Three-dimensional endoanal ultrasound is accurate and reproducible in determining type and height of anal fistulas.

AIM: Surgical treatment of high anal fistulas is associated with the potential risk of faecal incontinence and recurrence. The primary aim of this study was to determine the accuracy of three-dimensional endoanal ultrasound (3D-EAUS) in the assessment of height and type of anal fistulas, compared to the intra-operative findings (gold standard). The secondary aim was to evaluate the inter-observer reproducibility of 3D-EAUS. METHOD: The study design was a prospective analysis of retrospective data. 299 patients (202 men), mean age 45.3 years, who underwent surgery for anal fistulas, were included. All patients were preoperatively assessed by 3D-EAUS. Two readers independently reviewed the volumes to determine the type and height of fistulas. Sensitivity, specificity, positive and negative predictive values, proportion of agreements and Cohen's kappa coefficient (κ) were calculated for both examiners. Ultrasound findings were compared with intra-operative data (reference standard), evaluated blindly by the surgeons. RESULTS: At surgery, 201 (67%) were transsphincteric, 49 (16%) suprasphincteric, 47 (16%) intersphincteric and two (1%) extrasphincteric fistulas. Intra-operatively, 177 (59%) were low and 122 (41%) high fistulas. The overall accuracy of 3D-EAUS was 91% for fistula type (271/299 fistulas: 97% transsphincteric, 100% intersphincteric, 57% suprasphincteric, 0% extrasphincteric) and 92% for fistula height (275/299 fistulas: 80% high and 100% low). Both readers reported very good agreement with surgery in the assessment of fistula type (proportion of agreement 0.88, κ = 0.89) and height (proportion of agreement 0.90, κ = 0.91). CONCLUSIONS: 3D-EAUS is an accurate and reproducible modality for the assessment of type and height of anal fistulas.

Association of the Smad3 and NFATc2 gene polymorphisms and their serum levels with susceptibility to rheumatoid arthritis in Polish cohorts.

One among many factors involved in induction of rheumatoid arthritis (RA) are T cells, the differentiation of which depends upon a unique combination of stimulants and subsequent activation of diverse transcription factors. The aim of this study was to identify polymorphic variants in Smad3 and NFATc2 genes and their possible association with susceptibility to and severity of RA. A total of 272 RA patients, 321 for Smad3 and 304 for nuclear factor of activated T cells (NFAT)c2 healthy individuals, were examined for rs6494629 C/T and rs2289263 T/G Smad3 and rs880324 NFATc2 gene polymorphisms using the polymerase chain reaction-fragment length polymorphism (PCR-RFLP) method and TaqMan single nucleotide polymorphism (SNP) genotyping assay, respectively. Serum Smad3 and NFATc2 levels in RA patients and controls were measured by enzyme-linked immunosorbent assay (ELISA). The rs6494629 C/T Smad3 gene polymorphism under the recessive (TT versus CC+CT) and over-dominant (CC+TT versus CT) models were associated with RA (P=0.014 and P=0.008, respectively). Smad3 rs2289263 T/G revealed differences in the case-control distribution in co-dominant, recessive and over-dominant models (P=0.037, P=0.010, P=0.034). Overall, rs6494629 C/T and rs2289263 T/G Smad3 gene polymorphisms were in a weak linkage disequilibrium (LD) with D'=0.116 and r(2)=0.004. After Bonferroni correction, the genotype-phenotype analysis showed no significant correlation of the Smad3 rs6494629 C/T and rs2289263 T/G and NFATc2 rs2289263 TT polymorphisms with disease activity, joint damage and extra-articular manifestation in RA patients. Serum Smad3 and NFATc2 levels were significantly higher in RA patients than in control groups (both P=0 0000). The present findings indicated that Smad3 genetic polymorphisms may be associated with the susceptibility to RA in the Polish population.

Association of single nucleotide polymorphisms in the IL27 gene with rheumatoid arthritis.

