Procedures and operating instructions for diagnosis in vascular anomalies and pathology.

In the last 30 years a revolution has occurred in the diagnosis and management of vascular anomalies. The great changes began with Mulliken and Glowacki separation of hemangiomas and vascular anomalies. Their work has now morphed into the ISSVA classification. Subsequently the discovery of the significance of the presence of GLUT-1 in the diagnosis of the hemangiomas of infancy gave us a new marker in our quest for accurate classification. Now the genetic breakthroughs have led us into a "Star Wars" like environment in the experimental laboratory. During all these events the critical role of the pathologist has become more evident. Understanding the histopathology of anomalies has greatly aided in our approach to therapies. Moreover, genetic findings do not have full significance without the morphologic framework.

Cutaneous mucinosis of infancy: report of a rare case and review of the literature.

Cutaneous mucinosis of infancy (CMI) is a rare dermatologic condition, first reported in 1980 and currently classified within the complex group of papular mucinoses. We report a case of CMI and review the prior 13 cases in the literature. The patient was a 5-year-old girl who presented with asymptomatic dermal papules and plaques on her leg and back with no overlying color change. These lesions were first noticed during infancy and had become slightly more evident over time. The patient had a history of birthmarks and eczema. Her family history included eczema, allergies, photosensitivity, and Graves disease. Pre-biopsy clinical differential diagnosis included connective tissue nevus, granuloma annulare, myofibroma, lipofibroma, and lymphangioma. Biopsies revealed significant increase in interstitial mucin within the reticular and mid dermis, without significant sclerosis or fibroblastic proliferation. The relatively quiescent pattern of interstitial mucinosis with slight fibrocyte hyperplasia presenting as dermal papules-plaques on the trunk and extremities was most consistent with a diagnosis of CMI. We report another case of CMI in an otherwise healthy patient. Our patient is unique as she is the first CMI patient with a family history of Graves disease, although our patient appeared euthyroid. We will also review the literature on this rare entity.

Coexistence of Eph receptor B1 and ephrin B2 in port-wine stain endothelial progenitor cells contributes to clinicopathological vasculature dilatation.

BACKGROUND: Port-wine stain (PWS) is a vascular malformation characterized by progressive dilatation of postcapillary venules, but the molecular pathogenesis remains obscure. OBJECTIVES: To illustrate that PWS endothelial cells (ECs) present a unique molecular phenotype that leads to pathoanatomical PWS vasculatures. METHODS: Immunohistochemistry and transmission electron microscopy were used to characterize the ultrastructure and molecular phenotypes of PWS blood vessels. Primary culture of human dermal microvascular endothelial cells and in vitro tube formation assay were used for confirmative functional studies. RESULTS: Multiple clinicopathological features of PWS blood vessels during the development and progression of the disease were shown. There were no normal arterioles and venules observed phenotypically and morphologically in PWS skin; arterioles and venules both showed differentiation impairments, resulting in a reduction of arteriole-like vasculatures and defects in capillary loop formation in PWS lesions. PWS ECs showed stemness properties with expression of endothelial progenitor cell markers CD133 and CD166 in non-nodular lesions. They also expressed dual venous/arterial identities, Eph receptor B1 (EphB1) and ephrin B2 (EfnB2). Co-expression of EphB1 and EfnB2 in normal human dermal microvascular ECs led to the formation of PWS-like vasculatures in vitro, for example larger-diameter and thick-walled capillaries. CONCLUSIONS: PWS ECs are differentiation-impaired, late-stage endothelial progenitor cells with a specific phenotype of CD133+ /CD166+ /EphB1+ /EfnB2+ , which form immature venule-like pathoanatomical vasculatures. The disruption of normal EC-EC interactions by coexistence of EphB1 and EfnB2 contributes to progressive dilatation of PWS vasculatures.

Neuropeptide Y expression in primary cutaneous melanoma.

