Faecal calprotectin effectively excludes inflammatory bowel disease in 789 symptomatic young adults with/without alarm symptoms: a prospective UK primary care cohort study.

BACKGROUND: Primary care faecal calprotectin testing distinguishes inflammatory bowel disease (IBD) from functional gut disorder in young patients presenting with abdominal symptoms; however, previous evaluations have excluded patients with alarm symptoms. AIMS: We sought to evaluate the diagnostic accuracy of calprotectin to distinguish IBD from functional gut disorder in young adults in whom general practitioners (GPs) suspected IBD; including patients reporting gastrointestinal alarm symptoms. We hypothesised that calprotectin would reduce secondary care referrals and healthcare costs. METHODS: We undertook a prospective cohort study of 789 young adults (18-46 years old) presenting with gastrointestinal symptoms to 49 local general practices that had undergone calprotectin testing (1053 tests: between Jan 2014 and May 2016) because of suspected IBD. We considered calprotectin levels of ≥100 µg/g positive. Primary and secondary care records over 12 months from the point of calprotectin testing were used as the reference standard. RESULTS: Overall, 39% (308/789) patients reported gastrointestinal alarm symptoms and 6% (50/789) tested patients were diagnosed with IBD. The positive and negative predictive values of calprotectin testing for distinguishing IBD from functional gut disorder in patients with gastrointestinal alarm symptoms were 50% (95% CI 36%-64%) and 98% (96%-100%): and in patients without gastrointestinal alarm symptoms were 27% (16%-41%) and 99% (98%-100%), respectively. We estimate savings of 279 referrals and £160 per patient. CONCLUSIONS: Calprotectin testing of young adults with suspected IBD in primary care accurately distinguishes IBD from functional gut disorder, even in patients with gastrointestinal alarm symptoms and reduces secondary care referrals and diagnostic healthcare costs.

Melanocyte transformation requires complete loss of all pocket protein function via a mechanism that mitigates the need for MAPK pathway activation.

Deregulation of p16INK4A is a critical event in melanoma susceptibility and progression. It is generally assumed that the major effect of loss of p16 function is mediated through the CDK-cyclin pathway via its influence on the pocket protein (PP) pRb. However, there are also two other PPs, p107 and p130, which, when phosphorylated by CDK-cyclin complexes, play a role in permitting cell progression. Cohorts of mice carrying melanocyte-specific knockouts (KOs) of various combinations of the three PPs were generated. Mice null for pRb, p107, p130 or any combination of double mutants did not develop melanoma. Surprisingly, melanocyte-specific loss of all three PPs facilitated melanoma development (median age of onset 308 days, penetrance 40% at 1 year). Tumorigenesis was exacerbated by Trp53 co-deletion (median age of onset 275 days, penetrance 82% at 1 year), with cell culture studies indicating that this difference may result from the apoptotic role of Trp53. Melanomas in PP;Trp53-deficient mice lacked either Ras or Braf mutations, and hence developed in the absence of constitutive MAPK pathway activation. The lag period between induction of total PP or PP/Trp53 KO and melanoma development indicates that additional genetic or epigenetic alterations may account for neoplastic progression. However, exome sequencing of PP;Trp53 KO melanomas failed to reveal any additional recurrent driver mutations. Analysis of the putative mutation signature of the PP;Trp53 KO melanomas suggests that melanocytes are primed for transformation via a mutagenic mechanism involving an excess of T>G substitutions, but not involving a preponderance of C>T substitutions at CpG sites, which is the case for most spontaneous cancers not driven by a specific carcinogen. In sum, deregulation of all three PPs appears central to neoplastic progression for melanoma, and the customary reference to the p16INKA/CDK4/pRB pathway may no longer be accurate; all PPs are potentially critical targets of CDK-cyclins in melanoma.

Murine melanomas accelerated by a single UVR exposure carry photoproduct footprints but lack UV signature C>T mutations in critical genes.

