidney differed from previously described cases, which were uniformly fatal, in that there were marked arterial changes in addition to arteriolar and glomerular microangiopathy. A high rate of frequency of apoptotic cells within glomeruli was found. These findings are discussed in terms of their pathogenetic and prognostic relevance.

Background: Until recently, the lack of clinical outcomes information for rheumatoid arthritis (RA) in administrative claims databases limited their use in comparative effectiveness research. A validated claims-based algorithm has been developed to estimate the effectiveness of biologics for RA, allowing for estimation of cost and effectiveness in the same database. Objectives: To implement a validated claims-based effectiveness algorithm in a US managed care claims database to compute the 1-year biologic cost per effectively treated patient among first-line biologics approved for moderate-to-severe RA (abatacept, adalimumab, certolizumab pegol, etanercept, golimumab, and infliximab). Methods: This retrospective cohort study used administrative claims data for individuals in the HealthCore Integrated Research Database (HIRDSM). The first claim for a first-line biologic between July 1, 2009, and January 31, 2013, after 6 months of continuous enrollment, was defined as the index event and date. Patients were aged 18-63 years on the index date and had at least one claim for RA in the 6-month pre- index period. Biologic costs included plan and patient paid amounts on claims for the biologic drug and administration. The algorithm defined effectiveness during the 12-month post-index period as achieving all six of the following: high adherence (medication possession ratio ≥80% or infusions consistent with the product label); no increase in biologic dose or decrease in dosing interval; no new biologic; no new nonbiologic disease-modifying antirheumatic drug; no new or increased oral glucocorticoid use; and ≤1 glucocorticoid injection. Cost per effectively treated patient was calculated as the total biologic cost (drug and administration) divided by the number of patients categorized by the algorithm as effectively treated. Results: The cohort comprised 4844 patients (mean age 48.6 years, 76.4% female). Average first-year biologic cost ranged from $14 795 (golimumab) to $19 520 (abatacept). Average first-year biologic cost per effectively treated patient was significantly lower for etanercept ($50 217) than for golimumab ($56 427, p

Online educational videos disseminated content for a university pre-service teacher internship program. Placed within an online course management system, the videos were accessed by 202 interns located in several Western U.S. states. To ascertain the interns’ subjective experiences of the online course and videos to assist in the evaluation of the course, the researcher analyzed qualitative survey data in the form of constructed response items using a new qualitative-to-quantitative protocol. Based on phenomenological and grounded theory methods, this protocol was designed to handle the large amount of subjective constructed responses, allowing the inductive understanding of the overall experiences of a common phenomenon. The responses provided critical information that is useful for instructional designers, online educators, and educational video producers. The data suggest:1) Universities must carefully consider video hosting options to ensure access.2) Online videos should be carefully planned to create high quality, concise videos of less than ten minutes in length, yet contain enough educational content to reduce the overall number of required videos.3) Students appreciate the flexibility online course delivery offered in terms of scheduling and eliminating the need to come to campus to attend seminars.4) Self-paced online courses require progress indicators to alleviate confusion.

The mechanisms by which the hepatitis B x protein (HBx) contributes to hepatocarcinogenesis remain unclear. However, interaction with the tumor suppressor gene p53 and inhibition of p53-dependent cellular functions, including nucleotide excision repair, could be central to this process. We studied the levels of global repair (removal of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts) and transcription-coupled repair (removal of CPDs in both strands of the dihydrofolate reductase gene) in primary wild-type and p53-null mouse hepatocytes. We show that global repair of CPDs appears to be more efficient in mouse hepatocytes than in other commonly studied rodent cells and approaches the levels of human cells and that p53 is required for global genomic DNA repair of CPDs but not for transcription-coupled repair. We then investigated the effect of HBx expression on hepatocyte nucleotide excision repair. We demonstrate that HBx expression affects DNA repair in a p53-dependent manner. Transient HBx expression reduces global DNA repair in wild-type cells to the level of p53-null hepatocytes and has no effect on the repair of a transfected damaged plasmid. Therefore, in viral hepatitis, the hepatitis B virus could inhibit the p53-dependent component of global repair leading, over time, to accumulation of genetic defects and fostering carcinogenesis.

