Katarzyna Bogunia-Kubik

From molecular biology to nanotechnology and nanomedicine

Great progress in the development of molecular biology techniques has been seen since the discovery of the structure of deoxyribonucleic acid (DNA) and the implementation of a polymerase chain reaction (PCR) method. This started a new era of research on the structure of nucleic acids molecules, the development of new analytical tools, and DNA-based analyses. The latter included not only diagnostic procedures but also, for example, DNA-based computational approaches. On the other hand, people have started to be more interested in mimicking real life, and modeling the structures and organisms that already exist in nature for the further evaluation and insight into their behavior and evolution. These factors, among others, have led to the description of artificial organelles or cells, and the construction of nanoscale devices. These nanomachines and nanoobjects might soon find a practical implementation, especially in the field of medical research and diagnostics. The paper presents some examples, illustrating the progress in multidisciplinary research in the nanoscale area. It is focused especially on immunogenetics-related aspects and the wide usage of DNA molecules in various fields of science. In addition, some proposals for nanoparticles and nanoscale tools and their applications in medicine are reviewed and discussed.

Clinical significance of the HLA-E and CD94/NKG2 interaction

HLA-E belongs to the non-classical HLA (class Ib family) broadly defined by a limited polymorphism and a restricted pattern of cellular expression. So far, only two functional alleles differing at only one amino acid position (non-synonymous mutation) in the α2 heavy chain domain, where an arginine in position 107 in HLA-E*0101 is replaced by a glycine in HLA-E*0103, have been reported. The interaction between non-classical HLA-E molecule and CD94/NKG2A receptor plays a crucial role in the immunological response involving natural killer (NK) cells and cytotoxic T lymphocytes. All proteins forming CD94/NKG2 receptors are encoded by genes situated in the same cluster on chromosome 12, allowing tight control over the order of their expression. The inhibitory members of the NKG2 receptor family are available on the cell surface before activating the members to prevent autoimmune incidents during immune cells’ ontogenesis. In the present review, the potential role of this interaction in viral infection, pregnancy and transplantation of allogeneic hematopoietic stem cells (HSC) is presented and discussed. The review will also include the effect of HLA-E polymorphism on the outcome of HSC transplants in humans.

HSP70-hom gene polymorphism in allogeneic hematopoietic stem-cell transplant recipients correlates with the development of acute graft- versus-host disease

Background. A number of genetic polymorphisms have been shown to be associated with the outcome after allogeneic hematopoietic stem-cell transplantation (HSCT). In the present study, HSP70-hom polymorphism (+2763 G/A) was analyzed in the patients and donors of allogeneic HSCT in relation to transplantation outcome, susceptibility for generation of severe toxic lesions, and acute (a) graft-versus-host disease (GVHD). Methods. One hundred thirty-three recipients of allogeneic hematopoietic stem cells and 64 haploidentical and matched unrelated donors were investigated. All these individuals were typed for dimorphism within the HSP70-hom gene (+2763 G/A) with the use of amplification refractory mutation system technique. Results. Patients with the HSP-AA homozygous genotype presented more frequently with grade II to IV toxic lesions (12 of 14 vs. 61 of 105, P=0.039) and aGVHD (12 of 16 vs. 56 of 114, P=0.045). Conversely, DRB1*11 was associated with a lower risk of aGVHD manifestation (10 of 31 vs. 58 of 99, P=0.009). These contrary associations of HSP-AA and DRB1*11 with the risk of aGVHD were confirmed using logistic regression modeling in multivariable analysis (HSP-AA, odds ratio [OR]=3.833, P=0.004; DRB1*11, OR=0.224, P=0.048). None of donor HSP genotypes or patient-donor incompatibility within HSP alleles was associated with susceptibility to toxic complications or aGVHD. Conclusions. Polymorphism of the HSP70-hom gene is associated with the development of posttransplant complications. Recipient HSP-AA homozygous genotype is a risk factor for aGVHD.

MiR-29a Reduces TIMP-1 Production by Dermal Fibroblasts via Targeting TGF-β Activated Kinase 1 Binding Protein 1, Implications for Systemic Sclerosis