Ultraviolet radiation (UVR) exposure increases malignant melanoma (MM) risk, but in the context of acute, not cumulative exposure. C>T and CC>TT changes make up the overwhelming majority of single base substitutions (SBS) in MM DNA, as both precursor melanocytes and melanocytic lesions have incurred incidental exposures to sunlight. To study the mutagenic mechanisms by which acute sunburn accelerates MM, we sequenced the exomes of spontaneous and neonatal UVB-induced Cdk4-R24C::Tyr-NRASQ61K mouse MMs. UVR-induced MMs carried more SBSs than spontaneous MMs, but the levels of genomic instability, reflected by translocations and copy number changes, were not different. C>T/G>A was the most common SBS in spontaneous and UVR-induced MMs, only modestly increased in the latter. However, they tended to occur at the motif A/GpCpG (reflecting C>T transition due to spontaneous deamination of cytosine at CpG) in spontaneous MMs, and T/CpCpC/T (reflecting the effects of pyrimidine dimers on either side of the mutated C) in UVR-induced MMs. Unlike MMs associated with repetitive exposures, we observed no CC>TT changes. In addition, we also found UVR 'footprints' at T>A/A>Ts (at NpTpT) and T>C/A>G (at CpTpC). These footprints are also present in MMs from a chronic UVR mouse model, and in some human MMs, suggesting that they may be minor UVR signature changes. We found few significantly somatically mutated genes (~6 per spontaneous and 15 per UVR-induced melanoma) in addition to the Cdk4 and NRAS mutations already present. Trp53 was the most convincing recurrently mutated gene; however, in the UVR-induced MMs no Trp53 mutations were at C>T/G>A, suggesting that it was probably mutated during tumour progression, not directly induced by UVR photoproducts. The very low load of recurrent mutations convincingly induced by classical UVB-induced dimer photoproducts may support a role for cell extrinsic mechanisms, such as photoimmunosuppression and inflammation in driving MM after acute UVB exposure.

Melanoma susceptibility as a complex trait: genetic variation controls all stages of tumor progression.

Susceptibility to most common cancers is likely to involve interaction between multiple low risk genetic variants. Although there has been great progress in identifying such variants, their effect on phenotype and the mechanisms by which they contribute to disease remain largely unknown. We have developed a mouse melanoma model harboring two mutant oncogenes implicated in human melanoma, CDK4(R24C) and NRAS(Q61K). In these mice, tumors arise from benign precursor lesions that are a recognized strong risk factor for this neoplasm in humans. To define molecular events involved in the pathway to melanoma, we have for the first time applied the Collaborative Cross (CC) to cancer research. The CC is a powerful resource designed to expedite discovery of genes for complex traits. We characterized melanoma genesis in more than 50 CC strains and observed tremendous variation in all traits, including nevus and melanoma age of onset and multiplicity, anatomical site predilection, time for conversion of nevi to melanoma and metastases. Intriguingly, neonatal ultraviolet radiation exposure exacerbated nevus and melanoma formation in most, but not all CC strain backgrounds, suggesting that genetic variation within the CC will help explain individual sensitivity to sun exposure, the major environmental skin carcinogen. As genetic variation brings about dramatic phenotypic diversity in a single mouse model, melanoma-related endophenotype comparisons provide us with information about mechanisms of carcinogenesis, such as whether melanoma incidence is dependent upon the density of pre-existing nevus cells. Mouse models have been used to examine the functional role of gene mutations in tumorigenesis. This work represents their next phase of development to study how biological variation greatly influences lesion onset and aggressiveness even in the setting of known somatic driver mutations.

No effect of fluid intake on neutrophil responses to prolonged cycling.