Rheumatoid arthritis (RA) is one of the autoimmune diseases, where different polymorphisms in cytokine genes play a pathogenic role. Interleukin 27 (IL-27) is a novel pro-/anti-inflammatory cytokine, an excellent candidate for chronic inflammatory disease studies. The aim of the study was to identify polymorphisms in the IL-27 gene and their possible association with susceptibility to and severity of RA. Two hundred and seventy-four patients with RA and of 295 healthy individuals were examined for -924A/G and 4730T/C IL27 gene polymorphisms using PCR-RFLP method and TaqMan SNP genotyping assay, respectively. Haplotype frequencies of IL-27 polymorphisms were estimated using SHEsis platform. Frequencies of the -924GG genotype and the -924G allele were statistically higher in RA patients comparing with the healthy control group (P = 0.008 and P = 0.004, respectively). Overall, strong LD was observed between the IL27 gene -924A/G and 4730 T/C polymorphisms (D' = 0.613, r2 = 0.199). From four possible haplotypes, frequencies of two (CA and CG) showed significant differences between both examined groups (respectively: P < 0.001 and P = 0.001062). The genotype-phenotype analysis showed significant association between the IL-27 4730 T/C polymorphism and HAQ score and means value of the ESR, additionally they revealed that individuals with the polymorphic allele -924G had more advanced disease than wild-type allele carriers. Present findings indicated that IL27 -924A/G polymorphism may be involved in susceptibility to RA in the Polish population.

MicroRNAs, Aging, Cellular Senescence, and Alzheimer's Disease.

Aging is a normal process of living being. It has been reported that multiple cellular changes, including oxidative damage/mitochondrial dysfunction, telomere shortening, inflammation, may accelerate the aging process, leading to cellular senescence. These cellular changes induce age-related human diseases, including Alzheimer's, Parkinson's, multiple sclerosis, amyotrophic lateral sclerosis, cardiovascular, cancer, and skin diseases. Changes in somatic and germ-line DNA and epigenetics are reported to play large roles in accelerating the onset of human diseases. Cellular mechanisms of aging and age-related diseases are not completely understood. However, recent discoveries in molecular biology have revealed that microRNAs (miRNAs) are potential indicators of aging, cellular senescence, and Alzheimer's disease (AD). The purpose of our chapter is to highlight recent advancements in miRNAs and their involvement in cellular changes in aging, cellular senescence, and AD. This chapter also critically evaluates miRNA-based therapeutic drug targets for aging and age-related diseases, particularly Alzheimer's.

Association of HLA-DRB1 alleles with susceptibility to mixed connective tissue disease in Polish patients.

Mixed connective tissue disease (MCTD) is a systemic autoimmune disease, originally defined as a connective tissue inflammatory syndrome with overlapping features of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), polymyositis/dermatomyositis (PM/DM) and systemic sclerosis (SSc), characterized by the presence of antibodies against components of the U1 small nuclear ribonucleoprotein (U1snRNP). The aim of the study was to assess the frequency of (high-resolution-typed) DRB1 alleles in a cohort of Polish patients with MCTD (n = 103). Identification of the variants potentially associated with risk and protection was carried out by comparison with the DKMS Polish Bone Marrow Donor Registry (41306 alleles). DRB1*15:01 (odds ratio (OR): 6.06; 95% confidence interval (CI) 4.55-8.06), DRB1*04 (OR: 3.69; 95% CI 2.69-5.01) and *09:01 (OR: 8.12; 95% CI 2.15-21.75) were identified as risk alleles for MCTD, while HLA-DRB1*07:01 allele was found to be protective (OR: 0.50; 95% CI 0.28-0.83). The carrier frequency of the DRB1*01 was higher in MCTD patients compared with controls, although the differences were not statistically significant. Our results confirm the modulating influence of HLA-DRB1 genotypes on development of connective tissue diseases such as MCTD.

Characterization of two major isoforms of juvenile hormone esterase from Trichoplusia ni (Lepidoptera).

