Pavlína Čejková

Immunomodulatory role of prolactin in diabetes development

Pituitary hormone and cytokine prolactin (PRL) is one of the mediators of the bidirectional communication between neuroendocrine and immune systems. It participates in many immunomodulatory activities, affects differentiation and maturation of both, B and T lymphocytes and enhances inflammatory responses and production of immunoglobulins. Hyperprolactinemia has been described in many autoimmune diseases, both systemic (SLE, RA, PsA) and organ-specific (T1D, CD and others) and the activity of PRL has been intensively studied. Nevertheless, no data on PRL contribution to pathogenesis of diabetes mellitus is available, although the effect of PRL on beta cells of the pancreas and insulin secretion has been observed.

HLA DRB1, DQB1 and insulin promoter VNTR polymorphisms: interactions and the association with adult‐onset diabetes mellitus in Czech patients

Both the human leucocyte antigen (HLA) DRB1 and the HLA DQB1 gene loci play a role in the development and progression of autoimmune diabetes mellitus (T1DM). Similarly, the insulin promoter variable number tandem repeats (INS‐VNTR) polymorphism is also involved in the pathogenesis of diabetes mellitus (DM). We studied the association between each of these polymorphisms and DM diagnosed in patients older than age 35 years. Furthermore, we analysed possible interactions between HLA DRB1/DQB1 and INS‐VNTR polymorphisms. Based on C‐peptide and GADA levels we were able to distinguish three types of diabetes: T1DM, latent autoimmune diabetes in adults (LADA) and T2DM. INS‐VNTR was genotyped indirectly by typing INS–23HphI A/T polymorphism. The genotype and allele frequencies of INS–23HphI did not differ between each of the diabetic groups and group of healthy subjects. We did, however, observe an association between the INS–23HphI alleles, genotypes and C‐peptide secretion in all diabetic patients: A allele frequency was 86.2% in the C‐peptide‐negative group vs. 65.4% in the C‐peptide‐positive group (Pcorr. < 0.005); AA genotype was found to be 72.4% in the C‐peptide‐negative group vs. 42.6% in the C‐peptide‐positive groups (Pcorr. < 0.01). The HLA genotyping revealed a significantly higher frequency of HLA DRB1*03 allele in both T1DM and LADA groups when compared to healthy subjects: T1DM (25.7%) vs. control group (10.15%), odds ratio (OR) = 3.06, P < 0.05; LADA (27.6%) vs. control (10.15%), OR = 3.37, P < 0.01. The simultaneous presence of both HLA DRB1*04 and INS–23HphI AA genotype was detected in 37.5% of the T1DM group compared to only 9.2% of the healthy individuals group (OR = 5.9, Pcorr. < 0.007). We summarize that in the Central Bohemian population of the Czech Republic, the INS–23HphI A allele appears to be associated with a decrease in pancreatic beta cell secretory activity. HLA genotyping points to at least a partial difference in mechanism, which leads to T1DM and LADA development as well as a more diverse genetic predisposition in juvenile‐ and adult‐onset diabetes. The simultaneous effect of HLA and INS‐VNTR alleles/genotypes predispose individuals to an increased risk of diabetes development.

Autoimmune Diabetes Mellitus with Adult Onset and Type 1 Diabetes Mellitus in Children Have Different Genetic Predispositions

Abstract:  Type 1 diabetes with manifestation after 35 years of age is defined by CP <200 pmol/L and institution of insulin therapy within 6 months after diagnosis. Latent autoimmune diabetes mellitus in adults (LADA) manifesting after 35 years of age is defined by minimum 6 months after diagnosis without insulin therapy and C peptide (CP) >200 pmol/L and antiGAD > 50 ng/mL. We aimed to find a possible genetic discrimination among different types of autoimmune diabetes. To accomplish this goal, we analyzed DNA samples from 31 LADA patients, 75 patients with adult‐onset type 1 diabetes mellitus, 188 type 1 diabetic children, and 153 healthy adult individuals. We studied five genetic loci on chromosomes 6, 11, 4, and 14: HLA DRB1 and DQB1 alleles, major histocompatibility complex (MHC) class I–related gene‐A (MIC‐A) microsatellite polymorphism, interleukin (IL)‐18 single nucleotide polymorphism, the microsatellite polymorphism of nuclear factor kappa B gene (NF‐κB1), and the single nucleotide polymorphism of a gene for its inhibitor (NF‐κBIA). HLA‐DR3 was detected as the predisposition allele for LADA (OR = 4.94, P < 0.0001). Further we found a statistically significant increase of NF‐κBIA AA genotype (OR = 2.68, P < 0.01). On the other hand, DRB1*04, which is linked with DQB1*0302, was observed as a risk factor in patients with type 1 diabetes mellitus (T1DM) onset after 35 years of age (OR = 10.47, P < 0.0001 and OR = 9.49, P < 0.0001, respectively). There was also an association with MIC‐A5.1 (OR = 2.14, P < 0.01). Statistically significant difference was found in the distribution of IL‐18 promoter –607 (C/A) polymorphism between LADA and T1DM in adults (P < 0.01). We conclude that all subgroups of autoimmune diabetes have partly different immunogenetic predisposition.