Ingesting carbohydrate beverages during prolonged exercise is associated with fewer numbers of circulating neutrophils and attenuated neutrophil functional responses, yet there is little information about the effect of fluid intake alone on immune responses to prolonged exercise. The aim of this study was to examine the influence of regular fluid ingestion compared with no fluid ingestion on plasma cortisol, circulating neutrophil and lipopolysaccharide (LPS)-stimulated neutrophil degranulation responses to prolonged cycling. In a randomized design, nine recreationally active males cycled for 2 h at 65% VO2max on two occasions with either fluid ingestion (lemon-flavoured water, fluid trial) before and during the exercise, or with no fluid intake at all (no fluid trial). Venous blood samples were obtained at rest, immediately after exercise and 1 h after exercise. Immediately after exercise, the plasma cortisol concentration was significantly higher in the no fluid trial than in the fluid trial (592 +/- 62 vs 670 +/- 63 nmol x l(-1), P < 0.05). Circulating numbers of neutrophils increased 4.5-fold (P < 0.01) and LPS-stimulated elastase release per neutrophil decreased 34 +/- 7% (P < 0.01) immediately after exercise; there were no differences between trials. These results suggest that in ambient environmental conditions, fluid ingestion alone has a negligible effect on circulating neutrophil and LPS-stimulated neutrophil degranulation responses to prolonged exercise.

Activating point mutations in cyclin-dependent kinase 4 are not seen in sporadic pituitary adenomas, insulinomas or Leydig cell tumours.

Cell cycle dysregulation is one of the defining features of cancer. Cyclin-dependent kinase 4 (CDK4), together with its regulatory subunit cyclin D, governs cell cycle progression through the G1 phase. Cyclin-dependent kinase inhibitors, including p16(INK4A) (encoded by CDKN2A), in turn regulate CDK4. In particular, dysregulation of the p16/CDK4/cyclin D complex has been established in a variety of types of human tumours. Dominant activating mutations affecting codon 24 of the CDK4 gene (replacement of Arg24 by Cys or His) render CDK4 insensitive to p16(INK4) inhibition and are responsible for melanoma susceptibility in some kindreds. However, 'knock-in' mice homozygous for the CDK4(R24C) mutation were noted to develop multiple neoplasia, most commonly including endocrine tumours: pituitary adenomas, insulinomas and Leydig cell testicular tumours. We therefore speculated that sporadic human endocrine tumours might also harbour such mutations. The aim of the current study was to analyze the CDK4 gene for the two characterized activating mutations, R24C and R24H, in sporadic human pituitary adenomas, insulinomas and Leydig cell tumours. We used DNA extracted from 61 pituitary adenomas, and paired tumorous and neighboring normal genomic DNA extracted from 14 insulinoma and 6 Leydig cell tumour samples. Genomic DNA from patients with familial melanoma harbouring the R24C or the R24H mutations served as positive controls. All samples were subjected to PCR, mutation-specific restriction digests and/or sequencing. Both methodologies failed to detect mutations at these two sites in any of the sporadic endocrine tumours including pituitary adenomas, benign or malignant insulinomas or Leydig cell tumours, while the positive controls showed the expected heterozygote patterns. Protein expression of CDK4 was demonstrated by immunohistochemistry and Western blotting in pituitary and pancreatic samples. These data suggest that the changes in the regulatory 'hot-spot' on the CDK4 gene, causing various endocrine tumours in CDK4(R24C/R24C )mice, are not a major factor in sporadic pituitary, insulin beta-cell or Leydig cell tumorigenesis.

PTEN inactivation is rare in melanoma tumours but occurs frequently in melanoma cell lines.

Deletions detected in cytogenetic and loss of heterozygosity (LOH) studies indicate that at least one tumour suppressor gene maps to the long arm of chromosome 10. Previous deletion mapping studies have observed LOH on 10q in about 30% of melanomas analysed. The PTEN gene, mapping to chromosome band 10q23.3, encodes a protein with both lipid and protein phosphatase activity. Somatic mutations and deletions in have been detected in a variety of cell lines and tumours, including melanoma samples. We performed mutation analyses and extensive allelic loss studies to investigate the role this gene plays in melanoma pathogenesis. We found that a total of 34 out of 57 (60%) melanoma cell lines carried hemizygous deletions of chromosome 10q encompassing the PTEN locus. A further three cell lines carried smaller deletions excluding PTEN. Inactivation of both PTEN alleles by exon-specific homozygous deletion or mutation was observed in 13 out of 57 (23%) melanoma cell lines. The mutation spectrum observed does not indicate an important role for ultraviolet radiation in the genesis of these mutations, and evidence from three cell lines supports the acquisition of PTEN aberrations in culture. Ten out of 49 (20%) matched melanoma tumour/normal samples harboured hemizygous deletions of either the whole chromosome or most of the long arm. Mutations within were detected in only one of the 10 tumours demonstrating LOH at 10q23 that were analysed. These results suggest that PTEN inactivation may be important for the propagation of melanoma cells in culture, and that another chromosome 10 tumour suppressor gene may be important for melanoma pathogenesis.

