Combining spore germination and heat inactivation to decontaminate materials contaminated with Bacillus anthracis spores.

AIMS: To add a spore germination step in order to reduce decontamination temperature and time requirements compared to the current hot, humid air decontamination parameters, which are 75-80°C, ≥72 h, 70-90% RH, down to ≤60°C and ≤24 h total decontamination time. METHODS AND RESULTS: Bacillus anthracis spore germination with l-alanine+inosine+calcium dipicolinate (CaDPA) was quantified at 0-40°C, several time points and spore concentrations of 5-9 log10 per ml. Germination efficiency at 0-40°C was >99% at <8 log10 spores per ml. The temperature optimum was 20°C. Germination efficiency was significantly higher but slower at 0°C compared to ≥30°C at ≥8 log10 spores per ml. A single germinant application followed by 60°C, 1-h treatment consistently inactivated >2 log10 (>99%) of spores. However, a repeat application of germinant was needed to achieve the objective of ≥6 log10 spore inactivation out of a 7 log10 challenge (≥99·9999%) for ≤24 h total decontamination time for nylon and aircraft performance coating. CONCLUSIONS: l-alanine+inosine+CaDPA stimulated germination across wide temperature and spore concentration ranges. SIGNIFICANCE AND IMPACT OF THE STUDY: Germination expands the scope of spore decontamination to include materials from any industry sector that can be sprayed with an aqueous germinant solution.

Effect of animal sera on Bacillus anthracis Sterne spore germination and vegetative cell growth.

AIMS: The aims of this work were to investigate the effects of sera on B. anthracis Sterne germination and growth. Sera examined included human, monkey and rabbit sera, as well as sera from eight other species. METHODS AND RESULTS: Standard dilution plate assay (with and without heat kill) was used as a measure of germination, and spectroscopy was used to measure growth. In addition, a Coulter Counter particle counter was used to monitor germination and growth based on bacterial size. Spores germinated best in foetal bovine and monkey sera, moderately with human sera and showed limited germination in the presence of rabbit or rat sera. Vegetative bacteria grew best in foetal bovine sera and moderately in rabbit sera. Human and monkey sera supported little growth of vegetative bacteria. CONCLUSION: The data suggested sera can have a significant impact on germination and growth of Sterne bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: These data should be considered when conducting in vitro cell culture studies and may aid in interpreting in vivo infection studies.

Preliminary evidence for involvement of the tumour suppressor gene CHD5 in a family with cutaneous melanoma.

BACKGROUND: Cutaneous melanoma is rapidly increasing in incidence worldwide and approximately 5% of melanomas are hereditary. Deletions in chromosome 1p36 have been detected in melanoma but no candidate melanoma tumour suppressor gene has yet been found in this area. Recently, strong evidence has been reported that CHD5 is a tumour suppressor gene in this region. OBJECTIVES: To investigate CHD5 involvement in familial melanoma. METHODS: Peripheral blood DNA from 47 melanoma families who do not carry mutations in any of the three currently recognized melanoma genes, 398 patients with sporadic melanoma and 398 geographically matched nonmelanoma-bearing controls were studied. Linkage investigation, single nucleotide polymorphism (SNP) genotyping and mutation screening studies were carried out on the CHD5 locus. RESULTS: The CHD5 gene was not excluded by linkage analysis in any of the families. On SNP genotyping, the CHD5 rs7513548 SNP was found to be significantly associated with sporadic melanoma (odds ratio 1·53, 95% confidence interval 1·13-2·06). The AG genotype was found in 208 cases and 169 controls (cf. 141 and 175 cases and controls, respectively, for the AA genotype). On CHD5 mutation screening, a total of 50 single-base substitutions were detected. Of these, 39 were intronic and 11 were exonic. While 32 were previously recognized variants, 18 were newly identified. Three, in exons 4, 31 and 32, led to nonsynonymous substitutions. A p.Met1576Ile substitution was identified in a mother and daughter, both with invasive cutaneous melanoma. CONCLUSIONS: This study appears to be the first report of CHD5 variants in familial cutaneous melanoma. Such CHD5 variants could block or alter the ability of CHD5 to regulate the cell cycle pathway and to effect cellular control. As only one of the 47 families studied has this variant, it appears to be a rare event and further screening of melanoma families is required to confirm whether or not CHD5 is involved in melanoma pathogenesis.

Identification of A- and B-site cation vacancy defects in perovskite oxide thin films.