BACKGROUND: Neuropeptide Y (NPY) is involved in the carcinogenesis of different tumours, especially neural crest-derived tumours. OBJECTIVE: The aim of our study is to investigate the expression of NPY on melanoma and its relation with prognostic histological parameters and survival. METHODS: This is a retrospective observational study of two independent series, with a total of 79 primary melanomas, diagnosed in two independent University Hospitals in Spain, from January 2000 to December 2004. RESULTS: We found a significant higher expression of NPY on superficial spreading melanoma and lentigo maligna (40%) (P = 0.030). Thinner tumours were associated with higher NPY expression (Clark level, P = 0.003; Breslow level, P = 0.012). Melanomas with low NPY expression were associated with intense cell proliferation (Ki-67, P = 0.034), high density of peritumoral mast cell infiltrates (P = 0.033) and low E-cadherin expression (P = 0.031). Melanomas with high NPY expression exhibited significant differences in terms of relapse time (median: 114 vs. 68 months, P = 0.008) and overall survival (114 vs. 74 months, P = 0.004). CONCLUSION: High expression of NPY was associated with better prognostic histological parameters, low peritumoral mast cells density, presence of adhesion proteins and better outcome.

Topical axitinib suppresses angiogenesis pathways induced by pulsed dye laser.

BACKGROUND: The recurrence of port-wine stain (PWS) blood vessels by pulsed dye laser (PDL)-induced angiogenesis is a critical barrier that must be overcome to achieve a better therapeutic outcome. OBJECTIVES: To determine whether PDL-induced angiogenesis can be suppressed by topical axitinib. METHODS: The mRNA expression profiles of 86 angiogenic genes and phosphorylation levels of extracellular signal regulated kinases (ERKs), phosphorylated protein kinase B (AKT) and ribosomal protein S6 kinase (p70S6K) in rodent skin were examined with or without topical axitinib administration after PDL exposure. RESULTS: The PDL-induced increased transcriptional levels of angiogenic genes peaked at days 3-7 post-PDL exposure. Topical application of 0·5% axitinib effectively suppressed the PDL-induced increase in mRNA levels of the examined angiogenic genes and activation of AKT, P70S6K and ERK from days 1 to 7 post-PDL exposure. After topical administration, axitinib penetrated into rodent skin to an approximate depth of 929·5 µm. CONCLUSIONS: Topical application of 0·5% axitinib can systematically suppress the PDL-induced early stages of angiogenesis via inhibition of the AKT/mammalian target of rapamycin/p70S6K and Src homology 2 domain containing transforming protein-1/mitogen-activated protein kinase kinase/ERK pathway cascades.

Large agminated cellular 'plaque-type' blue nevus surrounding the ear: a case and review.

Large or giant cellular blue nevi are usually congenital and represent a challenge for the physician. Close anatomic structures may be altered by the size of the moles. In this article, we report the case of an uncommon large, agminated, cellular blue nevus of the 'plaque type' in a 42-year-old female. Due to the risks of malignant melanoma development on a large or giant blue nevus, we highlight the importance of proper histopathological diagnosis. Furthermore, because of the possibility that the nevus may invade the bone and cerebral tissues, we discuss the indication of a radiological diagnosis. The accurate correlation to clinical and histopathological findings and appropriate multidisciplinary management can save the lives of patients.

Effect of the metabolic environment at key stages of follicle development in cattle: focus on steroid biosynthesis.

