Direct immunofluorescence diagnosis of pemphigus without biopsy

Background: Direct immunofluorescence (DIF) is a necessary examination tool for the diagnosis of pemphigus. The suction-blister-method splits the skin at the lamina lucida and it is possible with a scalpel to separate the entire epidermis from the dermis. Objective: The study was to determine whether DIF is reliable on epidermal sheets separated using a suction apparatus. Methods: Thirteen patients were selected for this study: (nine with pemphigus vulgaris (PV), one with paraneoplastic pemphigus (PP), and three with pemphigus erythematosus (PE). Frozen epidermal sheets, separated from the dermis with a scalpel, were used as a substrate. Diagnosis with routine fluorescein isothiocyanate (FITC) antibodies was made. Results: In all patients a pericellular deposition of IgG was evident and in eight of these patients a pericellular deposition of C3 was present. In two cases of PE and one of PP, the C3 deposits were also present in the lower part of basal keratinocytes. Conclusion: This diagnostic method without skin biopsy is easy to perform and, together with the histology and clinical aspects, could be a useful tool in the diagnosis of pemphigus. We recommend this method when the patient is allergic to local anaesthetics, the patient easily produces hypertrophic scars, or in follow-up of already biopsied patients

Direct immunofluorescence diagnosis of pemphigus without biopsy

Background: Direct immunofluorescence (DIF) is a necessary examination tool for the diagnosis of pemphigus. The suction-blister-method splits the skin at the lamina lucida and it is possible with a scalpel to separate the entire epidermis from the dermis. Objective: The study was to determine whether DIF is reliable on epidermal sheets separated using a suction apparatus. Methods: Thirteen patients were selected for this study: (nine with pemphigus vulgaris (PV), one with paraneoplastic pemphigus (PP), and three with pemphigus erythematosus (PE). Frozen epidermal sheets, separated from the dermis with a scalpel, were used as a substrate. Diagnosis with routine fluorescein isothiocyanate (FITC) antibodies was made. Results: In all patients a pericellular deposition of IgG was evident and in eight of these patients a pericellular deposition of C3 was present. In two cases of PE and one of PP, the C3 deposits were also present in the lower part of basal keratinocytes. Conclusion: This diagnostic method without skin biopsy is easy to perform and, together with the histology and clinical aspects, could be a useful tool in the diagnosis of pemphigus. We recommend this method when the patient is allergic to local anaesthetics, the patient easily produces hypertrophic scars, or in follow-up of already biopsied patients

Gelatinases A and B are expressed in late lesions in autoimmune bullous disorders

Gelatinases (or metalloproteinase or collagenases) are involved in remodelling the extracellular matrix in several skin disorders. Previous reports show that the 72 kDa (Gelatinase A), the 92 kDa collagenase (Gelatinase B) and their inhibitors TIMP-2 and TIMP-1 respectively are overexpressed in tumor invasion and metastasis, granuloma annulare, necrobiosis lipoidica diabeticorum and bullous pemphigoid. Usually their natural inhibitors, TIMP-1 and TIMP-2, are inversely related to the production of the 72 kDa and 92 kDa proteins. 8 patients affected by Bullous Pemphigoid (BP) and 8 patients affected by Pemphigus Vulgaris (PV) were biopsized in perilesional areas of young lesions and in lesional areas of old lesions. Materials were snap frozen in liquid nitrogen until use. Antibodies to 72 kDa, 92 kDa, TIMP-1, TIMP-2, CD44, laminin, collagen type I, III, IV, VII were used and evidentiated by the avidin-biotin immunoperoxidase technique. mRNA expression for Gelatinases and their inhibitors were also analyzed by RT-PCR. In all patients we found gelatinases expression only in the late lesions. A different expression was found between the two diseases, the 92 kDa protein and its inhibitor TIMP-1 were positive in both PB and PV whereas the 72 kDa form and its inhibitor TIMP-2 were evident only in PB. By RT-PCR we show that the 72 kDa mRNA was expressed exclusively in the dermis, on the contrary the 92 kDa was present in epidermis and dermis. No signals were detected in the early phase of blistering suggesting a role of gelatinases in re-epithelization but not in blistering where other proteases play a major role. The differential expression of Gelatinases and their inhibitors is probably under a cytokine network control

