Polymorphism of hla-dr and hla-dq alleles in patients with pemphigus vulgaris

Uvod: Pemphigus vulgaris (PV) pripada grupi organ specifičnih autoimunskih oboljenja, kod koga se stvaraju autoantitela usmerena na specifične proteine (antigene) kože i sluzokoža. Autoantitela kod pemfigusa su usmerena prema komponentama međućelijskih spojeva (dezmozomima). To su dezmoglein 3 (Dsg 3), u manjoj meri dezmoglein 1 (Dsg 1) i drugi proteini. Kao posledica vezivanja cirkulišućih antitela za proteine dezmozoma dolazi do cepanja međućelijskih spojeva (akantolize) i pojave plikova (bula) na koži i sluzokožama. Autoimunski pemfigus, kao i druge autoimunske bolesti, predstavlja multifaktorsko oboljenje, rizik od nastanka bolesti zavisi od složenih interakcija gena i faktora spoljašnje sredine. U okviru naslednih faktora u etiopatogenezi pemfigusa najviše je ispitivan kompleks gena tkivne podudarnosti koji kodira sistem humanih leukocitnih antigena (HLA). HLA je najpolimorfniji deo humanog genoma. Složenost polimorfizma HLA je posledica postojanja više genskih lokusa, velikog broja alela za većinu gena i kombinacija produkata ovih alela. HLA molekuli II klase su posebno značajni jer prikazuju peptide CD4+ T-limfocitima pod čijom kontrolom je sekrecija anti Dsg 3 i anti Dsg 1 antitela od strane B-limfocita. Do sada je dokazana udruženost pojedinih HLA alela sa većim brojem autoimunskih oboljenja uključujući i pemfigus. Ciljevi istraživanja: Ciljevi ovog istraživanja su bili da se ustanovi učestalost alela i haplotipova u HLA-DR i DQ lokusima kod pacijenata sa PV u Srbiji, da se ustanovi stepen genetske sličnosti sa obolelima od PV u drugim populacijama, kao i da se ispita korelacija između genotipova, težine bolesti i koncentracije Dsg1 i Dsg3 antitela. Ispitanici i metode: Ispitivanje je izvršeno kod 72 pacijenta sa dokazanim PV. Kod ispitanika je određena alelska učestalost u HLA-DR i DQ lokusima (DRB1*, DQB1* i DQA1*). Izolacija DNK je rađena upotrebom QIAamp DNA Blood Mini Kit-a (QIAGEN, Germany). Određivanje grupa alela ispitivanih lokusa je rađeno testovima niske/srednje rezolucije, a potom su testovima visoke rezolucije određivani aleli upotrebom prajmera specifičnih za alelsku sekvencu PCR-SSP (engl. Polymerase chain reaction with sequence-specific primers) prema preporukama proizvođača testova (BAG lich Germany i Invitrogen)...Introduction: Pemphigus vulgaris (PV) belongs to the group of organ specific autoimmune disorders, characterized by the production of pathogenic autoantibodies against keratinocyte adhesion molecules- desmogleins, mostly Desmoglein 3 (Dsg3) and 1 (Dsg1), or other proteins. As a result of bounding autoantibodies to desmosomal proteins, separation of keratinocytes occur with clinical manifestation of blisters on the skin and mucosal membranes. PV, as other autoimmunes diseases belongs to the family of polygenic disorders, the risk of developing the disease depends on interactions of endogenous genetic factors and environmental factors. Inheritance of certain human leukocyte antigen (HLA) alleles has been suggested to be the most probable predisposing factor. HLA is the most polymorphous part of the human genome. The complexity of HLA polymorphism is due to the existence of a big number of genetic loci, alleles for most of the genes and combination of these alleles. HLA II class genes are particularly important due to peptide presentation to CD4+ T-cells and consequent secretion by B- cells of anti-Dsg3 and anti-Dsg1 antibodies. So far, associations of some HLA alleles with a great number of autoimmune diseases have been shown, including pemphigus. Aims of the investigation: Aims of this study were to determine HLA-DR and HLA-DQ allelic and haplotypic frequencies in patients with pemphigus vulgaris in Serbia; to establish similarities with other PV patients in different populations; to establish correlation between genotypes, disease activity and anti-Dsg3 and anti-Dsg1 antibodies. Individuals and methods: Investigation of allelic frequency of HLA-DR and HLA-DQ was conducted on 72 patients with diagnosed PV. DNA was extracted from blood samples using the Qiagen QIAamp DNAMini Kit (Qiagen, Germany). Allele groups were determined with low/intermediary resolution tests. High resolution was performed using polymerase chain reaction sequence-specific primers (PCRSSP) according to the manufacturer’s instructions (BAG lich Germany and Invitrogen). ARLEQUIN software package, version 3.11 was applied for estimated allele and haplotype frequencies, as well as for testing Hardy–Weinberg equilibrium..

