Correlation of TNF-α and TGF-ß polymorphisms with protein levels in pancreatic ductal adenocarcinoma and colorectal cancer.

AIM OF THE STUDY: To determine the correlation of protein serum levels of two cytokines and their polymorphisms, which have an influence on their expression. MATERIAL AND METHODS: The study group consisted of 65 patients (33 men, 31 women) who met the criteria for inclusion and exclusion of pancreatic cancer, and 41 patients (25 men, 16 women) with colorectal cancer. The control group consisted of 100 healthy volunteers (63 men, 37 women). Detection of polymorphisms was performed using TaqMan probes, and concentration of proteins by ELISA method. RESULTS: The mean TNF-α concentration in patients with colorectal cancer was significantly higher compared to the control group, p< 0.0001. A statistically significant difference was noted when comparing both study groups, p = 0.0009. The analyses show that the occurrence of the polymorphic genotype -308AA of the TNF-α gene was not correlated with the increased concentration of the examined protein in patients with both pancreatic and colorectal cancer. It was also noted that the concentration of TGF-ß protein was significantly higher in patients with colorectal cancer than in patients with pancreatic cancer. These results also proved to be statistically significant, p = 0.0353. CONCLUSIONS: The only statistically significant effects were the correlations between patients belonging to a specific group (pancreatic cancer/colorectal cancer/control) and average protein levels. There was no effect of sex or genotype on the occurrence of elevated levels of TNF-α and TGF-ß protein control, despite their variability in particular types of cancer.

Predilection to Dermato-Lymphangio-Adenitis in Obstructive Lymphedema of Lower Limbs Depending on Genetic Polymorphisms at TNFα-308G>A, CCR2-190G>A, CD14-159C>T, TLR2 2029C>T, TLR4 1063A>G, TLR4 1363C>T, TGFß 74G>C, and TGFß 29T>C.

BACKGROUND: Infection is the most common type of complication observed in lymphedema and is promoted by lymphatic system dysfunction, which causes locoregional immune disorders. Infectious complications are primarily bacterial and most commonly cellulitis (dermato-lymphangio-adenitis, DLA) caused by patients' own skin Staphylococci epidermidis and aureus. The clinical course and outcomes in the immune response to infection have been shown to be associated with genetic polymorphisms. AIM: To investigate polymorphism of TNFα-308G>A, CCR2-190G>A, CD14-159C>T, TLR2 2029C>T, TLR4 1063A>G, TLR4 1363C>T, TGFß 74G>C, and TGFß 29T>C. The second part of study was the correlation of levels of TNFα and TGFß with their genes polymorphism in one hundred patients with lower limb postdermatitis lymphedema. RESULTS: (a) High percentage of TNFα homozygotes, no differences in genotypes of CD14-159C>T, CCR2-190G>A, TGFß 74G>C, TGFß 29T>C, and TLR4 1063A>G, low percentage of TLR2 2029C>T heterozygotes and homozygotes TT, and a high percentage of TLR4 1363C>T homozygotes TT, (b) low serum levels of TGFß and TNFα in 19% and 43% of patients, respectively, however, lack of correlation between low levels of these cytokines and frequency of homozygotes CC and AA, respectively. CONCLUSIONS: The practical implications of finding high frequency of genotype TT of TLR4 1363C>T are indications for testing this gene in patients with obstructive lymphedema of lower limbs and early antibiotic prophylaxis of recurrent attacks of DLA, and during elective surgery of lymphedema. The obtained data are also important as a contribution to mapping of genetic variations in acquired lymphedema of lower limbs.

An Overview of Genetic Changes and Risk of Pancreatic Ductal Adenocarcinoma.

The pancreatic carcinoma is a leading cause of death in cancer carriers worldwide. The early diagnostic is difficult due to late stage during diagnosis, lack of characteristic symptoms and also multifactor basis. In cancer development take part both, environmental and genetic factors, alone or in conjunction with each other. The nonspecific biomarkers of cancers are a reason for the search for more accurate factors which allow for fast and personalized diagnostics. Some of cancers have identified molecular (metabolic, biochemical or genetic) markers but in most cases the only clue is patient`s interview and abnormal levels of organ functions markers. Possible genetic basis of cancer suggests to widen studies on connection between environmental factors with both, nuclear and mitochondrial, genes changes.

Bacteria of leg atheromatous arteries responsible for inflammation.