Antibiotics for syphilis diagnosed during pregnancy.

BACKGROUND: Congenital syphilis is an increasing problem in many developing countries and in the transitional economies of Eastern Europe and the former Soviet Union. In several countries this increase has been aggravated by HIV/AIDS. While the effectiveness of penicillin in the treatment of syphilis in pregnant women and the prevention of congenital syphilis was established shortly after the introduction of penicillin in the 1940s, there is uncertainty about the optimal treatment regimens. OBJECTIVES: To identify the most effective antibiotic treatment regimen (in terms of dose, length of course and mode of administration) of syphilis with and without concomitant infection with HIV for pregnant women infected with syphilis. SEARCH STRATEGY: MEDLINE 1966 to March 2000; EMBASE 1974 to March 2000, the Cochrane Controlled Trials Register (last searched March 2001), the Cochrane Pregnancy and Childbirth group trials register (last searched March 2001) and the references of traditional reviews were searched. Experts in specialist units were contacted. SELECTION CRITERIA: It was planned that any trial in which an attempt is made to allocate treatment for syphilis during pregnancy by a random or quasi-random method would be included in this review. DATA COLLECTION AND ANALYSIS: Information was extracted using a data extraction sheet and this included entry criteria, the source of controls, and whether the authors stratified by the stage of pregnancy when the diagnosis of syphilis was made. MAIN RESULTS: Twenty six studies met the criteria for detailed scrutiny. However, none of these met the pre-determined criteria for comparative groups and none included comparisons between randomly allocated groups of pregnant women. REVIEWER'S CONCLUSIONS: While there is no doubt that penicillin is effective in the treatment of syphilis in pregnancy and the prevention of congenital syphilis, uncertainty remains about what are the optimal treatment regimens. Further studies are needed to evaluate treatment failure cases with currently recommended regimens and this should include an assessment of the role of HIV infection in cases of prenatal syphilis treatment failure. The effectiveness of various antibiotic regimens for the treatment of primary and secondary syphilis in pregnant women need to be assessed using randomised controlled trials which compare them with existing recommendations.

Interventions for treating scabies.

BACKGROUND: Scabies is a common public health problem with an estimated global prevalence of 300 million. Infestation can cause considerable discomfort and intense itching. Severe adverse effects have been reported for some drugs used to treat scabies. OBJECTIVES: The objective of this review is to assess the effects and toxicity of topical and systemic drug treatment for scabies. SEARCH STRATEGY: We searched the Cochrane Infectious Diseases Group trials register, the Cochrane Controlled Trials Register, MEDLINE, EMBASE, military records, traditional medicine databases. We also contacted international specialist centres and drug manufacturers. SELECTION CRITERIA: Randomised controlled trials of any drug treatment for scabies. Tolerability and toxicity were sought in any study of humans taking any drug treatments for scabies. DATA COLLECTION AND ANALYSIS: Two reviewers assessed trial quality and extracted data. MAIN RESULTS: Thirteen trials were included (nine compared drug treatments, two compared treatment regimens, one compared the drug vehicle, and one was a community intervention). In one small trial, ivermectin was associated with a significant higher clinical cure rate at seven days when compared with placebo. Permethrin appeared to be more effective than crotamiton for clinical and parasitic cure rates. Permethrin appeared to be better than lindane for clinical cure rates in two small trials, but had no advantage in the largest trial (test for heterogeneity P < 0.001). Permethrin also appeared more effective in reducing itch persistence than lindane. There appeared to be no difference in clinical cure rates between crotamiton and lindane or benzyl benzoate and sulphur. Two trials assessed: the effectiveness of oral versus topical treatment (ivermectin versus benzyl benzoate and ivermectin ); single trial assessed treatment vehicle (pork fat versus cold cream); and mass community treatment (ivermectin), but all were too small to demonstrate an effect. No randomised trials of malathion were identified. Serious adverse drug reactions (including death and convulsions), most notably to lindane, permethrin and ivermectin, have been reported elsewhere. REVIEWER'S CONCLUSIONS: The evidence that permethrin is more effective than lindane is inconsistent. Lindane, permethrin, and ivermectin appear to be associated with rare but serious drug reactions although this is not derived from trial data. More research is needed on the safety and effectiveness of ivermectin and malathion compared to permethrin, on community management, and on different regimens and vehicles for topical treatment.