Correlation of antibodies against desmogleins 1 and 3 with indirect immunofluorescence and disease activity in 72 patients with pemphigus vulgaris

The enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) have both been used for testing of antibodies to desmogleins 1 and 3 (anti-Dsg1 and anti-Dsg3) and for the serologic diagnosis of pemphigus. IIF values and antibody concentrations and profile do not always correlate with a specific clinical phenotype and with the disease activity. The purpose of the present study was to correlate the clinical phenotype of patients with pemphigus vulgaris (PV) and the disease activity with anti-Dsg1 and anti-Dsg3 antibodies and IIF titers. A total of 72 patients with PV underwent ELISA serum testing for the presence and titers of anti-Dsg1 and anti-Dsg3 and IIF which were correlated with the severity of the disease (evaluated using the Pemphigus Disease Area Index, PDAI), clinical phenotype, and clinical course. In 79.2% patients there was a perfect correlation between the clinical phenotype and antibody profiles; in 20.8% patients, clinical features and antigenic findings were discordant. A statistically significant correlation was found between disease activity and a) anti-Dsg3 and anti-Dsg1 concentrations (Rho=0.679, P&lt;0.001 and Rho=0.363, P=0.02, respectively) and b) IIF titers (Rho=0.426, P&lt;0.01), as well between IIF titers and anti-Dsg3 and anti-Dsg1 antibodies (Rho=0.742, P&lt;0.01 and Rho=0.372, P=0.02, respectively).  This study supports the previous observations that the disease severity in most patients with pemphigus correlates with IIF titers, which in turn is determined by the quantities of Dsg1 and Dsg3 antibodies, as well as the previous observation that the clinical phenotype and antibody profile are not always in correlation. </p

Atypical pyoderma gangrenosum in a patient with osteomyelofibrosis

Background. Atypical forms of pyoderma gangrenosum generally appear on the upper extremities; most frequently they are associated with myeloproliferative disorders, including osteomyelofibrosis. A response to systemic steroids is more pronounced than in classical form. Sometimes it may be the first sign of an underlying malignancy. Case report. We reported a patient with atypical pyoderma gangrenosum developed during the course of a myeloid malignancy - osteomyelofibrosis. The lesions occurred after a minor trauma. Painful blistering plaques, with an elevated, bluish-gray border were located on the dorsal aspect of hands. No skin malignancy was found. The lesions resolved rapidly to systemic steroids. Conclusion. Considering the unusual clinical presentation which makes the diagnosis difficult, as well as the fact that atypical forms of pyoderma gangrenosum can be the first sign of malignancies, especially myeloproliferative ones, recognizing this entity enables timely guiding future investigations toward their prompt detection