Cation vacancies on both sublattices (V(Ti), V(Sr)) have been identified in homoepitaxial pulsed laser deposited SrTiO3 films using high intensity variable energy positron annihilation lifetime spectroscopy (PALS) measurements. Film nonstoichiometry was varied by varying laser fluence. PALS showed that on increasing the fluence above the Ti/Sr∼1 value, the concentration ratio [V(Sr)]/[V(Ti)] systematically increased. Reducing the fluence into the Ti-poor region below resulted in additional vacancy cluster defect formation. Vacancy concentrations greater than ∼50 ppm were observed in all films.

Epidemiology of invasive cutaneous melanoma.

Data are presented on the current incidence of melanoma with recent and predicted future trends illustrating a likely continuing increase in incidence. Risk factors for developing melanoma are discussed, including current known melanoma susceptibility genes. Phenotypic markers of high-risk subjects include high counts of benign melanocytic naevi. Other risk factors considered include exposure to natural and artificial ultraviolet radiation, the effect of female sex hormones, socioeconomic status, occupation, exposure to pesticides and ingestion of therapeutic drugs including immunosuppressives and non-steroidal anti-inflammatory drugs. Aids to earlier diagnosis are considered, including public education, screening and use of equipment such as the dermatoscope. Finally, the current pattern of survival and mortality is described.

Utility of adjuvant systemic therapy in melanoma.

The lack of effective drugs in stage IV melanoma has impacted the effectiveness of adjuvant therapies in stage II/III disease. To date, chemotherapy, immunostimulants and vaccines have been used with minimal success. Interferon (IFN) has shown an effect on relapse-free survival (RFS) in several clinical trials; however, without a clinically significant effect on overall survival (OS). A recently conducted meta-analysis demonstrated prolongation of disease-free survival (DFS) in 7% and OS benefit in 3% of IFN-treated patients when compared with observation-only patients. There were no clear differences for the dose and duration of treatment observed. Observation is still an appropriate control arm in adjuvant clinical trials. Regional differences exist in Europe in the adjuvant use of IFN. In Northwest Europe, IFN is infrequently prescribed. In Central and Mediterranean Europe, dermatologists commonly prescribe low-dose IFN therapy for AJCC stage II and III disease. High-dose IFN regimens are not commonly used. The population of patients that may benefit from IFN needs to be further characterised, potentially by finding biomarkers that can predict response. Such studies are ongoing.

An evidence base for reconsidering current follow-up guidelines for patients with cutaneous melanoma less than 0.5 mm thick at diagnosis.

BACKGROUND: Despite current guidelines, there is uncertainty about the required duration and frequency of follow-up visits for patients with invasive primary cutaneous melanoma < 0.5 mm thick. OBJECTIVES: To review patients with invasive melanoma thinner than 0.5 mm followed for at least 5 years to provide an evidence base for considering modification of guidelines. METHODS: A retrospective review of 430 patients diagnosed in the west of Scotland during 1992-2001 with melanoma < 0.5 mm was carried out. Recurrence, deaths from melanoma and second primary melanomas were all identified. RESULTS: From 1992 to 2001, 430 melanomas < 0.5 mm thick at diagnosis were diagnosed out of a total of 3036 primary cutaneous melanomas. To date there have been 593 deaths from melanoma (19%) in the whole group. Five of these deaths were reported in patients with melanomas < 0.5 mm, but on pathological review two were thicker than 0.5 mm at diagnosis (1.5 and > 3 mm), and the remaining three patients all developed thicker second primary melanomas (2.7, 12.0 and 19.0 mm) with a recurrence pattern and timing indicating that these thicker primaries were the cause of death. Fourteen further patients developed a second primary melanoma, and 13 are currently alive and disease free, one dying of other causes. CONCLUSIONS: Our data indicate that recurrence and subsequent death from melanomas < 0.5 mm is a very rare event, and that quarterly follow-up for 3 years will yield very few events. Modification of current guideline recommendations are suggested to include a period of patient education concentrating on recognition of second primary tumours followed by rapid access to an expert opinion if required.

Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth.