BACKGROUND--Glutathione S-transferases (GSTs) are involved in the detoxification of xenobiotics by conjugation with glutathione. One of the mu class genes of this superfamily of enzymes, GSTM1, is polymorphic because of a partial gene deletion. This results in a failure to express GSTM1 in approximately 50% of individuals. Several studies have linked GSTM1 null status to an increased risk of lung carcinoma. This study investigated the expression and distribution of GST isoenzymes in human lung, and developed a polymerase chain reaction (PCR) assay which would allow genotyping of archival, paraffin embedded lung tissue. METHODS--Distribution was examined using a panel of polyclonal anti-GST antibodies for immunohistochemistry in normal tissue of 21 tumour-bearing lungs. DNA for PCR was extracted from paraffin blocks and a control group of 350 blood lysates. As a positive control each assay amplified part of GSTM4, a mu class gene which is not polymorphic but which shows strong sequence homology to GSTM1. The presence of GST in bronchoalveolar lavage fluid was sought by Western analysis. RESULTS--Proximal airways contained pi class GST, alpha class GST, and mu class GST with expression concentrated in the brush border. In distal airspaces no alpha GST was expressed but pi GST and mu GST were present in alveolar cells and also alveolar macrophages. Pi class GST was present in bronchoalveolar lavage fluid. The PCR assay enabled genotypic determination using DNA extracted from archival material. Of the control group 56% were null at the GSTM1 locus. CONCLUSIONS--The distribution of GST isoenzymes in the lung is heterogeneous with an apparent decrease in GST in distal lung. Since GSTM1 status has already been associated with susceptibility to disease, the PCR assay developed will allow further studies of the relation between genotype and structural disorders in the lung using archival pathological material.

The tumor suppressor proteins IRF-1 and p53 are involved in response pathways after DNA damage. In different cell types, IRF-1 and p53 can cooperate to produce cell cycle arrest (embryo fibroblasts) or can independently trigger apoptosis (lymphoid cells). p53 may also regulate DNA repair, but there is no information on IRF-1 and repair. The cell lineage dependency of these effects precludes extrapolation of findings to other tissues of relevance to human cancer. Here, we report the consequences of IRF-1 deficiency for apoptosis, cell cycle arrest, and DNA repair in primary hepatocytes after DNA damage and extend previous work on the role of p53 in hepatocytes. IRF-1-deficient hepatocytes showed reduced DNA repair activity compared with wild-type, as assessed by unscheduled DNA synthesis after UV irradiation (10J/m2) and by host reactivation of a UV-damaged reporter construct. p53-deficient hepatocytes also showed reduced unscheduled DNA synthesis after UV, but there was no impairment of specific repair in host reactivation assays. IRF-1 deficiency did not affect the p53-dependent G1/S arrest after UV irradiation. Hepatocyte apoptosis after UV treatment, previously reported to be independent of p53, was also independent of IRF-1. However, IRF-1 deficiency produced dysregulation of p53, manifested as increased transactivation of a p53-reporter plasmid in undamaged hepatocytes, and accelerated p53 stabilization after DNA damage. Hence, in hepatocytes, IRF-1 is not required for growth arrest or apoptosis after DNA damage, but the results suggest for the first time a role in DNA repair regulation.

To determine whether the tissue distribution of glutathione S-transferase (GST) isoenzymes could define the precise nature of renal injury, 13 adult kidneys were studied, using specific antibodies raised against purified isoenzymes. Basic GST stained strongly proximal convoluted tubules and some medullary tubules; acidic GST stained strongly distal convoluted tubules and medullary tubules; neutral GST stained similarly to acidic GST, but weaker, and microsomal GST stained glomerular and interstitial endothelium and collecting ducts deep in the medulla, although there was considerable variation in staining intensity among cases. It is suggested that the measurement of these isoenzymes in serum and urine may help to elucidate the localisation of tissue damage, which may be particularly valuable in patients with cyclosporine toxicity following renal transplantation.

The use of serum antibodies to neutrophil cytoplasmic antigens (ANCA) as a diagnostic marker for Wegener's granulomatosis and other forms of vasculitis has been assessed. Although ANCA have been described by several groups the precise antigenic targets are unknown, and detection of ANCA still relies on an indirect immunofluorescence assay technique. Several different patterns of fluorescence have been produced by using sera from different groups of patients, and insufficient information is available on the frequency of positive results and of the patterns of immunofluorescence obtained when serum from patients with vasculitis as a part of a generalised connective tissue disease is used. A study was carried out on serum from 240 patients, including 23 patients with Wegener's granulomatosis, 12 with microscopic polyarteritis, and 30 with various connective tissue diseases. Three patterns of fluorescence were observed: bright coarsely granular cytoplasmic, bright non-granular cytoplasmic, and weak diffuse cytoplasmic. The bright, coarsely granular pattern was 86% specific for Wegener's granulomatosis in this series and was observed in 18 of 23 cases. Other patterns of fluorescence were found in various conditions and were not of diagnostic value. The technique is simple, inexpensive, rapid, and reproducible.

ON previous occasions free-living and parasitic amœbae have been obtained free from their associated bacteria by sterilization of the cysts either by washing in bactericidal chemicals1,2 or in antibiotics3,4. The technique I have used for isolating clones of soil amœbae and growing them in sterile peptone solution involves the treatment of the trophozoite stage with antibiotics, making use of the fact that the amœbae are more tolerant to many antibiotics than are certain bacteria.