The "Azzopardi phenomenon" in high-grade astrocytoma with a PNET-like component.

Guidance molecules have attracted interest by demonstration that they regulate patterning of the blood vascular system during development. However, their significance during postnatal angiogenesis has remained unknown. Here, we demonstrate that endothelial cells of human malignant brain tumors also express guidance molecules, such as EphB4 and its ligand ephrinB2. To study their function, EphB4 variants were overexpressed in blood vessels of tumor xenografts. Our studies revealed that EphB4 acts as a negative regulator of blood vessel branching and vascular network formation, switching the vascularization program from sprouting angiogenesis to circumferential vessel growth. In parallel, EphB4 reduces the permeability of the tumor vascular system via activation of the angiopoietin-1/Tie2 system at the endothelium/pericyte interface. Furthermore, overexpression of EphB4 variants in blood vessels during (i) vascularization of non-neoplastic cell grafts and (ii) retinal vascularization revealed that these functions of EphB4 apply to postnatal, non-neoplastic angiogenesis in general. This implies that both neoplastic and non-neoplastic vascularization is driven not only by a vascular initiation program but also by a vascular patterning program mediated by guidance molecules.

Chapter 8 - Diseases of small and medium-sized blood vessels

Chapter 4 - Diseases of Small and Medium-sized Blood Vessels

Tissue kallikrein releases kinins by specific proteolysis, an activity inhibited by kallistatin. In this study, kallikrein and kallistatin were localized to endothelial and smooth muscle cells of large, medium, and small normal blood vessels by immunohistochemical techniques. Immunostaining for both proteins was strong in the endothelium of all sizes of blood vessels and was more intense in medial smooth muscle cells of small and medium-sized blood vessels than in elastic arteries. The sites of synthesis by endothelial and smooth muscle cells were demonstrated in normal blood vessels of all sizes by in situ hybridization histochemistry. Kallikrein and kallistatin levels were measured by immunoassays in homogenates of human aorta, vena cava, and iliac artery and vein. Tissue kallikrein and kallistatin transcripts were identified in human blood vessels by RT-PCR followed by Southern blot analysis with specific oligonucleotide probes. The results demonstrated the expression and co-localization of tissue kallikrein and kallistatin in human vessels and suggest a potential role of kallistatin in regulating tissue kallikrein in blood vessels.

Angiotropic large cell lymphoma (ALCL) is characterized by the intravascular proliferation of malignant lymphoid cells in small and medium-sized blood vessels. In the current study, the authors report an unusual case in which the initial presentation of the ALCL was that of superior vena cava (SVC) syndrome. The case is presented, followed by a general review of the literature regarding ALCL. Surgical intervention was required for diagnosis in this case. Successful treatment with chemotherapy followed by involved field radiation ensued with a maintained disease remission at 48 months of follow-up. Although usually presenting in small blood vessels, ALCL can present initially with large blood vessel involvement and should be considered in the differential diagnosis of this condition, even in the absence of extravascular lymph node involvement. Aggressive treatment with antineoplastic therapy is warranted and may result in long term recurrence free survival.

Objective To discuss the expression patterns of hypoxia inducible factor(HIF)-1α, HIF- 2α and vascular endothelial growth factor(VEGF)in metastatic lung cancer tissues. Methods The immunohistochemical streptavid-in- peroxidase(SP) method was applied to detect the expression of HIF-1α, HIF- 2α and VEGF in 93 cases of surgicall resected metastatic lung cancer, and 57 cases of primary non- small- cell lung carcinoma(NSCLC)as blank control group. Results HIF-1α, HIF-2α and VEGF proteins were expressed in 72 cases of metastatic lung cancer and 58 cases of primary NSCLC with the positive rate being 77.4%, 62.3% and 74.2% respectively(P 0.05). Conclusion HIF-1α, HIF-2α and VEGF expression in lung metastases is increased, and HIF-1α expression was positively correlated with VEGF expression. Key words: Metastatic lung cancer; Hypoxia inducible factor-1α; Hypoxia inducible factor-2α; Vascular endothelial growth factor

