Near infrared photoimmunotherapy targeting the cutaneous lymphocyte antigen for mycosis fungoides.

Background: Mycosis fungoides (MF) is a low-grade T-cell lymphoma with primary cutaneous involvement accounting for more than half of all primary cutaneous lymphomas. The treatment of MF is very challenging due to the limited therapies available. Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and highly selective cancer treatment that employs a monoclonal antibody conjugated to a photo-absorber dye, the hydrophilic phthalocyanine IRdye 700DX® (IR700), and near infrared light. In this study, we investigated the effect of NIR-PIT on MF targeting the cell-surface antigen cutaneous lymphocyte antigen (CLA)Matherial and methods: MF derived My-La CD4+ cells were incubated with the anti-CLA antibody conjugated to IR700 and then irradiated with a 690 nm near-infrared light. Cell death was evaluated by propidium iodide staining and flow cytometry 24 hours after irradiation.Results: Treatment with anti-CLA or light irradiation exhibited very modest pro-death effects, whereas treatment with the anti-CLA antibody conjugated to IR700 and then irradiation with a 690 nm near-infrared light induced a substantial increase in death in the MF cell line.Conclusions: NIR-PIT targeting CLA to treat MF showed marked antitumour effects. As such, CLA-targeted NIR-PIT could be a promising treatment for MF and, possibly, other cutaneous diseases characterized by CLA+ skin infiltrating T-cells.

Ultra-low dose rituximab for refractory pemghigus vulgaris: a pilot study.

Background: Pemphigus vulgaris is an autoimmune blistering disease affecting the skin and mucous membranes. Current treatments for pemphigus vulgaris include anti-inflammatory and immunosuppressive agents. Rituximab, an anti-CD20 monoclonal antibody, has been shown to be effective for the treatment of pemphigus vulgaris. However, the optimal dosage of rituximab for the treatment of this autoimmune bullous disease has not been clearly defined.The aim of this study was to investigate the clinical efficacy and adverse effects of an ultra-low dosage regimen of rituximab for pemphigus vulgaris.Methods: We performed a prospective non-randomized open case series including eight patients affected by pemphigus vulgaris. Patients were treated with an ultra-low dosage of rituximab (a single infusion of 200 mg).Results: All patients had a positive response after infusion. At the end of the follow-up period, 5 patients achieved a complete remission and 3 a partial remission. Except for one case of sepsis due to Citrobacer freundii and a pneumonia due to Haemophilus influenzae, no adverse events were documented in our patients.Conclusions: Data from our study suggest that an ultra-low dosage of rituximab could be an effective treatment for pemphigus vulgaris. Consequently, there is a need for a larger, confirmatory, randomized, multicenter trial.

Biochip detection of BP180 autoantibodies in blister fluid for the serodiagnosis of bullous pemphigoid: A pilot study.

Bullous pemphigoid is an autoimmune subepidermal blistering skin disease immunologically defined by autoantibodies directed against basement membrane zone antigens, the main of which is BP180. Laboratory tests are essential for diagnosis and include direct immunofluorescence and serologic assessments with indirect immunofluorescence and ELISA. Serology may be performed on blister fluid, in alternative to blood serum. This study investigated the use of a Biochip-based indirect immunofluorescence approach for the serum diagnosis of bullous pemphigoid on blister fluid. We compared the results using the Biochip-method with the ELISA detection of bullous pemphigoid-180 autoantibodies in blister fluid and observed a perfect correlation between these 2 methods in our group of 13 patients with clinical and direct immunofluorescence diagnosis of bullous pemphigoid. The Biochip is a simple, standardized and inexpensive diagnostic tool and its use on blister fluid may facilitate the diagnosis of this and other autoimmune bullous disorders. Our results suggest that the Biochip assay on serum of bullae is a non-invasive screening technique for the early diagnosis of bullous pemphigoid that is practical for fragile elderly patients and achievable even in small laboratory settings.

Evaluation of anti-desmoglein-1 and anti-desmoglein-3 autoantibody titers in pemphigus patients at the time of the initial diagnosis and after clinical remission.

It has been suggested that anti-desmoglein autoantibody titers could be helpful in follow-up and therapeutic management of pemphigus patients. However, there is no consensus regarding the relationship between anti-desmoglein autoantibody titers and clinical activity of pemphigus.The aim of our study was to evaluate if clinical remission of pemphigus relates to the presence of anti-desmoglein autoantibodies.Thirty patients with pemphigus vulgaris and 7 patients with pemphigus foliaceous were included in the study. Assessment of autoantibody titers was carried out at the time of the initial diagnosis and after the clinical remission using an enzyme-linked immunosorbent assay-based assay.Our results indicate that pemphigus clinical remission did not necessarily imply a serological remission, and consequently it is necessary to establish if withdrawal of the immunosuppressive regimen in pemphigus should be based exclusively on the achievement of clinical remission or also on the serological findings.

