Influence of sampling technique and bedding type on the milk microbiota: Results of a pilot study.

The objective of this pilot study was to evaluate the influence of sampling technique and exposure to different bedding types on the milk microbiome of healthy primiparous cows. Primiparous Holstein cows (n = 20) with no history of clinical mastitis or monthly somatic cell counts >150,000 cells/mL were selected for this study. From each enrolled cow, a composite milk sample was aseptically collected from all 4 mammary quarters (individual quarter somatic cell counts <100,000 cells/mL), 1 individual quarter milk sample was collected using conventional aseptic technique, and 2 individual quarter milk samples were collected directly from the gland cistern using a needle and vacuum tube. All milk samples were cultured using standard milk microbiological techniques and DNA was extracted. Extracted DNA was subjected to PCR and next-generation sequencing for microbiota determination. All samples yielded relatively little total DNA. Amplification of PCR was successful in 45, 40, and 83% of composite, conventional, and cisternal samples, respectively. Bacteria were successfully cultured from 35% of composite milk samples but from none of the quarter milk samples collected using conventional or cisternal sampling techniques. Bacterial DNA sequences were assigned to operational taxonomic units (OTU) based on 97% sequence similarity, and bacterial richness and diversity were determined. Most samples were dominated by low-prevalence OTU and of the 4,051 identified OTU, only 14 were prevalent at more than 1% each. These included bacteria typically recovered from environmental sources. Chao richness was greatest in composite samples and was 636, 347, and 356 for composite, conventional quarter, and cisternal milk samples, respectively. Shannon diversity was similar among sample types and ranged from 3.88 (quarter) to 4.17 (composite). Richness and diversity did not differ by bedding type among cisternal samples, but the power of this pilot study was limited due to small sample size. Despite the small sample size, for milk samples collected from the gland cistern, overall bacterial community composition differed among bedding types. These results demonstrate that sampling technique and bedding type may be associated with the microbiota detected in bovine milk, and we suggest that these variables should be considered in designing and reporting studies about the milk microbiota.

Short communication: antimicrobial susceptibility and frequency of resistance genes in Escherichia coli isolated from bovine mastitis.

Escherichia coli isolated from bovine milk samples submitted to the Ohio Agricultural Research and Development Center Mastitis Laboratory (Wooster) in 1985 to 1987 and in 2009 were compared for antimicrobial susceptibility and prevalence of antimicrobial resistance genes. Forty-four isolates from 1985 to 1987 and 55 isolates from 2009 were tested. Minimum inhibitory concentrations of 15 antimicrobials were determined using a commercially available broth microdilution system. Multiplex polymerase chain reaction was performed for gene detection. The percentage of isolates susceptible to trimethoprim/sulfamethoxazole, ampicillin, and kanamycin was lower in those collected in 1985 to 1987 than in isolates collected in 2009. Susceptibility did not differ between isolates from 1985 to 1987 and isolates from 2009 for the 12 other antimicrobials tested. A trimethoprim/sulfamethoxazole resistance gene was detected more frequently in isolates from 1985 to 1987 than in isolates from 2009. Gene frequencies for streptomycin resistance and tetracycline resistance were similar among 1985 to 1987 isolates and 2009 isolates. Resistance to most antimicrobials did not differ between isolates submitted to a mastitis diagnostic laboratory in 1985 to 1987 and those submitted in 2009. Changes observed indicated an increase in frequency of susceptibility in isolates to trimethoprim/sulfamethoxazole, ampicillin, and kanamycin in 2009 isolates compared with 1985 to 1987 isolates.

Quantitative measurement of genome-wide DNA methylation by a reliable and cost-efficient enzyme-linked immunosorbent assay technique.

