Stephanie F. MacCallum

Atopic Dermatitis and Disease Severity Are the Main Risk Factors for Food Sensitization in Exclusively Breastfed Infants

Filaggrin (FLG) loss-of-function skin barrier gene mutations are associated with atopic dermatitis (AD) and transepidermal water loss (TEWL). We investigated whether FLG mutation inheritance, skin barrier impairment, and AD also predispose to allergic sensitization to foods. Six hundred and nineteen exclusively breastfed infants were recruited at 3 months of age and examined for AD and disease severity (SCORing Atopic Dermatitis (SCORAD)), and screened for the common FLG mutations. TEWL was measured on unaffected forearm skin. In addition, skin prick testing was performed to six study foods (cows milk, egg, cod, wheat, sesame, and peanut). Children with AD were significantly more likely to be sensitized (adjusted odds ratio (OR)=6.18, 95% confidence interval (CI): 2.94-12.98, P<0.001), but this effect was independent of FLG mutation carriage, TEWL, and AD phenotype (flexural vs. non-flexural). There was also a strong association between food sensitization and AD severity (adjusted ORSCORAD<20=3.91, 95% CI: 1.70-9.00, P=0.001 vs. adjusted ORSCORAD20=25.60, 95% CI: 9.03-72.57, P<0.001). Equally, there was a positive association between AD and sensitization with individual foods (adjusted ORegg=9.48, 95% CI: 3.77-23.83, P<0.001; adjusted ORcows milk=9.11, 95% CI: 2.27-36.59, P=0.002; adjusted ORpeanut=4.09, 95% CI: 1.00-16.76, P=0.05). AD is the main skin-related risk factor for food sensitization in young infants. In exclusively breastfed children, this suggests that allergic sensitization to foods can be mediated by cutaneous antigen-presenting cells.

Filaggrin breakdown products determine corneocyte conformation in patients with atopic dermatitis.

Background Loss-of-function (LOF) mutations in the filaggrin gene (FLG) are a well-replicated risk factor for atopic dermatitis (AD) and are known to cause an epidermal barrier defect. The nature of this barrier defect is not fully understood. Patients with AD with FLG LOF mutations are known to have more persistent disease, more severe disease, and greater risk of food allergies and eczema herpeticum. Abnormalities in corneocyte morphology have been observed in patients with AD, including prominent villus-like projections (VP); however, these ultrastructural features have not been systematically studied in patients with AD in relation to FLG genotype and acute and convalescent status. Objective We sought to quantitatively explore the relationship between FLG genotype, filaggrin breakdown products (natural moisturizing factor [NMF]), and corneocyte morphology in patients with AD. Methods We studied 15 children at first presentation of AD and after 6 weeks of standard therapy. We applied atomic force microscopy to study corneocyte conformation in patients with AD stratified by FLG status and NMF level. By using a new quantitative methodology, the number of VPs per investigated corneocyte area was assessed and expressed as the Dermal Texture Index score. Corneocytes were also labeled with an anti-corneodesmosin antibody and visualized with scanning electron microscopy. Results We found a strong correlation between NMF levels and Dermal Texture Index scores in both acute and convalescent states (respective r = −0.80 and −0.75, P < .001 and P = .002). Most, but not all, VPs showed the presence of corneodesmosin abundantly all over the cell surface in homozygous/compound heterozygous FLG patients and, to a lesser extent, in heterozygous and wild-type patients. Conclusions NMF levels are highly correlated with corneocyte morphology in patients with AD. These corneocyte conformational changes shed further insight into the filaggrin-deficient phenotype and help explain the barrier defect in patients with AD with FLG LOF mutations.

Dysregulation of autophagy in chronic lymphocytic leukemia with the small-molecule Sirtuin inhibitor Tenovin-6

Tenovin-6 (Tnv-6) is a bioactive small molecule with anti-neoplastic activity. Inhibition of the Sirtuin class of protein deacetylases with activation of p53 function is associated with the pro-apoptotic effects of Tnv-6 in many tumors. Here, we demonstrate that in chronic lymphocytic leukemia (CLL) cells, Tnv-6 causes non-genotoxic cytotoxicity, without adversely affecting human clonogenic hematopoietic progenitors in vitro, or murine hematopoiesis. Mechanistically, exposure of CLL cells to Tnv-6 did not induce cellular apoptosis or p53-pathway activity. Transcriptomic profiling identified a gene program influenced by Tnv-6 that included autophagy-lysosomal pathway genes. The dysregulation of autophagy was confirmed by changes in cellular ultrastructure and increases in the autophagy-regulatory proteins LC3 (LC3-II) and p62/Sequestosome. Adding bafilomycin-A1, an autophagy inhibitor to Tnv-6 containing cultures did not cause synergistic accumulation of LC3-II, suggesting inhibition of late-stage autophagy by Tnv-6. Thus, in CLL, the cytotoxic effects of Tnv-6 result from dysregulation of protective autophagy pathways.

Generation and characterisation of keratin 7 (K7) knockout mice.