BACKGROUND: Ischaemia of the lower limbs is frequently followed by inflammation and, in advanced cases, necrosis of peripheral tissues. Whether this is caused by arterial hypoperfusion only or by the presence of bacteria in the arterial walI as well remains unclear. The aim of the study was to prove the presence and source of bacteria in arterial specimens and evaluate their chemotactic properties resulting in the formation of periarterial cellular infiltrates. MATERIALS AND METHODS: Bacterial culture and testing for 16sRNA were performed in fragments of popliteal artery harvested from amputated limbs. Carotid artery plaques served as controls. Fragments of arteries were transplanted into scid mice to evaluate their chemotactic activity for macrophages. RESULTS: a) higher prevalence of isolates and 16sRNA in atherosclerotic popliteal than carotid arteries, b) high density of plaque and periarterial infiltrates and mRNA level for pro-inflammatory cytokines in popliteal arteries, c) prevalent microbes were Staphylococcus aureus, S. epidermidis and Enterococci, d) foot skin and arterial bacterial phenotypes and DNA revealed evident similarities, and e) more intensive mouse macrophage accumulation in popliteal than carotid implants into scid mice. CONCLUSIONS: The presence of bacteria in the lower limb arterial wall was documented. They may predispose to inflammation secondary to ischaemic changes.

Genetic Polymorphism and Messenger Ribonucleic Acid Concentrations of TNFα and TGFß Genes in Patients with Chronic Lower Limb Infections.

BACKGROUND: The number of chronic lower limb infections and their complications as venous and diabetic ulcers and chronic calf dermatitis is increasing worldwide. The clinical course and outcome in the immune responses to infection have been shown to be associated with genetic polymorphisms. The aim of study was to investigate frequencies of chosen single nucleotide polymorphisms (SNPs) in TNFα and TGFß genes in patients with chronic lower limb infections and evaluate expression of messenger ribonucleic acid (mRNA) concentrations in chronic leg ulcers. METHODS: Patients were divided into three groups: (group A) chronic venous leg ulcers, (group B) chronic post-traumatic non-healing wounds, and (group C) infected ischemic necrosis of the foot. Blood donors comprised the control group. Detection of polymorphisms was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and gene expression by real-time PCR methods. RESULTS: Patients in all groups showed higher frequency of TNFα gene polymorphism -308GG and lower frequency of -308GA genotypes than controls. The mutated homozygote AA was higher in groups A and B than in controls. The TGFß74GG genotype was represented at highest values in group B. The GC genotype was found in all groups at a similar concentration lower than in controls. Genotypes TGFß29TT and TC were represented at similar concentrations as controls. Analyses showed that the presence of the polymorphic allele -308A of TNFα gene was correlated with an increased concentration of gene expression in patients with chronic leg ulcers (group A). In the case of both TGFß gene polymorphisms the presence of polymorphic allele C resulted in increased TGFß gene expression. CONCLUSIONS: Comparison of genotypes in polymorphic sites in TNFα and TGFß genes with their expression concentrations showed that the presence of polymorphic alleles could predispose to increased production of their proteins. Patients with prolonged non-healing wounds should have their genotypes studied, and in cases of mutation, long-term antibiotic and immune protein supply should be considered.

Lack of effect of the CD14 promoter gene C-159T polymorphism on nutritional status parameters in hemodialysis patients.

BACKGROUND: CD14 is a membrane glycoprotein that acts as a co-receptor for the detection of bacterial lipopolysaccharide (LPS). Mutual interaction between CD14 and LPS plays an important role in the innate immune system. Increased serum soluble CD14 levels have been described in hemodialysis (HD) patients, and linked to increased mortality risk, inflammation and protein-energy wasting. The expression of CD14 may be influenced by CD14 promoter gene C-159T polymorphism. This study aimed to clarify the possible association between CD14 promoter gene C-159T polymorphism and nutritional status in hemodialysis patients. MATERIAL/METHODS: The study population consisted of 185 (104 males; 81 females) long-term HD patients treated in 5 dialysis centers. The control group consisted of 112 apparently healthy volunteers (32 males and 80 females). Nutritional status was assessed using a modified SGA scale, and anthropometric methods (BMI, WHR, waist, hip and mid-arm circumferences, biceps, triceps, subocular and subscapular skinfolds). Biochemical parameters evaluated included: CRP, albumin, creatinine, urea, cholesterol, triglycerides and TIBC. CD14 promoter gene C-159T polymorphism was determined by restriction fragment length polymorphism, after digestion of the PCR product with Hae III restriction endonuclease. RESULTS: Genotype and allele frequencies were similar to controls and compliant with Hardy-Weinberg equilibrium. No between-group differences were detected in measured variables with the exception of lower triglyceride levels in carriers of C allele in comparison to TT genotype. CONCLUSIONS: CD14 promoter gene C-159T polymorphism does not seem to be associated with nutritional status parameters in HD patients. It does seem, however, to influence triglyceride blood levels.

Genetic factors responsible for long bone fractures non-union.