The two major isoforms of juvenile hormone (JH) esterase isolated from Trichoplusia ni were fragmented by cyanogen bromide and trypsin digestion. The resulting CNBr or CNBr/trypsin fragments were characterized and compared biochemically by SDS-PAGE, isoelectric focusing, two-dimensional electrophoresis and HPLC. Similar and unique fragments were examined for sequence, antigenic determinants and carbohydrate moieties. The studies identified small regions of the proteins which possess either potentially different sequences or different post-translational modifications. The location of a glycosylated asparagine residue was determined, as well as a region containing an epitope probably composed of a linear sequence of residues. An N-terminal region was identified that contained charge variation between the two isoforms and the sequence was obtained for the only unique CNBr/trypsin fragment detected from that region. These are the first data on mapping of regions of charge variation, epitope location and glycosylation sites for this enzyme from any insect species.

Epitope recognition and T cell receptors in recurrent autoimmune anterior uveitis in Lewis rats immunized with myelin basic protein.

Lewis rats immunized with myelin basic protein (MBP) develop experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU). Rats recover and become resistant to further reinduction of EAE. We investigated whether the resistance to reinduction of EAE was associated with the resistance to AU in LEW rats reinjected with MBP. We demonstrated that while rats remained resistant to EAE, they become susceptible to uveitis after recovery, and suffered a second episode of disease. The susceptibility to reinduced disease was associated with the recognition of new MBP epitopes. In contrast to the initial episode of AU, TCR Vbeta8.2 predominance was not observed in the iris/ciliary body. Our results suggest that T cells specific for MBP, which are rapidly reactivated when re-exposed to antigen, are sufficient to induce clinical uveitis in LEW rats. This process may involve a shifting of T cell specificity from the major encephalitogenic peptide utilizing the Vbeta8.2 receptor to a more diverse cell repertoire.

Expression of CC chemokines and their receptors in the eye in autoimmune anterior uveitis associated with EAE.

PURPOSE: To determine the pattern of expression of CC chemokines and their receptors in the eyes of Lewis rats and to establish their role in autoimmune anterior uveitis (AU) associated with experimental autoimmune encephalomyelitis (EAE). METHODS: EAE/AU was induced in Lewis rats with myelin basic protein in complete Freund's adjuvant (CFA). The rats were scored for the development of clinical EAE and AU. The expression of CCL5/regulated on activation normal T-cell expressed and secreted (RANTES), CCL2/monocyte chemotactic protein (MCP)-1, CCL3/macrophage inflammatory protein (MIP)-1alpha, and CCL4/MIP-1beta and their receptors was examined at the preclinical stage, onset, peak, and recovery by RT-PCR and ELISA. EAE/AU rats were treated with neutralizing polyclonal antibodies against CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL2/MCP-1, and CCL5/RANTES and tested for the suppression of onset of clinical AU and EAE. The control group received normal rabbit IgG at the same dose. RESULTS: The gene expression of those chemokines was upregulated concurrently with symptom onset of EAE/AU and correlated with the intensity of inflammatory changes in the eye and central nervous system (CNS). The highest expression of CCL4/RANTES, CCL2/MCP-1, and CCL3/MIP-1alpha in the eye was detected at onset of clinical uveitis, whereas CCL4/MIP-1beta was elevated at the peak of AU. The expression of chemokine receptors associated with T-helper (Th)1-type response, CCR1 and CCR5, correlated with their appropriate ligands and was the highest at the peak of AU, whereas CCR2, the receptor for CCL2/MCP-1, was present before the onset of the disease. Treatment of anti-MIP-1beta and anti-MCP-1 significantly delayed the onset and shortened the duration of AU and EAE. Anti-MIP-1alpha treatment had no effect on clinical EAE but inhibited the clinical signs of AU. Although CCL5/RANTES expression was observed during the entire course of the disease, anti-RANTES treatment had no effect on clinical disease progression. CONCLUSIONS: The data suggest that CCL2/MCP-1, CCL3/MIP-1alpha, and CCL4/MIP-beta contribute to the recruitment of inflammatory cells into the eye and CNS and to disease activity.