Localization of multiple melanoma tumor-suppressor genes on chromosome 11 by use of homozygosity mapping-of-deletions analysis.

Loss-of-heterozygosity (LOH) studies have implicated one or more chromosome 11 tumor-suppressor gene(s) in the development of cutaneous melanoma as well as a variety of other forms of human cancer. In the present study, we have identified multiple independent critical regions on this chromosome by use of homozygosity mapping of deletions (HOMOD) analysis. This method of analysis involved the use of highly polymorphic microsatellite markers and statistics to identify regions of hemizygous deletion in unmatched melanoma cell line DNAs. Regions of loss were defined by the presence of an extended region of homozygosity (ERH) at > or =5 adjacent markers and having a statistical probability of < or =.001. Significant ERHs were similar in nature to deletions identified by LOH analyses performed on uncultured melanomas, although a higher frequency of loss (24 [60%] of 40 vs. 16 [34%] of 47) was observed in the cell lines. Overall, six small regions of overlapping deletions (SROs) were identified on chromosome 11 flanked by the markers D11S1338/D11S907 (11p13-15.5 [SRO1]), D11S1344/D11S11385 (11p11.2 [SRO2]), D11S917/D11S1886 (11q21-22.3 [SRO3]), D11S927/D11S4094 (11q23 [SRO4]), AFM210ve3/D11S990 (11q24 [SRO5]), and D11S1351/D11S4123 (11q24-25 [SRO6]). We propose that HOMOD analysis can be used as an adjunct to LOH analysis in the localization of tumor-suppressor genes.

Interventions for treating scabies.

BACKGROUND: Scabies is a common public health problem with an estimated prevalence of 300 million. Infestation can cause considerable discomfort and intense itching. Severe adverse effects have been reported for some drugs used to treat scabies. OBJECTIVES: The objective of this review is to assess the effects and toxicity of topical and systemic drug treatment for scabies. SEARCH STRATEGY: We searched the Cochrane Infectious Diseases Group trials register, the Cochrane Controlled Trials Register, Medline, Embase, military records, traditional medicine databases. We also contacted international specialist centres and drug manufacturers. SELECTION CRITERIA: Randomised controlled trials of any drug treatment for scabies. Tolerability and toxicity were sought in any study of humans taking any drug treatments for scabies. DATA COLLECTION AND ANALYSIS: Two reviewers assessed trial quality and extracted data. MAIN RESULTS: Eleven trials were included (7 compared drug treatments, 2 compared treatment regimes, 1 compared the drug vehicle and 1 was a community intervention). Compared with placebo in one small trial, ivermectin was associated with a significant higher clinical cure rate at seven days. Permethrin appeared to be more effective than crotamiton for clinical and parasitic cure rates. Permethrin appeared to be better than gamma benzene hexachloride for clinical cure rates in two small trials but had no advantage in the largest trial (test for heterogeneity p< 0.001). Permethrin also appeared more effective in reducing itch persistance than gamma benzene hexachloride. There appeared to be no difference in clinical cure rates between crotamiton and gamma benzene hexachloride. Single trials assessed: the effectiveness of oral versus topical treatment (ivermectin versus benzyl benzoate); treatment vehicle (pork fat versus cold cream); and mass community treatment (ivermectin) but were too small to demonstrate an effect. No randomised trials of malathion were identified. Serious adverse drug reactions (including death and convulsions) have been reported in other studies of scabies drugs, most notably gamma benzene hexachoride and ivermectin. REVIEWER'S CONCLUSIONS: The evidence that permethrin is more effective than gamma benzene hexachloride is inconsistent. Permethrin appears to have less potential serious drugs reactions than gamma benzene hexachloride although this is not derived from trial data. More research is needed of the safety and effectiveness of ivermectin and malathion compared to permethrin, on community management, and on different regimes and vehicles for topical treatment.

