Ian D. Odell

Rules of Engagement for Base Excision Repair in Chromatin

Most of the DNA in eukaryotes is packaged in tandemly arrayed nucleosomes that, together with numerous DNA‐ and nucleosome‐associated enzymes and regulatory factors, make up chromatin. Chromatin modifying and remodeling agents help regulate access to selected DNA segments in chromatin, thereby facilitating transcription and DNA replication and repair. Studies of nucleotide excision repair (NER), single strand break repair (SSBR), and the homology‐directed repair (HDR), and non‐homologous end‐joining (NHEJ) double strand break repair pathways have led to an “access‐repair‐restore” paradigm, in which chromatin in the vicinity of damaged DNA is disrupted, thereby enabling efficient repair and the subsequent repackaging of DNA into nucleosomes. When damage is extensive, these repair processes are accompanied by cell cycle checkpoint activation, which provides cells with sufficient time to either complete the repair or initiate apoptosis. It is not clear, however, if base excision repair (BER) of the ∼20,000 or more oxidative DNA damages that occur daily in each nucleated human cell can be viewed through this same lens. Until recently, we did not know if BER requires or is accompanied by nucleosome disruption, and it is not yet clear that anything short of overwhelming oxidative damage (resulting in the shunting of DNA substrates into other repair pathways) results in checkpoint activation. This review highlights studies of how oxidatively damaged DNA in nucleosomes is discovered and repaired, and offers a working model of events associated with BER in chromatin that we hope will have heuristic value. J. Cell. Physiol. 228: 258–266, 2013.

Nucleosome Disruption by DNA Ligase III-XRCC1 Promotes Efficient Base Excision Repair

ABSTRACT Each day, approximately 20,000 oxidative lesions form in the DNA of every nucleated human cell. The base excision repair (BER) enzymes that repair these lesions must function in a chromatin milieu. We have determined that the DNA glycosylase hNTH1, apurinic endonuclease (APE), and DNA polymerase β (Pol β), which catalyze the first three steps in BER, are able to process their substrates in both 601- and 5S ribosomal DNA (rDNA)-based nucleosomes. hNTH1 formed a discrete ternary complex that was displaced by the addition of APE, suggesting an orderly handoff of substrates from one enzyme to the next. In contrast, DNA ligase IIIα-XRCC1, which completes BER, was appreciably active only at concentrations that led to nucleosome disruption. Ligase IIIα-XRCC1 was also able to bind and disrupt nucleosomes containing a single base gap and, because of this property, enhanced both its own activity and that of Pol β on nucleosome substrates. Collectively, these findings provide insights into rate-limiting steps that govern BER in chromatin and reveal a unique role for ligase IIIα-XRCC1 in enhancing the efficiency of the final two steps in the BER of lesions in nucleosomes.

Non-specific DNA binding interferes with the efficient excision of oxidative lesions from chromatin by the human DNA glycosylase, NEIL1

Although DNA in eukaryotes is packaged in nucleosomes, it remains vulnerable to oxidative damage that can result from normal cellular metabolism, ionizing radiation, and various chemical agents. Oxidatively damaged DNA is repaired in a stepwise fashion via the base excision repair (BER) pathway, which begins with the excision of damaged bases by DNA glycosylases. We reported recently that the human DNA glycosylase hNTH1 (human Endonuclease III), a member of the HhH GpG superfamily of glycosylases, can excise thymine glycol lesions from nucleosomes without requiring or inducing nucleosome disruption; optimally oriented lesions are excised with an efficiency approaching that seen for naked DNA [1]. To determine if this property is shared by human DNA glycoylases in the Fpg/Nei family, we investigated the activity of NEIL1 on defined nucleosome substrates. We report here that the cellular concentrations and apparent k(cat)/K(M) ratios for hNTH1 and NEIL1 are similar. Additionally, after adjustment for non-specific DNA binding, hNTH1 and NEIL1 proved to have similar intrinsic activities toward nucleosome substrates. However, NEIL1 and hNTH1 differ in that NEIL1 binds undamaged DNA far more avidly than hNTH1. As a result, hNTH1 is able to excise both accessible and sterically occluded lesions from nucleosomes at physiological concentrations, while the high non-specific DNA affinity of NEIL1 would likely hinder its ability to process sterically occluded lesions in cells. These results suggest that, in vivo, NEIL1 functions either at nucleosome-free regions (such as those near replication forks) or with cofactors that limit its non-specific binding to DNA.

Somatic p.T771R KDR (VEGFR2) Mutation Arising in a Sporadic Angioma During Ramucirumab Therapy.

