Immune responses to Aspergillus antigen in IL‐4‐/‐ mice and the effect of eosinophil ablation

BACKGROUND: Exposure to Aspergillus fumigatus allergens results in enhanced total serum IgE and peripheral blood eosinophils in mice. The associated pulmonary inflammation and immunologic responses are comparable to those detected in human allergic bronchopulmonary aspergillosis. Allergen-induced cytokines are thought to regulate the inflammatory and immune responses in these animals. METHODS: In the present study, we exposed C57BL/6 and BALB/c mice to A. fumigatus antigen. Both wild-type and IL-4 knockout phenotypes of animals of both strains were used. Some animals were also treated with anti-IL-5 or anti-IFN-gamma. Total serum IgE, Aspergillus species IgG subclass, peripheral blood eosinophils, and lung histology were studied. RESULTS: The results demonstrate similar lung inflammation in all wild-type and IL-4-/- animals exposed to A. fumigatus antigen. Similarly, in spite of the diverse immune response produced by the anticytokine treatment, no major differences were detected among any of the animal groups studied. CONCLUSIONS: It can be concluded that A. fumigatus exposure in an immunologically unaltered host is predominantly of a Th2 type, and that depletion of the Th2 cytokine leads to a similar lung inflammation but with a characteristic Th1 response, suggesting that the pathogenesis of allergic aspergillosis is the result of multiple induction pathways.     

Screening of Twenty-Four South African Combretum and Six Terminalia Species (Combretaceae) for Antioxidant Activities

The dried leaves of Combretum and Terminalia species (Combretaceae) were extracted with acetone, hexane, dichloromethane and methanol. Thin layer chromatography (TLC) plates were developed under saturated conditions and sprayed with 0.2% 2,2-diphenyl-1-picryl hydrazyl (DPPH) in methanol for antioxidant screening. Visualization of separated bands exhibiting antioxidant activities enabled the localization and the subsequent identification of the potential active compounds. The acetone and methanol extracts displayed the presence of antioxidant activity after spraying the chromatogram with DPPH. Hexane and dichloromethane extracts did not have any antioxidant activity. C. hereroense had the highest number of active compounds, followed by C. collinum ssp. taborense, which were 16 and 10, respectively. Acetone extracts of all tested Combretum species had 53 active bands and methanol had 55. All Terminalia species extracted with acetone and methanol had antioxidant activity. T. gazensis and T. mollis methanol extracts had 11 and 14 active compounds respectively in one of the solvent systems used. The qualitative DPPH assay on TLC was successfully used in this study to systematically assess the total antioxidant activity of the Combretum and Terminalia species extracts.     

Germline mutations of the CDKN2 gene in UK melanoma families

Germline mutations in CDKN2 on chromosome 9p21, which codes for the cyclin D kinase inhibitor p16, and more rarely, mutations in the gene coding for CDK4, the protein to which p16 binds, underlie susceptibility in some melanoma families. We have sequenced all exons of CDKN2 and analysed the CDK4 gene for mutations in 27 UK families showing evidence of predisposition to melanoma. Five different germline mutations in CDKN2 were found in six families. Three of the mutations (Met53Ile, Arg24Pro and 23ins24) have been reported previously. We have identified two novel CDKN2 mutations (88delG and Ala118Thr) which are likely to be associated with the development of melanoma, because of their co-segregation with the disease and their likely functional effect on the CDKN2 protein. In binding assays the protein expressed from the previously described mutation, Met53Ile, did not bind to CDK4/CDK6, confirming its role as a causal mutation in the development of melanoma. Ala118Thr appeared to be functional in this assay. Arg24Pro appeared to bind to CDK6, but not to CDK4. No mutations were detected in exon 2 of CDK4, suggesting that causal mutations in this gene are uncommon. The penetrance of these mutant CDKN2 genes is not yet established, nor is the risk of non-melanoma cancer to gene carriers.      