DNA methylation, the conversion of cytosine to 5-methylcytosine, is an important epigenetic modification involved in gene regulation. DNA methylation is essential for normal development whereas abnormal methylation has been implicated in pathological conditions including cancer. To evaluate the extent and variation of genome-wide DNA methylation and its changes during cellular differentiation and tumorgenesis as well as the interplay with histone modifications, accurate and reproducible quantification of the genomic DNA methylation level is required. These measurements have so far been achieved only by sophisticated and costly techniques. Here we report the generation of an enzyme-linked immunosorbent assay (methDNA-ELISA) for the accurate quantification of global DNA methylation levels. The linear region of this methDNA-ELISA ranges from 1 to 10%, making it highly suitable for the typical ranges from 2 to 6% in mammalian genomes. This method requires 10 ng of isolated DNA per sample, thus permitting investigation with minimal amounts of DNA previously not applicable for global DNA methylation analysis, e.g., clinical biopsies or cells collected by microdissection.

[Medication reconciliation in a department of internal medicine and infectious and tropical diseases: Feedback after one year practice]. / Conciliation médicamenteuse d'entrée en service de médecine interne : retour d'expérience après un an de pratique.

Since April 2015, medication reconciliation is performed in our Department. The objective of this study is to assess the impact of this activity on patients' care after one year of practice. METHODS: All patients who received medication reconciliation between April-October 2015 and June-December 2016 were included in this retrospective study. Undocumented unintentional discrepancies (DNIND) which result from the comparison between the patient's usual treatments and the medication prescribed at admission were collected. Then, a multidisciplinary discussion was initiated to correct them. The gravity of each DNIND was determined a posteriori. RESULTS: A statistical comparison between the two studies (2015 vs. 2016) showed the following significant results: decrease in DNIND (0.9 vs. 0.43), in percentage of patients with at least one DNIND (43% vs 31% P <5.10-6), in reconciliation time (43min vs. 23min) and no significant difference in the distribution of DNIND typology. The main therapeutic classes are: metabolism-diabetes-nutrition (21%), cardiology (18%), pneumology (17%) and neurology-psychiatry (15%). Drugs mainly concerned with DNIND are inhaled anti-asthmatics (13% of the medicines with DNIND), vitamins (8% of DNIND) and the levetiracetam antiepileptic drug (5% of DNIND). CONCLUSION: The implementation of the reconciliation medication allowed a significant reduction of the DNIND that permits to improve the patient healthcare pathway.

Common genetic variation in the promoter of the human apo CIII gene abolishes regulation by insulin and may contribute to hypertriglyceridemia.

Overexpression of plasma apolipoprotein CIII (apo CIII) causes hypertriglyceridemia in transgenic mice. A genetically variant form of the human apo CIII promoter, containing five single base pair changes, has been shown to be associated with severe hypertriglyceridemia in a patient population. In animals and in cultured cells the apo CIII gene is transcriptionally downregulated by insulin. In this study we demonstrate that, unlike the wild-type promoter, the variant promoter was defective in its response to insulin treatment, remaining constitutively active at all concentrations of insulin. The loss of insulin regulation was mapped to polymorphic sites at -482 and -455, which fall within a previously identified insulin response element. Loss of insulin regulation could result in overexpression of the apo CIII gene and contribute to the development of hypertriglyceridemia. The variant apo CIII promoter is common in the human population and may represent a major contributing factor to the development of hypertriglyceridemia.

Direct identification of major histocompatibility complex class I-bound tumor-associated peptide antigens of a renal carcinoma cell line by a novel mass spectrometric method.