Polymorphism of the extrapituitary prolactin promoter and systemic sclerosis

Sirs, Increased serum prolactin (PRL) levels have been demonstrated in several autoimmune diseases including polymyositis (PM) and systemic sclerosis (SSc) [1–3]. PRL is produced by the lactotrophs of the pituitary gland, but the immune cells can produce PRL as well, and PRL acts as a cytokine with role in B, T, and dendritic cell maturation [1]. Czuwara-Ladykowska et al. [4] demonstrated higher PRL mRNA synthesis in lymphocytes from patients suVering from SSc than in healthy individuals. Multiple promoters regulate PRL gene transcription. Unlike the pituitary production controlled by the pituitary promoter under the inXuence of transcriptional factor Pit-1, the extrapituitary (including that by immune cells) PRL secretion is regulated by the Pit-1 independent alternative promoter located 5840 bp distal to the start of the PRL transcriptional site [5]. This extrapituitary promoter contains functional single nucleotide polymorphism (SNP) ¡1149 G/T (rs1341239) in the GATA sequence; G allele leads to higher PRL mRNA in lymphocytes synthesis [6]. GG genotype has been associated with systemic lupus erythematosus (SLE) [6, 7], while TT genotype was identiWed as protective for non-Hodgkin and follicular lymphoma, and rheumatoid arthritis development [8, 9]. In this study, we looked for distribution of allele and genotype of the ¡1149 G/T SNP of the extrapituitary PRL promoter in 75 patients with SSc (average age 61.4), in 47 subjects with PM (average age 57.8), in 68 patients with dermatomyositis (DM) (average age 58.3), and in 123 healthy Czech individuals (blood donors and medical students, average age 39.7). SSc and PM/DM patients met classiWcation criteria for these illnesses [10, 11]. PCR– RFLP methodology was used for ¡1149G/T SNP detection [7]. BrieXy, we ampliWed 137 bp region of the PRL extrapituitary promoter and for restriction used ApoI endonuclease. We determined the following genotypes: the homozygote TT characterized by 120 + 17 bp, the homozygote GG by 85 + 35 + 17 bp, and the heterozygote GT by 120 + 85 + 35 + 17 bp DNA fragments. Results were evaluated by 2 test with Bonferroni correction. Control group was in Hardy–Weinberg equilibrium. The genotype and allele distribution did not show any signiWcant diVerences in groups of PM, DM, and SSc patients compared to healthy individuals (Table 1), and we detected neither gender distribution diVerences (data not shown) nor correlation with speciWc clinical or serological manifestation of these diseases (lung Wbrosis, type of scleroderma, antiScl-70 and antiJo-1 positivity) (Table 1). However, we found a signiWcant inverse association between TT genotype in SSc patients with disease onset after 45 years compared to individuals with SSc and diseases onset prior to 45 years (2/47 = 4.1 vs. 6/18 = 25%, respectively; P corrected = 0.02; OR = 0.13; CI (95%): 0.02–0.69). M. Fojtíková (&) · R. Bebvál · J. Vencovský · J. Tomasová Studýnková Institute of Rheumatology, Na Slupi 4, 12850 Prague 2, Czech Republic e-mail: fmar@centrum.cz