The patient flow of marine disease and nonbattle injury conditions within a multi-echelon system of care.

Hospitalization data were extracted for Marines who incurred disease and nonbattle injuries in Vietnam from 1965 through 1969, and the inter-echelon movement of each patient who was hospitalized at an echelon II or III facility was tracked until the treatment was completed or until the patient was moved to a continental U.S. facility. The inter-echelon flow of treatment for different types of diagnosis categories was also examined. Results showed that approximately three-fourths of the patients admitted to echelon II or III facilities had no further treatment recorded at a higher echelon of care. Less than one-fifth of the patients required treatment at an echelon IV or echelon V facility. Of the major diagnostic categories, those with infective or parasitic diseases had the lowest percentage of patients treated at echelon IV or V facilities.

Functional reassessment of P16 variants using a transfection-based assay.

CDKN2A appears to be the major melanoma susceptibility gene, and is also mutated/deleted in sporadic tumours of various types including melanoma. Thus far most approaches to assessing the functionality of mutations in this gene have used in vitro methods such as CDK4 binding and kinase inhibition assays, with sometimes disparate conclusions about functional significance of some variants between studies. We have used a melanoma cell line (MM96L) with no functional p16, as the basis for a "semi-in vivo" transfection-based assay for exogenous p16 functionality based on the growth parameters of the cells and the behaviour of variant proteins after transfection of different CDKN2A cDNAs. Colony counts performed on these transfectants revealed that all but the wild type, + 24 bp ad A148T variants have a diminished ability to inhibit cell growth. All other variants detected either constitutionally in familial melanoma patients (I49T, R87P, G101W and V126D) or somatically in melanomas (N71S, and P81L), appeared functionally impaired in this assay. This diminution of function was independent of CDK4 and CDK6 binding ability. Furthermore, the predominant localization of these variants within the cell was different from that of wt p16. This mislocalization may provide an explanation for their lack of function, or alternatively, it may also be an indicator that the cells are processing unstable, misfolded p16 proteins. This novel assay for assessment of functionality of p16 variants may better reflect the role of some of these mutations in vivo, and as such is a useful adjunct to other in vitro assays.

The patient flow of wounded marines within a multi-echelon system of care.

Hospitalization data were extracted for Marines wounded in Vietnam from 1965 to 1969 to examine the echelon flow of treatment care for different types of injuries. The inter-echelon movement of each patient who was hospitalized at an echelon II or III facility was tracked until the treatment was completed or until the patient was moved to a facility in the continental United States. Results showed that approximately half of the admissions to echelon II or III facilities had no further treatment recorded at a higher echelon of care. Almost one-fourth of the patients required treatment at an echelon IV facility, and more than one-third were admitted to an echelon V facility.

Virtually 100% of melanoma cell lines harbor alterations at the DNA level within CDKN2A, CDKN2B, or one of their downstream targets.

