Mária Kiss

Effects of the neuropeptides substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide and galanin on the production of nerve growth factor and inflammatory cytokines in cultured human keratinocytes

Neuropeptides released from the cutaneous sensory nerve endings have neurotransmitter and immunoregulatory roles; they exert mitogenic actions and can influence the functions of different cell types in the skin. The aims of this study were a systematic investigation of the effects of the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and galanin (GAL) on the inflammatory cytokine production (IL-1alpha, IL-8 and TNF-alpha) of the keratinocytes, and a study of their role in the production and secretion of nerve growth factor (NGF) and its precursor molecule (proNGF). Cultures of normal human keratinocytes were treated with 10(-8)M SP, CGRP, VIP or GAL for 30 min. After different time intervals, cells were harvested for total RNA isolation; in addition, cell lysates and supernatants were collected. The effects of the neuropeptides on the mRNA expressions of the different cytokines and NGF were investigated by Q-RT-PCR and the protein levels were studied by means of ELISA assays and Western blotting. Each of the four neuropeptides induced increases in the expressions of IL-1alpha, IL-8 and TNF-alpha mRNA. Increases appeared in the amount of the IL-1alpha protein in the supernatants of neuropeptide-treated cells, and the IL-8 secretion was mildly elevated, while secretion of TNF-alpha remained undetectable. The four neuropeptides increased the NGF mRNA expression to different extents. In the cell lysates of the keratinocytes, only proNGF could be detected, its concentration in the neuropeptide-treated cells being approximately twice that in the time-matched controls. Both control cultures and neuropeptide-treated cultures were found to secrete proNGF and mature NGF, but neuropeptide-treated cell cultures produced markedly higher (3-7-fold) amounts of NGF-like immunoreactive materials. The results demonstrated that neuropeptides released from cutaneous nerves after an injurious stimulus are able to induce an upregulation of IL-1alpha and IL-8 production; they are additionally able to influence the expressions of proNGF/NGF and their secretion from the keratinocytes. These findings may contribute toward an understanding of the neural influence on skin health and disease.

Detection of Merkel cell polyomavirus DNA in Merkel cell carcinomas

Background  Merkel cell carcinoma (MCC) is a rare, aggressive tumour for which an increasing incidence has been reported. A new human polyomavirus, Merkel cell polyomavirus (MCV), was recently isolated from these tumours by applying digital transcriptome subtraction methodology.

Kaposi’s sarcoma–associated herpesvirus/human herpesvirus-8: A new virus in human pathology☆☆☆

The discovery of a new human herpesvirus in Kaposis sarcoma (KS) tissue of patients with AIDS has opened up new vistas in virology and oncology. This herpesvirus was first descriptively named KS-associated herpesvirus (KSHV), but was recently renamed human herpesvirus 8 (HHV8). KSHV/HHV8 DNA has been found in all forms of KS, suggesting that it might be involved in the pathogenesis of KS. In addition, KSHV/HHV8 can be detected in both malignant and benign lymphoproliferative disease. KSHV/HHV8 was also found in patients with angiosarcoma of the face and angiolymphoid hyperplasia with eosinophilia. Although only a limited portion of the virus has been sequenced, KSHV/HHV8 is equipped with genes that could confer oncogenic potential. The virus can now be cultured, providing the possibility for studies of viral replication and the mode of transmission. The recently developed serologic assays for antiviral antibodies suggest that infection with KSHV/HHV8 is not ubiquitous because KSHV/HHV8 seropositivity is limited to a small proportion of the population.

Galanin receptor expression in cultured human keratinocytes and in normal human skin