This study tested the hypothesis that a host mucogenic response to an intestinal coccidial infection promotes the onset of necrotic enteritis (NE). A chick NE model was used in which birds were inoculated with Eimeria acervulina and E. maxima and subsequently with Clostridium perfringens (EAM/CP). A second group of EAM/CP-infected birds was treated with the ionophore narasin (NAR/EAM/CP). These groups were compared to birds that were either non-infected (NIF), or infected only with E. acervulina and E. maxima (EAM), or C. perfringens (CP). The impact of intestinal coccidial infection and anti-coccidial treatment on host immune responses and microbial community structure were evaluated with histochemical-, cultivation- and molecular-based techniques. Barrier function was compromised in EAM/CP-infected birds as indicated by elevated CFUs for anaerobic bacteria and C. perfringens in the spleen when compared to NIF controls at day 20, with a subsequent increase in intestinal NE lesions and mortality at day 22. These results correlate positively with a host inflammatory response as evidenced by increased ileal interleukin (IL)-4, IL-10 and IFN-gamma RNA expression. Concurrent increases in chicken intestinal mucin RNA expression, and goblet cell number and theca size indicate that EAM/CP induced an intestinal mucogenic response. Correspondingly, the growth of mucolytic bacteria and C. perfringens as well as alpha toxin production was greatest in EAM/CP-infected birds. The ionophore narasin, which directly eliminates coccidia, reduced goblet cell theca size, IL-10 and IFN-gamma expression, the growth of mucolytic bacteria including C. perfringens, coccidial and NE lesions and mortality in birds that were co-infected with coccidia and C. perfringens. Collectively the data support the hypothesis that coccidial infection induces a host mucogenic response providing a growth advantage to C. perfringens, the causative agent of NE.

"Candidatus Paraporphyromonas polyenzymogenes" encodes multi-modular cellulases linked to the type IX secretion system.

BACKGROUND: In nature, obligate herbivorous ruminants have a close symbiotic relationship with their gastrointestinal microbiome, which proficiently deconstructs plant biomass. Despite decades of research, lignocellulose degradation in the rumen has thus far been attributed to a limited number of culturable microorganisms. Here, we combine meta-omics and enzymology to identify and describe a novel Bacteroidetes family ("Candidatus MH11") composed entirely of uncultivated strains that are predominant in ruminants and only distantly related to previously characterized taxa. RESULTS: The first metabolic reconstruction of Ca. MH11-affiliated genome bins, with a particular focus on the provisionally named "Candidatus Paraporphyromonas polyenzymogenes", illustrated their capacity to degrade various lignocellulosic substrates via comprehensive inventories of singular and multi-modular carbohydrate active enzymes (CAZymes). Closer examination revealed an absence of archetypical polysaccharide utilization loci found in human gut microbiota. Instead, we identified many multi-modular CAZymes putatively secreted via the Bacteroidetes-specific type IX secretion system (T9SS). This included cellulases with two or more catalytic domains, which are modular arrangements that are unique to Bacteroidetes species studied to date. Core metabolic proteins from Ca. P. polyenzymogenes were detected in metaproteomic data and were enriched in rumen-incubated plant biomass, indicating that active saccharification and fermentation of complex carbohydrates could be assigned to members of this novel family. Biochemical analysis of selected Ca. P. polyenzymogenes CAZymes further iterated the cellulolytic activity of this hitherto uncultured bacterium towards linear polymers, such as amorphous and crystalline cellulose as well as mixed linkage ß-glucans. CONCLUSION: We propose that Ca. P. polyenzymogene genotypes and other Ca. MH11 members actively degrade plant biomass in the rumen of cows, sheep and most likely other ruminants, utilizing singular and multi-domain catalytic CAZymes secreted through the T9SS. The discovery of a prominent role of multi-modular cellulases in the Gram-negative Bacteroidetes, together with similar findings for Gram-positive cellulosomal bacteria (Ruminococcus flavefaciens) and anaerobic fungi (Orpinomyces sp.), suggests that complex enzymes are essential and have evolved within all major cellulolytic dominions inherent to the rumen.

Differential susceptibility of macrophage cell lines to Bacillus anthracis-Vollum 1B.

