A novel SERPINA12 variant and first European patients with diffuse palmoplantar keratoderma.

BACKGROUND: Hereditary palmoplantar keratodermas (hPPKs) comprise a heterogeneous group of skin disorders characterized by persistent palmoplantar hyperkeratosis. Loss-of-function variants in a serine peptidase inhibitor, SERPINA12, have recently been implicated in autosomal recessive diffuse hPPK. The disorder appears to share similarities with another hPPK associated with protease overactivity, namely Nagashima-type PPK (NPPK) caused by biallelic variants in SERPINB7. OBJECTIVES: The aim of this study was to enhance the understanding of the clinical and genetic characteristics of serine protease-related hPPKs caused by variants in SERPINA12 and SERPINB7. METHODS: Whole-exome sequencing (WES) was performed for hPPK patients. Haplotype analysis was completed for the patients with identified recessive SERPINA12 variants and their available family members. In addition, the current literature of SERPINA12- and SERPINB7-related hPPKs was summarized. RESULTS: The phenotype of SERPINA12-related hPPK was confirmed by reporting three new SERPINA12 patients, the first of European origin. A novel SERPINA12 c.1100G>A p.(Gly367Glu) missense variant was identified confirming that the variant spectrum of SERPINA12 include both truncating and missense variants. The previously reported SERPINA12 c.631C>T p.(Arg211*) was indicated enriched in the Finnish population due to a plausible founder effect. In addition, SERPINA12 hPPK patients were shown to share a similar phenotype to patients with recessive variants in SERPINB7. The shared phenotype included diffuse transgradient PPK since birth or early childhood and frequent palmoplantar hyperhidrosis, aquagenic whitening and additional hyperkeratotic lesions in non-palmoplantar areas. SERPINA12 and SERPINB7 hPPK patients cannot be distinguished without genetic analysis. CONCLUSIONS: Recessive variants in SERPINA12 and SERPINB7 leading to protease overactivity and hPPK produce a similar phenotype, indistinguishable without genetic analysis. SERPINA12 variants should be assessed also in non-Asian patients with diffuse transgradient PPK. Understanding the role of serine protease inhibitors will provide insights into the complex proteolytic network in epidermal homeostasis.

Head and neck arteriovenous malformations: results of ethanol sclerotherapy.

BACKGROUND AND PURPOSE: Peripheral AVM is a locally aggressive disease with a high tendency to recur; its treatment is complex, especially in the anatomically delicate head and neck area. Here, we report results of ethanol sclerotherapy for head and neck AVM and discuss its potential use for peripheral AVM. MATERIALS AND METHODS: We retrospectively assessed degree of AVM eradication, complications, and clinical or imaging signs of recurrence for 19 patients treated with ethanol sclerotherapy for head and neck AVM (1 intraosseous, 18 soft-tissue AVMs). RESULTS: Of the 19 patients, 11 had complete eradication of arteriovenous shunting at DSA, with 1 recurrence (mean follow-up 15 months), and for 7 patients, treatment is ongoing. During 59 treatment sessions, 12 patients experienced 14 complications, 1 leading to permanent functional damage. CONCLUSIONS: Ethanol sclerotherapy has potential for complete eradication of head and neck AVM with low recurrence within the first year after completion of treatment. Complete eradication may require several treatment sessions during which complications should be minimized with careful techniques.

Effect of temporin A modifications on its cytotoxicity and antimicrobial activity.

Temporin A (TA), a short alpha-helical antimicrobial peptide isolated from the skin of the frog Rana temporaria, is effective against a broad spectrum of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium strains. TA interacts directly with the cell membrane of the microorganism and it has been reported to be non-toxic to erythrocytes at concentrations that are antimicrobial. Less is known about the effects on the viability and growth of nucleated eukaryotic cells. In this study we have tested antibacterial and growth-inhibitory properties of TA, its dimeric analogue (TAd), and all-L (TAL L512) and all-D (TAD L512) enantiomeric derivatives of modified TA towards S. aureus and cultured human keratinocytes, respectively. All molecules were antibacterial at concentrations from 1.5 microM to 10 microM. In keratinocyte cultures, TAD L512, as well as TAd, showed cytotoxicity. The original TA and TAL L512 did not affect the viability of the cells at their bacteriolytic concentrations. The growth of keratinocytes in low- and high-calcium media was only slightly inhibited by temporins at concentrations which were antibacterial to S. aureus. This suggests that original TA and its modification, TAL L512, are promising molecules against multiresistant bacterial infections.