The cyclin-dependent kinase inhibitor 2A (CDKN2A), or p16INK4a, gene on 9p21 is important in the genesis of both familial and sporadic melanoma. Homozygous deletions and intragenic mutations of this gene have been identified in both melanoma cell lines and uncultured tumors, although the frequency of these alterations is higher in the cell lines. A proportion of melanoma cell lines and tumors without deletion/mutation of CDKN2A have also been determined to harbor transcriptionally inactive CDKN2A alleles or carry alterations in other components of the pathway through which p16INK4a acts on pRb to mediate cell cycle arrest. We sought to determine the frequency of these alternative events (in relationship to those that specifically inactivate CDKN2A) in a panel of 45 melanoma cell lines. Surprisingly, at the DNA level alone, 96% (43/45) of melanoma cell lines examined were found to be deleted/mutated/methylated for CDKN2A (34/45), homozygously deleted for CDKN2A's neighbor and homolog CDKN2B (6/45), and/or mutated/amplified for CDK4 (5/45). In two of these 43 cases, homozygous deletions of CDKN2A were detected along with a CDK4 mutation or amplification of the cyclin D1 (CCND1) gene. The latter discoveries were made in two of three cell lines which harbored extremely large (3-6 Mb) homozygous deletions on 9p21; all other homozygous deletions in similarly affected cell lines (N = 23) were confined to a region immediately surrounding the CDKN2A/CDKN2B loci. These results suggest that (1) only melanoma cells with alterations in this pathway can be propagated in culture, and (2) the homozygous deletions on 9p21 in the cell lines, which are also mutated/amplified for CDK4 or CCND1, could serve to target tumor suppressor genes other than CDKN2A.

Low frequency of p16/CDKN2A methylation in sporadic melanoma: comparative approaches for methylation analysis of primary tumors.

Methylation of the 5' CpG island of the p16 tumor suppressor gene represents one possible mechanism for inactivation of this cell cycle regulatory gene that is also a melanoma predisposition locus. We have investigated the potential contribution of somatic silencing of the p16 gene by DNA methylation in 30 cases of sporadic cutaneous melanoma. The methylation status of the 5' CpG island of p16 was initially determined by Southern analysis and then reevaluated (in a blinded manner) using methylation-specific PCR, methylation-sensitive single nucleotide primer extension, and bisulfite genomic sequencing. All methodologies yielded concordant results, and significant levels of methylation were observed in 3 of the 30 (10%) melanoma DNAs analyzed. Of the three tumors found to be methylated, two were also positive for LOH on 9p21 (where the p16 gene resides), implying that both p16 alleles were inactivated, one via deletion and the other via methylation-associated transcriptional silencing. The association between methylation and transcriptional silencing of p16 was also further supported by inducing p16 expression with a DNA demethylating agent (5-aza-2'-deoxycytidine) in a melanoma cell line known to harbor a methylated p16 allele. Although methylation-associated gene silencing does not represent a common mechanism for p16 inactivation in sporadic melanoma, our findings provide support that PCR-based techniques, such as methylation-specific PCR and methylation-sensitive single nucleotide primer extension, can be reliably used for the accurate detection and quantitation of aberrant levels of DNA methylation in tumor specimens.

Adsorption of Streptococcus sobrinus dextranase inhibitor to water-insoluble alpha-D-glucans of oral streptococci.

A low molecular weight dextranase inhibitor from Streptococcus sobrinus has previously been identified and purified. The range of conditions under which inhibition occurs, and the situations in which dextranase activity of S. sobrinus can reappear, have been examined in the chemostat. These studies have revealed that when dextranase production exceeds that of the inhibitor, all the inhibitor is tightly bound into enzyme-inhibitor complexes, and the excess enzyme remains active. Another factor that influences the activity of dextranase inhibitor has now been identified, namely the ability of the inhibitor to bind to water-insoluble glucans. Adsorption to water-insoluble alpha-D-glucans, produced by oral streptococci that were grown in batch culture, increased with their proportion of alpha-1,3-linked sequences of glucose residues. Studies with water-insoluble dextrans of Leuconostoc mesenteroides strains showed that alpha-1,6-linked sequences were also important for binding. The inhibitor was not active when adsorbed to glucan, but active inhibitor was released by incubation with soluble dextran. The interactions of sucrose, alpha-D-glucosyltransferases, alpha-D-glucans, dextranase and dextranase inhibitor are discussed in relation to the growth rate of S. sobrinus. At low growth rate in the chemostat the predominant alpha-D-glucosyltransferase (GTF) is a GTF-S that converts sucrose into soluble dextran, and the activity of free dextranase inhibitor in the culture filtrate is high. By contrast, at high growth rate the streptococci produce GTFs capable of synthesizing water-insoluble alpha-D-glucans, and no free inhibitor is found in culture filtrate. Thus the activity of free, extracellular dextranase inhibitor is controlled by (i) the extent of binding to dextranase and (ii) the extent of adsorption to water-insoluble alpha-D-glucan.