Melanoma and renal cell carcinoma (RCC) are thought to be the most immunogenic human tumors. Presently a series of tumor-specific peptides of melanoma is being tested in clinical trials with different immunotherapy protocols. In contrast, only one decameric peptide (SPSSNRIRNT) derived from one (ORF2) of three possible open reading frames (ORFs) of a gene named RAGE (Renal tumor AntiGEn) was shown to be the target for tumor-specific CTLs on renal carcinoma cells. One reason for the lack of identification of tumor antigens on RCC compared with melanoma may be the difficulty in generating tumor-specific CTLs as screening instruments. Therefore, our approach was directly to isolate and identify peptides bound to HLA class I molecules of the HLA-A2 and -B8 homozygous RCC line A-498. High performance liquid chromatography-fractionated peptides eluted with acid from immunoaffinity-purified HLA class I-peptide complexes were sequenced and identified for the first time by the novel and highly sensitive mass spectrometric method matrix-assisted laser desorption ionization-post source decay (MALDI-PSD) from minute amounts of 100 fmol to 1.5 pmol of the fractionated peptide samples. Fourteen peptide sequences first deduced from interpretations of the mass spectra were also shown to fulfill other reliability criteria such as matching the mass spectra of the respective synthetic peptides. Some peptides were identified to be derived from genes preferentially activated in malignant tissues or resulted from a possibly mutated gene. The most promising candidate for a CTL epitope is a decameric peptide (PASKKTDPQK) derived from another possible ORF (ORF5) of the RAGE gene and probably presented in association with HLA-B8. This peptide was synthesized and used for the in vitro induction of CTLs that lysed the A-498 cells and another HLA-B8-positive RCC line significantly more strongly than either other RAGE-positive but HLA-B8-negative RCC lines or K562 cells. Sensitive sequencing by MALDI-PSD thus may provide a powerful method of identifying potentially tumor-specific and HLA-restricted antigens, even on native malignant cells and tissues.

Advanced Modeled Iterative Reconstruction in Low-Tube-Voltage Contrast-Enhanced Neck CT: Evaluation of Objective and Subjective Image Quality.

BACKGROUND AND PURPOSE: Dose-saving techniques in neck CT cause increased image noise that can be counteracted by iterative reconstruction. Our aim was to evaluate the image quality of advanced modeled iterative reconstruction (ADMIRE) in contrast-enhanced low-tube-voltage neck CT. MATERIALS AND METHODS: Sixty-one patients underwent 90-kV(peak) neck CT by using third-generation 192-section dual-source CT. Image series were reconstructed with standard filtered back-projection and ADMIRE strength levels 1, 3, and 5. Attenuation and noise of the sternocleidomastoid muscle, internal jugular vein, submandibular gland, tongue, subscapularis muscle, and cervical fat were measured. Signal-to-noise and contrast-to-noise ratios were calculated. Two radiologists assessed image noise, image contrast, delineation of smaller structures, and overall diagnostic acceptability. Interobserver agreement was calculated. RESULTS: Image noise was significantly reduced by using ADMIRE compared with filtered back-projection with the lowest noise observed in ADMIRE 5 (filtered back-projection, 9.4 ± 2.4 Hounsfield units [HU]; ADMIRE 1, 8.3 ± 2.8 HU; ADMIRE 3, 6.7 ± 2.0 HU; ADMIRE 5, 5.4 ± 1.7 HU; all, P < .001). Sternocleidomastoid SNR and internal jugular vein-sternocleidomastoid contrast-to-noise ratios were significantly higher for ADMIRE with the best results in ADMIRE 5 (all, P < .001). Subjective image quality and image contrast of ADMIRE 3 and 5 were consistently rated better than those for filtered back-projection and ADMIRE 1 (all, P < .001). Image noise was rated highest for ADMIRE 5 (all, P < .005). Delineation of smaller structures was voted higher in all ADMIRE strength levels compared with filtered back-projection (P < .001). Global interobserver agreement was good (0.75). CONCLUSIONS: Contrast-enhanced 90-kVp neck CT is feasible, and ADMIRE 5 shows superior objective image quality compared with filtered back-projection. ADMIRE 3 and 5 show the best subjective image quality.

Critical analysis of the extinction coefficient of chloroplast cytochrome f.

