Norbert Sepp

Endothelial cells from cord blood CD133+CD34+ progenitors share phenotypic, functional and gene expression profile similarities with lymphatics

The existence of endothelial progenitor cells (EPC) with high cell‐cycle rate in human umbilical cord blood has been recently shown and represents a challenging strategy for therapeutic neovascularization. To enhance knowledge for future cellular therapy, we compared the phenotypic, functional and gene expression differences between EPC‐derived cells generated from cord blood CD34+ cells, and lymphatic and macrovascular endothelial cells (EC) isolated from human foreskins and umbilical veins, respectively. Under appropriate culture conditions, EPC developed into fully matured EC with expression of similar endothelial markers as lymphatic and macrovascular EC, including CD31, CD36, von Willebrand factor FVIII, CD54 (ICAM‐1), CD105 (endoglin), CD144 (VE‐cadherin), Tie‐1, Tie‐2, VEGFR‐1/Flt‐1 and VEGFR‐2/Flk‐1. Few EPC‐derived cells became positive for LYVE‐1, indicating their origin from haematopoietic stem cells. However they lacked expression of other lymphatic cell‐specific markers such as podoplanin and Prox‐1. Functional tests demonstrated that the cobblestone EPC‐derived cells up‐regulated CD54 and CD62E expression in response to TNF‐α, incorporated DiI‐acetylated low‐density liproprotein and formed cord‐ and tubular‐like structures with capillary lumen in three‐dimensional collagen culture – all characteristic features of the vascular endothelium. Structures compatible with Weibel‐Palade bodies were also found by electron microscopy. Gene microarray profiling revealed that only a small percentage of genes investigated showed differential expression in EPC‐derived cells and lymphatic EC. Among them were adhesion molecules, extracellular matrix proteins and cytokines. Our data point to the close lineage relationship of both types of vascular cells and support the theory of a venous origin of the lymphatic system.

Recruitment of plasmacytoid dendritic cells in ultraviolet irradiation‐induced lupus erythematosus tumidus

SIR, We report a rare case of inhaler-induced burn in a 13-year-old boy caused by applying the nozzle of the inhaler to his hand and discharging the inhaler. A 13-year-old boy presented with a 4-day history of blisters around the edge of an erythematous patch on the dorsum of his left hand, which had been present for a year. His sister suffered from asthma. On examination, there was a welldemarcated erythematous patch on the dorsum of his left hand with crusts from ruptured blisters around the edge of the patch (Fig. 1). On further questioning, he admitted having developed this red patch 1 year previously after he had applied the nozzle of his sister’s salbutamol inhaler onto his hand and discharged it a few times playfully. The repetition of this practice had resulted in the inhaler-induced burns we see in this picture. Inhaler-induced burn is very rare, and to our knowledge there are only three cases reported in the literature. All three patients were asthmatic. Chemical burn from the pharmaceutical ⁄preservative ⁄propellant aerosol, cold injury to the skin or mechanical abrasive effect of the aerosol blasts were the suggested mechanisms of injury. Burge et al. suggested that the physical signs of inhaler-induced burn are similar to those seen after a standardized cryotherapy freeze injury to the skin. Children who gain access to inhalers may find them to be interesting toys. They could then sustain chemical burns due to inappropriate discharge onto the skin. We need to be aware of this effect of commonly used inhalers and warn parents, teachers and children in order to avoid these injuries.

Oral mucous squamous cell carcinoma-an anticipated consequence of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED).

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomal recessive disease caused by mutations in the AIRE gene. We report the case of a female patient with a 967-979del13 mutation in the AIRE gene. Her medical history included autoimmune hypoparathyroidism, Addison disease, and chronic mucocutaneous candidiasis. At the age of 40, she developed multiple white verrucous plaques on the oral mucosa. Histologically, the lesions appeared as moderately differentiated squamous cell carcinomas. The patient subsequently developed multiple local recurrences and therefore required repeated surgery. Notably, a higher incidence rate of oral and esophageal squamous cell carcinoma has been observed in this syndrome. However, the critical pathogenetic pathways implicated in squamous cell carcinoma development in APECED are far from being well understood.