Background Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterised by skin and internal organs fibrosis due to accumulation of extra cellular matrix (ECM) proteins. Tissue inhibitor of metalloproteinases 1 (TIMP-1) plays a key role in ECM deposition. Aim To investigate the role of miR-29a in regulation of TAB1-mediated TIMP-1 production in dermal fibroblasts in systemic sclerosis. Methods Healthy control (HC) and SSc fibroblasts were cultured from skin biopsies. The expression of TIMP-1, MMP-1 and TGF-β activated kinase 1 binding protein 1 (TAB1) was measured following miR-29a transfection using ELISA, qRT-PCR, and Western Blotting. The functional effect of miR-29a on dermal fibroblasts was assessed in collagen gel assay. In addition, HeLa cells were transfected with 3′UTR of TAB1 plasmid cloned downstream of firefly luciferase gene to assess TAB1 activity. HC fibroblasts and HeLa cells were also transfected with Target protectors in order to block the endogenous miR-29a activity. Results We found that TAB1 is a novel target gene of miR-29a, also regulating downstream TIMP-1 production. TAB1 is involved in TGF-β signal transduction, a key cytokine triggering TIMP-1 production. To confirm that TAB1 is a bona fide target gene of miR-29a, we used a TAB1 3′UTR luciferase assay and Target protector system. We showed that miR-29a not only reduced TIMP-1 secretion via TAB1 repression, but also increased functional MMP-1 production resulting in collagen degradation. Blocking TAB1 activity by pharmacological inhibition or TAB1 knockdown resulted in TIMP-1 reduction, confirming TAB1-dependent TIMP-1 regulation. Enhanced expression of miR-29a was able to reverse the profibrotic phenotype of SSc fibroblasts via downregulation of collagen and TIMP-1. Conclusions miR-29a repressed TAB1-mediated TIMP-1 production in dermal fibroblasts, demonstrating that miR-29a may be a therapeutic target in SSc.

HSP70-hom gene single nucleotide (+2763 G/A and +2437 C/T) polymorphisms in sarcoidosis.

In the present study, two coding polymorphisms within the heat shock protein 70‐hom gene (HSP70‐hom) were analysed. One hundred and thirty‐eight individuals were studied, including 42 Polish patients with sarcoidosis, 13 of which presented with Löfgrens syndrome (LS), and 94 control subjects. Dimorphisms at positions +2763 (A/G) and +2437 (C/T) of the HSP70‐hom gene were typed using amplification refractory mutation system and polymerase chain reaction–restriction fragment length polymorphism technique, respectively.

The presence of IFNG 3/3 genotype in the recipient associates with increased risk for Epstein–Barr virus reactivation after allogeneic haematopoietic stem cell transplantation

Recent studies have shown that interferon‐γ gene (IFNG) polymorphism constitutes a risk factor for acute and chronic graft‐versus‐host disease (GvHD) after allogeneic haematopoietic stem cell transplantation (HSCT). Patients with IFNG 3/3 have been found to be more prone to GvHD. This rather puzzling result, as 3/3 genotype is associated with a decreased IFN‐γ production, was investigated in the present study in the context of Epstein–Barr virus (EBV) reactivation. Microsatellite polymorphism (CA)n within the first intron of IFNG gene was assessed in 83 HSCT recipients and related to EBV load. Quantification of EBV copies was performed by a real‐time polymerase chain reaction in peripheral blood cells taken from the patients 2–3 months after HSCT. It was found, that patients having IFNG 3/3 genotype presented with a high number of EBV copies (over 10/105 blood cells) when compared with the recipients with other IFNG genotypes (10/14 vs. 17/69, P < 0·001). This association was independent of recipients age, underlying disease, conditioning regimen, type of donor, source of stem cells or pretransplant donor and recipient EBV serological status. Thus IFNG 3/3 genotype, known to be associated with a decreased IFN‐γ production, appeared as a factor significantly contributing to the risk of EBV reactivation after allogeneic HSCT.

Lack of association between the TNF-α promoter gene polymorphism and susceptibility to B-cell chronic lymphocytic leukaemia

B‐cell chronic lymphocytic leukaemia (B‐CLL) is a lymphoproliferative disorder characterized by clonal expansion of B lymphocytes. The present study aimed to determine whether there is an association between the polymorphic features located within the promoter/enhancer region of tumour necrosis factor‐α (TNFA) gene and susceptibility to B‐CLL.

Analysis of associations between polymorphisms within genes coding for tumour necrosis factor (TNF)-alpha and TNF receptors and responsiveness to TNF-alpha blockers in patients with rheumatoid arthritis

INTRODUCTION Despite the fact that therapy with TNF-α inhibitors constitutes a breakthrough in rheumatoid arthritis management, no improvement is still achieved in approximately 30% of cases. The aim of the study was to evaluate whether single nucleotide polymorphisms (SNPs) within the TNF-α and TNF receptor encoding genes affect the efficacy of therapy with TNF-α inhibitors in patients with RA. METHODS Five SNPs within the TNF-α and TNF receptor encoding genes (TNFA: G-308A, G-238A, C-857T; TNFR1A G36A; TNFR1B T676G) were determined in 280 RA patients who had been treated with TNF-α inhibitors for at least 6 months or they stop therapy because of adverse events. The association between the relative change in DAS28 and SNP genotypes was tested by linear regression. RESULTS At week 24, low disease activity or remission was achieved by 45% of the patients. After 6 months remission of the disease or low disease activity were more frequently observed among patients homozygous for the TNFR1A 36A allele than among those who were GG homozygotes (52% vs. 34%, P=0.04). At week 24 DAS28 was significantly lower in the subgroup of patients homozygous for the TNFA-857T variant compared to the C allele carriers (P=0.045). The other polymorphisms were not found to be significantly associated with EULAR response at week 12 and 24 of the anti-TNF treatment. CONCLUSIONS Homozygosity for the TNFR1A 36A allele and the TNFA-875T variant could act as a genetic factor associated with better response to anti-TNF treatment.