In oxygenic photosynthesis the cytochrome bf complex links electron transport between photosystem II and photosystem I. The largest subunit of the complex is cytochrome f, a 32-kDa polypeptide that is anchored in the membrane by a transmembrane alpha helix located near the carboxyl end. The three-dimensional structure of the soluble domain of cytochrome f isolated from turnip has been determined by X-ray crystallography to 1.96 A resolution. The structure revealed several novel features compared to previously solved soluble c-type cytochrome structures, including a predominant beta-strand motif, the N-terminal alpha-amino group of a tyrosyl residue as an orthogonal ligand, and a bound internal water chain. Here we report a novel and unprecedented extinction coefficient for cytochrome f. Using the pyridine hemochrome assay, the reduced minus oxidized extinction coefficient for the soluble domain of turnip cytochrome f was 26 +/- 1 mM-1 cm-1 for the alpha-band wavelength peak at 554 nm relative to the isosbestic wavelengths (534, 543.5 and 560.5 nm), and 25 +/- 1 mM-1 cm-1 for spinach cytochrome f relative to the isosbestic wavelengths (533.5, 543.3 and 560.2). The extinction coefficients reported here are significantly higher than previously published values for cytochrome f. We believe earlier determinations underestimated the cytochrome f extinction coefficient and that the same is likely true for commonly used extinction coefficients of cytochrome b6. The cytochrome f extinction coefficient is large compared to most other c-type cytochromes, which could be due to the unique axial ligand of the cytochrome f heme. Polarographic measurements show the midpoint potential of soluble turnip cytochrome f to be 362 +/- 5 mV at pH 7.5. The midpoint potential was pH-independent from 5.0 to 8.5, and pH-dependent from pH 8.5 to 10.5 (-58 mV/pH unit) with a pK on the oxidized from near 9. Storage of some samples of purified turnip and spinach cytochrome f at -20 degrees C modified the heme environment in a fraction of the protein, shifting the midpoint potential to near -165 mV (pH 7.5) and the peak of the alpha-band absorption spectrum from 554 nm to 552 nm.

Modification of inhibitor binding sites in the cytochrome bf complex by directed mutagenesis of cytochrome b(6) in Synechococcus sp. PCC 7002.

The cytochrome bf complex, which links electron transfer from photosystem II to photosystem I in oxygenic photosynthesis, has not been amenable to site-directed mutagenesis in cyanobacteria. Using the cyanobacterium Synechococcus sp. PCC 7002, we have successfully modified the cytochrome b(6) subunit of the cytochrome bf complex. Single amino acid substitutions in cytochrome b(6) at the positions D148, A154, and S159 revealed altered binding of the quinol-oxidation inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), myxothiazol, and stigmatellin. Cytochrome bf and mitochondrial-type cytochrome bc(1) complexes are closely related in structure and function but exhibit quite different inhibitor specificities. Cytochrome bf complexes are insensitive to myxothiazol and sensitive to DBMIB, whereas cytochrome bc(1) complexes are sensitive to myxothiazol and relatively insensitive to DBMIB. Measurements of flash-induced and steady-state electron transfer rates through the cytochrome bf complex revealed increased resistance to DBMIB in the mutants A154G and S159A, increased resistance to stigmatellin in A154G, and created sensitivity to myxothiazol in the mutant D148G. Therefore these mutations made the cytochrome bf complex more like the cytochrome bc(1) complex. This work demonstrates that cyanobacteria can be used as effective models to investigate structure-function relationships in the cytochrome bf complex.

Structure determination of the small ubiquitin-related modifier SUMO-1.