Bacillus anthracis (BA) is a spore forming bacterium and the causative agent of anthrax disease. Macrophages (Mphis) play a central role in anthrax disease. An important step in disease progression is the ability of BA to secrete lethal toxin (LeTx) that kills Mphis. LeTx is a heterodimer composed of protective antigen (PA) and lethal factor (LF). Researchers have shown that Mphi cell lines demonstrate differential susceptibility to purified LeTx; for example RAW264.7 and J774A.1 Mphis are sensitive to LeTx whereas IC-21 Mphis are resistant. Research has also suggested that exogenous factors, including other BA proteins, can influence the activity of LeTx. For this reason, the objective of the current work was to examine if RAW264.7, J774A.1, and IC-21 Mphis demonstrated differential susceptibility when cultured with a LeTx-producing strain of BA. Here, we co-cultured Mphis with LeTx+ Vollum 1B (V1B) spores for >15 h and assayed for Mphi cell death by morphology, trypan blue (TB) staining, neutral red (NR) activity, and lactate dehydrogenase (LDH) activity in the culture media. Following the addition of V1B spores, necrosis (approximately 50% mortality) was observed in RAW264.7 and J774A.1 Mphis at 7.5 and 10 h, respectively. By 15 h, both RAW264.7 and J774A.1 Mphis demonstrated 100% mortality. In contrast, IC-21 Mphis, under identical culture conditions, remained viable (98%) and activated throughout the course of the experiment (>24 h). The mechanism of RAW264.7 cell death appeared to involve LeTx because the V1B-induced cytotoxicity was dose-dependently reversed by the addition of anti-PA antibody to the culture media. These observations suggest there is differential susceptibility of Mphi cell lines to the LeTx+ V1B strain of BA. Further development of this in vitro model may be useful to further characterize the interactions between Mphis and BA spores.

Quantitation of dendritic cells in normal and abnormal human epidermis using monoclonal antibodies directed against Ia and HTA antigens.

Two monoclonal antibodies, NAI 34 and DA6 231, have been used as surface markers to count epidermal Langerhans cells in normal skin, allergic contact dermatitis, and mycosis fungoides of the plaque and poikilodermatous varieties. The antibodies recognized the HTA (human thymocyte antigen) and Ia antigens, respectively. In all situations the numbers of cells labeled by the two antibodies differed. In normal skin 75% of dendritic cells were labeled by both antibodies and 25% were labeled by NAI 34 alone. In contact dermatitis there is an increase in dendritic cells labeled by both antibodies and this pattern is also seen in untreated plaque stage mycosis fungoides. In poikilodermatous mycosis fungoides, striking staining of the epidermal keratinocytes with DA6 231 is seen with no dendritic cells labeled. Dendritic cells are, however, clearly seen with NAI 34 staining. Numbers of dendritic cells labeled by both antibodies fall during PUVA therapy.

Immunophenotyping of the cutaneous infiltrate and of the mononuclear cells in the peripheral blood in patients with atopic dermatitis.

Fourteen adult patients with chronic atopic dermatitis and active skin lesions had a skin biopsy and venous blood sample taken on the same day. Absolute numbers of circulating lymphocytes were normal in all patients. Fluorescence-activated cell sorter (FACS) analysis revealed normal numbers of total T lymphocytes and T-helper and T-suppressor subsets (helper:suppressor ratio, 2:1) in the atopic patients' peripheral blood, but an increase in circulating B lymphocytes and in HLA-D-related antigen-bearing cells. The skin biopsy showed a dermal infiltrate of predominantly T-helper lymphocytes (helper:suppressor ratio, 7:1). These cells showed strong HLA-DR plasma membrane staining. There was no HLA-DR staining in the membranes of epidermal keratinocytes. Using a monoclonal antihuman IgE, positive staining was observed in the dermis, though none was identified in the epidermis. The dermal anti-IgE staining was concentrated around clusters of T lymphocytes.

DNA repair in cells from patients with actinic keratosis.

UV-induced DNA repair was studied in peripheral blood lymphocytes from patients with multiple actinic keratoses (AK) requiring surgical therapy and from age-matched normal control individuals. The DNA repair activity in lymphocytes from AK patients at 4 h after UV-irradiation was 50% of that in control lymphocytes, but at 21 h the extent of DNA repair synthesis was similar to that in control cells.

CDKN2A germline mutations in U.K. patients with familial melanoma and multiple primary melanomas.

We report six of 16 U.K. melanoma families and two of 17 patients with multiple primary melanomas and a negative family history who have between them four different functionally damaging mutations of the CDKN2A (p16) gene: an Arg 24 Pro substitution in exon 1 in one family, a stop codon at codon 44 of exon 1 in one family, and a Met 53 Ile substitution in exon 2 in four families. One multiple primary melanoma patient also has the Met 53 Ile mutation and a second has a G-T substitution at the IVS2 + 1 splice donor site. Our data together with other recent publications from France and the U.S.A. indicate that screening melanoma kindreds with only two affected family members for CDKN2A mutations is justified.

The phototumorigenic potential of broad-band (270-350 nm) and narrow-band (311-313 nm) phototherapy sources cannot be predicted by their edematogenic potential in hairless mouse skin.

