The aim of the study was to detect prevalence of MBL2 exon 1 (codons 52, 54 and 57) genetic polymorphism in postmenopausal women in Bosnia and Herzegovina and its possible role as genetic risk factor for susceptibility to occurrence of osteoporosis in this study group. Also, we investigated association between MBL serum concentrations and osteoporosis in postmenopausal women. Genetic codons' variations were determined by PCR-RFLP and MBL in serum was measured by ELISA method in 75 postmenopausal women (37 with osteoporosis and 38 apparently healthy, non-osteoporotic women serving as a control). Serum MBL levels were not significantly different between osteoporosis and control group (492 (37-565.1) and 522.6 (477-559.4) ng/mL respectively, p=0.206). Genotype frequencies were not significantly different (p=0.997) between the studied groups of postmenopausal women. Genotype frequencies A/A, A/0 and 0/0 in osteoporosis group were 0.576; 0.405; 0.018 and in control group 0.562; 0.412; 0.026, respectively. Frequencies of A and 0 allele were 0.78 and 0.22 in osteoporosis and 0.77 and 0.23 in control group. The results do not suggest association of functional polymorphism of MBL2 gene and MBL serum concentration with osteoporosis in postmenopausal females.

Pyrosequencing represents one of the most thorough methods used to analyze polymorphisms. One advantage of using pyrosequencing for genotyping is the ability to identify not only single-nucleotide polymorphisms (SNPs) but also tri-allelic variations, insertions and deletions (InDels). In contrast to most other genotyping assays the sequence surrounding the polymorphism provides an internal control making this method highly reliable.

Lung infections are the leading cause of morbidity and mortality in cystic fibrosis (CF). Mannose-binding lectin (MBL) is a key factor in innate immunity. We therefore investigated whether MBL2 gene variants are associated with pulmonary function or susceptibility to Pseudomonas aeruginosa and Burkholderia cepacia infection in Slovak patients affected with CF. DNA polymorphisms in exon 1 and the promoter region were typed by single base primer extension assay in 91 patients and 100 healthy controls. The concentrations of MBL protein were determined in 34 patients by a sandwich enzyme-linked immunosorbent assay, and spirometric and microbiological data were collected from medical records. In this study we found that MBL2 genotypes were associated neither with earlier acquisition of P. aeruginosa or B. cepacia nor with reduced pulmonary function among patients. Although MBL2 genotypes were associated with the MBL2 protein serum level, results were statistically significant only for polymorphisms in exon 1, with p = 0.0008. The role of the MBL2 gene in lung disease severity in CF patients represents a very complex phenomenon where both genetic and environmental factors play an important role in addition to that of the MBL2 gene. Understanding this complexity requires further studies based on a broader scale of genetic factors involving both a whole-genome approach and a larger patient cohort.

BACKGROUND

Rheumatic fever (RF) is the result of an autoimmune response to pharyngitis caused by infection with Streptococcus pyogenes. RF is most prevalent in Africa and the Middle East. Rheumatic heart disease (RHD) is the most serious complication of RF. Mannose-binding lectin 2 gene (MBL2) has been reported to be correlated with different cardiac conditions. In Egyptian patients as a new studied ethnic population, it is the first time to evaluate the association between MBL2 gene polymorphism rs1800450 and RF with and without RHD.

METHODS

One hundred and sixty RF patients (80 with RHD and 80 without RHD) and eighty healthy ethnically matched controls were studied. MBL2 (rs1800450) was genotyped by real-time PCR using TaqMan® allele discrimination assay. The MBL level was measured by ELISA. Westergren erythrocytes sedimentation rate (ESR), anti-streptolysin O titer (ASOT), C-reactive protein (CRP) and complements (C3 and C4) were determined.

RESULTS

The AA genotype with high production of MBL was associated with increased risk of RHD more than the B allele carrying subjects. However, MBL2 genotype related to the low production of MBL was more frequently observed in those patients without RHD.

CONCLUSIONS

Our results suggested the involvement of MBL2 (rs1800450) polymorphism and its protein in RHD pathogenesis. Also, it might be a promising future strategy to utilize this polymorphism to help differentiate patients with RHD from those without RHD.