INTRODUCTION: Approximately 10-15% of all fractures of long bones heal with delay, prolonged immobilization and repetitive operative interventions. Despite intense investigations, the pathomechanism of impaired healing of skeletal tissue remains unclear. An important role in the pathomechanism of mal-union of close fractures plays subclinically proceeding infections. AIM: The question arises whether colonization and proliferation of bacteria in the fracture gap could be related to the mutation of genes for factors regulating local antimicrobial response, such as pathogen recognizing receptors (PRR), cytokines and chemokines. METHODS: We carried out studies in patients with delayed long bone fractures estimating the frequency of mutation of genes crucial for pathogen recognition (TLR2, TLR4 and CD14), and elimination (CRP, IL-6, IL-1ra), as well as wound healing (TGF-ß). The molecular milieu regulating healing process (IGF-1, COLL1a, TGF-ß, BMP-2, and PDGF) was validated by Western blot analysis of the gap tissue. RESULTS: Microbiological investigations showed the presence of viable bacterial strains in 34 out of 108 gaps in patients with non-healing fractures (31.5%) and in 20 out of 122 patients with uneventful healing (16.4%) (P < 0.05). The occurrence of mutated TLR4 1/W but not 2/W gene was significantly higher (P < 0.05) in the non-healing infected than sterile group. In the non-healing infected group 1/W mutated gene frequency was also higher than in healing infected. In the TGF-ß codon 10 a significantly higher frequency of mutated homozygote T and heterozygote C/T in the non-healing infected versus non-healing sterile subgroup was observed (P < 0.05). Similar difference was observed in the non-healing infected versus healing infected subgroup (P < 0.05). The CRP (G1059C), IL1ra (genotype 2/2), IL-6 (G176C), CD14 (G-159T), TLR2 (G2259A) and TLR4/2 (Thr399Ile) polymorphisms did not play evident role in the delay of fracture healing. CONCLUSIONS: Individuals bearing the mutant TLR 4 gene 1/W (Asp299Gly) and TGF-ß gene codon 10 mutant T and T/C allele may predispose to impaired pathogen recognition and elimination, leading to prolonged pathogen existence in the fracture gaps and healing delays.

Microchimerism after pancreas and kidney transplantation--a review.

The main cause of a negative response to grafting is immune rejection connected with a reaction to the donor's antigens. The process of rejecting transplanted organs is a whole-body process. The reaction of the organism begins when the donor's antigens reach the recipient's lymphatic organs via blood or lymph. The donor's genetic material (DNA) is detected in the recipient's blood and lymphoid tissues even a few months after transplant rejection. Microchimerism occurs when in an individual patient the cells and genetic material from both the donor and recipient are present and the cell count of one of these is overrepresented. Such situations can occur after blood transfusions, grafts or pregnancies. It is suggested that this phenomenon could have an influence on tolerance, prolonged graft survival or the rejection process. The results of many experiments are thus far equivocal; therefore further research is needed elucidate the process and mechanisms of graft rejection and its molecular aspects.

[Genetic factors predisposing to bone fracture non-union. A role of single point mutation Asp299Gly TLR4 on pathogen-evoked healing]. / Genetyczna predyspozycja do zaburzen zrostu kostnego--wplyw punktowej mutacji Asp299Gly TLR4 na bakteriozalezne zaburzenia gojenia.

UNLABELLED: We previously reported the presence of the bacterial genetic material (16S rRNA) and viable pathogens in fracture gaps specimens, which suggests an impaired pathogen recognition and/or elimination. The aim of study was to validate the hypothesis that patients with delayed bone fracture healing express the higher frequency of TLR4 mutations. Observations were performed in 295 patients treated due to closed fractures of the long bones of the lower extremity; in 151 with delayed bone union (group A), and in 144 with uneventful healing (group B). Control group consisted of 125 healthy blood donors from ethnically the same as investigations groups polish population. Fracture gaps and deep tissue biopsies served for microbiological studies, and DNA isolated from venous blood leukocyteswas used for analysis of mutations of TLR4 gene at Asp299Gly (1/W) and Thr399Ile (2/W). RESULTS: Microbiological studies revealed positive isolates in 31.5% fracture gaps in group A and 16.4% in group B (p < 0.05). The most frequent isolates were S. epidermidis, S. aureus and S. warneri, capitis, sciuri and lentus, in lower percentage micrococci and enterococci. Amplification of 16S rRNA was positive in 56.8 and 65.2% of fracture gaps in both groups respectively. The frequency of occurrence of 1/W was significantly higher (p <0.05) in subgroups of patients with non-healing infected vs. sterile fractures. In all subgroups with viable pathogens isolated from fracture gaps the frequency of 1/W allele was higher when compared with subgroups, where fracture gaps occurred sterile. DISCUSSION: Performed investigations supported our previously reported observations that gaps of closed bone fractures are not sterile and are positive for 16S rRNA. Genetic predisposal to infection and inflammatory response evoked by a single TLR4 mutation may be one of the factors affecting bone union. Observed coexistence of bacterial colonization with decreased inflammatory reaction observed in individuals bearing TLR4 mutations have to be mentioned as a possible, etiologic factor responsible for delayed healing