Michele Fimiani

Targeted next-generation sequencing appoints c16orf57 as clericuzio-type poikiloderma with neutropenia gene.

Next-generation sequencing is a straightforward tool for the identification of disease genes in extended genomic regions. Autozygosity mapping was performed on a five-generation inbred Italian family with three siblings affected with Clericuzio-type poikiloderma with neutropenia (PN [MIM %604173]), a rare autosomal-recessive genodermatosis characterised by poikiloderma, pachyonychia, and chronic neutropenia. The siblings were initially diagnosed as affected with Rothmund-Thomson syndrome (RTS [MIM #268400]), with which PN shows phenotypic overlap. Linkage analysis on all living subjects of the family identified a large 16q region inherited identically by descent (IBD) in all affected family members. Deep sequencing of this 3.4 Mb region previously enriched with array capture revealed a homozygous c.504-2 A>C mismatch in all affected siblings. The mutation destroys the invariant AG acceptor site of intron 4 of the evolutionarily conserved C16orf57 gene. Two distinct deleterious mutations (c.502A>G and c.666_676+1del12) identified in an unrelated PN patient confirmed that the C16orf57 gene is responsible for PN. The function of the predicted C16orf57 gene is unknown, but its product has been shown to be interconnected to RECQL4 protein via SMAD4 proteins. The unravelled clinical and genetic identity of PN allows patients to undergo genetic testing and follow-up.

Extracorporeal photochemotherapy restores Th1/Th2 imbalance in patients with early stage cutaneous T‐cell lymphoma

Extracorporeal photochemotherapy (ECP) has been shown to be a potent activator of peripheral blood macrophages because it causes a marked release of macrophage‐dependent proinflammatory cytokines, and it is therefore currently considered to be a safe and non‐toxic immunomodulatory treatment. On this basis we studied the function of peripheral blood mononuclear cells (PBMC) in eight patients with early stage (Ib) cutaneous T‐cell lymphoma (CTCL), before and 1 year after ECP, together with their clinical and histological responses. In particular we evaluated in vitro phytohaemagglutinin (PHA)‐stimulated proliferation and production of interleukin‐4 (IL‐4) and interferon‐&ggr; (IFN‐&ggr;) as well as lipopolysaccharide (LPS)‐induced production of IL‐12. Before treatment we observed that PBMC of patients produced significantly higher levels of IL‐4 and lower levels of IFN‐&ggr; and IL‐12 than those of healthy control subjects. After 1 year of ECP, IL‐4, IFN‐&ggr; and IL‐12 production no longer differed from that of control subjects. Moreover, we observed a good clinical result matched by histological response. Our data confirm that early‐ stage CTCL patients show a predominantly type‐2 immune response that might be responsible for several immunological abnormalities found in this disease. We have demonstrated that ECP reverses the T‐helper type 1/T‐helper type 2 (Th1/Th2) imbalance and may therefore be considered an efficient biological response modifier.

Use of donor bone marrow mesenchymal stem cells for treatment of skin allograft rejection in a preclinical rat model.

Recent studies indicate that mesenchymal stem cells (MSC) exhibit a degree of immune privilege due to their ability to suppress T cell mediated responses causing tissue rejection; however, the impact of allogeneic MSC in the setting of organ transplantation has been poorly investigated so far. The aim of our study was to evaluate the effect of intravenous donor MSC infusion for clinical tolerance induction in allogeneic skin graft transplantations in rats. MSC were isolated from Wistar rats and administered in Sprague-Dawley rats receiving Wistar skin graft with or without cyclosporine A (CsA). Graft biopsies were performed at day 10 post transplantation in all experimental groups for histological and gene expression studies. Intravenous infusion with donor MSC in CsA-treated transplanted rats resulted in prolongation of skin allograft survival compared to control animals. Unexpectedly, donor MSC infusion in immunocompetent rats resulted in a faster rejection as compared to control group. Cytokine expression analysis at the site of skin graft showed that CsA treatment significantly decreased pro-inflammatory cytokines IFN-γ and IL-2 and reduced TNF-α gene expression; however, the level of TNF-α is high in MSC-treated and not immunosuppressed rats. Results of our study in a rat tissue transplantation model demonstrated a possible immunogenic role for donor (allogeneic) MSC, confirming the need of adequate preclinical experimentation before clinical use.

Dysplastic naevus vs. in situ melanoma: digital dermoscopy analysis.

Background  To date, much confusion exists about the biological significance of dysplastic naevi and about the relationship between melanocytic dysplasia and clinical atypia.

Role of extracorporeal photochemotherapy in patients with refractory chronic graft-versus-host disease

Recent studies suggest that extracorporeal photochemotherapy (ECP) may be beneficial in patients with steroid‐refractory chronic graft‐versus‐host disease (cGvHD). However, it is not yet clear whether certain conditions, such as age, mode of onset of cGvHD etc., influence clinical response and whether certain affected organs are more sensitive to ECP than others. We analysed the main clinical and laboratory parameters related to evolution of the disease in 32 steroid‐refractory cGvHD patients, to identify any useful response predictors to ECP. ECP affected the course of the disease positively in 78% (25/32) of our cases.