Polymorphism of hla-dr and hla-dq alleles in patients with pemphigus vulgaris

Uvod: Pemphigus vulgaris (PV) pripada grupi organ specifičnih autoimunskih oboljenja, kod koga se stvaraju autoantitela usmerena na specifične proteine (antigene) kože i sluzokoža. Autoantitela kod pemfigusa su usmerena prema komponentama međućelijskih spojeva (dezmozomima). To su dezmoglein 3 (Dsg 3), u manjoj meri dezmoglein 1 (Dsg 1) i drugi proteini. Kao posledica vezivanja cirkulišućih antitela za proteine dezmozoma dolazi do cepanja međućelijskih spojeva (akantolize) i pojave plikova (bula) na koži i sluzokožama. Autoimunski pemfigus, kao i druge autoimunske bolesti, predstavlja multifaktorsko oboljenje, rizik od nastanka bolesti zavisi od složenih interakcija gena i faktora spoljašnje sredine. U okviru naslednih faktora u etiopatogenezi pemfigusa najviše je ispitivan kompleks gena tkivne podudarnosti koji kodira sistem humanih leukocitnih antigena (HLA). HLA je najpolimorfniji deo humanog genoma. Složenost polimorfizma HLA je posledica postojanja više genskih lokusa, velikog broja alela za većinu gena i kombinacija produkata ovih alela. HLA molekuli II klase su posebno značajni jer prikazuju peptide CD4+ T-limfocitima pod čijom kontrolom je sekrecija anti Dsg 3 i anti Dsg 1 antitela od strane B-limfocita. Do sada je dokazana udruženost pojedinih HLA alela sa većim brojem autoimunskih oboljenja uključujući i pemfigus. Ciljevi istraživanja: Ciljevi ovog istraživanja su bili da se ustanovi učestalost alela i haplotipova u HLA-DR i DQ lokusima kod pacijenata sa PV u Srbiji, da se ustanovi stepen genetske sličnosti sa obolelima od PV u drugim populacijama, kao i da se ispita korelacija između genotipova, težine bolesti i koncentracije Dsg1 i Dsg3 antitela. Ispitanici i metode: Ispitivanje je izvršeno kod 72 pacijenta sa dokazanim PV. Kod ispitanika je određena alelska učestalost u HLA-DR i DQ lokusima (DRB1*, DQB1* i DQA1*). Izolacija DNK je rađena upotrebom QIAamp DNA Blood Mini Kit-a (QIAGEN, Germany). Određivanje grupa alela ispitivanih lokusa je rađeno testovima niske/srednje rezolucije, a potom su testovima visoke rezolucije određivani aleli upotrebom prajmera specifičnih za alelsku sekvencu PCR-SSP (engl. Polymerase chain reaction with sequence-specific primers) prema preporukama proizvođača testova (BAG lich Germany i Invitrogen)...Introduction: Pemphigus vulgaris (PV) belongs to the group of organ specific autoimmune disorders, characterized by the production of pathogenic autoantibodies against keratinocyte adhesion molecules- desmogleins, mostly Desmoglein 3 (Dsg3) and 1 (Dsg1), or other proteins. As a result of bounding autoantibodies to desmosomal proteins, separation of keratinocytes occur with clinical manifestation of blisters on the skin and mucosal membranes. PV, as other autoimmunes diseases belongs to the family of polygenic disorders, the risk of developing the disease depends on interactions of endogenous genetic factors and environmental factors. Inheritance of certain human leukocyte antigen (HLA) alleles has been suggested to be the most probable predisposing factor. HLA is the most polymorphous part of the human genome. The complexity of HLA polymorphism is due to the existence of a big number of genetic loci, alleles for most of the genes and combination of these alleles. HLA II class genes are particularly important due to peptide presentation to CD4+ T-cells and consequent secretion by B- cells of anti-Dsg3 and anti-Dsg1 antibodies. So far, associations of some HLA alleles with a great number of autoimmune diseases have been shown, including pemphigus. Aims of the investigation: Aims of this study were to determine HLA-DR and HLA-DQ allelic and haplotypic frequencies in patients with pemphigus vulgaris in Serbia; to establish similarities with other PV patients in different populations; to establish correlation between genotypes, disease activity and anti-Dsg3 and anti-Dsg1 antibodies. Individuals and methods: Investigation of allelic frequency of HLA-DR and HLA-DQ was conducted on 72 patients with diagnosed PV. DNA was extracted from blood samples using the Qiagen QIAamp DNAMini Kit (Qiagen, Germany). Allele groups were determined with low/intermediary resolution tests. High resolution was performed using polymerase chain reaction sequence-specific primers (PCRSSP) according to the manufacturer’s instructions (BAG lich Germany and Invitrogen). ARLEQUIN software package, version 3.11 was applied for estimated allele and haplotype frequencies, as well as for testing Hardy–Weinberg equilibrium..