In this study, a 27-kDa ribonuclease (RNase) was purified from the dried fruiting bodies of the mushroom, Hohenbuehelia serotina. The isolation protocol involved anion exchange chromatography, affinity chromatography, cation exchange chromatography and gel filtration in succession. The RNase was unadsorbed on DEAE-cellulose, but was adsorbed on Affi-gel blue gel and CM-cellulose. The N-terminal amino acid sequence was TVGGSLAEKGN which showed homology to other fungal RNases to a certain degree. The RNase exhibited maximal RNase activity at pH 5 and 80˚C. It demonstrated the highest ribonucleolytic activity toward poly(C), a relatively high activity toward poly(U), and a considerably weaker activity toward poly(A) and (G). The RNase inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase with an IC50 of 50 µM and reduced [3H-methyl]-thymidine uptake by L1210 leukemia cells and MBL2 lymphoma cells with an IC50 of 25 µM and 40 µM, respectively.

The presence of the single nucleotide polymorphisms in exon 1 of the mannose-binding lectin 2 (MBL2) gene was evaluated in a sample of 159 patients undergoing coronary artery bypass surgery (71 patients undergoing valve replacement surgery and 300 control subjects) to investigate a possible association between polymorphisms and heart disease with Chlamydia infection. The identification of the alleles B and D was performed using real time polymerase chain reaction (PCR) and of the allele C was accomplished through PCR assays followed by digestion with the restriction enzyme. The comparative analysis of allelic and genotypic frequencies between the three groups did not reveal any significant difference, even when related to previous Chlamydia infection. Variations in the MBL plasma levels were influenced by the presence of polymorphisms, being significantly higher in the group of cardiac patients, but without representing a risk for the disease. The results showed that despite MBL2 gene polymorphisms being associated with the protein plasma levels, the polymorphisms were not enough to predict the development of heart disease, regardless of infection with both species of Chlamydia.

Endurance running training can lead to gradual accumulation of inflammation and soreness ultimately resulting in overuse injuries. Management of soreness and inflammation with pharmaceuticals (i.e. non-prescription pain relievers) during long-term training is not a suitable solution due to known side effects (e.g. gastrointestinal complications, etc.). Dietary polyphenols (i.e. curcumin, pomegranate, etc.) have been purported to reduce inflammation and muscle soreness, without these negative side effects making them ideal for use in an exercise model. The purpose of the present feasibility study was to explore the combined effect of optimized curcumin and pomegranate extract supplementation prior to (PRE) and after (4H and 24H) an organized half-marathon race on blood inflammatory proteins and inflammation-associated RNA. Daily supplementation (1000 mg/d) started 26 days before a half-marathon which doubled on days 27-31. Data were analyzed with R software and Welch t-test, significance set at p < 0.05. At both 4H and 24H, supplementation was associated with alterations in protein (IL-10, IL-13, IL-4, ITAC, MIP-1alpha, MIP-3alpha, BDNF, sIL-2Ralpha, and TNF-alpha; p < 0.05) and RNA (CCL22, GUSB, IL-6, LINC00305, NKILA, PTGES, THRIL, TRAF6, ARG2, CD1A, CD55, CFI, CSF2, CXC3CL1, CX3CR1, EDNRB, GATA3, LILRB5, THY1, CD3D, MRC1, GPR183, HAMP, MBL2, CASP3, B2M, KLRF2, PDCD1LG2, IL-10, PTGS2, TLR2, IL-6R, IL-8, IL-7R, MASP1, MYD88, TNFRSF1B, TNFRSF1A, and TIRAP; p < 0.05) biomarkers compared to control. Pathway classification of these biomarkers indicated supplementation may be associated with a more favorable muscle recovery profile. Our findings support the notion that combined curcumin and pomegranate supplementation may represent a useful addition to a comprehensive exercise training plan.

Keywords: inflammation; muscle injury; polyphenols; running.

OBJECTIVE

To study the association between cervical insufficiency and single nucleotide polymorphisms in seven genes coding for pro- and anti-inflammatory cytokine-related factors, mannose-binding lectin 2 (MBL2), collagen1α1 (COL1A1), factor II and factor V Leiden genes.

