Diagnosis and Management of Microscopic Colitis.

Microscopic colitis (MC) is a relatively common cause of chronic watery diarrhea, especially in older persons. Associated symptoms, including abdominal pain and arthralgias, are common. The diagnosis is based upon characteristic histological findings in the presence of diarrhea. The two types of MC, collagenous and lymphocytic colitis, share similar clinical features, with the main difference being the presence or absence of a thickened subepithelial collagen band. There are several treatment options for patients with MC, although only budesonide has been well studied in multiple controlled clinical trials. This review will describe the clinical features, epidemiology, pathophysiology, diagnostic criteria, and treatment of patients with MC.

IL28B rs12980275 and HLA rs4273729 genotypes as a powerful predictor factor for rapid, early, and sustained virologic response in patients with chronic hepatitis C.

Single-nucleotide polymorphisms (SNPs) in the Interleukin-28B (IL28B) gene and rs4273729 in the human leukocyte antigen (HLA) gene in chronic hepatitis C (CHC) virus infection are important for predicting treatment outcome. In this study, the distribution of IL28B SNPs (rs12979860 and rs12980275) and HLA rs4273729 in rapid virologic response (RVR), complete early virologic response (cEVR) and sustained virologic response (SVR) in HCV Iranian patients with CHC virus infection was assessed. IL28B genotyping and rs4273729 were performed using the amplification refractory mutation system (ARMS)-PCR and direct sequencing in 190 CHC virus infections, respectively. RVR, cEVR, and SVR were 53.2 %, 78.9 %, and 65.8 %, respectively. Multivariate regression analysis demonstrated that the responses significantly predicted SVR in patients with age <40 years (p = 0.008), HCV genotypes (p = 0.032), IL28B rs12979860 CC genotype (p < 0.001), rs12980275 AA genotype (p < 0.001), rs4273729 GG genotype (p < 0.001), RVR (p < 0.001) and cEVR (p = 0.024). Three critical predictor factors based on RVR response were rs12979860 CC genotype (p = 0.033), rs12980275 AA genotype (p < 0.001) and rs4273729 GG genotype (p < 0.001), while rs12980275 AA (p = 0.003) and rs4273729 GG genotypes (p < 0.001) predicted cEVR. For the first time in Iran, these results revealed that the rs12980275 and HLA rs4273729 are important for the treatment of CHC infection. These findings may help predict responses to CHC infection treatment and reduce the cost and side effects of therapy.

Chronic graft-versus-host-disease in CD34(+)-humanized NSG mice is associated with human susceptibility HLA haplotypes for autoimmune disease.

Chronic graft-versus-host disease (cGVHD) is a significant hurdle to long-term hematopoietic stem-cell transplantation success. Insights into the pathogenesis and mechanistical investigations of novel therapeutic strategies are limited as appropriate animal models are missing. The immunodeficient NSG mouse - when humanized with human bone marrow, fetal liver and thymus (BLT NSG) - is prone for cGVHD, yet mainly affects the skin. In contrast, the NSG mouse humanized exclusively with CD34(+)-selected, CD3(+)-depleted stem cells (CD34(+)NSG) has neither been described for acute nor chronic GVHD so far. This is the first report about the development of systemic autoimmune cGVHD ≥24 weeks post stem cell receipt involving lung, liver, skin, gingiva and intestine in two NSG cohorts humanized with CD34(+) grafts from different donors. Affected mice presented with sclerodermatous skin, fibrotic lung, severe hepatitis, and massive dental malformation/loss. CD4(+)-dominated, TH2-biased, bulky T-cell infiltrates featured highly skewed T cell receptor (TCR) repertoires, clonal expansions, and autoreactive TCRs. In affected tissues profibrotic IL-13 and -4 dominated over TH1 cytokines IFN-γ and TNF-α. Thus, the time point of manifestation and the phenotype match human systemic pleiotropic sclerodermatous GVHD. The CD34(+)NSG-model's intrinsic deficiency of thymus, thymus-derived regulatory T cells (nTreg) and B cells emphasizes the role of the genetic polymorphism and the cytokines in the pathogenesis of cGVHD. Importantly, the only factor discriminating diseased versus non-diseased CD34(+)NSG cohorts were two risk HLA haplotypes that in human mediate susceptibility for autoimmune disease (psoriasis). Thus, the CD34(+)NSG model may serve as a platform for addressing issues related to the pathophysiology and treatment of human autoimmunity and chronic GVHD.

HLA Haplotypes and Genotypes Frequencies in Brazilian Chronic Periodontitis Patients.