KSHV mRNA expression in tissue samples from Kaposi's sarcoma

Kaposi's sarcoma (KS) etiology has generally been thought to be of multifactorial origin involving genetic predisposition, geographical factors, and endogenous influences such as angiogenic factors. Recently a new herpesvirus later called with the descriptive name of Kaposi's sarcoma associated Herpes Virus (KSHV), had been discovered in tissue samples of KS skin lesions and in peripheral blood of these patients. KSHV has been found in all types of Kaposi's sarcoma and many studies give strong basis to affirm that this virus has a role in the etiology of this disease. We performed a molecular biology study to investigate the presence and the replication of this virus in four patients affected by KS; 3 with Classical KS form (CKS) and 1 with Iatrogenic KS associated with multiple myeloma (IKS). Two specimens of CKS and the IKS one were taken from recent onset lesions of rapidly progressive disease at a papule-nodules stage, while another specimen of CKS was from a long-standing lesion (more than 20 years) without any tendency for progression. All patients were HIV negative. Control skin samples were obtained from patients undergoing plastic surgery. Both for the Polymerase chain reaction (PCR) and for the reverse transcriptase-poymerase chain reaction (RT-PCR) the primers KS1 and KS2 were used, which amplify a 233 bp sequence from KSHV genome. All three specimens from recent onset lesion resulted positive for KHSV presence at the PCR and also at the RT-PCR demonstrating an active replication of KSHV in these lesions. Instead both the PCR and the RT-PCR of the long standing CKS lesion failed to demonstrate respectively the presence and the replication of the virus even if the numbers of the cycles were raised. Our report is the first in which not only DNA but also KSHV mRNA was demonstrated, showing the active replication of the virus in lesions of recent onset. The negative result in the long standing KS lesion could be just a false negative, giving the extreme sensibility of PCR, on the other hand it could be that either the presence of the virus nor its replication is necessary for the disease to maintain itself after long periods and a particular cytokine network is responsible for the long lasting 'slowly developing' disease sometimes seen in Classic KS. Our findings on the replication of KSHV in recent and progressive disease further support the hypothesis that KSHV reactivation or infection could take place in particular immunological conditions and thus that KSHV could have an early causative role in the development of KS of different origin

Gelatinases A and B are expressed in late lesions in autoimmune bullous disorders

Gelatinases (or metalloproteinase or collagenases) are involved in remodelling the extracellular matrix in several skin disorders. Previous reports show that the 72 kDa (Gelatinase A), the 92 kDa collagenase (Gelatinase B) and their inhibitors TIMP-2 and TIMP-1 respectively are overexpressed in tumor invasion and metastasis, granuloma annulare, necrobiosis lipoidica diabeticorum and bullous pemphigoid. Usually their natural inhibitors, TIMP-1 and TIMP-2, are inversely related to the production of the 72 kDa and 92 kDa proteins. 8 patients affected by Bullous Pemphigoid (BP) and 8 patients affected by Pemphigus Vulgaris (PV) were biopsized in perilesional areas of young lesions and in lesional areas of old lesions. Materials were snap frozen in liquid nitrogen until use. Antibodies to 72 kDa, 92 kDa, TIMP-1, TIMP-2, CD44, laminin, collagen type I, III, IV, VII were used and evidentiated by the avidin-biotin immunoperoxidase technique. mRNA expression for Gelatinases and their inhibitors were also analyzed by RT-PCR. In all patients we found gelatinases expression only in the late lesions. A different expression was found between the two diseases, the 92 kDa protein and its inhibitor TIMP-1 were positive in both PB and PV whereas the 72 kDa form and its inhibitor TIMP-2 were evident only in PB. By RT-PCR we show that the 72 kDa mRNA was expressed exclusively in the dermis, on the contrary the 92 kDa was present in epidermis and dermis. No signals were detected in the early phase of blistering suggesting a role of gelatinases in re-epithelization but not in blistering where other proteases play a major role. The differential expression of Gelatinases and their inhibitors is probably under a cytokine network control