Cellular mechanisms that contribute to low estradiol concentrations produced by the preovulatory ovarian follicle in cattle with a compromised metabolic status are largely unknown. To gain insight into the main metabolic mechanisms affecting preovulatory follicle function, two different animal models were used. Experiment 1 compared Holstein-Friesian nonlactating heifers (n = 17) and lactating cows (n = 16) at three stages of preovulatory follicle development: 1) newly selected dominant follicle in the luteal phase (Selection), 2) follicular phase before the LH surge (Differentiation), and 3) preovulatory phase after the LH surge (Luteinization). Experiment 2 compared newly selected dominant follicles in the luteal phase in beef heifers fed a diet of 1.2 times maintenance (M, n = 8) or 0.4 M (n = 11). Lactating cows and 0.4 M beef heifers had higher concentrations of ß-hydroxybutyrate, and lower concentrations of glucose, insulin, and IGF-I compared with dairy heifers and 1.2 M beef heifers, respectively. In lactating cows this altered metabolic environment was associated with reduced dominant follicle estradiol and progesterone synthesis during Differentiation and Luteinization, respectively, and in 0.4 M beef heifers with reduced dominant follicle estradiol synthesis. Using a combination of RNA sequencing, Ingenuity Pathway Analysis, and qRT-PCR validation, we identified several important molecular markers involved in steroid biosynthesis, such as the expression of steroidogenic acute regulatory protein (STAR) within developing dominant follicles, to be downregulated by the catabolic state. Based on this, we propose that the adverse metabolic environment caused by lactation or nutritional restriction decreases preovulatory follicle function mainly by affecting cholesterol transport into the mitochondria to initiate steroidogenesis.

Agreement of dermatopathologists in the evaluation of clinically difficult melanocytic lesions: how golden is the 'gold standard'?

BACKGROUND: The 'gold standard' for the diagnosis of melanocytic lesions is dermatopathology. Although most of the diagnostic criteria are clearly defined, the interpretation of histopathology slides may be subject to interobserver variability. OBJECTIVES: The aim of this study was to determine the variability among dermatopathologists in the interpretation of clinically difficult melanocytic lesions. METHODS: This study used the database of MelaFind®, a computer-vision system for the diagnosis of melanoma. All lesions were surgically removed and sent for independent evaluation by four dermatopathologists. Agreement was calculated using kappa statistics. RESULTS: A total of 1,249 pigmented melanocytic lesions were included. There was a substantial agreement among expert dermatopathologists: two-category kappa was 0.80 (melanoma vs. non-melanoma) and three-category kappa was 0.62 (malignant vs. borderline vs. benign melanocytic lesions). The agreement was significantly greater for patients ≥40 years (three-category kappa = 0.67) than for younger patients (kappa = 0.49). In addition, the agreement was significantly lower for patients with atypical mole syndrome (AMS) (kappa = 0.31) than for patients without AMS (kappa = 0.76). LIMITATIONS: The data were limited by the inclusion/exclusion criteria of the MelaFind® study. This might represent a selection bias. The agreement was evaluated using kappa statistics. This is a standard method for evaluating agreement among pathologists, but might be considered controversial by some statisticians. CONCLUSIONS: Expert dermatopathologists have a high level of agreement when diagnosing clinically difficult melanocytic lesions. However, even among expert dermatopathologists, the current 'gold standard' is not perfect. Our results indicate that lesions from younger patients and patients with AMS may be more problematic for the dermatopathologists, suggesting that improved diagnostic criteria are needed for such patients.

Basophilic leukocytes in allergic contact dermatitis.

A variety of cell-mediated hypersensitivity reactions in experimental animals include a prominent infiltrate of basophilic leukocytes. This form of reactivity has been designated cutaneous basophil hypersensitivity and is favored when sensitization to several types of antigen is accomplished without the use of complete Freund's adjuvant. A similar type of hypersensitivity response was sought in man using morphologic techniques which permit identification of basophilic leukocytes. Eight individuals with allergic contact dermatitis to a variety of allergens were studied and six of these developed typical contact reactions with erythema, edema, and epidermal vesicles. The microscopic findings in 3-day biopsies from these individuals differed significantly from classic descriptions of tuberculin hypersensitivity and showed, in addition to mononuclear cells and the characteristic epidermal changes, a substantial infiltrate of basophilic leukocytes and evidence of altered vascular permeability with vascular compaction, dermal edema, and fibrin deposition. Serial biopsies from one individual permitted analysis of the microscopic pathology as it unfolded at successive intervals after patch test. The initial lesion consisted of perivascular accumulations of lymphocytes; this was followed by an influx of basophils and, subsequently, of eosinophils. These findings associate contact allergy in man with the parallel reactions of cutaneous basophil hypersensitivity in animals and provide further evidence for the heterogeneity of the cellular immune response. The data are consistent with the hypothesis that interaction between sensitized lymphocytes and antigen, at a local test site, is responsible for the attraction of basophils. They also directly implicate the clotting system in delayed-type reactions and suggest the possibility of a synergistic relationship between cellular immunity and reactions mediated by basophil-bound, homocytotrophic antibody.