Isolation and characterization of CD133 + CD34 + VEGFR-2 + CD45 − fetal endothelial cells from human term placenta☆

The phenotypes and functions of endothelial cells (EC), a heterogeneous cell population, vary along the vascular tree and even in the same organ between different vessels. The placenta is an organ with abundant vessels. To enhance further knowledge concerning placenta derived EC, we develop a new method for isolation, purification and culture of these EC. Moreover, in order to investigate the peculiarity of placenta derived EC we compare their phenotypic and functional characteristics with human dermal lymphatic endothelial cells (HDLEC) and human umbilical vein endothelial cells (HUVEC). Freshly isolated placenta derived EC displayed an elongated shape with pale cytoplasm and showed the typical cobblestone pattern of EC but also a swirling pattern when confluent. FISH-analyses of the isolated EC from placentae of male fetus revealed an XY genotype strongly indicating their fetal origin. Characterisation of placenta derived fetal EC (fEC) underlined their blood vessel phenotype by the expression of vWF, Ulex europaeus lectin-1, HLA-class I molecules, CD31, CD34, CD36, CD51/61, CD54, CD62E, CD105, CD106, CD133, CD141, CD143, CD144, CD146, VEGFR-1, VEGFR-2, EN-4, PAL-E, BMA120, Tie-1, Tie-2 and α-Tubulin. In contrast to previous reports the expression of lymphatic markers, like VEGFR-3, LYVE-1, Prox-1 and Podoplanin was consistently negative. Haematopoietic surface markers like CD45 and CD14 were also always negative. Various functional tests (Dil-Ac-LDL uptake, Matrigel assay and TNF-α induced upregulation of CD62E and CD54) substantiated the endothelial nature of propagated fEC. At the ultrastructural level, fEC harboured numerous microvilli, micropinocytic vesicles at their basis, were rich in intermediate filaments and possessed typical Weibel - Palade bodies. In conclusion, the placenta is a plentiful source of fetal, microvascular, blood EC with an expression profile (CD34+, CD133+, VEGFR-2+, CD45-) suggestive of an endothelial progenitor phenotype.

Chondrodermatitis nodularis chronica helicis – a conservative therapeutic approach by decompression

Background: Chondrodermatitis nodularis chronica helicis is a rather common condition which substantially affects quality of life causing pain and sleeping disturbances. Surgical treatment is connected with a tendency to recurrence. Mechanical pressure is probably the main etiological factor.

Infantile hemangioma is a proliferation of LYVE-1-negative blood endothelial cells without lymphatic competence

Infantile hemangiomas are common benign vascular tumors that exhibit a characteristic history of rapid proliferation in the first year of life and slow spontaneous involution during early childhood. The causative pathogenic event responsible for the abnormal endothelial proliferation remains elusive. The recent discovery of an immature phenotype of proliferating hemangioma endothelial cells due to the exclusive expression of the lymphatic endothelial hyaluronan receptor LYVE-1 led to the proposal that infantile hemangiomas are the result of a primary defect in endothelial cell maturation. To test this hypothesis, we looked for the expression of the lymphatic endothelial cell-specific markers LYVE-1, Prox-1, podoplanin and D2-40 in β4 integrin-negative proliferating and β4 integrin-positive involuting infantile hemangiomas. As β4 integrin proved to be a suitable marker for staging infantile hemangiomas, we used it in combination with clinical and histological criteria to objectively determine the proliferative and involutional phases. In immunohistochemical and immunofluorescent stains, hemangioma vessels were negative for all lymphatic endothelial cell-specific markers tested during both proliferation and involution. LYVE-1 immunoreactivity, however, was found in the dense network of perivascular HLA-DR-positive cells with dendritic cell morphology that are supposed to play a role in hemangiogenesis by releasing pro- and antiangiogenic factors. Notably, this LYVE-1 staining failed to correlate with the growth status of infantile hemangiomas. Our results do not support the notion that LYVE-1 expression was restricted to the proliferative phase and downregulated during involution. Thus, LYVE-1 does not seem to be a reliable marker for proliferating infantile hemangiomas. We conclude that the suggested intrinsic defect in endothelial cell maturation is unlikely the cause for the post-natal rapid growth in infantile hemangiomas. In addition, the lack of lymphatic endothelial cell-specific markers implies that infantile hemangiomas are tumors of blood vessels without lymphatic competence.

Human herpesvirus-8 infection of umbilical cord-blood-derived CD34+ stem cells enhances the immunostimulatory function of their dendritic cell progeny

Abstract:  CD34+ progenitor cells carrying human herpesvirus‐8, Kaposis sarcoma‐associated herpesvirus (HHV‐8/KSHV), have been described in the peripheral blood of AIDS patients suffering from Kaposis sarcoma (KS). In this study, we investigated the influence of HHV‐8 on the differentiation of CD34+ progenitor cells. Native CD34+ cells derived from cord blood could be infected by a laboratory strain of HHV‐8, as shown by immunofluorescence staining and polymerase chain reaction, but no significant initial maturation/differentiation effects were observed. In addition, these infected cells were differentiated into immature and mature dendritic cells (DCs) using cytokine induction with recombinant human granulocyte‐macrophage colony‐stimulating factor (rhGm‐CSF), recombinant human tumor necrosis factor (rhTNF‐α) and recombinant human stem cell factor (rhSCF). Double immunofluorescence and flow cytometry studies demonstrated that virus infection did not impair the development of immature and mature DC populations. Subsequently, the immunostimulating capacity of DC populations was tested in a mixed lymphocyte reaction using allogeneic T‐cells. The HHV‐8‐infected CD34+ progenitor cell‐derived mature DC population showed a significantly enhanced antigen‐presenting capacity, compared to non‐infected DCs, which was not observed with the immature DCs. This suggests stimulation of DC function by HHV‐8 infection. Because there are only a small percentage of HHV‐8‐positive DCs in the preparations and because it is not clear whether infection is abortive or productive to some extent, this seems to be most likely due to an indirect viral effect.