Infra-inguinal percutaneous transluminal angioplasty for limb salvage: a retrospective analysis in a single center.

PURPOSE: To review the feasibility of infra-inguinal angioplasty in the management of critical limb ischemia (CLI). MATERIAL AND METHODS: Data on 221 patients with 230 critically ischemic limbs, treated with consecutive percutaneous transluminal angioplasty (PTA) at Helsinki University Central Hospital between January 2000 and December 2002 were collected and analyzed retrospectively. Patency, limb salvage, and survival rates were calculated on an intention-to-treat basis. Comparisons were done with univariate (Kaplan-Meier) and multivariate analysis (Cox regression). RESULTS: Overall primary patency, secondary patency, limb salvage, and survival rates were 47%, 59%, 92%, and 76%, respectively, at 12 months. In the multivariate analysis, low toe pressure (< or =30 mmHg) was a significant risk factor for poor patency. Uremia with hemodialysis, low toe pressure (< or =30 mmHg), and hemodynamic failure of the endovascular procedure were found to increase significantly the risk of amputation. Uremia with hemodialysis, coronary artery disease, tissue loss as indication for PTA (Fontaine stage IV), and age over 70 years were all found to increase significantly the risk of death. CONCLUSION: Infra-inguinal PTA is feasible in patients with CLI and resulted in good limb salvage.

Contribution of winter to the annual CH4 emission from a eutrophied boreal lake.

The springtime methane (CH4) emission from a small, eutrophied boreal lake was assessed during the winter ice-cover by measurement of gas ebullition and CH4 accumulation in the water column in association with the development of oxygen depletion after ice formation. The winter CH4 production was estimated to result in a loss of 3.6-7.9 g CH4 m(-2) from the lake to the atmosphere during the short period of ice melt. This could account for 22-48% of the annual CH4 emission from the pelagic zone of the lake. The contribution of winter to the annual CH4 release can be similar or even higher in seasonally ice-covered northern aquatic ecosystems than in northern terrestrial wetlands, thus winter must be considered in any studies into the aquatic CH4 emissions. The trophic state and wintertime oxygen conditions, linked to the changes in land-use in the catchments and climate, are important factors controlling the springtime lake CH4 emissions.

A novel sediment gas sampler and a subsurface gas collector used for measurement of the ebullition of methane and carbon dioxide from a eutrophied lake.

A novel sediment bubble gas sampler and a subsurface bubble gas collector were designed to measure the ebullition of gases from profundal sediments of aquatic ecosystems. The sediment gas sampler was constructed to collect bubble gas samples directly from the uppermost sediment layers for gas composition analysis. The floating subsurface gas collector, designed to trap the bubbles released naturally from sediments, permitted the measurement of both the volume and the composition of the bubble gas. Due to its low cost, light weight and rapid sampling capability, the gas collector is ideal for studies requiring many replicate collectors. These devices were used for measurement of the ebullition of methane (CH4) and carbon dioxide (CO2) during an open water period from hypereutrophic Lake Postilampi, situated within the midboreal zone in Finland. The bubble gas obtained from the sediment with the sediment gas sampler had higher concentrations of CH4 and CO2 than the bubbles trapped in the gas collectors. This indicated that the bubble gas composition changed, either naturally during the migration of the bubbles from the sediment through the water column to the gas collectors, and/or during their storage in the collectors prior to sampling. The mean CH4 ebullition from Lake Postilampi was estimated to be in the range from 36 to 46 mg m(-2 d(-1), based on the bubble gas CH4 concentrations measured from the gas collectors and sediment, respectively. The bubbles contained only 0.02-0.57% of CO2 and thus, the ebullition had no significance in the release of CO2 from the lake.