The recently discovered small ubiquitin-related modifier SUMO-1 belongs to the growing family of ubiquitin-related proteins involved in postranslational protein modification. Unlike ubiquitin, SUMO-1 does not appear to target proteins for degradation but seems to be involved in the modulation of protein-protein interactions. Independent studies demonstrate an essential function of SUMO-1 in the regulation of nucleo-cytoplasmic transport, and suggest a role in cell-cycle regulation and apoptosis. Here, we present the first three-dimensional structure of SUMO-1 solved by NMR. Although having only 18% amino acid sequence identity with ubiquitin, the overall structure closely resembles that of ubiquitin, featuring the betabetaalphabetabetaalphabeta fold of the ubiquitin protein family. In addition, the position of the two C-terminal Gly residues required for isopeptide bond formation is conserved between ubiquitin and SUMO-1. The most prominent feature of SUMO-1 is a long and highly flexible N terminus, which protrudes from the core of the protein and which is absent in ubiquitin. Furthermore, ubiquitin Lys48, required to generate ubiquitin polymers, is substituted in SUMO-1 by Gln69 at the same position, which provides an explanation of why SUMO-1 has not been observed to form polymers. Moreover, the hydrophobic core of SUMO-1 and ubiquitin is maintained by conserved hydrophobic residues, whereas the overall charge topology of SUMO-1 and ubiquitin differs significantly, suggesting specific modifying enzymes and target proteins for both proteins.

Recombination and replication of plasmid-like derivatives of a short section of the mitochondrial chromosome of Neurospora crassa.

The 21-kbp mitochondrial chromosome of the stp-ruv strain of Neurospora crassa undergoes regional amplification yielding plasmid-like supercoiled circles varying in size from subunit length to very high multimers. A comparison of the base sequence of the five plasmids studied, with the region of the chromosome from which they were derived, indicated that the amplified chromosomal segments were determined by a recombination-excision process near or within two structurally distinctive regions. One of these, consisting of nearly uninterrupted strings of Cs and Gs straddling tandem PstI site direct repeats, could form an extended hairpin loop with only a few mismatches. It was found at or near the 5' exchange point of all of the plasmids. An extended 35-bp sequence containing 17-bp direct repeats was the primary 3' site of exchange. Base sequence changes were found in the vicinity of exchange points. Most notable of these was a G insertion and T to C transition within a section of the 5' region likely to form a hairpin loop, suggesting the involvement of a mismatch repair-like mechanism in the recombination process. The sequence, TATATAGACATATA, was identified as a likely candidate for the site of replication initiation. A nearly identical sequence was found common to all of the corresponding plasmids of Podospora anserina and was reported near the presumed replication origin of the Drosophila yakuba mitochondrial chromosome. A search of GenBank revealed a remarkable association of the consensus sequence, TATATAGAXATATA, with the plus strand of organelle DNA.

Molecular properties and involvement of heparanase in cancer progression and normal development.

Heparan sulfate proteoglycans (HSPGs) play a key role in the self-assembly, insolubility and barrier properties of basement membranes and extracellular matrices. Hence, cleavage of heparan sulfate (HS) affects the integrity and functional state of tissues and thereby fundamental normal and pathological phenomena involving cell migration and response to changes in the extracellular microenvironment. Here, we describe the molecular properties, expression and function of a human heparanase, degrading HS at specific intrachain sites. The enzyme is synthesized as a latent approximately 65 kDa protein that is processed at the N-terminus into a highly active approximately 50 kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and human tumor tissues. Overexpression of the heparanase cDNA in low-metastatic tumor cells conferred a high metastatic potential in experimental animals, resulting in an increased rate of mortality. The heparanase enzyme also releases ECM-resident angiogenic factors in vitro and its overexpression induces an angiogenic response in vivo. Heparanase may thus facilitate both tumor cell invasion and neovascularization, both critical steps in cancer progression. The enzyme is also involved in cell migration associated with inflammation and autoimmunity. The unexpected identification of a single predominant functional heparanase suggests that the enzyme is a promising target for drug development. In fact, treatment with heparanase inhibitors markedly reduces tumor growth, metastasis and autoimmune disorders in animal models. Studies are underway to elucidate the involvement of heparanase in normal processes such as implantation, embryonic development, morphogenesis, tissue repair, inflammation and HSPG turnover. Heparanase is the first functional mammalian HS-degrading enzyme that has been cloned, expressed and characterized. This may lead to identification and cloning of other glycosaminoglycan degrading enzymes, toward a better understanding of their involvement and significance in normal and pathological processes.