Studies on binding of HIV-1 p24gag peptide to HLA-Cw3+ cells.

Human major histocompatibility complex class I antigens, HLA-C, are expressed on the cell surface at approximately a tenfold lower level than HLA-A and -B. We hypothesized that the expression of HLA-C is limited by the quantity of high affinity peptides which bind to these molecules, thus allowing only a small fraction of HLA-C molecules to be transported and/or to remain stable on the cell surface. If this assumption is correct, then the addition of exogenous peptide should increase cell surface HLA-C expression. To verify the hypothesis, we pulsed lymphoblastoid cell line PAJ (HLA-Cw3+) with synthetic HIV-1 p24gag 145-152 peptide, known to be presented to T-lymphocytes by HLA-Cw3 molecule. PAJ (HLA-Cw3+) cells bound approximately two times more of the peptide than HAJ (HLA-Cw3-), and four times more than 500/C9 (HLA-Cw3-) cells. Accordingly, overnight pulsing of PAJ cells with the p24gag 145-152 peptide caused an increase in class I HLA expression detected on the cell surface by flow cytofluorimetric analysis with anti-HLA-B,C monoclonal antibodies but not by anti-HLA-A antibody. In contrast, HLA-Cw3- cells treated in the same manner did not show any increase of HLA class I expression. Our data suggest that low concentration of high affinity peptides within the cell may be one of the factors limiting cell surface expression of HLA-C molecules.

Human in vitro cell lines verification by minisatellite DNA restriction fragment length polymorphism.

Four families of human in vitro cell lines were tested for minisatellite restriction fragment length polymorphism (RFLP) using multilocus probes MZ1.3 and/or 33.15 after digestion of DNA with restriction enzymes HinfI or HaeIII. These results confirmed that (i) the RFLP pattern is relatively stable in established cell lines and, therefore, could be used as a specific marker of a cell line identity, (ii) the use of MZ1.3 and 33.15 probes permits the identification of hybridomas and (iii) one of the cell lines tested, a lymphoblastoid cell line HAJ, may possess a hot spot of mutation.

Transcriptional regulation of an unusual trypsin-related protein expressed during insect metamorphosis.

The full-length cDNA for a trypsin-related protein expressed during larval-pupal metamorphosis was obtained. The encoded N-terminal half of the protein possessed similarity to trypsin proteases, including the correctly positioned histidine and aspartic acid members of the catalytic triad. However, the remainder of the encoded protein bore little resemblance to trypsin, and was altogether missing the canonical sequence containing the catalytic serine residue. The product of in vitro transcription/translation of the cDNA was a 28 kDa protein. Under normal conditions of a declining juvenile hormone titer, transcription of the mRNA as a proportion of total genomic transcription steadily increased for the first 3 days of the final stadium, but this increase was delayed and suppressed by maintenance of a high juvenile hormone titer. During the normal increase in transcription on day 3 of the final stadium, a sharp decline was observed in the steady-state abundance of the transcript, measured relative to abundance of the remaining mRNAs, suggesting that the stability of the message decreases after tissue commitment for a pupal molt.

Polymorphism of C3 component of complement in the Polish population. II. Rare phenotypes in C3 system.

In a sample of the Polish population including 4741 adults, 15 phenotype variants were found in 22 of them. These variants are determined by 12 rare alleles of the mean frequency 0.0023. Family studies of several probands with C3 phenotype variants have confirmed their genetic determination. They have been observed to be heterozygotes in which beside one common gene, rare codominant alleles are situated. Studies on polymorphism of C3 component carried out on numerous populations, allowed the discovery of new phenotype variants within the C3 group system. Family studies have confirmed their hereditary character and that they are determined by the alleles codominant in relation to the commonly occurring C3S and C3F. The paper presents the results of studies on the rare C3 phenotype variants encountered in the Polish population.

Polymorphism of C3 component of complement in the Polish population. I. Population and family studies.