To the Editor: A prominent vascular component has been rarely described in melanocytic lesions. Diaz-Cascajo et al1 were the first to report a particular variant of desmoplastic Spitz nevus with a striking fibrous stroma and many densely arranged small blood vessels. Due to its similarity to a vascular tumor, they proposed the term of “angiomatoid nevus” for this condition. Since then, few similar cases have been described, corresponding almost exclusively to Spitz nevi.2,3 To the best of our knowledge, there is only one report of angiomatoid cellular blue nevus,4 and we have not found any case of junctional, compound, or intradermal angiomatoid melanocytic nevus. We recently observed a 53-year-old woman, with a progressively enlarging nodule, located on her right lumbar area, which was present for 7 years. The lesion measured 13 mm in diameter. It had a slightly pigmented and shiny surface. Telangiectasias could also be appreciated. Dermoscopically, the lesion showed a background of light brownish pigment, with a prominent vascular pattern composed by arborizing vessels and hemorrhagic areas that were surrounded by a striking whitish veil, which was especially located in the central part of the lesion (Fig. 1).FIGURE 1: Dermoscopy shows a lesion with a multicomponent pattern, with multiple big-sized vessels and a prominent whitish veil especially located in the centre.We excised the lesion, and histologically, an intradermal melanocytic congenital type nevus was found. Several medium size vessels with thin fibrous wall, and red blood cell content were also found (Fig. 2). Significant subepidermal collagenous fibrosis was present above the vessels (Fig. 3). A cystic sudoriparous duct was present in association with the melanocytic proliferation in the lower part of the lesion.FIGURE 2: Low-power magnification showing medium sized blood vessels within a congenital intradermal melanocytic nevus, with superficial fibrosis and dilated sweat ducts (hematoxylin and eosin stain, ×40).FIGURE 3: High-power view showing subepidermal fibrosis above medium sized vessels with no endothelial atypia (hematoxylin and eosin stain, ×400).A prominent vascular component is present histologically in a variety of tumors. In spite of this, a vascular striking component is not frequent in melanocytic lesions, although they commonly display pseudovascular features.4 Angiomatoid nevus is characterized by its distinctive fibrovascular stroma. It was initially described in Spitz nevus. To date, 13 cases have been characterized in the literature (12 corresponding to Spitz nevus and 1 to cellular blue nevus).1-4 In angiomatoid Spitz nevus, there is a proliferation of numerous small blood vessels with plump endothelia, resembling a vascular tumor. Melanocytes are embedded in a fibrous stroma composed of thick collagen bundles distributed haphazardly. In all cases, the stroma showed large numbers of small thick-walled blood vessels with round to oval lumina lined by plump endothelial cells with monomorphous nuclei devoid of atypia. Mitotic figures were rare or absent. The vessels were distributed in clusters or as solitary units throughout the stroma, and they were preferentially located in the upper part of the lesions. A variably prominent, mostly perivascular, or diffusely distributed lymphoplasmacytic infiltrate, was noted in association with these prominent vessels. This lymphocytic infiltrate was composed predominantly of small, mature lymphocytes.1,2 The reported case of cellular blue nevus displayed a prominent angioma-like appearance due to the presence of numerous ectatic vessels throughout the neoplasm. Vessels were lined by flattened endothelium, incompletely filled with red blood cells and fibrinous material, and also showed fibrinous hyalinized walls of variable thickness. They were associated with irregular pseudovascular spaces, devoid of endothelial lining and directly delimited by neoplastic melanocytes. The size of these vessels was higher and they were less numerous compared with those appreciated in angiomatoid Spitz nevus.4 The ectatic vessels found in our case are similar to those reported by Urso and Tinacci,4 and they were surrounded by a striking fibrous stroma that was also present in the subepidermal area. Clinically, the lesion was also a large nodule located in the lumbar area. Interestingly, dermoscopically, it showed a striking vascular pattern composed of large-sized vessels, which were embedded in a whitish veil that corresponded histologically with the significant fibrosis present in the papillary dermis and surrounding vessels. Regarding its developmental origin one might consider the combination of what appears to be the equivalent of a vascular malformation or anomaly, for example, a superficial arteriovenous malformation, and a dermal melanocytic nevus. Perhaps the lesion could be a developmental anomaly with 2 components: vascular and melanocytic rather than 1 alone. Such a lesion might fall, therefore, under the rubric of a chimeric nevus. In this patient, the vascular pattern was only present in the raised lesion, and the adjacent back was devoid of any vascular structure suggestive of an extensive vascular malformation. Moreover, although histological features suggest a congenital origin, the patient reported that it appeared 7 years ago. Nonetheless, many nevi exhibit relatively prominent vascular components and this simply may be an exaggerated version of a common nevus with prominent vascularity, although the combination of the prominent vascularity with the striking fibrosis is extremely rare. The causes that would provoke this fibrosis and vascular proliferation remain unknown. In our case, the location of the raised lesion on the lumbar region, made it susceptible to repeated trauma. There are entities such as atypical decubital fibroplasia in which there is also a prominent myxoid stroma rimmed by ectatic, thin-walled vascular channels as the consequence of continued pressure of the skin against the subjacent bone.5 We do not have an explanation for the fact that angiomatoid nevi affect almost exclusively women (13/14 cases). Hormonal factors might be involved as benign vascular lesions such as acquired angiomas can be affected by hormonal alterations due to pregnancy or contraceptive treatments.6 Although sometimes angiomatoid nevi may enter the differential diagnosis of melanoma, clinically, dermoscopically, or histologically,2,3 the absence of recurrences or metastases after complete excision in all cases reported supports the benign behavior of this lesion. José M. Martín, MD Luis Calduch, MD Verónica López, MD Esperanza Jordá, MD, PhD Department of Dermatology, Hospital Clínico Universitario, Valencia, Spain Carlos Monteagudo, MD, PhD Department of Pathology, Hospital Clínico Universitario, Valencia, Spain