Abstract  Galanin (GAL) is a biologically active neuropeptide that is widely distributed in the nervous system. GAL exerts diverse action via the GAL receptors (GALR1, GALR2, and GALR3), which belong in the superfamily of G‐protein‐coupled transmembrane receptors. In human skin, GAL‐like immunoreactivity has been reported in free nerve endings and fibers of the dermis. The extraneuronal expression of GAL has also been demonstrated. Although the GALRs are essential for biological functions, the expressions of different GALR subtypes in cultured human keratinocytes have not yet been investigated. The aim of our study was to investigate the mRNA and protein expressions of the different GALRs in the HaCaT immortalized keratinocyte cell line and in cultured human keratinocytes. When reverse transcription (RT)‐polymerase chain reaction (PCR) was used with different GALR‐specific primers, only GALR2 mRNA was identified in cultured HaCaT cells and keratinocytes. Sequencing of the PCR products proved the presence of GALR2 mRNA in the keratinocytes. The presence of GALR2 protein was next investigated, using a polyclonal antibody against human GALR2. Both the HaCaT cells and the cultured keratinocytes displayed specific immunohistochemical staining, with higher intensity on the surface of the keratinocytes. Immunohistochemical investigations of normal human skin specimens revealed that GALR2 was expressed with high intensity in the basal layer of the epidermis and also around the hair follicles in the dermis. GAL treatment of the keratinocytes resulted in an increase in cytosolic Ca2+ concentration, suggesting that GALR2 is a functional receptor. Further studies are necessary to clarify the biological effects of GAL in the skin.

Presence of antidrug antibodies correlates inversely with the plasma tumor necrosis factor (TNF)-α level and the efficacy of TNF-inhibitor therapy in psoriasis

Antidrug antibodies have been shown to be associated with a loss of response during biologic therapy. Despite the potential association, there has been no report on the simultaneous monitoring of the following parameters in psoriasis: presence of neutralizing antibodies, plasma tumor necrosis factor (TNF)‐α concentration, TNFi concentration and disease activity. Plasma concentrations of adalimumab, infliximab, etanercept and their respective antidrug antibodies, as well as plasma concentrations of TNF‐α were measured in 77 psoriasis patients receiving biologic therapy, and the values were correlated with the clinical activity of the skin disease. Antidrug antibodies were identified in the plasma of 25% of infliximab‐treated patients and 29.6% of adalimumab‐treated patients, but not in the etanercept group. Clinical severity scores were significantly higher in the antibody‐positive patients. In patients receiving infliximab or adalimumab therapy, the presence of antidrug antibodies was directly associated with reduced plasma TNF‐inhibitor concentration and elevated plasma TNF‐α level.

Higher levels of autoantibodies targeting mutated citrullinated vimentin in patients with psoriatic arthritis than in patients with psoriasis vulgaris.

Antibodies against citrullinated proteins/peptides (ACPAs), and especially antibodies targeting mutated citrullinated vimentin (anti-MCVs), are novel biomarkers of rheumatoid arthritis (RA). Whereas ACPAs are specific and sensitive markers for RA, there have hardly been any reports relating to ACPAs in psoriatic arthritis (PsA) or in psoriasis without joint symptoms (PsO). The aim of the present study was to investigate the prevalence of anti-MCVs in PsA and PsO. Serum anti-MCV titers were measured in 46 PsA and 42 PsO patients and in 40 healthy controls by means of a commercial enzyme-linked immunosorbent assay. The potential correlations of the serum autoantibody levels with several clinical and laboratory parameters were examined. The anti-MCV levels in the PsA patients were significantly higher than those in the PsO group. Among the clinical variables, the presence of tender knee joints and nail psoriasis was significantly associated with anti-MCV positivity in the PsA patients. Higher anti-MCV titers in the PsO patients were associated with a more severe disease course and with the early onset of psoriatic skin symptoms. Our results suggest that anti-MCVs can be used as novel markers in the diagnosis of PsA and in a subset of PsO patients.

Autoantibodies to Human α6 Integrin in Patients with Bullous Pemphigoid

Abstract: Bullous pemphigoid (BP) is characterized immunologically by tissue‐bound and circulating autoantibodies targeting the hemidesmosomal proteins BP230 and BP180. Recent evidence suggests a pathophysiological role for autoantibodies against α6 integrin in the subepidermal blister formation of oral pemphigoid. The objective of our study was to investigate the presence of anti‐α6 integrin antibodies in patients with classical BP. The autoantibody profiles of 30 patients with BP, 10 patients with pemphigus vulgaris, and 20 healthy persons were identified. With the use of PeptideStructure and PlotStructure software, four different antigenic epitopes for α6 integrin were predicted, and their fusion recombinant constructs were prepared in an E. coli expression system. Sera were tested for α6 integrin autoantibodies by an ELISA technique. Altogether, 52% of the patients with BP displayed circulating antibodies against at least one recombinant protein. Our findings provide the first evidence for the presence of anti‐α6 integrin antibodies in patients with classical BP.