The role of size, sequence and haplotype in the stability of FRAXA and FRAXE alleles during transmission

Factors involved in the stability of trinucleotide repeats during transmission were studied in 139 families in which a full mutation, premutation or intermediate allele at either FRAXA or FRAXE was segregating. The transmission of alleles at FRAXA, FRAXE and four microsatellite loci were recorded for all individuals. Instability within the minimal and common ranges (0-40 repeats for FRAXA, 0-30 repeats for FRAXE) was extremely rare; only one example was observed, an increased in size at FRAXA from 29 to 39 repeats. Four FRAXA and three FRAXE alleles in the intermediate range (41-60) repeats for FRAXA, 31-60 for FRAXE) were unstably transmitted. Instability was more frequent for FRAXA intermediate alleles that had a tract of pure CGG greater than 37 although instability only occurred in two of 13 such transmissions: the changes observed were limited to only one or two repeats. Premutation FRAXA alleles over 100 repeats expanded to a full mutation during female transmission in 100% of cases, in agreement with other published series. There was no clear correlation between haplotype and probability of expansion of FRAXA premutations. Instability at FRAXA or FRAXE was more often observed in conjunction with a second instability at an independent locus suggesting genomic instability as a possible mechanism by which at least some FRAXA and FRAXE mutations arise.      

Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP)

Mutations in the PEX gene at Xp22.1 (phosphate-regulating gene with homologies to endopeptidases, on the X-chromosome), are responsible for X-linked hypophosphataemic rickets (HYP). Homology of PEX to the M13 family of Zn2+ metallopeptidases which include neprilysin (NEP) as prototype, has raised important questions regarding PEX function at the molecular level. The aim of this study was to analyse 99 HYP families for PEX gene mutations, and to correlate predicted changes in the protein structure with Zn2+ metallopeptidase gene function. Primers flanking 22 characterised exons were used to amplify DNA by PCR, and SSCP was then used to screen for mutations. Deletions, insertions, nonsense mutations, stop codons and splice mutations occurred in 83% of families screened for in all 22 exons, and 51% of a separate set of families screened in 17 PEX gene exons. Missense mutations in four regions of the gene were informative regarding function, with one mutation in the Zn2+-binding site predicted to alter substrate enzyme interaction and catalysis. Computer analysis of the remaining mutations predicted changes in secondary structure, N-glycosylation, protein phosphorylation and catalytic site molecular structure. The wide range of mutations that align with regions required for protease activity in NEP suggests that PEX also functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism.      

High-dose etoposide and cyclophosphamide without bone marrow transplantation for resistant hematologic malignancy

Seventy-five patients with resistant acute leukemia or lymphoma received high-dose cyclophosphamide and etoposide to explore the activity of this combination in resistant hematologic malignancies, and to determine the maximum doses of these drugs that can be combined without bone marrow transplantation. Etoposide was administered over 29 to 69 hours by continuous infusion corresponding to total doses of 1.8 g/m2 to 4.8 g/m2. Cyclophosphamide, 50 mg/kg/d, was administered on 3 or 4 consecutive days total 150 to 200 mg/kg ideal body weight). At all dose levels myelosuppression was severe but reversible. Mucosal toxicity was dose-limiting with the maximum tolerated dose level combining etoposide 4.2 g/m2 with cyclophosphamide 200 mg/kg. Continuous etoposide infusion produced stable plasma levels that were lower than would be achieved after administration by short intravenous infusion, and this could explain our ability to escalate etoposide above the previously reported maximum tolerated dose. There were 28 complete (35%) and 12 partial (16%) responses. Median duration of complete response (CR) was 3.5 months (range 1.1 to 20+). Seventeen of 40 patients (42%) with acute myelogenous leukemia (AML) achieved CR, including 6 of 20 (30%) with high-dose cytosine arabinoside resistance. We conclude that bone marrow transplantation is not required after maximum tolerated doses of etoposide and cyclophosphamide. This regimen is active in resistant hematologic neoplasms, and the occurrence of CR in patients with high-dose cytosine arabinoside-resistant AML indicates a lack of complete cross-resistance between these regimens.     