METHODS

In a case-control study, potential maternal biomarkers for cervical insufficiency were investigated in 30 women with a history of second-trimester miscarriage or preterm birth due to cervical insufficiency and in 70 control women.

RESULTS

Homozygous carriers of the interleukin 6 (IL6) -174 genotype GG had an odds ratio (OR) of 3.1 [95% confidence interval (95% CI) 1.3-7.4, p = 0.01] and MBL2 genotypes coding for low or intermediate levels of plasma MBL had an OR of 3.3 (95% CI 1.2-9.0, p = 0.01) for cervical insufficiency compared with controls. Serum MBL levels were lower in women with cervical insufficiency than in controls (median 408 and 1,985 ng/ml, respectively, p < 0.01).

CONCLUSIONS

Single nucleotide polymorphisms in the IL6 gene and the MBL2 gene and low MBL levels related to the latter polymorphism may increase the risk of preterm birth due to cervical insufficiency.

Decades of artificial selection have significantly improved performance and efficiency of animal production systems. However, little is known about the microevolution of genomes due to intensive breeding. Using whole-genome sequencing, we document dynamic changes of chicken genomes under divergent selection on adiposity over 19 generations. Directional selection reduced within-line but increased between-line genomic differences. We observed that artificial selection tended to result in recruitment of preexisting variations of genes related to adipose tissue growth. In addition, novel mutations contributed to divergence of phenotypes under selection but contributed significantly less than preexisting genomic variants. Integration of 15 generations genome sequencing, genome-wide association study, and multi-omics data further identified that genes involved in signaling pathways important to adipogenesis, such as autophagy and lysosome (URI1, MBL2), neural system (CHAT), and endocrine (PCSK1) pathways, were under strong selection. Our study provides insights into the microevolutionary dynamics of domestic animal genomes under artificial selection.

Keywords: Biological Sciences; Evolutionary Biology; Genetics; Genomics.

A 17.5-kDa trypsin inhibitor was purified from Phaseolus vulgaris cv. "gold bean" with an isolation protocol including ion exchange chromatography on DEAE-cellulose (Diethylaminoethyl-cellulose), affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-sepharose (Sulfopropyl-sepharose), and gel filtration by FPLC (Fast protein liquid chromatography) on Superdex 75. It dose-dependently inhibited trypsin with an IC50 value of 0.4 μM, and this activity was reduced in the presence of dithiothreitol in a dose- and time-dependent manner, signifying the importance of the disulfide linkage to the activity. It inhibited [methyl-³H] thymidine incorporation by leukemia L1210 cells and lymphoma MBL2 cells with an IC50 value of 2.3 μM and 2.5 μM, respectively. The inhibitor had no effect on fungal growth and the activities of various viral enzymes when tested up to 100 μM.

Traumatic brain injury (TBI) is the leading cause of death in the global population. Disturbed inflammatory processes after TBI exacerbate secondary brain injury and contribute to unfavorable outcomes. Multiple inflammatory events that accompany brain trauma, such as glial activation, chemokine release, or the initiation of the complement system cascade, have been identified as potential targets for TBI treatment. However, the participation of chemokines in the complement activation remains unknown. Our studies sought to determine the changes in the expression of the molecules involved in the CCL2/CCL7/CCL12/CCR2 pathway in the injured brain and the effect of CCL2, CCL7, and CCL12 (10, 100, and 500 ng/mL) on the classic and lectin complement pathways and inflammatory factors in microglial cell cultures. Brain injury in mice was modeled by controlled cortical impact (CCI). Our findings indicate a time-dependent upregulation of CCL2, CCL7, and CCL12 at the mRNA and protein levels within the cortex, striatum, and/or thalamus beginning 24 h after the trauma. The analysis of the expression of the receptor of the tested chemokines, CCR2, revealed its substantial upregulation within the injured brain areas mainly on the mRNA level. Using primary cortical microglial cell cultures, we observed a substantial increase in the expression of CCL2, CCL7, and CCL12 after 24 h of LPS (100 ng/mL) treatment. CCL2 stimulation of microglia increased the level of IL-1β mRNA but did not influence the expression of IL-18, IL-6, and IL-10. Moreover, CCL2 significantly increased the expression of Iba1, a marker of microglia activation. CCL2 and CCL12 upregulated the expression of C1qa but did not influence the expression of C1ra and C1s1 (classical pathway); moreover, CCL2 increased ficolin A expression and reduced collectin 11 expression (lectin pathway). Additionally, we observed the downregulation of pentraxin 3, a modulator of the complement cascade, after CCL2 and CCL12 treatment. We did not detect the expression of ficolin B, Mbl1, and Mbl2 in microglial cells. Our data identify CCL2 as a modulator of the classical and lectin complement pathways suggesting that CCL2 may be a promising target for pharmacological intervention after brain injury. Moreover, our study provides evidence that CCL2 and two other CCR2 ligands may play a role in the development of changes in TBI.