Keratin 7 (K7) is a Type II member of the keratin superfamily and despite its widespread expression in different types of simple and transitional epithelia, its functional role in vivo remains elusive, in part due to the lack of any appropriate mouse models or any human diseases that are associated with KRT7 gene mutations. Using conventional gene targeting in mouse embryonic stem cells, we report here the generation and characterisation of the first K7 knockout mouse. Loss of K7 led to increased proliferation of the bladder urothelium although this was not associated with hyperplasia. K18, a presumptive type I assembly partner for K7, showed reduced expression in the bladder whereas K20, a marker of the terminally differentiated superficial urothelial cells was transcriptionally up-regulated. No other epithelia were seen to be adversely affected by the loss of K7 and western blot and immunofluorescence microscopy analysis revealed that the expression of K8, K18, K19 and K20 were not altered in the absence of K7, with the exception of the kidney where there was reduced K18 expression.

TRAF4 is potently induced by TAp63 isoforms and localised according to differentiation in SCCHN

p63, a member of the p53 family, is overexpressed in squamous cell carcinoma of the head and neck (SCCHN) and some other tumors of epithelial origin. As a transcription factor, p63 can bind to p53-type response elements and there is some overlap between p53 family transcriptional targets. Tumor necrosis factor receptor associated factor 4 (TRAF4) is a p53 regulated gene which is overexpressed in many human carcinomas. We investigated the involvement of p63 in regulation of TRAF4 and the expression of the TRAF4 protein in SCCHN. Disrupting endogenous p63 expression resulted in downregulation of TRAF4 mRNA and protein in an SCCHN cell line. Endogenous p63 bound to the TRAF4 promoter in vivo and reporter assays showed that p63, p73 and p53 can all transactivate TRAF4, with TAp63 isoforms being the most potent activators. The level of TRAF4 activation by TAp63 was two-fold higher than by p53, and TRAF4 was 10-fold more responsive to TAp63 than another p63-target, IGFBP3. Nuclear expression of TRAF4 was seen in normal oral epithelium and highly/moderately differentiated SCCHN, whereas cytoplasmic expression of TRAF4 was seen in poorly differentiated SCCHN. These results indicate that TRAF4 is a common target of p53 family members and that localization of TRAF4 is associated with differentiation of SCCHN cells.

Reliability and validity of genotyping filaggrin null mutations.