Role of the clotting system in cell-mediated hypersensitivity. I. Fibrin deposition in delayed skin reactions in man.

The expression of delayed-type hypersensitivity in animals has been inhibited by a variety of anticoagulants, but direct evidence for activation of clotting in the evolution of these reactions has been lacking. Using the fluorescent antibody technique we here demonstrate that fibrin deposition is a prominent and consistent feature of both allergic contact dermatitis and classic delayed hypersensitivity skin reactions in man. Fib was detected in 55 of 58 delayed reactions studied at the peak of their intensity. The characteristic distribution of Fib-principally in the intervascular portions of the reticular dermis with sparing of vessels and their associated cuffs of mononuclear cells-is unusual and quite different from that described in antibody-mediated lesions in animals or man. Fib was found in vessel walls in only 2 of 94 biopsies studied. With a single exception, deposition of immunoglobulins and complement was not observed. The pathogensis and significance of Fib deposition in these reactions are not yet clear. Fib is ultimately derived from circulating fibrinogen, and its accumulation provides additional evidence for locally increased vascular permeability in delayed hypersensitivity. Polymerization of extravascular fibrinogen could be triggered nonspecifically by dermal elements (e.g., collagen) or by a product of sensitized lymphocytes. The appearance of Fib early in the development of these reactions (4-8 h after epicutaneous test with DNCB) and inhibition studies with anticoagulants together suggest that clotting may have a role in their pathogenesis, possibly by the release of bioactive peptides from fibrinogen/fibrin or by contributing to the induration characteristic of delayed hypersensitivity.

Rejection of first-set skin allografts in man. the microvasculature is the critical target of the immune response.

Recent reports of microvascular injury in delayed hypersensitivity skin reactions prompted us to reexamine the pathogenesis of first-set skin allograft rejection in man using morphologic techniques that allowed both extensive vessel sampling and unequivocal evaluation of microvascular endothelium. We here report that widespread microvascular damage is a characteristic, early consequence of the cellular immune response to first-set human skin allografts and is qualitatively similar to, but substantially more extensive than, that occurring in delayed hypersensitivity reactions. Microvascular damage in invariably preceded significant epithelial necrosis and affected initially and primarily those venules, arterioles, and small veins enveloped by lymphocytes. Vessels of both the allograft itself and the underlying graft bed (recipient tissue) were equally affected. These data suggest that endothelial cells of the microvasculature are the critical target of the immune response in first-set vascularized skin allograft rejection in man and that rejection can be attributed largely to ischemic infarction resulting from extensive microvascular damage. Other mechanisms, such as direct cellular contacts between infiltrating lymphocytes and epithelium, apparently played only a minor role. The findings presented here indicate that the rejection of first-set vascularized skin allografts, though induced by immunologically specific mechanisms, is primarily effected by final pathways that are relatively nonspecific and that may cause damage to both foreign and host vessels and cells. Rather than contradicting studies demonstrating the exquisite specificity of allograft rejection in other systems, these findings provide a further example of the heterogeneity of the cellular immune response. Recognition of the critical role of immunologically mediated microvascular injury may prove important both for an understanding of the biology of allograft rejection and for strategies aimed at prolonging allograft survival.

Differential expression of signal transduction factors in ovarian follicle development: a functional role for betaglycan and FIBP in granulosa cells in cattle.