Danger lurks in the Mediterranean.

In February, 2009, a 52-year-old woman presented to us with a 2-month history of progressive swelling of the right cheek, which did not aff ect the eyelid or the lips. She had no previous medical history, recent trauma, surgery, medication (particularly no ACE inhibitors, which can cause facial swelling) and no history of skin tumours or autoimmune diseases. She was otherwise well, with no dyspnoea, dysphagia, xerostomia, pain, or itching. At another hospital, dental radiography and CT of the head showed right cheek soft tissue swelling. Histological examination of a biopsy sample of the buccal mucosa showed non-specifi c infl ammation. No exact diagnosis was established. The patient was prescribed amoxicillin with clavulanic acid for 3 weeks, followed by corticosteroids for 1 week, without clinical improvement. She was then admitted to our department for further investigations. On examination, she had distinct, soft swelling of her right cheek without infl ammation or scaling of the overlying skin. A subcutaneous mobile nodule was palpable enorally in the centre of the swelling. Saliva could be expressed from the salivary gland. Possible causes of unilateral cheek swelling (surgery, trauma, congenital disorders, drugs, dental disorders, salivary gland disorders, tumours, vasculitis, allergy, infections, and chronic infl ammatory disorders) were considered. ESR, blood count, aspartate and alanine aminotransferases, glomerular fi ltration rate, creatinine concentrations, and creatine kinase were normal. Serum tests for viruses, stool tests for parasites, and antinuclear antibodies were all negative. MRI showed diff use lymphoedema of the right cheek with a central distinct infl ammatory process. A deep incisional biopsy was done enorally at the centre of the right cheek swelling. Histology showed a chronic infl ammatory process with epitheloid granulomas. In the centre of one granuloma, we found an eosinophilic sickle-shaped fi gure, which was a section of a nematode identifi ed as Dirofi laria repens (fi gure). PCR confi rmed the diagnosis. Our patient was started on albendazole 400 mg twice daily for 3 weeks. The cheek swelling resolved within 4 weeks. On questioning about travel history, our patient recalled an insect bite 6 months previously, in Florence, Italy. At follow-up in June, 2010, the swelling had not recurred. D repens, a nematode of dogs and other domestic and wild carnivores, is endemic in southern and eastern Europe, south and central Asia, and Africa. It is transmitted by mosquitoes. People are only accidental hosts, and the parasites may be destroyed by the immune response. Pre-adult worms migrate subcutaneously and cause non-specifi c infl ammatory symptoms such as subcutaneous oedema due to obstructed lymph vessels. Subcutaneous nodules are a result of foreign-body granulomas around the worm. Infection is most common in the upper body, especially the face. The epidemiology of human dirofi lariasis is related to the prevalence of infected dogs, the presence of mosquitoes, suitable climate, humidity, and human activities that lead to exposure. The incidence in Europe has grown substantially in the past 50 years. Diagnosis is made on morphological examination of the worm, after surgical excision. Eosinophilia or raised IgE concentrations are rare. Ultrasonography and MRI can diff erentiate subcutaneous dirofi larial lesions from those of other aetiologies. Treatment of cutaneous dirofi lariasis consists of total excision of the nodule. The goal of anthelmintic therapy (ivermectin or albendazole) is to eradicate the infection if secondary lesions or incomplete excision are suspected. With global warming, fi larial infection might spread into previously infection-free areas. Dirofi lariasis must be considered in particular in cases with chronic progressive swelling.

Analysis of 303 Ro/SS-A antibody-positive patients: is this antibody a possible marker for malignancy?

Background  The risk of cancer in patients with autoimmune diseases has been investigated in several studies. Ro/SS‐A antibodies are frequent and specific autoantibodies among patients with various autoimmune diseases.

Hepatitis C virus and autoimmunity

Hepatitis C virus infection is associated with several extrahepatic manifestations. About 60% of patients infected with HCV develop at least one extrahepatic manifestation. The majority of these diseases seem to be triggered through autoimmune mechanisms, such as autoantibody production, autoreactive T cells and complex autoimmune mechanisms leading to systemic autoimmune disorders. In this review we categorize these diseases into three groups according to the main pathogenetic process involved, in particular B-cell-mediated, T-cell-mediated and complex autoimmune systemic diseases.