Systemic vasculitis is a heterogeneous disorder characterized by chronic or acute inflammation, and is classified into groups by the size of the affected blood vessels: large vessel vasculitis, medium vessel vasculitis, or small vessel vasculitis. The pathogenesis of systemic vasculitis is not yet understood and except for Anti-neutrophil Cytoplasmic Antibodies (ANCA) for a subset of small vessel vasculitis, biomarkers for diagnosis and disease activity in systemic vasculitis have still to be discovered. Kawasaki Disease (KD) is an acute systemic vasculitis of infancy and early childhood. It mainly affects small and medium size blood vessels, and can cause cardiovascular complications like coronary artery aneurysm. Anti-endothelial Cell Antibodies (AECA) have been detected in a variety of diseases associated with vascular injury,and KD is one of them. Further, the presence of AECA has been correlated with disease activity, and/or its clinical manifestations [1,2]. A number of mechanisms for AECA, for example, activation and apoptosis of vascular Endothelial Cells (ECs), have been proposed [3]. In KD patients, IgM AECA mediate complement-dependent cytotoxicity against Human Umbilical Vein Endothelial Cells (HUVEC) and gammaglobulin, a standard therapy for KD, may reduce their effect [4]. Grunebaum et al. [5] demonstrated that AECA increased the secretion of Interleukin-6 (IL-6) from HUVEC, in KD patients. However, a significant role for AECA in systemic vasculitis has yet to be found. To evaluate the roles of AECA in detail, it is essential that their target antigens are identified and that the role of individual target antigens is assessed. Various approaches, such as expression libraries and proteomic, have been developed to identify biomarkers [6-11]. In KD, two target antigens for the AECA, tropomyosin and T-plastin, were identified using serological analysis of a recombinant cDNA expression library [11]. However, target antigens suitable for clinical applications, including Myeloperoxidase (MPO) and proteinase 3 of ANCA, remain to be discovered. Proteomics is a powerful tool for detecting and identifying biomarker proteins, because proteomics techniques allow the global analyses of protein function, modifications, composition and dynamics to be performed. In addition, current proteomic techniques can be used to analyze several samples from, for example, serum, urine, cerebrospinal fluid, and various tissues and cells. These kinds of analyses may lead to clinical applications. To identify the target proteins for AECA in patients with vasculitis, we used a proteomics approach that included 2-dimensional electrophoresis and Western Blotting, followed by mass spectrometry [9]. Briefly, we extracted the proteins from HUVEC and HeLa cells (control), and separated them by 2-dimensional electrophoresis to detect AECA antigens specific for ECs. We then analyzed the antigens by Western Blotting against serum samples from patients with vasculitis. We selected the spots that were detected only in the HUVEC samples, and not in HeLa cell samples. This procedure should identify candidate antigens, specific for ECs. We identified the detected proteins by peptide mass fingerprinting. Currently, we have detected more than 150 candidate antigenic spots, and identified more than 50 protein spots. In our earlier study, we identified Peroxiredoxin-2 (Prx2), a member of the Peroxiredoxin (Prx) family of peroxidases, as a target antigen for AECA [9]. In mammalian cells, the Prx family has at least six members (Table 1), and Prx2 is one of the most rapid and potent responders to oxidative stress. Furthermore, oxidative stress has been implicated as a pathogenic factor, and/or a progression-related factor in various diseases, including vasculitis. For these reasons, Prx2 was selected for further investigation. We investigated the clinical significance of anti-Prx2 antibodies in patients with KD. The titers of IgG antibodies to recombinant Prx2 were evaluated by ELISA (Enzymelinked Immunosorbent Assay), using 30 untreated patients with KD, including three patients with Coronary Artery Lesions (CALs), and 15 age- and sex-matched controls. The optical density value of the average ± 2 Standard Deviations (SDs) in the control individuals was defined as 100 AU, and values more than 100 AU, were regarded as positive. IgG antibodies to recombinant Prx2 were detected in 60% (18 of 30) of the untreated patients with KD, whereas no IgG antibodies were detected in the control individuals (Figure 1). We also evaluated the titers of IgM and IgA antibodies to recombinant Prx2 by ELISA, in patients with KD and in controls. IgM antibodies to recombinant Prx2 were

ANNA-1 Paraneoplastic Encephalomyelitis Presenting as Rapidly Progressing Dementia in a Man With Undiagnosed Small Cell Lung Cancer