Ovarian follicles develop in groups yet individual follicles follow different growth trajectories. This growth and development are regulated by endocrine and locally produced growth factors that use a myriad of receptors and signal transduction pathways to exert their effects on theca and granulosa cells. We hypothesize that differential growth may be due to differences in hormonal responsiveness that is partially mediated by differences in expression of genes involved in signal transduction. We used the bovine dominant follicle model, microarrays, quantitative real-time PCR and RNA interference to examine this. We identified 83 genes coding for signal transduction molecules and validated a subset of them associated with different stages of the follicle wave. We suggest important roles for CAM kinase-1 and EphA4 in theca cells and BCAR1 in granulosa cells for the development of dominant follicles and for betaglycan and FIBP in granulosa cells of regressing subordinate follicles. Inhibition of genes for betaglycan and FIBP in granulosa cells in vitro suggests that they inhibit estradiol production in regressing subordinate follicles.

Mechanisms for dominant follicle selection in monovulatory species: a comparison of morphological, endocrine and intraovarian events in cows, mares and women.

The selection of a single ovarian follicle for further differentiation and finally ovulation is a shared phenomenon in monovulatory species from different phylogenetic classes. The commonality of dominant follicle (DF) development leads us to hypothesize that mechanisms for DF selection are conserved. This review highlights similarities and differences in follicular wave growth between cows, mares and women, addresses the commonality of the transient rises in FSH concentrations, and discusses the follicular secretions oestradiol and inhibin with their regulatory roles for FSH. In all three species, rising FSH concentrations induce the emergence of a follicle wave and cohort attrition occurs during declining FSH concentrations, culminating in DF selection. Cohort secretions are initially responsible for declining FSH, which is subsequently suppressed by the selected DF lowering it below the threshold of FSH requirements of all other cohort follicles. The DF acquires relative FSH-independence in order to continue growth and differentiation during low (cow, mare) or further declining FSH concentrations (women), and thus may be the one cohort follicle with the lowest FSH requirement due to enhanced FSH signalling. In all three monovulatory species a transition from FSH- to LH-dependence is postulated as the mechanism for the continued development of the selected DF. In addition, FSH and IGF enhance each other's ability to stimulate follicle cell function and access of IGF-I and -II to the type 1 receptor is regulated by IGF binding proteins that are in turn regulated by specific proteases; all of which have been ascribed a role in DF development. No fundamental differences in DF selection mechanisms have been identified between the different species studied. Thus functional studies of the selection of DFs in cattle and mares are also valuable for identifying genes and pathways regulating DF development in women.

Dietary lutein/zeaxanthin partially reduces photoaging and photocarcinogenesis in chronically UVB-irradiated Skh-1 hairless mice.

Lutein and zeaxanthin are xanthophyll carotenoids with potent antioxidant properties protecting the skin from acute photodamage. This study extended the investigation to chronic photodamage and photocarcinogenesis. Mice received either a lutein/zeaxanthin-supplemented diet or a standard nonsupplemented diet. Dorsal skin of female Skh-1 hairless mice was exposed to UVB radiation with a cumulative dose of 16,000 mJ/cm(2) for photoaging and 30,200 mJ/cm(2) for photocarcinogenesis. Clinical evaluations were performed weekly, and the animals were sacrificed 24 h after the last UVB exposure. For photoaging experiments, skin fold thickness, suprapapillary plate thickness, mast cell counts and dermal desmosine content were evaluated. For photocarcinogenesis, samples of tumors larger than 2 mm were analyzed for histological characterization, hyperproliferation index, tumor multiplicity, total tumor volume and tumor-free survival time. Results of the photoaging experiment revealed that skin fold thickness and number of infiltrating mast cells following UVB irradiation were significantly less in lutein/zeaxanthin-treated mice when compared to irradiated animals fed the standard diet. The results of the photocarcinogenesis experiment were increased tumor-free survival time, reduced tumor multiplicity and total tumor volume in lutein/zeaxanthin-treated mice in comparison with control irradiated animals fed the standard diet. These data demonstrate that dietary lutein/zeaxanthin supplementation protects the skin against UVB-induced photoaging and photocarcinogenesis.

Delayed type IV hypersensitivity reaction to porcine acellular dermal matrix masquerading as infection resulting in multiple debridements.