IMPORTANCE Inhibition of angiogenesis is an effective anticancer strategy because neoplasms require a rich blood supply. Ramucirumab, approved by the US Food and Drug Administration in 2014 to treat gastric adenocarcinomas and non-small cell lung carcinomas, targets vascular endothelial growth factor 2 (VEGFR2). We identified a patient prescribed a regimen of irinotecan hydrochloride, cetuximab, and ramucirumab for metastatic rectal cancer (diagnosed in November 2013 and treated through early January 2015) who developed a new-onset, expanding vascular lesion on his right leg. Via exome sequencing, we found that the lesion contained a single somatic mutation in KDR (encodes VEGFR2), possibly in response to ramucirumab. Vascular tumors are not a known complication of antiangiogenic therapeutics. OBSERVATIONS Exome sequencing of the well-demarcated, blanching vascular lesion on the lateral right shin revealed a somatic p.T771R mutation in KDR, without evidence of other somatic mutations or loss of heterozygosity. Histological features included lobules of small vessels within the dermis, resembling a tufted angioma. CONCLUSIONS AND RELEVANCE A potential adverse effect of ramucirumab in combination therapy is the development of sporadic angiomas. The p.T771R mutation was previously implicated in autophosphorylation of VEGFR2 and reported in angiosarcomas alongside other driver mutations. Our observations suggest that this mutation confers a proliferative advantage in the setting of ramucirumab therapy. Patients receiving ramucirumab should be monitored for the development of new vascular lesions.

Treatment of melanoma in-transit metastases with combination intralesional interleukin-2, topical imiquimod, and tretinoin 0.1% cream

The treatment of in-transit and satellite melanoma metastases is challenging. Treatment options for these cutaneous and subcutaneous lesions include surgical excision, radiotherapy, isolated limb infusion/perfusion, electrochemotherapy, cryotherapy, laser therapy (pulsed dye or carbon dioxide), systemic treatment with interferon-α or interleukin-2 (IL-2), topical imiquimod, dinitrochlorobenzene, and intralesional immunotherapy with bacillus Calmette-Guerin vaccine, granulocyte macrophage colony-stimulating factor, IL-2, or talimogene laherparepvec.1 Response rates for these therapies are often suboptimal. Topical imiquimod has been used for the treatment of both melanoma in situ (in patients who are either poor surgical candidates or have positive margins after excision) and in-transit metastases.1, 2 There are reports of regression of locoregional melanoma metastases after topical imiquimod to cutaneous lesions.1, 2 Combination therapy using intralesional IL-2 together with topical imiquimod and tretinoin may increase the efficacy of IL-2.3 Here we report the case of a patient with in-transit metastatic melanoma treated with intratumoral IL-2 together with topical imiquimod and tretinoin cream.