Tumor necrosis factor inhibits the transcriptional rate of glucose-6-phosphatase in vivo and in vitro.

Recombinant human tumor necrosis factor-alpha (TNF) injection in mice was associated with a reduced blood glucose level, already manifest 6 hours following cytokine administration. Insulin levels were not affected. Glycogen content was decreased in a dose-dependent and time-response manner. The activity of glucose-6-phosphatase (G6Pase) was already reduced 6 hours after TNF injection and was sustained 12 hours afterward. Phosphoenolpyruvate carboxykinase (PEPCK) activity was not affected initially (6 hours after injection), but a 50% reduction was observed 12 hours following cytokine administration compared with levels in fasting controls. Both liver G6Pase and PEPCK mRNAs were markedly reduced due to an inhibition of the transcriptional rate. A direct inhibitory effect of TNF on G6Pase promoter activity was demonstrated using HuH-7 cells transiently transfected with G6Pase promoter, fused to a reporter gene.

Detection of Plasmodium falciparum in blood using DNA hybridization.

A rapid and simple assay for detecting Plasmodium falciparum in human blood was developed. The assay is based on DNA-DNA spot hybridization, using radiolabeled P. falciparum DNA as a probe and finger prick blood as the assay sample. It is very sensitive, able to detect parasitemia levels of 0.0001% in 10 microliter of blood. The assay can be quantified and used to estimate parasitemia levels. Several hundred blood samples can be processed simultaneously, and the entire procedure is completed within 24 hr. This assay can be useful for epidemiological surveys, for screening of blood by blood banks and for health authorities examining immigrants and tourists coming from malaria infested areas.

Studies on the dephosphorylation of phosphotyrosine-containing peptides during post-source decay in matrix-assisted laser desorption/ionization.

Phosphorylation of tyrosine residues in proteins is a common regulatory mechanism, although it accounts for less than 1% of the total O-phosphate content in proteins. Whereas aromatic phosphorylation sites can be identified by a number of different analytical techniques, sequence analysis of phosphotyrosine-containing proteins at the low picomole or even femtomole level is still a challenging task. This paper describes the post-source decay in matrix-assisted laser desorption/ionization mass spectrometry of phosphotyrosine-containing model peptides by comparing their fragmentation behavior with sequence-homologous unphosphorylated peptides. Whereas the parent ions showed significant losses of HPO3, all phosphorylated fragment ions of the b- and y-series displayed only minor dephosphorylated signals, which often were not detectable. Surprisingly, one of the studied phosphotyrosine-containing sequences displayed, in addition to the [M + H - 80]+ ion, a more abundant [M + H - 98]+ ion, which could be explained by elimination of phosphoric acid. This dephosphorylation pattern was very similar to the patterns obtained for phosphoserine- and phosphothreonine-containing peptides. Because the dephosphorylation pattern of the parent ion is often used to identify modified amino acids in peptides, we investigated possible dephosphorylation mechanisms in detail. Therefore, we substituted single trifunctional amino acid residues and incorporated deuterated phosphotyrosine residues. After excluding direct elimination of phosphoric acid from tyrosine, we could show that the obtained loss of H3PO4 depends on aspartic acid and arginine residues. Most likely the HPO3 group is transferred to aspartic acid followed by cleavage of phosphoric acid forming a succinimide. On the other hand, arginine appears to induce the H3PO4 loss by protonation of phosphotyrosine leaving a phenyl cation.