Human leukocyte antigens (HLA) have a pivotal role in immune response and may be involved in antigen recognition of periodontal pathogens. However, the associations of HLA with chronic periodontitis (CP) have not been previously studied in the Brazilian population. In an attempt to clarify the issue of genetic predisposition to CP, we examined the distribution of HLA alleles, genotypes, and haplotypes in patients from Southern Brazil. One hundred and eight CP patients and 151 healthy and unrelated controls with age-, gender-, and ethnicity-matched were HLA investigated by polymerase chain reaction with sequence specific oligonucleotides. To exclude smoking as a predisposing factor, statistical analyses were performed in the total sample and in nonsmoking individuals. The significant results showed a positive association of the A∗ 02/HLA-B∗ 40 haplotype with CP (total samples: 4.2% versus 0%, Pc = 0.03; nonsmokers: 4.3% versus 0%, Pc = 0.23) and a lower frequency of HLA-B∗ 15/HLA-DRB1∗ 11 haplotype in CP compared to controls (total samples: 0.0% versus 4.3%, Pc = 0.04; nonsmokers: 0 versus 5.1%, P = 1.0). In conclusion, the HLA-A∗ 02/B∗ 40 haplotype may contribute to the development of CP, while HLA-B∗ 15/DRB1∗ 11 haplotype might indicate resistance to disease among Brazilians.

Exome capture from saliva produces high quality genomic and metagenomic data.

BACKGROUND: Targeted capture of genomic regions reduces sequencing cost while generating higher coverage by allowing biomedical researchers to focus on specific loci of interest, such as exons. Targeted capture also has the potential to facilitate the generation of genomic data from DNA collected via saliva or buccal cells. DNA samples derived from these cell types tend to have a lower human DNA yield, may be degraded from age and/or have contamination from bacteria or other ambient oral microbiota. However, thousands of samples have been previously collected from these cell types, and saliva collection has the advantage that it is a non-invasive and appropriate for a wide variety of research. RESULTS: We demonstrate successful enrichment and sequencing of 15 South African KhoeSan exomes and 2 full genomes with samples initially derived from saliva. The expanded exome dataset enables us to characterize genetic diversity free from ascertainment bias for multiple KhoeSan populations, including new exome data from six HGDP Namibian San, revealing substantial population structure across the Kalahari Desert region. Additionally, we discover and independently verify thirty-one previously unknown KIR alleles using methods we developed to accurately map and call the highly polymorphic HLA and KIR loci from exome capture data. Finally, we show that exome capture of saliva-derived DNA yields sufficient non-human sequences to characterize oral microbial communities, including detection of bacteria linked to oral disease (e.g. Prevotella melaninogenica). For comparison, two samples were sequenced using standard full genome library preparation without exome capture and we found no systematic bias of metagenomic information between exome-captured and non-captured data. CONCLUSIONS: DNA from human saliva samples, collected and extracted using standard procedures, can be used to successfully sequence high quality human exomes, and metagenomic data can be derived from non-human reads. We find that individuals from the Kalahari carry a higher oral pathogenic microbial load than samples surveyed in the Human Microbiome Project. Additionally, rare variants present in the exomes suggest strong population structure across different KhoeSan populations.

The K1K2 region of Lys-gingipain of Porphyromonas gingivalis blocks induction of HLA expression by gamma interferon.

In the context of periodontal disease, cysteine proteinases or gingipains from Porphyromonas gingivalis have been implicated in the hydrolysis of cytokines, including gamma interferon (IFN-γ). This cytokine plays a crucial role in host defenses, in part, by regulating expression of major histocompatibility complex molecules. Our recent analysis has identified three structurally defined modules, K1, K2, and K3, of the hemagglutinin region of the lysine gingipain Kgp. These three structurally homologous domains have a common ß-sandwich topology that is similar to that found in a superfamily of adhesins and carbohydrate binding domains. The three Kgp hemagglutinin modules are distinguished by variation in some of the loops projecting from the ß-sandwich core. Recombinant products corresponding to both single and multidomain regions as well as native Kgp bound IFN-γ with similar affinities. Among the adhesin domain constructs, only the K1K2 polypeptide inhibited the upregulation of HLA-1 expression in a human erythroleukemia (K562) line induced by both recombinant and native IFN-γ. The K1K2 polypeptide also inhibited HLA-DR expression induced by IFN-γ in human umbilical vein endothelial cells. These effects were competitively inhibited by coincubation with sodium or potassium chloride solution. The N-terminal residues of IFN-γ were implicated in mediating the effect of K1K2, while antibody binding to loop 1 of K2 blocked the action of K1K2. The findings indicate the potential significance of structurally defined Kgp adhesin modules in the inactivation of IFN-γ but also the potential of K1K2 in locating the target for the catalytic domain of Kgp.

Genes and hepatitis C: susceptibility, fibrosis progression and response to treatment.