Mechanism of action of extracorporeal photochemotherapy in chronic graft-versus-host disease

Chronic graft‐versus‐host disease (GvHD) affects 50% of long‐term bone marrow transplant survivors and remains a cause of major long‐term morbidity in these patients despite aggressive therapy. Extracorporeal photochemotherapy (ECP), considered as an effective treatment for patients with erythrodermic cutaneous T‐cell lymphoma (CTCL), has recently been used successfully in the treatment of GvHD. One of the most intriguing aspects of ECP is its ability to induce two apparently opposite effects: activation of the immune system against neoplastic cells (as in CTCL) and downregulation of the activity of T‐cell clones in autoimmune diseases (as in systemic sclerosis, systemic lupus erythematosus and pemphigus vulgaris) and autoallogeneic immune responses (as in GvHD and allograft rejection). Only a better and more complete understanding of the various mechanisms involved will enable this interesting new therapy to be made more effective and selective.

Evaluation of tumour-infiltrating CD4+CD25+FOXP3+ regulatory T cells in human cutaneous benign and atypical naevi, melanomas and melanoma metastases.

Background  CD4+CD25+FOXP3+ regulatory T cells (Tregs) are thought to induce immunotolerance in melanoma. They have not yet been investigated in the entire spectrum of melanocytic cutaneous lesions within a tumour site.

Extracorporeal photochemotherapy induces apoptosis of infiltrating lymphoid cells in patients with mycosis fungoides in early stages. A quantitative histological study

Extracorporeal photochemotherapy (ExP) is a well‐tolerated new form of chemoimmunotherapy, which is considered to be effective for cutaneous T‐cell lymphoma (CTCL) and the treatment of choice for Sézary syndrome. Improvements have also been seen in patients with non‐erythrodermic mycosis fungoides (MF) in the early stages, even when tumour cells are not detectable in the peripheral blood. In this study, we used ExP as a monotherapy in seven patients who had early stage (Ib) MF, and who were no longer responsive to or had contraindications for other therapies. We observed a clinical improvement in the disease after 12 months of treatment: one patient showed a complete response, five a partial response, and one remained stable. In each patient we compared skin biopsies of large plaque lesions before and after the treatment. We undertook a histological evaluation of the infiltrate. The lymphoid cell proliferation and death rates were quantified using the following parameters: lymphoid cell density (LCD), Ki67+ lymphoid cell nuclei percentage (Ki67+ Lcn percentage), and apoptotic index (AI). Significant decreases in the lymphoid cell infiltrate and in cell proliferation, and a significant increase in AI were observed after therapy. The mean LCD decreased from 187 ± 33 to 34 ± 17·7, Ki67+ Lcn mean percentage decreased from 16·9 ± 3·9 to 4·9 ± 2·4, and the AI mean value increased from 0·05 ± 0·03 to 2·41 ± 1·54. Our results suggest a role for apoptosis in the improvement of the skin lesions and are in line with some reports on the mode of action of ExP. Although the way in which ExP works needs to be clarified further, it does seem to stimulate a CD8+ cell‐mediated anticlonotypic activity against circulating pathogenic clones. Furthermore, a release of tumour necrosis factor α (TNF‐α) by circulating monocytes has been demonstrated after ExP. Both are known to induce cell death by apoptosis.

Extracorporeal photopheresis affects co‐stimulatory molecule expression and interleukin‐10 production by dendritic cells in graft‐versus‐host disease patients

Graft‐versus‐host disease (GVHD) is a major complication of allogeneic bone marrow transplantation. Extracorporeal photochemotherapy (ECP) has been introduced as an alternative treatment for GVHD refractory to conventional immunosuppressive treatment, although its mechanism of action is not yet clear. We investigated, in seven GVHD patients, the effects of ECP on dendritic cell maturation and cytokine production in an in vitro model that could mimic the potential in vivo effect of reinfusion of ECP‐treated peripheral blood mononuclear cells. The model was based on co‐culture of ECP‐treated lymphocytes with monocyte‐derived dendritic cells (DCs) of the same patient. We found that the co‐culture of ECP‐treated lymphocytes with immature DCs reduced CD54, CD40 and CD86 mean fluorescence intensity (MFI) significantly after lipopolysaccharide (LPS) stimulation, without affecting human leucocyte antigen D‐related and CD80 MFI. In the same co‐culture model, DCs produced increased amounts of interleukin (IL)‐10 when co‐cultured with ECP‐treated lymphocytes and stimulated with LPS, while IL‐12 and tumour necrosis factor‐α production were not affected. These results suggest that reinfusion of large numbers of autologous apoptotic lymphocytes is significant for the therapeutic outcome of ECP through down‐regulation of co‐stimulatory molecules on DCs, inducing non‐fully mature DCs with a low signal 2 and up‐regulation of IL‐10, which is an immunosuppressive cytokine.

Extracorporeal photochemotherapy in the treatment of eosinophilic fasciitis

Background Eosinophilic fasciitis (EF) is a rare connective tissue disorder characterized clinically by symmetrical swelling, induration and thickening of the skin and histologically by thickening of the fascia with chronic inflammatory infiltrate containing eosinophils. The disease is classified in the spectrum morphea/systemic sclerosis and treated with systemic steroids and other immunosuppressant drugs.