Leukaemic vasculitis with myelodysplastic syndrome

www.thelancet.com Vol 386 August 1, 2015 501 A 75-year-old black woman with insulin-dependent diabetes and obesity presented in January, 2015, with a 10 day history of pruritic lesions on her arms and legs, 2–3 months of night sweats, and 3 weeks of right-sided headache, sinus pain, and congestion. The skin lesions had developed while she was taking prednisone for presumed temporal arteritis. She had no associated fever, abdominal pain, diarrhoea, myalgia, or arthralgia. On examination she looked well. On the extensor elbows extending down her forearms bilaterally and on her knees and shins were dozens of 1–2 cm oedematous pink-red papules with a darker red-violet central zone, resembling atypical papular target lesions of erythema multiforme (fi gure). She had similar lesions on the knees and shins. Along Wallace’s lines on the medial aspect of her feet and the tips of her fi ngers and toes were about 20 red macules, 5–15 mm in diameter. She had one 2 mm erosion on the soft palate of her mouth; her face was otherwise spared. Our diff erential diagnosis was erythema multiforme, erythema elevatum diutinum (a small vessel vasculitis that occurs on the extensor surfaces of joints on the arms and legs), and Sweet’s syndrome. Initial blood tests showed pancytopenia with white blood cell count 2·7 × 109/L with 43% neutrophils, 37% lymphocytes, 8% monocytes, 1% eosinophils, 1% atypical lymphocytes, and 10% bands; haemoglobin 100 g/L and platelet count 129 × 109/L; and mild neutropenia with absolute neutrophil count 1·4 × 109/L. Liver function tests were normal; urinalysis showed 2+ protein and no blood. PCR for herpes simplex virus, antineutrophil cytoplasmic antibody, and cryoglobulins were negative, antinuclear antibody was less than 1:40, there were no discrete abnormal bands in serum or urine protein electrophoresis, and no evidence of a monoclonal component on immunofi xation electrophoresis. However, quantitative immunoglobulins showed IgA about twice the upper limit of normal (8860 mg/L, normal 700–4000 mg/L). Two skin biopsy specimens showed an infi ltrate of atypical mononuclear cells around and within small and medium-sized vessels with associated vascular damage (extravasated erythrocytes, leucocytoclasia, and fi brinoid degeneration). The atypical cells had blastic morphological features and were variably positive for myeloperoxidase, CD15, CD43, CD45, CD68, and TIA-1. The epidermis had scattered necrotic keratinocytes. Direct immunofl uorescence did not show any specifi c deposits of IgG, IgM, IgA, or complement. Histopathology fi ndings were consistent with leukaemic vasculitis mediated by atypical myeloid cells. Bone marrow biopsy specimen showed increased early myeloid forms with 5% CD34+ myeloblasts with abnormal localisation of immature precursors and dysme gakaryopoiesis in normocellular marrow without fi brosis. 11/18 bone marrow cells in metaphase showed an abnormal clone with a complex karyotype with an extra copy of chromosome 1, deletions in 1q, 3p, and 5q, and losses of one copy of chromosomes 7, 17, and 22 (45,XX,+1,del(1)(q21),del(3)(p14),del(5)(q13q31),-7,-17, -22[cp11]/46,XX[7]). FISH showed deletions of 5q and 7q in about 54% of bone marrow cells. These fi ndings were diagnostic of myelodysplastic syndrome (MDS) with refractory anaemia with excess blasts-1. By contrast with leucocytoclastic vasculitis, which is more commonly associated with MDS and is mediated by mature-appearing neutrophils, in rare circumstances leukaemic cells mediate blood vessel damage directly, causing leukaemic vasculitis. As in our case, the skin lesions are typically erythematous or purpuric papules, nodules, or plaques distributed on the limbs, which can mimic erythema multiforme. Leukaemic vasculitis is unusual as the presenting feature of MDS, because it is usually associated with acute myeloid leukaemia or myelodysplastic syndrome in transformation. On the basis of her RAEB-1 WHO category, complex karyotype, and no transfusion requirement, our patient’s WHO classifi cation-based Prognostic Scoring System Score at diagnosis was 4, placing her in the high-risk category with an expected median survival of 26 months. The presence of leukaemic vasculitis also portends a poor prognosis. Because she had not responded to corticosteroids we gave azacitidine 75 mg/m2 subcutaneously for 7 days, with great improvement in her white cell count and skin lesions (appendix), followed by three additional cycles of monthly azacitidine. At last follow-up in June, 2015, 4 months after her fi rst dose of azacitidine she remains well and has an excellent ongoing haematological response except for mild anaemia. Her skin lesions healed well leaving only postinfl ammatory pigmentation. She has not needed transfusion of any blood products since she started azacitidine treatment. Leukaemic vasculitis with myelodysplastic syndrome

Microbiome: Ecology of eczema

Patients with atopic dermatitis have fundamentally different skin microbial populations compared with people with healthy skin. Bacteria associated with atopic dermatitis express genes for survival in dry conditions and for ammonia production, modulating the pH of this distinct environment and driving complex ecological interactions.

Optimizing Direct Immunofluorescence

Immunofluorescence is a laboratory technique that utilizes a fluorophore-labeled antibody to detect immune complexes in tissue. Most of the labeled antibodies used in a clinical laboratory bind the conserved domains within each class of human antibodies, allowing them to detect a wide range of autoimmune complexes. Drawbacks to this technique mostly relate to proper handling of the specimen and the fluorophore-labeled antibodies. Therefore, having a basic understanding of fluorophores and antibodies is important for processing a specimen that yields a high signal-to-background ratio as well as troubleshooting problems, should they arise.

A case of subungual tumors of incontinentia pigmenti: A rare manifestation and association with bipolar disease

IP: incontinentia pigmenti NF-kB: nuclear factor-kB STIPs: subungual tumors of incontinentia pigment

Well-Differentiated Syringofibrocarcinoma in a Patient With Clouston Syndrome.

From the beginning, the therapy showed remarkable efficacy in both clinical symptoms and laboratory findings, with an almost complete disappearance of pustulation 48 hours after the first injection. Though 3 to 4 days after each injection, the patient developed minor flares of new pustules, he continuously improved. These small recurrences decreased over time and may be attributed to on-off phenomena of the antibody. In conclusion, the patient showed a rapid response to secukinumab with regard to the cutaneous and systemic manifestations of severe GPP, indicating that this new targeted therapy might also be used to treat PP. Prospective randomized clinical trials are required to further evaluate the efficacy and safety of IL-17 inhibitors for the treatment of GPP.