Although inflammation and altered barrier functions of the vasculature, due predominantly to the infection of endothelial cell lining of small and medium-sized blood vessels, represent salient pathological features of human rickettsioses, the interactions between pathogenic rickettsiae and microvascular endothelial cells remain poorly understood. We have investigated the activation of nuclear transcription factor-kappa B (NF-kappaB) and p38 mitogen-activated protein (MAP) kinase, expression of heme oxygenase 1 (HO-1) and cyclooxygenase 2 (COX-2), and secretion of chemokines and prostaglandins after Rickettsia rickettsii infection of human cerebral, dermal, and pulmonary microvascular endothelial cells in comparison with pulmonary artery cells of macrovascular origin. NF-kappaB and p38 kinase activation and increased HO-1 mRNA expression were clearly evident in all cell types, along with relatively similar susceptibility to R. rickettsii infection in vitro but considerable variations in the intensities/kinetics of the aforementioned host responses. As expected, the overall activation profiles of macrovascular endothelial cells derived from human pulmonary artery and umbilical vein were nearly identical. Interestingly, cerebral endothelial cells displayed a marked refractoriness in chemokine production and secretion, while all other cell types secreted various levels of interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in response to infection. A unique feature of all microvascular endothelial cells was the lack of induced COX-2 expression and resultant inability to secrete prostaglandin E(2) after R. rickettsii infection. Comparative evaluation thus yields the first experimental evidence for the activation of both common and unique cell type-specific host response mechanisms in macrovascular and microvascular endothelial cells infected with R. rickettsii, a prototypical species known to cause Rocky Mountain spotted fever in humans.

Spotted fever rickettsioses due to Rickettsia rickettsii and R. conorii and epidemic typhus caused by R. prowazekii have been known to humankind since the beginning of the 20th century. Alarmingly, new species/subspecies of arthropod-borne rickettsiae are still being recognized and described as globally emerging pathogens. The genus Rickettsia includes obligate intracellular α-proteobacteria with affinity to infect vascular endothelium of small and medium-sized blood vessels in humans. Endothelial cells, key immunoreactive cells involved in host defense and inflammation, are intimately involved in the manifestations of rickettsial infections. The present review features the current understanding of cell signaling, host response, and apoptotic death mechanisms during in vitro infection of cultured human umbilical vein endothelial cells or endothelial-like cell lines with pathogenic rickettsiae and summarizes critical roles for infectioninduced oxidative stress, nuclear factor-kappa B, and p38 MAP kinase pathways in the regulation of innate immune responses and in preventing apoptosis early during infection to ensure host cell survival for rickettsial replication/spread. However, in the context of emerging concept of ‘endothelial heterogeneity’, the existing knowledge pertaining to the interactions of these unique intracellular pathogens with endothelial cells of different vascular beds remains in its infancy. A complete definition of signaling interactions between organ-specific host endothelial cells and strains of varying virulence and further detailed characterization of direct in vivo models of disseminated vascular infection represent major steps in advancing our depth of understanding of rickettsial pathogenesis, which should allow the development of novel antiinflammatory strategies to combat the pathologic sequelae of debilitating human rickettsioses.

Central nervous system involvement in rheumatoid arthritis is infrequent. The most frequent neurological manifestations of rheumatoid arthritis are peripheral neuropathy and cervical spinal cord compression due to subluxation of the cervical vertebrae. Cerebral rheumatoid vasculitis is an uncommon and serious complication which can be life-threatening Neurological involvement in RA is rare, present in only 1% of patients. Disorders of the central nervous system (CNS) include cervical myelopathy, vasculitis, RNs located within the CNS, or meningitis. Stroke also occurs with increased frequency. CNS vasculitis is extremely rare. The diagnosis is supported by magnetic resonance imaging (MRI), alone or with magnetic resonance angiography (MRA), showing the segmental vascular stenosis characteristic of vasculitis. Peripheral neuropathy is usually manifested as sensorimotor neuropathy or mononeuritis multiplex. The underlying mechanism is small vessel vasculitis of the vasa vasorum of the nerves with ischaemic neuropathy and demyelinisation as part of the rheumatoid vasculitis (RV) syndrome. Rheumatoid vasculitis typically affects small and medium-size blood vessels. It is associated with high rates of premature mortality with up to 40% of patients dying by 5 years as well as significant morbidity due to both organ damage from vasculitis and consequences of the treatment. High levels of circulating immune complexes have been observed in atients with rheumatoid vasculitis, and in particular high serum levels of rheumatoid factor are often detected at the time of onset of vasculitis.Deposition of immune complexes most likely contributes to small vessel inflammation and organ damage. Anti CCP levels also tend to be higher in patients with RA who have systemic vasculitis than in those who do not. Histologically, rheumatoid vasculitis involves blood vessels of the small arteries, and all layers of the vessel wall are infiltrated by neutrophils, lymphocytes, and plasma cells.