A Th2-like cytokine response is involved in Bullous Pemphigoid. The role of IL-4 and IL-5 in the pathogenesis of the disease

Bullous Pemphigoid is an autoimmune bullous disorder characterized by production of IgG against an hemidesmosomal antigen (230 kDa, 180 kDa) responsible for blistering of the skin. In the past several mediators have been implicated in the pathogenesis of the disease such as proteases and collagenases secreted by local inflammatory cells. In order to investigate the role of cytokines in BP, the cytokine pattern was evaluated by an immunohistochemical analysis and by reverse transcriptase polymerase chain reaction procedure in 13 BP patients. Cytokines examined were interleukin (IL)-2, IL-4, IL-5, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. The T cell inflammatory infiltrate was also characterized by monoclonal antibodies showing CD3+, CD4+ T cells with a perivascular and scattered distribution in lesional skin. IL-4 and IL-5 were detected in a similar distribution to the inflammatory infiltrate. IL-4 and IL-5 mRNA levels were also revealed by RT-PCR. Proinflammatory cytokines such as TNF-α, IL-6 and Th1-like cytokines (IL-2 and INF-γ) were not detected neither as proteins nor as mRNA. Since IL-4 and IL-5 are important in eosinophil chemoattraction, maturation and functional activity, the presence of IL-4 and IL-5 in BP suggest that these cytokines could be important in the pathogenesis of the disease

'Suction split' as a routine method to differentiate epidermolysis bullosa acquisita from bullous pemphigoid

Background and Design: Epidermolysis bullosa acquisita (EBA) and bullous pemphigoid (BP) are diseases with similar clinical, histological, and immunofluorescent findings. Diagnosis requires the use of immunoelectron microscopy, immunoprecipitation or immunoblotting, but in recent years the differential diagnosis has been based on a cheaper technique named salt split skin. This study demonstrates that with a suction blister the fracture is at the same level as that obtained with the sodium split method anti that it is also faster and cheaper. Suction blisters on normal skin and autoimmune perilesional bullous lesions, obtained with a hand vacuum pump, were studied by direct immunofluorescence and electron microscopy to evaluate the level of the split on normal suction split skin. Normal human split skin was also used as a substrate for an indirect immunofluorescent study using sera of patients with BP (68 sera), EBA (10 sera) and cicatricial pemphigoid (CP) (16 sera). Direct immunofluorescent examination was also done on perilesional skin after artificial separation obtained with a hand-vacuum pump in patients with the same diseases listed above (32 BP, 11 CP, 6 EBA). Results: On normal human skin split by suction or sodium chloride (NaCl; 1 mol/l) direct immunofluorescence and electron microscopy demonstrated that the split is at the lamina lucida level. Indirect immunofluorescent study of both normal human skin and perilesional skin split using suction as a substrate showed IgG deposits localized on the floor of the suction blister in all cases of EBA, whereas in over 88% of cases of BP and in over 62% of CP the IgG were localized on the roof. Similar results were obtained with direct immunofluorescence in perilesional skin. Conclusions: 'Suction split' represents a simple technique to differentiate EBA from BP. This method provides final response in a few hours compared to at least 1-2 days with the sodium split method. Furthermore, the suction split method is cheaper and the tissue can be re-utilized for molecular biology and immunohistochemical studies