J-multiplied HSQC (MJ-HSQC): a new method for measuring 3J(HNHalpha) couplings in 15N-labeled proteins.

A new method for the measurement of homonuclear 3J(HNHalpha) coupling constants in 15N-labeled small proteins is described. The method is based on a modified sensitivity enhanced HSQC experiment, where the 3J(HNHalpha) couplings are multiplied in the f1-dimension. The J-multiplication of homonuclear 3J(HNHalpha) couplings is based on simultaneous incrementation of 15N chemical shift and homonuclear coupling evolution periods. The time increment for the homonuclear coupling evolution period is chosen to be a suitable multiple (2N x t1) of the corresponding increment for 15N-shift evolution. This results in the splitting of the HSQC correlation in the f1-dimension by 2N x 3J(HNHalpha). Because the pulse sequence has good sensitivity and water suppression properties, it is particularly useful for natural abundance samples.

Mantle cell lymphoma: clinical features, treatment and prognosis of 94 patients.

Mantle cell lymphoma (MCL) is a subtype of B-cell non-Hodgkin's lymphoma recently recognised as a distinct disease entity. Little is known about the prognostic factors and optimal treatment of MCL. The aim of this study was to analyse retrospectively the clinical features and effect of treatment in 94 MCL patients diagnosed and treated in one centre between 1980 and 1996, and to find out different factors influencing the treatment results and prognosis. The median age of the patients was 66 years, and 77% were over 60 years old. Of the patients, 76% had advanced disease, the performance status (PS) was WHO 0-1 in 86%, and B symptoms were present in 35% of the cases. Bone marrow infiltration was found in 61% and overt leukaemia in 12% of the patients. Of the patients, 47% achieved complete remission with first- or second-line therapy. The median duration of remission, time to treatment failure (TTF), and survival were 28, 18, and 41 months, respectively. In multivariate analyses, age, stage and leukaemic disease were significantly associated with TTF, and age, stage, leukaemic disease and lactate dehydrogenase (LDH) with survival. Long-term prognosis is poor in MCL. None of the conventional chemotherapies seems curative. A prospective randomised trial should be made to evaluate the benefit of anthracycline-containing regimens in MCL.

Exposure of gasoline road-tanker drivers to methyl tert-butyl ether and methyl tert-amyl ether.

Organic oxygenates, namely, methyl tert-butyl ether (MTBE) and methyl tert-amyl ether (MTAE), are added to gasoline to reduce carbon monoxide in exhausts and to enhance the octane number. The aim of this study was to investigate road-tanker drivers' exposure to oxygenate vapors during road-tanker loading and unloading as well as to evaluate the measurements of these ethers and their metabolites in the urine as a means of assessing the uptake of the ethers. A total of 11 drivers in different parts of Finland were trained to monitor their exposure with personal samplers, to report their working conditions, and to collect their whole-day urine samples. Charcoal tubes of the air samples were analyzed for MTBE, MTAE, benzene, toluene, and aliphatic hydrocarbons. For biological monitoring purposes the two main oxygenates, tertiary ethers MTBE and MTAE, as well as their main metabolites, tertiary alcohols tert-butanol (TBA) and tert-amyl alcohol (TAA), were determined in urine specimens. On average the drivers were exposed to vapors for short periods (21 +/- 14 min) three times during a work shift. The mean concentrations of MTBE and MTAE (mean +/- SD) were 8.1 +/- 8.4 and 0.3 +/- 0.4 mg/m3. The total MTBE uptake during the shift was calculated to be an average of 106 +/- 65 mumol. The mean concentrations of MTBE, TBA, MTAE and TAA detected in the first urine after the work shift were 113 +/- 76, 461 +/- 337, 16 +/- 21, and 40 +/- 38 nmol/l, and those found the next morning, 16 h later, were 18 +/- 12, 322 +/- 213, 9 +/- 10, and 20 +/- 27 nmol/l. The good relationship (r = 0.84) found between MTBE exposure and postshift excretion suggests that urinary MTBE can be used for biological monitoring of exposure, but at the present low level of exposure the corresponding metabolite TBA is not equally reliable. The determination of MTAE and its metabolite TAA in urine is sensitive enough to detect the low degree of exposure to MTAE, but in this study the data were too scarce to allow calculation of the correlations due to very low levels of MTAE exposure.