INTRODUCTION: Delayed type IV hypersensitivity reactions are well established in the surgical setting with respect to external exposure via topical antibiotics and internal exposure via synthetic materials. In contrast, biologic matrix is derived from decellularized human or animal tissues and is consequently believed to elicit a minimal host inflammatory response. OBJECTIVE: We report a case of delayed type IV hypersensitivity reaction secondary to a biologic comprised of porcine-derived acellular dermal matrix, [Strattice™]. CONCLUSIONS: While biologic matrix is often preferred over synthetic mesh due to its decreased risk for infection, this case emphasizes that potential for hypersensitivity to the material persists. Type IV hypersensitivity reactions should be included in the differential diagnosis for suspected post-operative infections.

Comparing the fatty acid composition of organic and conventional milk.

During a 12-mo longitudinal study, bulk-tank milk was collected each month from organic (n = 17) and conventional (n = 19) dairy farms in the United Kingdom. All milk samples were analyzed for fatty acid (FA) content, with the farming system type, herd production level, and nutritional factors affecting the FA composition investigated by use of mixed model analyses. Models were constructed for saturated fatty acids, the ratio of polyunsaturated fatty acids (PUFA) to monounsaturated fatty acids, total n-3 FA, total n-6 FA, conjugated linoleic acid, and vaccenic acid. The ratio of n-6:n-3 FA in both organic and conventional milk was also compared. Organic milk had a higher proportion of PUFA to monounsaturated fatty acids and of n-3 FA than conventional milk, and contained a consistently lower n-6:n-3 FA ratio (which is considered beneficial) compared with conventional milk. There was no difference between organic and conventional milk with respect to the proportion of conjugated linoleic acid or vaccenic acid. A number of factors other than farming system were identified which affected milk FA content including month of year, herd average milk yield, breed type, use of a total mixed ration, and access to fresh grazing. Thus, organic dairy farms in the United Kingdom produce milk with a higher PUFA content, particularly n-3 FA, throughout the year. However, knowledge of the effects of season, access to fresh grazing, or use of specific silage types could be used by producers to enhance the content of beneficial FA in milk.

Moles and site-specific risk of nonfamilial cutaneous malignant melanoma in women.

We examined the relationship between self-reported mole counts and cutaneous melanoma with respect to anatomic site in 110 case and 231 control female nurses. Counts of moles on the lower leg were better predictors of melanoma risk than were counts of moles on the arm. The relative risk for the highest quintile of lower leg mole counts versus no lower leg moles was 4.2. Mole counts at each site (arm, thigh, and lower leg) were associated with risk of melanoma of the trunk and lower leg, but none were associated with the risk of melanoma of the upper extremity. The absence of direct site-specificity suggests that mole counts primarily indicate systemic melanoma risk, rather than direct risk from the moles themselves.

Differences in dendritic cells stimulated in vivo by tumors engineered to secrete granulocyte-macrophage colony-stimulating factor or Flt3-ligand.

Both granulocyte-macrophage colony-stimulating factor (GM-CSF) and flt3-ligand (FL) induce the development of dendritic cells (DCs). To compare the functional properties of DCs stimulated by these cytokines in vivo, we used retroviral-mediated gene transfer to generate murine tumor cells secreting high levels of each molecule. Injection of tumor cells expressing either GM-CSF or FL resulted in the dramatic increase of CD11c+ cells in the spleen and tumor infiltrate. However, vaccination with irradiated, GM-CSF-secreting tumor cells stimulated more potent antitumor immunity than vaccination with irradiated, FL-secreting tumor cells. The superior antitumor immunity elicited by GM-CSF involved a broad T cell cytokine response, in contrast to the limited Thl response elicited by FL. DCs generated by GM-CSF were CD8alpha- and expressed higher levels of B7-1 and CD1d than DCs cells generated by FL. Injection sites of metastatic melanoma patients vaccinated with irradiated, autologous tumor cells engineered to secrete GM-CSF demonstrated similar, dense infiltrates of DCs expressing high levels of B7-1. These findings reveal critical differences in the abilities of GM-CSF and FL to enhance the function of DCs in vivo and have important implications for the crafting of tumor vaccines.