Atopic dermatitis (AD) is a common chronic waxing and waning disease 1. Of those with AD and European or Asian ancestry, more than 20% will have a FLG loss of function mutation (FLG null) resulting in the absence or near absence of filaggrin protein in the stratum corneum 2–5. It has been suggested that FLG null genetic testing might be a helpful clinical tool 2;4. The FLG gene is difficult to sequence because of its large, redundant, and repetitive structure limiting the availability of specific primer binding sites for PCR amplification 2;3;5. There are several techniques that are commonly used for genetic testing including the more time intensive TaqMan® allelic discrimination assays and high-throughput methods that utilize beadchip technology 3–6. The goal of this study was to compare the reliability (the consistency of and ability to replicate a measurement) and validity (the ability of a measurement to identify the correct event) of these two techniques. We obtained DNA from a cohort of more than 750 children 4;7Using both methods, we genotyped samples for the four most prevalent FLG null mutations found in European populations:R501X; 2282del4; R2447X and S3247X 2. Subjects were also categorized as: absence of a mutant allele, presence of one mutant allele, or presence of more than one mutant allele (i.e., null homozygotes or null compound heterozygotes (<0.1%)). Since one FLG null allele is sufficient to create a clinical phenotype, we also created a dichotomous variable for each mutation based on the presence or absence of a single FLG null allele 2;4;8. The reliability was expressed as Kendall’s Tau-b and Cohen’s Kappa and the validity as sensitivity and specificity. Reliability ranges are conventionally interpreted as: 0–0.20 as slight, 0.21–0.40 as fair, 0.41–0.60 as moderate, 0.61–0.80 as substantial, and 0.81–1 as almost perfect. When comparing two laboratory genetic tests, the common expectation is that the reliability should be almost perfect or perfect. Acceptable diagnostic tests often have sensitivity and specificity greater than 90% when compared to a gold standard. Validity of less than 95% is likely not acceptable for comparing non-invasive laboratory based tests. Our beadchip was established with assistance from Illumina using previously published data for all four mutations 3;9. Genotyping assays were conducted using the GoldenGate platform, a Bead Array Scanner, and the Genome Studio Software (Illumina Inc, San Diego, CA). About 15% of the samples were genotyped twice using the beadchip. The results were identical for both runs (i.e., perfect test-retest reliability). For our samples, custom-made TaqMan® assays (Applied Biosystems, Foster City CA) were designed using previously published protocols 3;9. TaqMan® genotyping for these mutations is the current gold standard and the application of this methodology to identify individuals who carry FLG null mutations and hence no or minimal filaggrin protein in the stratum corneum has been demonstrated using skin biopsy and Raman spectroscopy studies 2;3;8. Importantly, our TaqMan® assays have been used to screen more than 2000 cases of AD including verification by DNA sequencing 3;5, In our study, about 15% of samples were genotyped twice using TaqMan® and the results were identical for both runs (i.e., perfect test-retest reliability). Our a priori expectation was that there should be near perfect agreement between the two genotyping methods (inter-test reliability). However, this was not observed (Table 1). Pearson’s Chi squared tests indicated that statistically significant differences existed between the two techniques (p< 0.0001 for all mutations). The values for the two reliability measures, Cohen’s Kappa and Kendall’s tau-b are presented in Table 1. Reliability assessments were mostly “fair to moderate” and none were “almost perfect”. The sensitivity and specificity (assuming TaqMan® as the gold standard) ranged between 36% and 87% for sensitivity and between 84% and 100% for specificity. The positive predictive value (PPV) for the beadchip technique properly predicting the TaqMan® technique for the combined FLG null assessment was 53%. The results did not vary by race (results not shown). Table 1 Reliability and validity comparisons for FLG null mutations as assayed by TaqMan® and beadchip methods. Our study demonstrated that when the same genotyping method was repeated, the results were always identical (perfect test-retest reliability). However, when samples were assayed with both TaqMan® and beadchip technologies, the results were often different, with reliability values mostly suggesting only moderate agreement (inter-rater reliability). In addition, the validity measures for the beadchip method, assuming TaqMan® as the gold standard, were frequently poor. The PPV of the beadchip method was only 53%. In other words, only about half the time when the beadchip revealed a FLG null mutation, was a mutation truly present per TaqMan®. A potential limitation of this study could be the laboratory’s performance in the use of the beadchip technology. This is unlikely. First, the laboratory that conducted these tests is a University-based core facility that was established to perform genetic sequencing and is familiar with the technology. In addition, repeated assays were identical. Second, the chip was created by Illumina using the same information used to create the TaqMan® assays. All SNPs on the chip passed Illumina quality assurance. Third, the same chip was used to determine ancestry using 120 marker SNPs. The beadchip assays predicted self-reported race with more than 90% concordance to self-reported race4. Published results using beadchip technology have never fully captured the magnitude of association seen between the presence of FLG variants determined by TaqMan® and AD 6;10. Based on our findings this may be related to differences in the genotyping methods. It is likely that we should have expected our results in that the goal of high throughput techniques is often to find genes of interest using common tag SNPs and this technique is not ideal to assay redundent areas of the genome. TaqMan® techniques are often used to find specific causal mutations and can be more precisely customized. In conclusion, our study demonstrates that the beadchip technology may not be appropriate for genotyping the FLG gene. This technology should not be used for clinical or research purposes until futures studies are conducted better to better understand these differences and experimental proof are obtained with studies of filaggrin protein in the stratum corneum to verify bead chip results.

Heterogeneity of p53-pathway Protein Expression in Chemosensitive Chronic Lymphocytic Leukemia: A Pilot Study.

The presence of p53-pathway dysfunction in chronic lymphocytic leukemia (CLL) can be used to identify patients with chemotherapy-refractory disease. Therapeutic responses are known to vary between patients with chemosensitive CLL and may relate to differences in p53-pathway activity. We hypothesized that the magnitude or type of p53-pathway protein expression is heterogeneous in patients with chemosensitive disease and could associate with white cell responses. In this pilot study, changes in p53 and its transcriptional targets, p21/waf1 and MDM2 were analyzed by immunoblotting and densitometry in CLL cells from 10 patients immediately prior to the start of chemotherapy, and after culture for 24 hours (h) with fludarabine (n=7) or chlorambucil (n=3). The in vitro response was also compared to that in vivo in circulating cells pre-treatment, and at 24h and 96h of chemotherapy. Disease responses were evident in all patients after the first treatment-cycle. Significant p53 induction was observed in CLL cells treated in vitro and in vivo. Greater heterogeneity in the expression-intensity was observed in vivo (σ2=45.15) than in vitro (σ2=1.33) and the results failed to correlate (r2=0.18, p=0.22). p21/waf1 and MDM2 expression-profiles were also dissimilar in vitro and in vivo. Higher in vivo (but not in vitro) responses associated with changes in white cell count (p=0.026). Thus, heterogeneity of p53-pathway activity exists in chemosensitive CLL; in unselected patients, in vivo changes do not correlate with those in vitro, but may associate with post-treatment white cell responses.

Autoimmune pancytopenia following combination chemotherapy for chronic lymphocytic leukaemia

Autoimmune haemolysis or thrombocytopenia can complicate purine nucleoside monotherapy for chronic lymphocytic leukaemia (CLL), but Evans syndrome is rare. This is a report of the occurrence of pancytopenia secondary to a unique combination of red cell aplasia with autoimmune thrombocytopenia and neutropenia in a patient with CLL following treatment with fludarabine and cyclophosphamide. This case is unusual for the simultaneous targeting of three haemopoietic lineages by immune dysfunction following fludarabine and cyclophosphamide, which is a treatment regimen believed to reduce autoimmune haematological toxicity in CLL.