SP0149 Endothelial Damage in Anca-Associated Vasculitis (AAV)

A 44-year-old woman with a history of cocaine and heroin abuse presented with a painful, necrotic rash. Five weeks before presentation, the patient had experienced a cough and mild dyspnea. She was diagnosed with pneumonia, completed a 7-day course of ciprofloxacin and doxycycline, and her respiratory symptoms resolved. Three weeks before presentation, the patient had reported fevers, chills, diaphoresis, arthralgias, and myalgias, and developed a small erythematous lesion on her left breast. The lesion subsequently enlarged and became hemorrhagic. She simultaneously developed additional lesions on her chest, abdomen, arms, and back. The largest of these, located on her left arm, was 15 cm in diameter. All of the lesions were exquisitely tender. She was hospitalized at an outside hospital for 5 days and given a diagnosis of cocaine-induced vasculitis. Upon discharge, she was instructed to abstain from cocaine and was prescribed trimethoprim/sulfamethoxazole for a possible cellulitis involving the skin lesions. Ten days after discharge from the outside hospital, the patient presented to our institution for pain management and further evaluation. The rash had progressed since her earlier discharge. She had continued to use cocaine, heroin, and prescription opioids. She had last smoked cocaine 3 days before presentation and last used intranasal heroin 1 week before admission. She reported acquiring cocaine from a new supplier 2 weeks before her rash had begun. The patient had a history of nephrolithiasis and had undergone a cholecystectomy for gallstones. She had had 5 normal pregnancies. The patient had received ciprofloxacin, doxycycline, and trimethoprim/sulfamethoxazole in the weeks before her presentation. She also took combined oxycodone and acetaminophen for pain. She had no known drug allergies. Her youngest son had developed idiopathic thrombocytopenia at age 3 years. A paternal aunt had breast cancer and a nephew had cystinosis. She was unmarried, unemployed, and homeless. Three of her children were grown; the youngest two were in state custody. The patient had smoked 1 pack of cigarettes/day since age 15 years. She denied injection drug use and did not drink alcohol, but smoked cocaine and used intranasal heroin. The patient reported headache and fatigue secondary to poor pain control and disrupted sleep. She denied visual changes, sicca symptoms, jaw pain, oral ulcers, and dysphagia. She denied shortness of breath, cough, chest pain, palpitations, gastrointestinal symptoms, and abdominal pain. She had no genitourinary symptoms, arthritic pain, or musculoskeletal tenderness. She reported new upper extremity edema in her hands and arms bilaterally, more significant on the left than on the right, and equal bilateral lower extremity swelling. She had no sick contacts or recent travel. The patient was an obese woman in significant pain from her skin lesions. She was afebrile but had a pulse of 108/minute. Her blood pressure was 163/86 mm Hg, her respiratory rate was 22/minute, and her oxygen saturation was 97% on room air. She had a rash distributed in a symmetric pattern overlying fatty areas on the breasts, abdomen, upper arms, back, and flanks. The lesions consisted of stellate, purpuric plaques ranging in size from 5 cm to 15 cm (Figure 1A). The largest lesion was located on the left upper extremity (Figure 1B). The lesional borders were well demarcated and contained a reticular pattern surrounded by a 5-mm rim of erythema (Figure 1C). Small 1–2-cm lesions were also located on the earlobes bilaterally (Figure 1D). Many of the lesions were associated with flaccid bullae and exquisite tenderness to palpation. An additional dermatologic finding was a livedo pattern on her back and extremities. There was pitting edema in the upper and lower extremities bilaterally. Picture of the patient's necrotic rash. A, Multiple purpuric plagues were distributed symmetrically over the patient's back and flanks. Each lesion had reticulated borders consistent with a vasculopathic process. B, The largest lesion on the left arm had a necrotic appearance and contained central bullae. C, Borders were well demarcated and surrounded by a rim of erythema. D, Purpuric and necrotic lesions, 1–2 cm in diameter, were located on the earlobes. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)2151-4658. Examination of the head and neck was unremarkable, and there was no lymphadenopathy. The cardiovascular examination demonstrated a regular rate and rhythm, normal heart sounds, and no murmurs. The lungs were clear to auscultation. The abdomen was soft, nondistended, nontender, and without palpable masses. A neurologic examination showed intact cranial nerves and normal sensation and strength. Cerebellar signs were absent, and the gait was normal. The complete blood cell count, coagulation times, electrolytes, bilirubin levels, liver enzymes, cardiac biomarkers, and urinalysis were within normal limits. The patient had a mild normochromic anemia with a hematocrit of 35% and a mean corpuscular volume of 82 femtoliters (normal range 80–100). The serum iron saturation level was 4.5% and the ferritin was mildly elevated at 217 ng/ml (normal range 10–200). The erythrocyte sedimentation rate was 33 mm/hour (normal range 0–17). The patient's D-dimers were significantly elevated at 5,541 ng/ml (normal value <500), but the fibrinogen concentration was normal. Blood and urine cultures showed no growth. Assays for syphilis, hepatitis B and C, and the human immunodeficiency virus were nonreactive. An antinuclear antibody assay was positive at a titer of 1:160 (speckled). Assays for antibodies to both double-stranded DNA and histones were positive at titers of 1:20 (normal titer <1:10) and 2.2 units (normal