Carbamazepine-induced hypersensitivity syndrome in a chile with epilepsy

Carbamazepine is an effective anticonvulsant and is considered the drug of first choice for the treatment of partial and secondarily generalized seizures. Although carbamazepine is well tolerated, many side effects have been reported in the literature. The majority of these adverse effects are transient and do not lead to the discontinuation of the therapy. We present a case of a female child, aged 11 years and 6 months, who showed an anticonvulsant hypersensitivity syndrome induced by carbamazepine. This syndrome is a rare, potentially life-threatening adverse drug reaction. The patient developed a cutaneous nonpruritic rash, associated with high fever, diffuse lymphadenopathy, and arthralgias on the knees and the ankles with local signs of arthritis. Laboratory examination showed a lymphocytosis, mild thrombocytopenia, marked eosinophilia, and high transaminases. Corticosteroid therapy (betametasone 0.5 mg x 3 day) was started and carbamazepine was gradually withdrawn changing to valproic acid, with complete control of the seizures. The fever and the rash reduced gradually, beginning from the face and then disappearing completely after 10 days. Laboratory results showed a clear improvement: after 7 days the patient showed a complete normalization of the above parameters, except for transaminases. The complete normalization of these enzymes was observed after 2 weeks from the disappearance of the skin rash

The 72-kDa and the 92-kDa gelatinases, but not their inhibitors TIMP-1 and TIMP-2, are expressed in early psoriatic lesions

Psoriasis is histologically characterized by hyperkeratosis and papillomatosis with elongated vessels in the upper dermis. In order to evaluate the role of gelatinases in remodelling psoriatic skin in this study we examined the production of the 72-kDa (gelatinase A), 92-kDa collagenase (gelatinase B) and their tissue inhibitors TIMP-2 and TIMP-1. A total of 19 patients affected by different types of psoriasis were included in this study. An immunohistochemical study on cryosections was performed using antibodies to 72-kDa gelatinase, 92-kDa gelatinase, TIMP-1, TIMP-2, laminin, collagen types I, III, IV, VII. mRNA expression for gelatinases and their inhibitors were also analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). In 14 of 19 patients there was a positivity in 92-kDa protein expression in keratinocytes. The 92-kDa gelatinase protein was also present in the upper dermis with prevalence around blood vessels. In 15 of 19 patients the 72-kDa was localized in the upper dermis, almost exclusively in the papillary dermis but absent in epidermis. TIMP-1 and TIMP-2 were both negative in all cases in immunoperoxidase and RT-PCR. Using RT-PCR we show that the 72-kDa mRNA is expressed exclusively in the dermis, on the contrary the 92-kDa was present in epidermis and dermis. Type I, III, IV and VII collagens did not show any alteration or disruption. Overexpression and production of gelatinases without inhibitory effects suggest a role of these proteins in remodelling the psoriatic skin probably inducing the typical histological pattern of papillomatosis

Carbamazepine-induced hypersensitivity syndrome in a chile with epilepsy

Carbamazepine is an effective anticonvulsant and is considered the drug of first choice for the treatment of partial and secondarily generalized seizures. Although carbamazepine is well tolerated, many side effects have been reported in the literature. The majority of these adverse effects are transient and do not lead to the discontinuation of the therapy. We present a case of a female child, aged 11 years and 6 months, who showed an anticonvulsant hypersensitivity syndrome induced by carbamazepine. This syndrome is a rare, potentially life-threatening adverse drug reaction. The patient developed a cutaneous nonpruritic rash, associated with high fever, diffuse lymphadenopathy, and arthralgias on the knees and the ankles with local signs of arthritis. Laboratory examination showed a lymphocytosis, mild thrombocytopenia, marked eosinophilia, and high transaminases. Corticosteroid therapy (betametasone 0.5 mg x 3 day) was started and carbamazepine was gradually withdrawn changing to valproic acid, with complete control of the seizures. The fever and the rash reduced gradually, beginning from the face and then disappearing completely after 10 days. Laboratory results showed a clear improvement: after 7 days the patient showed a complete normalization of the above parameters, except for transaminases. The complete normalization of these enzymes was observed after 2 weeks from the disappearance of the skin rash