Polymorphism of the prolactin extrapituitary promoter in psoriatic arthritis

Psoriatic arthritis is characterized as seronegative arthritisthat aVects patients suVering from psoriasis [1]. Aetiologyof this condition is still not clear and therapeuticapproaches are not always successful. However, goodresponse to bromocriptine therapy decreasing prolactin lev-els in patients with psoriatic arthritis has been demonstratedin some reports [ 2, 3]. Prolactin acts as a cytokine and playsa role in pathogenesis of systemic autoimmune diseasessuch as rheumatoid arthritis and systemic lupus erythemat-osus [4, 5]. Moreover, high serum prolactin levels wereobserved in group of patients with psoriasis and linkbetween keratinocytes hyperproliferation and prolactin hasbeen proposed [6]. The peptide hormone prolactin is pro-duced from pituitary lactotrophs and extrapituitary tissuessuch as lymphocytes as well. Extrapituitary PRL produc-tion is regulated by an alternative promoter located 5.8 kbupstream from the pituitary one [7, 8]. A functional singlenucleotide polymorphism (SNP) G/T at the position i1149of this extrapituitary promoter has been observed and inlymphocytes higher PRL mRNA expression found to beconnected with G allele [9]. In our work we studiedi1149G/T SNP PRL in group of 83 Czech patients withpsoriatic arthritis (PsA).We genotyped i1149G/T SNP in 83 PsA patients and123 healthy individuals (control group). PsA patients: 43(51.8%) females, 40 (48.2%) males, average age 54.1 years.Control group: 40 (32.5%) females and 83 (67.5%) males,average age 38.7 years. This study was approved by theEthical Committee of the Third Faculty of Medicine,Charles University, Prague.PCR-RFLP methodology was used for i1149G/T SNPdetection. PCR: The 137 bp region of the PRL extrapitu-itary promoter was ampli Wed by employing following prim-ers: Forward primer 5 GCAGGTCAAGATAACCTGGAand reverse primer 5 CATCTCAGAGTTGAATTTATTTCCTT. The PCR reaction was run under these conditions:initial temperature 94°C for 2 min, then 32 cycles with94°C for 17 s, 55°C for 17 s, 72°C for 17 s, and Wnal tem-perature 72°C for 1 min. RFLP: ApoI restriction endonu-clease was used. The three genotypes were identiWed asindicated: the homozygote TT was characterized by120 + 17 bp, the homozygote GG by 85 + 35 + 17 bp, andthe heterozygote GT genotype by 120 + 85 + 35 + 17 bpDNA fragments. Results were evaluated by Chi square testwith Bonferroni correction.The genotypes and alleles frequency of i1149G/T SNPPRL extrapituitary promoter were similar in group of PsApatients and control group (Table1). We did not Wnd anydiVerences in genotype and allele distribution betweenhealthy female and male and between female and malepatients with psoriatic arthritis, respectively (data notshown). Moreover, we correlated genotype and allele distri-bution of i1149G/T SNP with clinical feature of psoriaticarthritis, such as age of onset psoriasis and radiologicaltype of psoriatic arthritis. In Table2 there are results of thiscorrelation, where no signiWcant association was detected.In conclusion, i1149G/T SNP of the extrapituitary pro-lactin promoter is not associated with psoriatic arthritis (seeTable 1) and its characteristic such as the onset of skinlesion and radiological type of arthritis (see Table2). OurWndings could indicate that i1149G/T SNP prolactin

TLR2 and TLR4 expression on CD14 ++ and CD14 + monocyte subtypes in adult-onset autoimmune diabetes

BACKGROUND Peripheral blood monocytes are key effectors of innate immunity. Dysfunction, changes in their counts or altered expression of cytokines and pattern-recognition receptors on monocytes may contribute to the development of the autoimmune type of diabetes mellitus (AD). AIMS We aimed to analyze the counts and proportions of the two main subtypes of monocyte cells, CD14(++) and CD14(+), and to look for potential changes in the expression of toll-like receptors 2 (TLR2) and 4 (TLR4) as well as cytokine prolactin (PRL) in adult-onset AD, including diabetes mellitus type 1 (T1DM) and latent autoimmune diabetes in adults (LADA). METHODS We examined 21 T1DM patients, 9 patients with LADA, 16 control patients with type 2 diabetes mellitus (T2DM) and 24 healthy individuals. All diabetic patients were diagnosed after the age of 18 years. Expression at the mRNA level was determined by quantitative PCR. Flow cytometry was used to ascertain membrane expression and cell counts. RESULTS T1DM patients had fewer CD14(++) (P < 0.01) and CD14(+) (P < 0.0001) monocytes whereas T2DM subjects showed decreased counts of CD14(+) monocytes compared to healthy controls (P < 0.001). TLR2 protein expression was significantly increased in T1DM CD14(+) monocytes compared to healthy controls (P < 0.05), while TLR4 expression in T1DM CD14(++) cells was significantly lower (P < 0.0001). There was no significant difference between the groups in terms of PRL mRNA expression in monocytes. CONCLUSIONS The observed changes in the proportions of both immune cell types and in the expression of functional pattern-recognition receptors on monocytes in the subjects examined may arise as a consequence of chronic inflammation that accompanies long-term diabetes.