Interferon Gamma 13-CA-Repeat Homozygous Genotype and a Low Proportion of CD4+ Lymphocytes Are Independent Risk Factors for Cytomegalovirus Reactivation with a High Number of Copies in Hematopoietic Stem Cell Transplantation Recipients

Cytomegalovirus (CMV) reactivation was analyzed in 92 recipients of allogeneic hematopoietic stem cell transplantation (HSCT) in relation to the proportion of CD4(+) lymphocytes in blood and a microsatellite polymorphism within the first intron of the interferon-gamma (IFNG) gene. CMV reactivation was found in 50% of the HSCT recipients; in 30% of these individuals, the level of CMV copies exceeded 100 per 10(5) peripheral blood (PB) cells on at least one occasion during the 100-day post-HSCT observation period. This high CMV copy level was most frequently found between 31 and 60 days post-HSCT (P = .021). Patients with > or = 100 CMV copies/10(5) cells were characterized by poorer overall survival (OS) compared with those lacking CMV copies or having < 100 CMV copies/10(5) cells (P = .04), and they suffered from severe post-HSCT complications, including acute graft-versus-host disease (aGVHD) and relapse. Thus, patients with > or = 100 CMV copies/10(5) cells were designated as having clinically significant CMV reactivation. Patients with < 10% CD4(+) lymphocytes had a higher number of CMV DNA copies than those with higher proportions of CD4(+) lymphocytes (0.62 vs 0.21, P = .001; mean +/- SEM, 4422 +/- 1667 vs 937 +/- 662 CMV copies/10(5) cells, P < .001, for the proportion of cases with reactivation and numbers of copies, respectively). Similarly, patients carrying 2 IFNG 13-CA-repeat alleles (homozygotes) had more frequent CMV reactivation (0.50 vs 0.26; P = .039) and a higher CMV load (4111 +/- 1699 vs 950+/-591 CMV copies/10(5) cells; P = .041) compared with those with other IFNG microsatellite allele constellations. Multivariate analysis demonstrated that the IFNG 13-CA-repeat homozygous genotype (odds ratio [OR] = 0.221; P = .044), a low proportion of CD4(+) lymphocytes (OR = 0.276; P = .050), and a lack of optimal (10/10 alleles) donor-recipient HLA match (OR = 15.19; P = .006) were independent risk factors for CMV reactivation with a high number of copies.

Interleukin‐10 gene polymorphisms influence the clinical course of non‐Hodgkin’s lymphoma

The pathophysiology of Non-Hodgkins lymphoma (NHL) is still unknown and clinical course is very unpredictable. Many cytokines, including interleukin-10 (IL-10), play a role in the perpetuation of this disease. The IL-10-producing capability has been found to be influenced by the IL-10 gene promoter polymorphisms. The aim of the present study was to assess whether any of IL-10 (-1082 A/G, -819 C/T and -592 A/C) genotypes prevails in Polish patients with NHL and whether IL-10 promoter polymorphisms may be associated with less or more favourable course of the disease. IL-10 gene promoter polymorphisms were assessed in 105 individuals, including 55 NHL patients and 50 ethically matched controls. The frequency of the IL-10 low-producing -1082 AA homozygous genotype was significantly higher in patients with aggressive NHL as compared with patients with indolent forms of the disease (0.57 vs 0.28, P < 0.05) and controls [0.57 vs 0.32, odds ratio (OR) = 2.69, P < 0.05]. Also, the presence of the ACC genotype was more frequently detected among patients with more aggressive disease than in those with indolent forms (0.74 vs 0.47, P < 0.05) and healthy controls (0.74 vs 0.42, OR = 3.69, P < 0.05). In multivariate analyses, the AA homozygosity (OR = 6.33, P < 0.05) and ACC genotype (OR = 3.57, P = 0.05) appeared as independent risk factors of more aggressive manifestation of NHL in addition to the elevated lactate dehydrogenase 480 level. Although no direct association was found between IL-10 promoter polymorphisms and NHL, IL-10 (-1082) AA homozygosity and IL-10 ACC genotype were found to be associated with unfavourable prognosis in patients with NHL.