Salivary Samples for the Diagnosis of Pemphigus vulgaris Using the BIOCHIP Approach: a Pilot Study.

Pemphigus vulgaris (PV) is a rare autoimmune intraepithelial blistering skin disease characterized by the presence of circulating autoantibodies against desmoglein 3 (DSG3) and desmoglein 1 (DSG1), resulting in loss of the normal epithelial cell-to-cell adhesion, through a process called acantholysis. In recent years, a BIOCHIP-based indirect immunofluorescence technique for the determination of anti-DSG3 and anti-DSG1 autoantibodies has been described. Even though, the use of saliva anti-DSG3 and anti-DSG1 ELISA for the diagnosis of PV has been already reported, there are no studies concerning the utilization of saliva by the BIOCHIP approach. In the present pilot study, ELISA and BIOCHIP were performed, using salivary and serum samples from the same patients to investigate if the detection of anti-desmoglein autoantibodies in salivary samples by BIOCHIP could be used as a test for the diagnosis of PV. There was a strong correlation between ELISA and BIOCHIP results both for anti-DSG3 and anti-DSG1 serum autoantibodies. Autoantibodies to DSG3 were detected in 8 out of 8 salivary samples by ELISA and in 6 out of 8 salivary samples by the BIOCHIP approach. Autoantibodies to DSG1 were negative in all salivary samples using both ELISA and BIOCHIP. There were no positive results in the negative control group. In conclusion, the results of this pilot study indicate lack of correlation between serum and salivary results using both ELISA and BIOCHIP, indicating that saliva may not be the ideal substrate for the laboratory diagnosis of PV using these approaches.

Association between Toll-like receptor 7 Gln11Leu single-nucleotide polymorphism and basal cell carcinoma.

Non-melanoma skin cancers (NMSC) are the most common form of human skin cancer. The majority of NMSC are basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) with a BCC:SCC incidence ratio of 4:1 in immunocompetent patients. Toll-like receptors (TLRs) are transmembrane glycoproteins that recognize pathogen-associated molecular patterns and damage-associated molecular patterns, against which they activate the innate immune response and initiate the adaptive immune response. Genetic variations of these receptors can alter the immune system and are involved in evolution and susceptibility of various diseases, including cancer. Imiquimod, an agonist of TLR7, is applied topically in the treatment of premalignant and malignant skin disorders, in particular BCC. The high efficacy of this TLR7 agonist toward BCC supports a possible role of this receptor in the induction of BCC and, consequently, polymorphisms of this receptor could be responsible for a greater or lesser susceptibility to BCC. The aim of the present study was to evaluate whether the presence of the functional TLR7 rs179008/Gln11Leu promoter polymorphism conferred an increased susceptibility to BCC. A case-control study with 177 BCC cases and 158 controls was performed to highlight the possible association between this polymorphism and the susceptibility to BCC. As the TLR7 gene is localized on chromosome X, the allelic frequency of this polymorphism was analyzed separately in males and females. The analysis of the distribution of frequencies of wild-type TLR7 and variant TLR7 carrying the single-nucleotide polymorphism (SNP) rs179008 in patients with BCC and healthy subjects did not reveal any statistically significant difference between cases and controls. This study does not suggest the involvement of the SNP rs179008 of TLR7 in the susceptibility to BCC, but cannot exclude a role for TLR7 in BCC carcinogenesis considering the high efficacy of the TLR7 agonist, imiquimod, in the treatment of this neoplastic disorder.

The use of biochip immunofluorescence microscopy for the diagnosis of Pemphigus vulgaris.

Pemphigus vulgaris is an autoimmune intraepithelial blistering skin disease characterized by the presence of circulating autoantibodies directed against surfaces of keratinocytes. Diagnosis is generally based on clinical features, histology, direct and indirect immunofluorescence and ELISA. This study describes a new BIOCHIP mosaic-based indirect immunofluorescence technique based on recombinant antigenic substrates and transfected cells. We investigated the diagnostic use of BIOCHIP for the serological diagnosis of Pemphigus vulgaris. Autoantibodies against desmoglein 3 were detected in 97.62% of patients (41/42) with P. vulgaris. There were no positive results in the negative control group. Our study revealed that BIOCHIP has high sensitivity and specificity comparable to that of the ELISA assays. Therefore the BIOCHIP technique seems to be an appropriate method for the diagnosis of P. vulgaris as it has been shown to be a simple, standardized and readily available novel tool, which could facilitate the diagnosis of this autoimmune bullous disease. We suggest that it could be used as an initial screening test to identify patients with P. vulgaris before using the ELISA approach.

Biochip technology for the serological diagnosis of bullous pemphigoid.