value <1.5), respectively. Assays for antibodies to the Ro, La, Sm, and RNP antigens were negative. The serum C3 and C4 levels were 137 mg/dl (normal range 86–184) and 10 mg/dl (normal range 20–58), respectively; cryoglobulins were not detected. An IgM anticardiolipin antibody (aCL) assay was strongly positive at 150 IgM phospholipid units (normal range 0–15), but the IgG aCL assay was negative at 4.2 IgG phospholipid units (normal range 0–15). Assays for a lupus anticoagulant and antibodies to β2-glycoprotein I were negative. The serum antithrombin IIIA level was 66% (range 80–130%). Because of the resemblance of the skin lesions to warfarin necrosis, a serum warfarin level was assayed and found to be normal. An immunofluorescence assay for antineutrophil cytoplasmic antibodies (ANCAs) was positive in a perinuclear ANCA (pANCA) pattern of staining. Enzyme-linked immunosorbent assays (ELISAs) confirmed the presence of antibodies to myeloperoxidase (MPO ANCA). Antibody activity was measured at 1,050 units (normal value ≤2.8). An ELISA for antibodies to proteinase 3 (PR3) was negative. A serum parathyroid hormone (PTH) was normal, and assays for PTH-related peptide and plasma activated inhibitor 1 were negative. A serum protein electrophoresis with immunofixation studies was normal. Cocaine, oxycodone, and opiates were identified in a urine toxicology screen. A chest radiograph was normal, and ultrasound studies of the lower extremities and left arm showed no deep venous thromboses. A diagnostic test was performed. The patient is a 44-year-old woman with active cocaine and heroin use who presented with a painful, bullous, necrotic rash that developed after receiving a short course of antibiotics. The skin lesions have persisted despite conservative management for 5 weeks. The physical examination is otherwise remarkable for bilateral upper and lower extremity swelling. Laboratory studies are significant for an IgM aCL and MPO ANCA. Here we discuss the differential diagnosis of the patient's necrotic rash in the context of her laboratory abnormalities. A primary concern on admission was a hypercoagulable state. Heritable disorders of coagulation typically are considered in patients with a history of venous or arterial thrombi or pregnancy loss, or a family history of such events. Our patient had no such history. Laboratory testing confirmed the absence of mutations in the prothrombin, antithrombin III, and factor V Leiden genes. Protein C and S activities and the homocysteine level were normal. Acquired hypercoagulable states were considered early in the management of this patient. The patient had no history of recent surgery, immobilization, cancer, pregnancy, or hormonal therapy. All of these risk factors are associated more strongly with venous thrombi than with arterial clots, and ultrasonography excluded evidence of venous thrombosis. In any event, the distribution and necrotic appearance of the rash were more consistent with an arterial process. One common hypercoagulable state that leads to arterial or venous thrombi (or both) is the antiphospholipid syndrome (APS). The APS can develop at any age, is more common among women, and has a diverse range of clinical manifestations (1). The APS is characterized by the detection within serum of antiphospholipid antibodies (aPL); specifically, aCL, antibodies to β2-glycoprotein I, or a lupus anticoagulant. By the Sapporo criteria, the diagnosis of APS is entertained in the setting of a typical rash (e.g., livedo racemosa) and the presence of aPL within the serum (2, 3). To fulfill the laboratory criteria, aPL must be detected on at least two occasions separated by more than 12 weeks. Overall, we had a high suspicion of APS, given the clinical manifestations and laboratory findings of IgM aCL. Polyarteritis nodosa (PAN) is a systemic necrotizing vasculitis of medium-sized arteries that can affect any organ system. Skin involvement is common. The characteristic skin findings include livedo reticularis (racemosa), tender erythematous nodules, bullae or vesicular eruptions, and ulcers (4). Palpable purpura can be present to a minor degree and when found, represents concomitant small-vessel involvement. Skin lesions can result in infarction and gangrene of the distal extremities or deep ulcerations that extend into the subcutaneous tissue. In PAN, the cutaneous involvement is often accompanied by renal disease (e.g., renin-mediated hypertension, kidney infarctions), an axonal sensorimotor mononeuritis multiplex, and mesenteric vasculitis. None of these other features was present in our patient. A form of PAN limited to the skin exists, but that condition is more subacute in its presentation and seldom leads to such dramatic bulla formation (5). PAN is a “seronegative” condition, i.e., one that is not associated with known autoantibodies. This fact poses a significant challenge in diagnosis compared with, for example, systemic lupus erythematosus and the ANCA-associated vasculitides (AAVs). A minority of PAN patients have been infected recently by the hepatitis B virus and have serologies consistent with that condition. Our patient had no history of hepatitis B exposure, and the titers of aCL and MPO ANCAs suggested alternative diagnoses. Cryoglobulinemia was considered in the differential diagnosis because the patient had palpable purpura and bilateral earlobe lesions. Both of these physical findings suggest small vessel involvement. Nonetheless, the absence of lymphadenopathy, hepatosplenomegaly, peripheral neuropathy, and renal insufficiency made cryoglobulinemia less likely. Secondary causes of type I cryoglobulinemia were considered. Electrophoresis studies of the serum and urine were negative, appearing to exclude a paraproteinemia. Mixed cryoglobulinemia (types II and III) can occur