The new Philips TL01 narrow-band (311-313 nm) and conventional broad-band (e.g., Philips TL12; 270-350 nm) sources are effective for psoriasis phototherapy, for which treatment regimens are based on a predetermined minimal erythema dose. TL01 phototherapy treatment times are approximately half those with TL12 for psoriasis, whereas the cumulative exposure doses at clearing are similar. We compared the phototumorigenic potential of TL01 and TL12 radiation in mouse skin. Groups of albino Skh-1 hairless mice were exposed for 5 d/week at three dose levels. At each dose level, TL12 and TL01 doses were equally edematogenic. At each dose level, TL01 radiation was significantly more effective at producing first tumors of 1 mm in diameter and multiple tumors. At the lower two dose levels, TL01 radiation produced a significantly greater proportion of squamous cell carcinomas. This study demonstrates that TL01 radiation is more phototumorigenic than TL12 radiation at equally edematogenically weighted doses. This is in contrast with previous reports that edema production by polychromatic sources is predictive of their phototumorigenic effect in Skh-1 mice. The absolute cumulative TL12 dose needed to induce tumors was much less than that for TL01 radiation. The possibility of increased tumor risk with TL01 phototherapy should be considered but must be balanced against the high phototherapeutic efficacy of this source, short treatment times, and the low cumulative doses necessary for clearing of psoriasis.

Somatic mutations in the Peutz-Jeghers (LKB1/STKII) gene in sporadic malignant melanomas.

Germline mutations in the LKB1/STK11 gene cause characteristic hamartomas and freckling to develop in patients with Peutz-Jeghers syndrome (PJS). The hamartomas arise as a result of somatic "second hits" at LKB1/STK11 and therefore contain a neoplastic element. The origin of the pigmented lesions in PJS is unknown and difficult to test, as these are hardly ever biopsied. PJS patients are at increased risk of benign and malignant tumors, particularly of the colon, breast, pancreas, testis, and ovary, although the increased risk for any one of these sites may be quite modest. Somatic LKB1/STK11 mutations have been found, albeit at a low frequency, in sporadic tumors of the colon, stomach, ovary, and testis. Although PJS patients are not known to have an excess of skin tumors, if the freckles of PJS patients are actually small, benign tumors, LKB1/STK11 mutations must provide these lesions with a selective advantage, and similar mutations might also give a selective advantage to related malignant tumors, such as melanomas. We have therefore screened 16 melanoma cell lines, 15 primary melanomas, and 19 metastases for LKB1/STK11 mutations. Two LKB1/STK11 mutations were found: a missense change (Y49D) accompanied by allele loss in a cell line; and a missense change (G135R), without a detected mutation in the other allele, in a primary tumor. Both these mutations are highly likely to be pathogenic. Novel polymorphisms, including an unusual heptanucleotide repeat, were also found in introns 2 and 3. LKB1/STK11 mutations occur in a significant minority of tumors of several sites, including malignant melanomas.

Localization of elevated transforming growth factor-alpha in psoriatic epidermis.

Biopsies of involved and uninvolved skin from five patients with plaque psoriasis and normal skin from four healthy volunteers were investigated for steady-state quantities of TGF-alpha RNA and protein by in situ hybridization and immunohistochemistry. Increased levels of TGF-alpha RNA were found only in the high-level keratinocytes of involved psoriatic skin (p less than 0.001). Elevated levels of TGF-alpha protein were seen in both the high-level and basal layers of involved psoriatic skin compared to uninvolved psoriatic and normal control skin. Elevated TGF-alpha gene expression is thus implicated in the hyperproliferation of keratinocytes and possibly the altered maturation pattern seen in psoriasis.

Partial purification and characterization of Ral8I, a class-IIS restriction endonuclease from Ruminococcus albus 8 which recognizes 5'-GGATC.

Heparin-agarose chromatography was used to isolate a restriction endonuclease (ENase) from the cellulolytic Gram+ anaerobe, Ruminococcus albus 8. The enzyme, Ral8I, was eluted from the column using 230-310 mM Na+. However, the preparation was active only with DNA substrates that were not Dam-methylated. Moreover, the restriction fragment pattern generated from simian virus 40 (SV40) DNA was not consistent with the expected number of Dam-methylation sites. Alignment of the Dam-methylation sites in SV40 DNA indicated that Ral8I may actually recognize the asymmetric sequence, GGATC. This was confirmed by nucleotide (nt) sequence analysis and, further, Ral8I was found to cause cleavage of the DNA approx. 5 nt downstream from the recognition sequence. Ral8I can therefore be classified as a type-IIS restriction endonuclease and is an isoschizomer of AlwI, BinI and BthII.