Analysis of the CDKN2A, CDKN2B and CDK4 genes in 48 Australian melanoma kindreds.

Germline mutations within the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene and one of its targets, the cyclin dependent kinase 4 (CDK4) gene, have been identified in a proportion of melanoma kindreds. In the case of CDK4, only one specific mutation, resulting in the substitution of a cysteine for an arginine at codon 24 (R24C), has been found to be associated with melanoma. We have previously reported the identification of germline CDKN2A mutations in 7/18 Australian melanoma kindreds and the absence of the R24C CDK4 mutation in 21 families lacking evidence of a CDKN2A mutation. The current study represents an expansion of these efforts and includes a total of 48 melanoma families from Australia. All of these families have now been screened for mutations within CDKN2A and CDK4, as well as for mutations within the CDKN2A homolog and 9p21 neighbor, the CDKN2B gene, and the alternative exon 1 (E1beta) of CDKN2A. Families lacking CDKN2A mutations, but positive for a polymorphism(s) within this gene, were further evaluated to determine if their disease was associated with transcriptional silencing of one CDKN2A allele. Overall, CDKN2A mutations were detected in 3/30 (10%) of the new kindreds. Two of these mutations have been observed previously: a 24 bp duplication at the 5' end of the gene and a G to C transversion in exon 2 resulting in an M531 substitution. A novel G to A transition in exon 2, resulting in a D108N substitution was also detected. Combined with our previous findings, we have now detected germline CDKN2A mutations in 10/48 (21%) of our melanoma kindreds. In none of the 'CDKN2A-negative' families was melanoma found to segregate with either an untranscribed CDKN2A allele, an R24C CDK4 mutation, a CDKN2B mutation, or an E1beta mutation. The last three observations suggest that these other cell cycle control genes (or alternative gene products) are either not involved at all, or to any great extent, in melanoma predisposition.

Loss of the p16INK4a and p15INK4b genes, as well as neighboring 9p21 markers, in sporadic melanoma.

Although homozygous deletions of the cyclin-dependent kinase inhibitor 2 gene p16INK4a on 9p21 have been reported frequently in metastatic melanoma cell lines, and intragenic mutations within the p16INK4a gene have been detected in familial melanoma kindreds, specific targeting of this gene in the development of sporadic melanoma in vivo remains controversial. Southern analyses were performed in this study to initially assess the frequency of hemi- or homozygous losses of p16INK4a, as well as its neighboring family member, p15INK4b, and other candidate regions within 9p21, in sporadic melanoma. Overall, 22 of 40 (55%) uncultured sporadic melanoma DNAs were determined to harbor deletions of 1-11 markers/genes located on 9p21. This included 10 tumors (25%; 10 of 40) with homozygous deletions limited to either the p16INK4a gene only (20%; 2 of 10), both the p16INK4a and p15INK4b genes (10%; 1 of 10), another novel 9p21 gene, FB19 (10%; 1 of 10), or all three of these genes plus surrounding markers (60%; 6 of 10). In subsequent single-strand conformation polymorphism and sequencing analyses, intragenic mutations in the p16INK4a gene were also revealed in two (10%; 2 of 21) melanoma DNAs that retained one copy of this locus. By comparison, the frequency of pl6INK4a and p15INK4b homozygous deletions, as well as p16INK4a mutations, in melanoma cell lines (analyzed in parallel) was 2-3-fold higher at 61 (23 of 38) and 24% (9 of 38), respectively. These findings indicate that (a) p16INK4a is inactivated in vivo in over one-fourth (27.5%; 11 of 40) of sporadic melanomas; (b) mutation/deletion of p16INK4a may confer a selective growth advantage in vitro; and (c) other 9p21 tumor suppressor genes could be targeted during the development of melanoma.