Elevated serum CD44 level is associated with unfavorable outcome in non-Hodgkin's lymphoma.

CD44 molecule is a cell surface glycoprotein involved in many cell-cell and cell-matrix interactions. Circulating serum CD44 (s-CD44) levels have been found to change in parallel with response to therapy, but little is known about the predictive or prognostic significance of s-CD44. In the present study, we measured s-CD44 levels in sera taken before treatment from 194 patients with non-Hodgkin's lymphoma using a chemiluminescence-enzyme immunoassay method. All except 1 patient were regularly followed-up after therapy at least for 60 months (range, 33 to 143 months). The median pretreatment s-CD44 level was 440 ng/mL (range, 13 to 1,220 ng/mL). Only 32% of the 92 patients with an International Prognostic Index (IPI) score of 0 or 1 had an s-CD44 concentration higher than the median as compared with 67% of the patients with an IPI score >/=2 (P < .0001). Patients with lower than the median s-CD44 achieved more often a complete remission to therapy (P = .0002) and had better survival (P = .007) than those with higher s-CD44 levels. However, in a multivariate analysis, only the IPI score had independent prognostic value (P < .001). The findings were similar if only the patients with diffuse large-cell lymphoma (n = 51) were included in the analysis, but among patients with low-grade lymphoma, the median s-CD44 level was not significantly associated with the IPI or survival. In conclusion, a high s-CD44 level at diagnosis is associated with a high IPI score, poor response to treatment, and unfavorable outcome in non-Hodgkin's lymphoma.

Intracellular distribution of oligonucleotides delivered by cationic liposomes: light and electron microscopic study.

Synthesized oligonucleotides are used in anti-sense and anti-gene technology to control gene expression. Because cells do not easily take up oligonucleotides, cationic liposomes have been employed to facilitate their transport into cells. Although cationic liposomes have been used in this way for several years, the precise mechanisms of the delivery of oligonucleotides into cells are not known. Because no earlier reports have been published on the liposomal delivery of oligonucleotides at the ultrastructural level, we performed a study, using electron microscopy, on the cellular uptake and intracellular distribution of liposomal digoxigenin-labeled oligodeoxynucleotides (ODNs) at several concentrations (0.1, 0.2, an 1.0 microM) in CaSki cells. Two cationic lipids (10 microM) were compared for transport efficiency: polycationic 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl -1-propanaminium trifluoroacetate (DOSPA) and monocationic dimethyl-dioctadecylammonium bromide (DDAB). Both liposomes contained dioleoyl-phosphatidylethanolamine (DOPE) as a helper lipid. Endocytosis was found to be the main pathway of cellular uptake of liposomal ODNs. After release from intracellular vesicles, ODNs were carried into the perinuclear area. The nuclear membrane was found to be a barrier against the penetration of ODNs delivered by liposomes into the nucleus. Release from vesicles and transport into the nuclear area was faster when the oligo-DDAB/DOPE complex had a positive net charge (0.1 and 0.2 microM ODN concentrations), and only under this condition were some ODNs found in nucleoplasm. Although DOSPA/DOPE could also efficiently deliver ODNs into the cytosol, no ODNs were found in nucleoplasm. These findings suggest that both the type of liposome and the charge of the oligo-liposome complex are important for determination of the intracellular distribution of ODNs.

Liposomal targeting of bcl-2 antisense oligonucleotides with enhanced stability into human myeloma cell lines.