The development of a post-mortem interval estimation for human remains found on land in the Netherlands

The decomposition process of human remains can be used to estimate the post-mortem interval (PMI), but decomposition varies due to many factors. Temperature is believed to be the most important and can be connected to decomposition by using the accumulated degree days (ADD). The aim of this research was to develop a decomposition scoring method and to develop a formula to estimate the PMI by using the developed decomposition scoring method and ADD.

A decomposition scoring method and a Book of Reference (visual resource) were made. Ninety-one cases were used to develop a method to estimate the PMI. The photographs were scored using the decomposition scoring method. The temperature data was provided by the Royal Netherlands Meteorological Institute. The PMI was estimated using the total decomposition score (TDS) and using the TDS and ADD. The latter required an additional step, namely to calculate the ADD from the finding date back until the predicted day of death.

The developed decomposition scoring method had a high interrater reliability. The TDS significantly estimates the PMI (R ² = 0.67 and 0.80 for indoor and outdoor bodies, respectively). When using the ADD, the R ² decreased to 0.66 and 0.56.

The developed decomposition scoring method is a practical method to measure decomposition for human remains found on land. The PMI can be estimated using this method, but caution is advised in cases with a long PMI. The ADD does not account for all the heat present in a decomposing remain and is therefore a possible bias.

     

Intratemporal vascular tumors: detection with CT and MR imaging

The diagnostic contributions of computed tomography (CT) and magnetic resonance (MR) imaging were compared in 12 patients with benign intratemporal vascular tumors (hemangioma or vascular malformation). The tumors included six in the internal acoustic canal and six in the geniculate ganglion region. Clinical and histologic correlations were made. Two of the six patients with tumors in the internal acoustic canal underwent CT, and both required gas cisternography to show the tumor. Five patients in that group underwent MR imaging, and all five studies showed the tumor. All six patients with geniculate ganglion tumors underwent CT. Results in one study were questionable, and five showed the tumor. Five patients in this group underwent MR imaging, but the MR findings were positive in only two cases. MR imaging should therefore be performed before CT in the evaluation of facial nerve dysfunction, as it demonstrated all tumors in the internal acoustic canal and some in the geniculate ganglion region. If MR findings are negative, CT should then be performed to rule out a possible geniculate ganglion lesion.     

MR imaging of hepatocellular carcinoma: relationship between lesion size and imaging findings, including signal intensity and dynamic enhancement patterns

PURPOSE: To assess the relationship between lesion size and MR imaging findings of pathologically-proven hepatocellular carcinoma (HCC). MATERIALS AND METHODS: In a retrospective, single-center study, 37 consecutive patients were identified between 1999 and 2005 that underwent preoperative MRI and surgical resection of HCC. A total of 47 lesions (mean size = 6.85 cm, range = 1-25 cm) were assessed for signal intensity (SI), enhancement patterns, and secondary morphologic features. Interobserver rating, percentage enhancement, and contrast-to-noise-ratio (CNR) were determined. Lesions were assessed for combinations of typical MRI features. Regression analysis was used to assess relations between MRI findings and tumor size. RESULTS: On fat-suppressed T2-weighted (T2w) fast-spin-echo, smaller lesions had lower SI compared to larger lesions (P < 0.05). In the arterial phase, smaller lesions showed significantly higher percentage enhancement compared to larger lesions (P < 0.05). In the delayed phase, smaller lesions showed less pronounced washout (P < 0.05). Heterogeneity of the lesions, including fatty infiltration, internal nodules, or mosaic pattern, was observed significantly more frequently in larger lesions (P < 0.001). The classic combination of high T2w signal, strong arterial enhancement, and delayed phase washout was present in 23 of 44 lesions (52%). CONCLUSION: Smaller HCC often showed lower SI on T2w, more intense arterial enhancement, and less pronounced delayed washout compared to larger HCC.