secondary to hepatitis C or human immunodeficiency virus infections, but testing for our patient was negative. Most importantly, careful attempts to isolate cryoglobulins from the serum were unsuccessful. The necrotic rash and positive ANCA assay are consistent with an AAV. There are two major ANCA immunofluorescence patterns: cytoplasmic ANCA (cANCA) describes diffuse staining throughout the cytoplasm and pANCA refers to perinuclear staining. In patients with systemic vasculitis, the preponderance of cANCA staining is caused by antibodies directed against serine PR3. In contrast, in a patient who has a primary vasculitic syndrome, the pANCA pattern is generally caused by antibodies to MPO. In our patient, the high-titer MPO ANCA suggested several possibilities: Wegener's granulomatosis (WG), microscopic polyangiitis (MPA), Churg-Strauss syndrome (CSS), and drug-induced AAV. The clinical presentations of WG and MPA are difficult to distinguish in some cases. In WG, skin lesions can range from mild erythematous purpura to severe ulcerations (6). Skin manifestations are reported in 40% of patients with WG and generally correspond to the presence of generalized disease (7). However, vasculitic involvement of the skin in WG is unlikely to involve the trunk and upper extremities in the absence of lower extremity disease. In addition, our patient had no sign of the classic extracutaneous disease manifestations that typify WG: specifically, upper respiratory tract dysfunction, pulmonary disease, and glomerulonephritis. The MPO ANCA detected in our patient is more typical of either MPA or CSS than of WG. In MPA, skin findings usually consist of purpura or livedo racemosa rather than bullae and extensive necrosis (8). Approximately 50% of CSS patients are ANCA positive, and in such cases, the antibody specificity is directed against MPO. However, the diagnosis of CSS is unlikely in the absence of renal or pulmonary involvement and eosinophilia. In summary, the diagnosis of an idiopathic form of AAV is tenuous without some sign of extracutaneous disease. The constitutional symptoms, necrotic rash, and high MPO ANCA titer are consistent with both drug-induced AAV and drug-induced lupus. The two entities, which have been described both together and separately in the literature, may represent a spectrum of the same disease process. Both syndromes are associated with arthralgia, myalgia, and rash at disease onset (9). Dramatically elevated MPO ANCA titers are more strongly associated with drug-induced AAV than with idiopathic AAV (10). In addition, antihistone and aPL antibodies (both present in this case) are observed more commonly in drug-induced processes than in idiopathic AAV (11, 12). In both drug-induced ANCA vasculitis and lupus, the syndromes occur after exposure to a drug over a prolonged period of time. This contrasts with the short exposures to medications usually linked to hypersensitivity vasculitis syndromes, of which antibiotics are the most common offenders (13). Numerous medications have been implicated in both drug-induced AAV and drug-induced lupus (Table 1). However, the strongest correlations are with medications used to treat hyperthyroidism (particularly propylthiouracil) and with hydralazine, D-penicillamine, and minocycline (10, 14, 15). Our patient had no exposure to any of these agents. She had been treated previously with a short course of doxycycline, but tetracyclines other than minocycline have not been associated with drug-induced AAV (15). Ciprofloxacin has been implicated in vasculitis case reports (16, 17). In the 5 most recent reports, ciprofloxacin exposure ranged from 3–14 days, similar to our patient's 7-day course. However, the skin findings in ciprofloxacin-induced cutaneous vasculitis are generally mild in comparison to our patient's lesions, and those patients do not develop ANCAs or antinuclear antibodies (17). In both drug-induced AAV and drug-induced lupus, early withdrawal of the offending agent usually leads to resolution of the clinical syndrome (9). Prolonged cases of AAV can occur if exposure to the medication has been lengthy, as in some cases of propylthiouracil-associated disease. In our patient's case, her doxycycline and ciprofloxacin had been stopped even before the onset of the rash. In summary, the likelihood that the antibiotics to which she had been exposed had caused her severe skin rash appeared low. Case reports correlating cocaine use with skin lesions emerged first in the 1980s (18, 19). Since that time, palpable purpura; violaceous papules, bullae, and blisters; livedo racemosa with cutaneous necrosis; and multifocal necrotic lesions of the skin and muscle have been associated with cocaine use (4, 20-22). Skin biopsy samples in such patients often demonstrated vasculopathic patterns rather than frank vasculitis (21, 22). Cocaine use is well known to trigger a disorder known as midline destructive lesion that mimics limited WG closely, even including the finding of cANCA positivity on immunofluorescence testing (21-23). In such cases, ELISA testing demonstrates ANCA directed against PR3 and/or human neutrophil elastase (HNE). It is not clear if ANCA contributes to the pathogenesis of these lesions. HNE and PR3 are both serine proteinases that share gene localization as well as structural and functional characteristics. It has been speculated that the true antigen in cases of cocaine-induced midline destructive lesion is HNE, and that the PR3 ANCA results from cross-reactivity with HNE (21, 24). However, investigators recently demonstrated the coexistence of distinct antigen-specific antibodies that recognize unique epitopes (25). Cocaine use is also associated with the APS. One prospective study showed a higher percentage of aCL positivity in cocaine users compared with controls (26). Whether this was an incidental finding or has broader clinical implications for the occurrence of APS in the setting of cocaine use remains unknown. Some cocaine users who have experienced acute strokes or myocardial infarctions have been reported to have aPL, suggesting the possibility of a synergistic effect between cocaine and aPL that propagates thromboembolic phenomena (27, 28). Based on the patient's clinic findings and serologic abnormalities, the two most likely causes of the patient's presentation appeared to be either drug-induced AAV or drug-induced APS, either of which might have been induced by cocaine. Skin biopsy was essential to distinguish between these two conditions, which require different approaches to treatment (in addition to cocaine cessation). Skin biopsy samples were obtained from the right proximal and distal forearm. Histologic examination revealed fibrin thrombi within small and medium-sized blood vessels in the superficial and deep dermis and the subcutaneous tissue (Figures 2A and B). There was no evidence of endothelial swelling, leukocytoclasis, or fibrinoid necrosis within blood vessel walls. The histopathology was thought to be highly consistent with APS. Skin pathology. A, Small and medium-sized vessels (arrows) occluded by fibrin thrombi in the superficial dermis, deep dermis, and subcutaneous tissue (original magnification × 40). Inset: medium-sized artery with the lumen almost completely occluded by an organizing fibrin thrombus (original magnification × 200). B, Multiple small arteries (arrows) with luminal fibrin thrombi (original magnification × 200). The most striking features of this case were the rapid appearance of the patient's bullous, necrotic rash and the high titers of both aPL and MPO ANCA in her blood. Although we believe that both autoantibodies were induced by her cocaine use, the histopathology of her skin lesions suggests that aPL were responsible for her skin rash. This conclusion is consistent with the current knowledge regarding drug-induced ANCA positivity. In several cross-sectional and prospective studies of propylthiouracil, between 20% and 60% of patients treated with this medication on a long-term basis developed ANCA, usually in high titers (29, 30). However, only a minority of patients who developed ANCA demonstrate clinical manifestations of drug-induced AAV. In one study (31), 8 (26.7%) of 30 patients receiving long-term antithyroid medication became ANCA positive. Among those, none developed frank AAV but 3 experienced myalgia and arthralgias after the appearance of ANCAs. Patients with idiopathic AAV are known to have a predisposition to venous thrombotic events (32, 33). Variable degrees of thrombosis can also be observed on lesional biopsy samples from patients with AAV, but such findings are accompanied by more classic patterns of injury to the blood vessel wall: endothelial swelling, leukocytoclasis, and fibrinoid necrosis. Although it is impossible to exclude an interaction between the patient's aPL and MPO ANCA that heightened the intensity of her cutaneous reaction, the major histopathologic findings implicate aPL as the primary etiology of the patient's skin disease. One theory of APS pathogenesis proposes that aPL develop in genetically susceptible individuals after exposure to an unknown infectious agent. Clinical manifestations subsequently occur after a “second hit” such as smoking, prolonged immobilization, pregnancy, hormone use, or cancer. Various medications are suspected triggers of APS (Table 1). IgM aCL in particular have been linked to drug-induced APS cases. Cocaine has also been implicated in the pathogenesis of APS. Although our patient was a chronic cocaine user, it is of interest that prior to the onset of her rash she had found a new cocaine supplier, perhaps leading to exposure to new contaminants in the cocaine. One commonly recognized contaminant in cocaine is levamisole. Levamisole, an alkaline phosphatase inhibitor, appears to potentiate the action of cocaine on dopaminergic pathways. Levamisole is widely available because it is used in agriculture worldwide as an antihelminthic drug. In humans, levamisole has been studied in various clinical settings for the treatment of autoimmune and malignant diseases (34). The compound has established efficacy in relapsing nephrotic syndrome in children. Most recently, levamisole has been reported to cause agranulocytosis in cocaine users (35, 36). Approximately 70% of seized cocaine entering the US contains levamisole (35). We conducted a literature search on levamisole-induced vasculitis and vasculopathies. Levamisole-induced vasculitis has only been reported in 4 adult cases (37-39). The histopathology reported in these describes vasculitis, but the results of ANCA or aPL testing were not In the literature, there are case reports of cutaneous vasculitis secondary to levamisole Five cases were characterized by earlobe purpura and necrosis, as in our case In 4 of the skin biopsy samples demonstrated a thrombotic ANCA serologies were reported in 4 pANCA patterns in 3 cases and a cANCA pattern in 1 aPL antibodies were detected in 3 We that levamisole is the active compound in cases of cocaine-induced vasculitis or cocaine-induced This further associated with antiphospholipid her was with a and was to warfarin prior to discharge. was for the diagnosis of APS. the high ANCA there was among the the patient from her acute presentation and the of the skin lesions, the patient was on and this her skin lesions and her was after 3 after her she had from cocaine and had no of her rash. She has developed of her necrotic skin rash. All were in the or it for and the to be for had to of the in the study and for the of the and the of the and of and of

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