Sequencing of peptides phosphorylated on serines and threonines by post-source decay in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

In the era of complete genome sequences, biochemical and medical research will focus more on the dynamic proteome of a cell. Regulation of proteins by post-translational modifications, which are not determined by the gene sequence, are already intensively studied. One example is phosphorylation of serines and threonines, probably the single most common cellular regulatory mechanism. In this paper we describe the sequencing of mono- and bisphosphorylated peptides, including identification of the phosphorylation sites, by post-source decay (PSD) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In addition to dephosphorylation of the parent ions, we studied the influence of the phosphate group on the fragmentation of peptides. Generally, peptides phosphorylated on serine and threonine residues displayed no difference in their fragmentation patterns. The intensities of the resulting fragment ion signals depend only on the peptide sequence and not on either the phosphorylated amino acid or its position in the peptide chain. Phosphorylation increased the bond cleavage C-terminal to the phosphorylation site more than 10-fold, resulting in abundant signals, which typically dominated the PSD spectra. The produced C-terminally phosphorylated b-type fragment ions showed characteristic dephosphorylated fragment ions b(n) -H(3)PO(4) (-98 Da) and b(n) -HPO(3) (-80 Da) of higher abundances than the phosphorylated fragment ion. As a second layer to identify the phosphorylation site, all internally phosphorylated fragment ions were accompanied by minor, but always detectable, signals of the dephosphorylated fragment ions. Interpretation of PSD spectra of phosphopeptides was not more complicated than for unphosphorylated peptides, despite the increased number of obtained fragment ion signals.

A biosensor based on magnetoresistance technology.

We are developing a biosensor that will measure, at the level of single molecules, the forces that bind DNA-DNA, antibody-antigen, or ligand-receptor pairs together. The Bead Array Counter (BARC) will use these interaction forces to hold magnetic microbeads to a solid substrate. Microfabricated magnetoresistive transducers on the substrate will indicate whether or not the beads are removed when pulled by magnetic forces. By adapting magnetoresistive computer memory technology, it may be possible to fabricate millions of transducers on a chip and detect or screen thousands of analytes. The multi-analyte capability of this portable sensor would be ideal for on-site testing, while the potential to directly gauge intermolecular interaction strengths suggests drug discovery applications.

Successful palliation of stenosing anorectal melanoma by intratumoral injections with natural interferon-beta.

Anorectal malignant melanoma is an uncommon tumour. Unlike for cutaneous melanoma, there are few guidelines for its optimal management. In particular, very few palliative treatment strategies have been described for patients with advanced disease. We report on an 80 year old patient with locally advanced anorectal melanoma nearly completely blocking the anal orifice and disseminated metastases. Complete regression of the primary tumour and partial remission of the metastases was achieved with intratumoral injections of natural interferon-beta and systemic administration of dacarbazine. The quality of life in this patient was improved markedly by providing relief from severe rectal pain and bleeding. We propose that conservative treatment strategies such as intratumoral injections with interferon-beta should be considered as a palliative treatment option for stenosing anorectal melanoma before an abdominoperineal resection is recommended.

Extent and consequences of physician delay in the diagnosis of acral melanoma.

The extent and consequences of professional delay in diagnosis were analysed in 83 patients with palmoplantar and subungual melanomas treated from January 1986 to March 1997 in our department. Seventeen (52%) out of 33 subungual melanomas and 10 (20%) out of 50 palmoplantar melanomas were clinically misdiagnosed by physicians. Three palmoplantar melanomas (6%) were initially misinterpreted by pathologists. In 23 of the 27 cases (85%) the clinical misdiagnosis was made by non-dermatologists. Misdiagnosis caused a median delay of 12 months in the diagnosis of palmoplantar melanomas and 18 months in the diagnosis of subungual melanomas. Delay in diagnosis was associated with increased tumour thickness, more advanced stage at time of melanoma diagnosis and a lower estimated 5-year survival rate (15.4% versus 68.9% for palmoplantar; 68.5% versus 90.9% for subungual). Acral melanomas are frequently misdiagnosed due to their less common locations and because plantar and subungual melanomas often do not fit the 'changing mole' pattern. To Improve the patient's prognosis it is necessary to increase the physicians' skill in the diagnosis of acral melanomas. Histological examination should always be performed in acral lesions that do not heal.