Bullous pemphigoid is an autoimmune blistering skin disease characterized by the presence of circulating autoantibodies which recognize specific proteins of the epidermis and dermoepidermal junction. Diagnosis is based on clinical criteria and laboratory investigations, notably histology, direct and indirect immunofluorescence, and ELISA. This study describes a new immunofluorescence assay for parallel determination of anti-BP180 and anti-BP230 based on recombinant antigenic substrates. The aim of the study was to detect BP180 and BP230 autoantibodies by BIOCHIP technology using both a specially designed recombinant BP180-NC16A protein and cells expressing the BP230-gc antigen fragment. 18 patients with bullous pemphigoid were included in the study. Autoantibodies to BP180 were detected by the BIOCHIP technique in 83.33% of patients with clinical, serological, and immunohistological confirmed bullous pemphigoid while autoantibodies against BP230-gC were detected only in 39% of patients. The detection of anti-BP180-NC16A and anti-BP230-gC by a new biochip-based immunoassay is a suitable alternative to indirect immunofluorescence and ELISA. This method has the advantage of easily discriminating the different autoantibody specificities. The BIOCHIP method is faster, cheaper, and easy to use when compared with the ELISA approach. For this reason, the new method could be used as an initial screening test to identify patients with bullous pemphigoid, and doubtful results could then be confirmed by ELISA.

The role of immunohistochemical analysis in the diagnosis of parapsoriasis.

Parapsoriasis is a chronic dermatosis whose biological distinction from early mycosis fungoides, the most frequent form of cutaneous T-cell lymphoma, is still not clearly defined. Two types of parapsoriasis have been delineated: large-plaque parapsoriasis and small-plaque parapsoriasis. The lack of clinical and histological features, which may allow distinguishing parapsoriasis from early mycosis fungoides has prompted several investigations to assess the role of immunohistochemistry in establishing a conclusive diagnosis of these conditions. However, the additional data obtained by immunohistochemical analysis concerning the CD4/CD8 ratio, the aberrant expression of T-cell antigens and the expression of proliferation markers has not generally helped establish a more definitive diagnosis. This review critically discusses these immunohistochemical markers and their use in diagnosis of parapsoriasis.

CD7 expression in reactive and malignant human skin T-lymphocytes.

CD7, a molecule normally expressed on 90% of CD4+ T cells, is often deficient on the malignant T cells of cutaneous T cell lymphoma. Therefore deletion of CD7 is considered a specific marker for the diagnosis of cutaneous T cell lymphomas. Because an expansion of CD4+CD7- cells may also be observed in benign lymphocyte-mediated dermatoses, we present our experience concerning CD7 expression in both cutaneous T cell lymphomas and a broad variety of T cell-mediated inflammatory dermatoses using an immunohistochemical approach on frozen sections from 45 patients. No, or at most scarce, expression of CD7 was detectable in the inflammatory skin conditions investigated as compared to CD3-positive cells. In cutaneous T cell lymphomas, a striking reduction of CD7 reactive cells was found in either reactive or malignant T cell components. Our findings indicate that CD7-negative T cells are more common within both benign and neoplastic T cell infiltrates than was previously demonstrated and suggest that, using a conventional immunoenzymatic technical approach on fresh-frozen sections, CD7 deletion is an unreliable criterion for the immunohistological diagnosis of T cell-mediated skin infiltrates.

Absent or low expression of T-cell receptor zeta-chain in T cells infiltrating human pathological skin conditions.

The T-cell receptor (TCR) zeta-chain is involved in signal transduction necessary for T-cell activation and subsequent proliferation. Expression of the TCR zeta-chain in vivo has been studied by a variety of technical approaches on different cell and tissue specimen. However, the in situ situation concerning the expression of the TCR zeta-chain has not yet been investigated on infiltrating T lymphocytes in neoplastic and inflammatory cutaneous diseases. In this study, we analysed the expression of the TCR zeta-chain in a number of skin tissues affected by established inflammatory and neoplastic conditions. Serial sections of different tissue specimens were stained immunoenzymatically for CD3 and TCR zeta-chain expression. No or at most scarce expression of TCR zeta-chain was detectable in the inflammatory and neoplastic skin conditions investigated as compared to CD3-positive cells. It is possible that this TCR zeta-chain deletion is induced by the skin microenvironment as an effect of local immunoregulatory influences. Alternatively, lymphocytes located in the skin may generally not express this molecule. In our study, tumour-infiltrating T lymphocytes of CTCL were negative for TCR zeta-chain expression. It has been hypothesized that downregulation of the TCR zeta-chain on tumour-infiltrating T lymphocytes is a mechanism by which neoplastic cells escape the cellular immune response. Our findings showing the absence or reduction of TCR zeta-chain expression also in inflammatory skin lymphocytic infiltrates is not consistent with a pivotal role of the TCR zeta-chain in the process of immune escape of tumour cells.