In a sample of the Polish population numbering 4741 subjects, the three common types C3S, C3F and C3FS and 15 phenotype variants were found with frequencies 0.0046. The frequencies of C3S and C3F genes determining the common types were 0.8227 and 0.1750, respectively. Examination of 40 newborns and their mothers has revealed that C3 types are formed during the fetal life. The results of studies on 76 families with 157 children and 2332 mother-child pairs have confirmed that 3C3 types are determined by a single genetic locus in which codominant autosomal alleles are situated.

Bf group system in the Polish population.

In a sample of the Polish population including 890 subjects, the five common types BfSO, BfF, BfFS, BfSSO 7, BfFSO 7 and one phenotypic variant BfF1S were met. The frequencies of genes determining the common types were: BfS = 0.8337, BfF = 0.1506, BfSO, 7 = 0.0112 and BfF1 = 0.0045, respectively. Examination of 38 newborns and their mothers has revealed that Bf types are formed during the fetal life. Inheritance of Bf types was studied on 84 families with 187 children and 449 mother-child pairs. The results obtained have confirmed that Bf system is determined by a single genetic locus in which multiple codominant, autosomal alleles are situated. The occurrence of phenotypic variant Bf FIS observed in two and three generations, confirmed its hereditary character and its dependence on the rare BfF1 gene occurring with the BfS gene.

GLO polymorphism in Polish population.

The determination of GLO types was carried out using two methods: on high voltage agarose gel and cellulose acetate foil. In the sample of the Polish population including 201 persons, three GLO types were encountered with the following frequencies: GLO 1-1 0.2438, GLO 2-1 0.4477, GLO 2-2 0.3035. Frequencies of GLO1 gene was 0.4726, GLO2 0.5724.

AK phenotypic changes observed in some hematologic diseases.

Determination of AK types in 372 patients of Hematologic Clinic, revealed in many cases changes in electrophoretic pattern of AK1 type namely the occurrence of an additional protein band. These changes were observed mostly in the acute granulocytic leukemia, lymphoblastic leukemia, and aplastic anemia. In the chronic granulocytic leukemia they were present as a rule, during the blastic crisis. Phenotypic changes were transient and the repeated examinations showed disappearance of an additional band in some patients. Etiology of the changes observed is still unclear.

6-PGD types in the Polish population.

6-PGD types were examined in 467 adult, nonrelated subjects, inhabiting various regions of Poland. In the investigated sample three phenotypes were encountered: A, AB and B, having the following frequencies: 0.9191, 0.0878 and 0.0021, respectively. Frequencies of determining genes were calculated from distribution of the phenotypes with the following results: 6-PGDA = 0.954, 6-PGDB = 0.046.

Atypical segregation in the GPT group system. Determination of types and activity of enzyme in families.

Examination of GPT system in 5418 paternity cases confirmed the elimination of maternity in 21 cases. The study of 12 probant families displayed the occurrence of opposite homozygous types in parents and children. The result obtained showed the presence of GPT0 gene in the families examined. Similar results were obtained by examining five families of alleged fathers whose paternity was eliminated on the same basis. In 12 families the activity of GPT was determined. In majority of subjects with GPT0 gene, markedly lowered activity was observed. This also concerned several homozygous subjects who were identical with their parents. The lowered activity of the enzyme is not present in all subjects with a "silent gene", and therefore, its examination cannot be used for detection of the gene.

Population studies on PLG group system in the Polish population.

Distribution of PLG types was studied in a sample of the Polish population numbering 230 subjects by the method of high-voltage agarose electrophoresis. Of three phenotypes encountered, PLG1, appeared with the frequency of 0.4870, PLG2-1 with 0.4391 and PLG2 with 0.0739. Assuming the hypothesis of PLG controlled by 2 alleles the frequencies of genotypes were calculated as follows: for PLG1 gene -0.71 and for PLG2 gene -0.29. PLG system in the Polish population was confirmed to be in a state of genetic equilibrium. The frequencies of PLG genes in the Polish population do not deviate from the frequencies encountered in other European populations.