Keywords: CCL2; complement system; lectin pathway; microglia.

The aim of this systematic review and meta-analysis was to pool the data on Single Nucleotide Polymorphisms (SNPs) in immune response genes associated with dental caries. Nineteen studies were included in the review and 18 in the meta-analysis. Twenty-two SNPs were evaluated, which are linked to six different genes (MBL2, LFT, MASP2, DEFB1, FCN2 and MUC5B). Most SNPs (81.8%) are related to the possible functional impact on protein coding. The MBL2 gene was associated with caries experience in the analysis of the homozygote (OR = 2.12 CI95%[1.12-3.99]) and heterozygote (OR = 2.22 CI95%[1.44-3.44]) genotypes. The MUC5B gene was associated according to an analysis of the heterozygous genotype (OR = 1.83 CI95%[1.08-3.09]). Thus, SNPs related to immune response genes are linked to the phenotype of caries experience. Although the meta-analysis showed that the genes MBL2 and MUC5B were associated with caries, these results should be interpreted with caution due to the quality of the evidence.

Keywords: Cariogenic; DMFT; genetics; immune system; oral habits.

Recent thymic emigrants that fail postpositive selection maturation are targeted by complement proteins. T cells likely acquire complement resistance during maturation in the thymus, a complement-privileged organ. To test this, thymocytes and fresh serum were separately obtained and incubated together in vitro to assess complement deposition. Complement binding decreased with development and maturation. Complement binding decreased from the double-positive thymocyte to the single-positive stage, and within single-positive thymocytes, complement binding gradually decreased with increasing intrathymic maturation. Binding of the central complement protein C3 to wild-type immature thymocytes required the lectin but not the classical pathway. Specifically, MBL2 but not MBL1 was required, demonstrating a unique function for MBL2. Previous studies demonstrated that the loss of NKAP, a transcriptional regulator of T cell maturation, caused peripheral T cell lymphopenia and enhanced complement susceptibility. To determine whether complement causes NKAP-deficient T cell disappearance, both the lectin and classical pathways were genetically ablated. This blocked C3 deposition on NKAP-deficient T cells but failed to restore normal cellularity, indicating that complement contributes to clearance but is not the primary cause of peripheral T cell lymphopenia. Rather, the accumulation of lipid peroxides in NKAP-deficient T cells was observed. Lipid peroxidation is a salient feature of ferroptosis, an iron-dependent nonapoptotic cell death. Thus, wild-type thymocytes naturally acquire the ability to protect themselves from complement targeting by MBL2 with maturation. However, NKAP-deficient immature peripheral T cells remain scarce in complement-deficient mice likely due to ferroptosis.

The complement system is essential for protection against infections in oncologic patients because of the chemotherapy-induced immunosuppression. One of the key elements in the activation of the complement system via the lectin pathway is the appropriate functioning of mannose-binding lectin (MBL) and mannose-binding lectin-associated serine protease 2 (MASP2) complex. The objective of our study was to find an association between polymorphisms resulting in low MBL level and activation of the MBL-MASP2 complex. Also, we aimed at finding a connection between these abnormalities and the frequency and severity of febrile neutropenic episodes in children suffering from hemato-oncologic diseases. Ninety-seven patients had been enrolled and followed from the beginning of the therapy for 8 months, and several characteristics of febrile neutropenic episodes were recorded. Genotypes of 4 MBL2 polymorphisms (-221C/G, R52C, G54D, G57E) were determined by real-time polymerase chain reaction. Activation of the MBL-MASP2 complex was evaluated by enzyme-linked immunosorbent assay at the time of diagnosis and during an infection. The number of febrile neutropenic episodes was lower, and the time until the first episode was longer in patients with normal MBL level than in patients with low MBL level coding genotypes. The MBL-MASP2 complex activation level correlated with the MBL genotype and decreased significantly during infections in patients with low MBL level. Our results suggest that infections after immunosuppression therapy in children suffering from hemato-oncologic diseases are associated with the MBL2 genotype. Our results may contribute to the estimation of risk for infections in the future, which may modify therapeutic options for individuals.