Clinical Relevance of Autoantibodies in Patients with Autoimmune Bullous Dermatosis

The authors present their experience related to the diagnosis, treatment, and followup of 431 patients with bullous pemphigoid, 14 patients with juvenile bullous pemphigoid, and 273 patients with pemphigus. The detection of autoantibodies plays an outstanding role in the diagnosis and differential diagnosis. Paraneoplastic pemphigoid is suggested to be a distinct entity from the group of bullous pemphigoid in view of the linear C3 deposits along the basement membrane of the perilesional skin and the “ladder” configuration of autoantibodies demonstrated by western blot analysis. It is proposed that IgA pemphigoid should be differentiated from the linear IgA dermatoses. Immunosuppressive therapy is recommended in which the maintenance dose of corticosteroid is administered every second day, thereby reducing the side effects of the corticosteroids. Following the detection of IgA antibodies (IgA pemphigoid, linear IgA bullous dermatosis, and IgA pemphigus), diamino diphenyl sulfone (dapsone) therapy is preferred alone or in combination. The clinical relevance of autoantibodies in patients with autoimmune bullous dermatosis is stressed.

Neuro-Immuno-Endocrine Modulation in Marathon Runners

Objective: Sports practice alters the homeostasis of athletes. To achieve homeostatic equilibrium, the integrated action of the neuroendocrine and immune systems is necessary. Here we studied the relation between cytokines, hormones and mood states in marathon runners. Methods: A total of 20 male recreational marathon runners (mean age = 35.7 ± 9 years) and 20 male sedentary individuals (mean age = 35.5 ± 7 years) were recruited. We compared the serum levels of growth hormone (GH), cortisol and interleukins 8 and 10 and the amounts of these two cytokines spontaneously produced by peripheral blood mononuclear cells. Blood samples of the sedentary group were collected at rest. Blood from the marathon runners was collected at rest (baseline: 24 h before the race), immediately after a marathon and 72 h after a marathon. Mood state analysis in both groups was performed using the 24-item Brunel Mood Scale (BRUMS). Results: Our results showed that, at rest, levels of interleukins 8 and 10 in the supernatant of culture cells, the serum concentration of GH, and tension and vigour (evaluated using the BRUMS), were significantly higher in athletes compared to sedentary people. Immediately after the race all serum parameters analysed were statistically higher than baseline values. At 72 h after the marathon, serum levels of hormones and interleukins returned to values at rest, but the concentrations of interleukins in the supernatant of culture cells showed a significant reduction compared to values at rest. Conclusion: The higher serum levels of GH in athletes at rest and the higher production of cytokines in culture without previous stimulus suggest that marathon runners present mechanisms that may be associated with preparing the body to perform prolonged strenuous exercise, such as a marathon.

Sortilin Is Expressed in Cultured Human Keratinocytes and Is Regulated by Cutaneous Neuropeptides

Sortilin, a member of the family of Vps10p domain receptors, has been shown to be able to bind the precursor peptide of nerve growth factor (proNGF). ProNGF interacts with sortilin and the p75(NTR) receptor on the cell surface to form a molecular complex capable of activating an apoptotic cascade. Keratinocytes can secrete proNGF and they have p75(NTR) on their surface. The expression of sortilin in normal human keratinocytes has not yet been clearly shown. In this study, we show that keratinocytes express sortilin mRNA, and the presence of sortilin protein is shown in cultured keratinocytes and in normal human skin. We have also shown that the cutaneous neuropeptides substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and galanin are able to reduce the expression of sortilin mRNA and sortilin protein in cultured human keratinocytes. In addition, each of the analyzed neuropeptides has the ability to arrest the proNGF-induced apoptosis of human keratinocytes. These results suggest that all the participants in the NGF/proNGF pathway are present in the keratinocytes, and cutaneous neuropeptides can modulate their expressions and actions. The NGF/proNGF balance and its regulation by neuropeptides may have an important role in skin homeostasis.