Primary systemic amyloidosis

Background. Systemic amyloidosis is a rare disorder which usually occurs in aged persons and has a poor prognosis. Systemic amyloidosis can be primary, occasionally associated with multiple myeloma, or secondary, associated with another disease. Case report. We presented a 72-year-old male patient with periocular purpura ("racoon sign") and waxy papules, petechiae and ecchymoses on the neck and thoracic area. Purpuric macules were present also on the lips and tongue which was edematous (macroglossia). The skin lesions occurred two years earlier, the patient lost more than 15 kilograms of the body mass for less than a year. Immunoelectrophoresis of urine and serum demonstrated the presence of immunoglobulin light chains of the circulating monoclonal protein. Histopathological examination of skin lesions showed Congo red positive deposits in the derm. Cardiac evaluation revealed the signs of heart failure, and renal evaluation revealed nephrotic syndrome, with excessive protein lost. He was treated with oral melphalan and prednisolone, and died 7 days after starting the therapy due to heart failure. Conclusion. This patient considered as a rare case with systemic amyloidosis highlights the importance of histopathological and physical examination in any cases with periocular purpura, petechiae, ecchymoses and macroglossia

Manuka Honey/2-Hydroxyethyl Methacrylate/Gelatin Hybrid Hydrogel Scaffolds for Potential Tissue Regeneration

Scaffolding biomaterials are gaining great importance due to their beneficial properties for medical purposes. Targeted biomaterial engineering strategies through the synergy of different material types can be applied to design hybrid scaffolding biomaterials with advantageous properties for biomedical applications. In our research, a novel combination of the bioactive agent Manuka honey (MHo) with 2-hydroxyethyl methacrylate/gelatin (HG) hydrogel scaffolds was created as an efficient bioactive platform for biomedical applications. The effects of Manuka honey content on structural characteristics, porosity, swelling performance, in vitro degradation, and in vitro biocompatibility (fibroblast and keratinocyte cell lines) of hybrid hydrogel scaffolds were studied using Fourier transform infrared spectroscopy, the gravimetric method, and in vitro MTT biocompatibility assays. The engineered hybrid hydrogel scaffolds show advantageous properties, including porosity in the range of 71.25% to 90.09%, specific pH- and temperature-dependent swelling performance, and convenient absorption capacity. In vitro degradation studies showed scaffold degradability ranging from 6.27% to 27.18% for four weeks. In vitro biocompatibility assays on healthy human fibroblast (MRC5 cells) and keratinocyte (HaCaT cells) cell lines by MTT tests showed that cell viability depends on the Manuka honey content loaded in the HG hydrogel scaffolds. A sample containing the highest Manuka honey content (30%) exhibited the best biocompatible properties. The obtained results reveal that the synergy of the bioactive agent, Manuka honey, with 2-hydroxyethyl methacrylate/gelatin as hybrid hydrogel scaffolds has potential for biomedical purposes. By tuning the Manuka honey content in HG hydrogel scaffolds advantageous properties of hybrid scaffolds can be achieved for biomedical applications

Manuka Honey/2-Hydroxyethyl Methacrylate/Gelatin Hybrid Hydrogel Scaffolds for Potential Tissue Regeneration

Scaffolding biomaterials are gaining great importance due to their beneficial properties for medical purposes. Targeted biomaterial engineering strategies through the synergy of different material types can be applied to design hybrid scaffolding biomaterials with advantageous properties for biomedical applications. In our research, a novel combination of the bioactive agent Manuka honey (MHo) with 2-hydroxyethyl methacrylate/gelatin (HG) hydrogel scaffolds was created as an efficient bioactive platform for biomedical applications. The effects of Manuka honey content on structural characteristics, porosity, swelling performance, in vitro degradation, and in vitro biocompatibility (fibroblast and keratinocyte cell lines) of hybrid hydrogel scaffolds were studied using Fourier transform infrared spectroscopy, the gravimetric method, and in vitro MTT biocompatibility assays. The engineered hybrid hydrogel scaffolds show advantageous properties, including porosity in the range of 71.25% to 90.09%, specific pH- and temperature-dependent swelling performance, and convenient absorption capacity. In vitro degradation studies showed scaffold degradability ranging from 6.27% to 27.18% for four weeks. In vitro biocompatibility assays on healthy human fibroblast (MRC5 cells) and keratinocyte (HaCaT cells) cell lines by MTT tests showed that cell viability depends on the Manuka honey content loaded in the HG hydrogel scaffolds. A sample containing the highest Manuka honey content (30%) exhibited the best biocompatible properties. The obtained results reveal that the synergy of the bioactive agent, Manuka honey, with 2-hydroxyethyl methacrylate/gelatin as hybrid hydrogel scaffolds has potential for biomedical purposes. By tuning the Manuka honey content in HG hydrogel scaffolds advantageous properties of hybrid scaffolds can be achieved for biomedical applications