OBJECTIVE

HIV patients have increased atherosclerotic coronary vascular disease (ASCVD), thought to be mediated through inflammatory mechanisms. We hypothesized that among asymptomatic HIV-infected patients with subclinical coronary plaque, statin therapy would modulate unique inflammatory and cardiovascular proteins in relation to change in subclinical coronary plaque volume. We tested this hypothesis using a novel proteomics approach.

METHODS

Forty HIV-infected participants were randomized to atorvastatin (40 mg/day) versus placebo, and underwent computed tomography coronary angiography to quantify plaque volume at baseline and 1 year.

METHODS

We used Olink Cardiovascular III and Cardiometabolic panels based on dual antibody epitope recognition with linked DNA amplification to compare change over time in 184 proteins in treatment versus placebo and in relation to change in coronary plaque volume.

RESULTS

Six proteins (TFPI, CCL24, NT-Pro BNP, MBL2, LTBR, PCOLCE) changed significantly in the atorvastatin versus placebo group, many in innate immune and other novel inflammatory pathways. Twenty-six proteins changed significantly in relationship to total coronary plaque volume over 1 year. Notably, many of these proteins changed only weakly in relationship to change in low-density lipoprotein (LDL). Overlapping these two broad discovery approaches, proteins involved in myocardial fibrosis/collagen formation and monocyte chemoattraction changed with statin treatment, in relationship to plaque volume, but not LDL.

CONCLUSIONS

This proof-of-concept study employing a proteomic discovery platform offers insight into statin effects on novel immune pathways relevant to ASCVD progression in HIV. Novel biomarker discovery may enhance precision medicine strategies to estimate the efficacy of targeted therapies to reduce ASCVD progression and events in HIV.

Trauma is a major public health problem worldwide. Infectious complications, sepsis, and multiple organ dysfunction syndrome (MODS) remain important causes for morbidity and mortality in patients who survive the initial trauma. There is increasing evidence for the role of genetic variation in the innate immune system on infectious complications in severe trauma patients. We describe a trauma patient with multiple infectious complications caused by multiple micro-organisms leading to prolonged hospital stay with numerous treatments. This patient had multiple single nucleotide polymorphisms (SNPs) in the MBL2, MASP2, FCN2 and TLR2 genes, most likely contributing to increased susceptibility and severity of infectious disease.

For thousands of years human beings have resisted life-threatening pathogens. This ongoing battle is considered to be the major force shaping our gene pool as every micro-evolutionary process provokes specific shifts in the genome, both that of the host and the pathogen. Past populations were more susceptible to changes in allele frequencies not only due to selection pressure, but also as a result of genetic drift, migration and inbreeding. In the present study we have investigated the frequency of five polymorphisms within innate immune-response genes (SLC11A1 D543N, MBL2 G161A, P2RX7 A1513C, IL10 A-1082G, TLR2 -196 to -174 ins/del) related to susceptibility to infections in humans. The DNA of individuals from two early Roman-Period populations of Linowo and Rogowo was analysed. The distribution of three mutations varied significantly when compared to the modern Polish population. The TAFT analysis suggests that the decreased frequency of SLC11A1 D543N in modern Poles as compared to 2nd century Linowo samples is the result of non-stochastic mechanisms, such as purifying or balancing selection. The disparity in frequency of other mutations is most likely the result of genetic drift, an evolutionary force which is remarkably amplified in low-size groups. Together with the FST analysis, mtDNA haplotypes' distribution and deviation from the Hardy-Weinberg equilibrium, we suggest that the two populations were not interbreeding (despite the close proximity between them), but rather inbreeding, the results of which are particularly pronounced among Rogowo habitants.