Cationic liposomes improve the delivery of antisense oligonucleotides (ODNs) into cells. However, there is marked variability in the cellular uptake of ODNs into different cell lines. We used liposomes containing dimethyloctadecylammonium bromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE) to increase the delivery of phosphodiester ODNs into four different myeloma cell lines. The delivery by cationic liposomes increased the delivery of bcl-2 antisense ODNs by a factor of 9 to 45 as compared to plain ODNs. The stability of ODNs was increased with liposomes both in the culture medium and within the cells. Intact liposomal ODNs were detected inside the cells up to 24 hours with gel electrophoresis and phosphor imager analysis. Antisense ODNs had no effect on bcl-2 mRNA levels. Also the proliferation of myeloma cells remained unchanged during the 3-day incubation period. Our study shows that liposomal antisense ODNs targeting bcl-2 of human myeloma cells result in increased stability of ODNs with minimal toxicity. However, further modifications are needed to gain biological effects of antisense ODNs on human myeloma cells.

Effects of liposomal antisense oligonucleotides on mRNA and protein levels of the HPV 16 E7 oncogene.

Despite the known association of human papillomavirus (HPV) infection with cervical cancer there is no specific antiviral treatment for HPV infection. Antisense oligode-oxynucleotides (AS-ODNs) may offer an effective way to treat HPV infections as the stability and delivery have been improved using modified ODNs or carrier systems. In this study we investigated the effects of liposomal AS-ODNs (0.1, 1 and 5 microM) on HPV 16 E7 mRNA and protein levels in CaSki cells. We used cationic liposomes (10 microM) containing dimethyldioctadecylammonium bromide (DDAB) or 2,3-dioleyloxy-N-[2(sperminecar-boxamido)ethyl]-N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA). Both these liposomes had dioleoylphosphatidyl-ethanolamine (DOPE) as a helper lipid. The target of the AS-ODNs was E7 protein because it is the one of the two oncoproteins of HPV 16. Only liposomal AS-ODNs which were targeted to the initiation codon of E7, had an effect on E7 mRNA expression; two shorter transcripts were detected, suggesting that RNase H degradation was activated. Liposomal random ODN or liposomal ODN targeted downstream from the initiation site of E7 did not affect the mRNA pattern. However, no change was found in the E7 protein levels detected by immunoprecipitation. Further studies showed that AS-ODNs inhibited the translation of E7 mRNA in a rabbit reticulocyte lysate assay. This data, together with the changes in mRNA levels, proved that the AS-ODNs reached the target mRNA. One possible explanation for the unchanged protein level of E7 in CaSki cells might be that immunoprecipitation is not sensitive enough to detect minor changes in protein levels. However, further progress is still needed in the field of carrier systems and modifications of AS-ODNs before non-sequence specific effects can be avoided.

Human papillomavirus in vulvar and vaginal carcinoma cell lines.

A number of reports associate human papillomavirus (HPV) with cervical cancer and cancer cell lines derived from this tumour type. Considerably fewer reports have focused on the role of HPV in carcinomas from other sites of female anogenital squamous epithelia. In this study we have tested for the presence of HPV in eight low-passage vulvar carcinoma cell lines and one extensively passaged cell line, A431. One cell line from a primary vaginal carcinoma was included. The presence of the HPV was evaluated by the polymerase chain reaction (PCR), by Southern blot analysis and by two-dimensional gel electrophoresis. General primer-mediated PCR was applied by using primers from the L1 region, E1 region and HPV 16 E7 region. Southern blot hybridisation was performed under low-stringency conditions (Tm = -35 degrees C) using a whole genomic HPV 6/16/18 probe mixture and under high stringency conditions (Tm = -18 degrees C) with the whole genomic probes of HPV 16 and 33. HPV 16 E6-E7 mRNA was assessed by ribonuclease protection assay (RPA). HPV was found in only one vulvar carcinoma cell line, UM-SCV-6. The identified type, HPV 16, was integrated in the cell genome and could be amplified with all primers used. Also E6-E7 transcripts were found in these cells. Five original tumour biopsies were available from the HPV-negative cell lines for in situ hybridisation. All these were HPV negative with both the HPV 6/16/18 screening probe mixture under low stringency and the HPV 16 probe under high stringency. The results indicate that vulvar carcinoma cell lines contain HPV less frequently than cervical carcinoma cell lines and suggest that a significant proportion of vulvar carcinomas may evolve by an HPV-independent mechanism.