Hepatitis C virus contact and infection show three different phenotypes: spontaneous viral clearance (SVC), chronic hepatitis C (CHC) and sustained virological response (SVR) following antiviral treatment. Many factors, including genetics, influence the evolution of these three phenotypes. We performed a literature search (PubMed) up to 31 January 2010 without language restriction to identify relevant studies on genes and hepatitis C. Additional studies were sought by reviewing the reference lists of the identified articles. Meta-analysis (using Meta-disk 1.4) was conducted to evaluate the association of single nucleotide polymorphism (SNP) in the IL28B region and SVR. The candidate gene approach showed strong relationships between human leucocyte antigen class II (DQB1(*) 0301 and DRB1(*) 1101) and SVC. A cirrhosis risk score involving 7 SNPs has been validated recently. The set of odds ratios of studies demonstrated an association between SNP (rs12987960/rs8099917) in the IL28B and SVR in CHC treated with peginterferon plus ribavirin (OR: 4.6; 95% CI: 2.9-7.3). The overall distribution of protective allele correlated with ethnic differences in SVR (Asians, Europeans, Hispanic and Afro-Americans) together with SVC, but not with fibrosis stage or viral load. These polymorphisms did not influence SVR in very-easy-to-treat patients such as genotype 2/3, rapid virological responders or patients with acute hepatitis C. While the genetic fingerprint for fibrosis progression remains elusive, IL28b polymorphism predicts SVC and SVR. However, nearly half of patients achieving SVR did not show favourable genotype. Further genetic signals are warranted to complete the puzzle of factors influencing hepatitis C.

Human leukocyte antigens are associated with salivary level of active MMP-8.

OBJECTIVES: This case control study examined the associations of HLA antigens and periodontitis with the salivary level of active MMP-8 (aMMP-8). MATERIALS AND METHODS: A total of 202 subjects, registered as Swiss bone marrow donors, participated in the study. HLA-A, -B, and -C types were determined by serology or PCR. Saliva samples were collected from subjects, followed by a periodontal examination. The salivary level of aMMP-8 was determined with immunofluorometric assay. RESULTS: The mean salivary level of aMMP-8 was directly comparable to the grade of periodontitis and increased from healthy to mild/moderate to severe (125.0 ± 132.1, 200.6 ± 170.2, 290.1 ± 202.3 ng/ml; p < 0.001 between each group, respectively). The only association between the HLA types and the salivary level of aMMP-8 was observed in subjects with HLA-A11. Subjects with healthy periodontium and HLA-A11 had a lower level of aMMP-8 (49.2 ± 32.5 ng/ml) compared with subjects without HLA-A11 (123.6 ± 119.2; p = 0.048). Among subjects with periodontitis, a higher level of aMMP-8 (394.2 ± 255.6 ng/ml) was observed in subjects with HLA-A11 compared with subjects without HLA-A11 (201.1 ± 146.1 ng/ml; p < 0.002). This finding was statistically significant also after adjusting for sex, age, smoking, tooth brushing and the number of medications (p < 0.05). CONCLUSIONS: HLA-A11 is associated with the salivary level of aMMP-8 which contributes to the subject's immune and inflammatory response in periodontium.

Antibody response against three widespread bovine viruses is not impaired in Holstein cattle carrying bovine leukocyte antigen DRB3.2 alleles associated with bovine leukemia virus resistance.

Due to the wide dissemination of bovine leukemia virus (BLV) infection among dairy cattle, control and eradication programs based on serological detection of infected cattle and subsequent culling face a major economic task. In Argentina, genetic selection of cattle carrying alleles of the bovine leukocyte antigen (BoLA) DRB3.2 gene associated with BLV-infection resistance, like *0902, emerges as the best additional tool toward controlling virus spread. A potential risk in expanding or segregating BoLA selected populations of cattle is that it might increase susceptibility to other common viruses. Special concern raises the strong association found between low proviral load and low antibody titer against major BLV structural proteins. This phenomenon might depend on host genetic factors influencing other viruses requiring, unlike BLV, strong and long-lasting humoral immune response to prevent infection. In this study, we demonstrate that there is no association among neutralizing antibody titers against foot and mouth disease virus, bovine viral diarrhea virus, or bovine herpesvirus type 1 and polymorphism of the BoLA DRB3.2 gene. Conversely, there is strong association between BoLA DRB3.2*0902 and low antibody titers against 2 BLV structural proteins--env gp51 and gag p24--to date, the best BLV resistance marker. There is also significant association between low antibody titers against gp51 and p24 and BoLA DRB3.2*1701 and low antibody titers against p24 and BoLA DRB3.2*1101 or 02. Our data suggest that increasing BoLA-selected BLV-resistant cattle or segregating BoLA-associated alleles to BLV susceptibility would not affect the resistance or the predisposition to bovine viral diarrhea virus, bovine herpesvirus type 1, or foot and mouth disease virus infection.

Horizons in Sjögren's syndrome genetics.

Sjögren's syndrome (SS) is a complex polygenic autoimmune disorder. A few major genetic effects have been identified. Historically, HLA and non-HLA genetic associations have been reported. Recently, the HLA region continued to reveal association findings. A new susceptibility region has been suggested by a study of a D6S349 microsatellite marker. Among non-HLA studies, recent association of immunoglobulin kappa chain allotype KM1 with anti-La autoantibodies in primary Sjögren's syndrome confirms findings in a study from two decades ago. Meanwhile, mouse models have been employed to study the genetic contribution to salivary lymphadenitis or dry eyes and mouth. Gene transfer exploration in mouse models shows promise. The authors review the HLA and non-HLA association studies and the mouse model work that has been reported. Newly developed genomic capacity will provide, in the future, a much closer approximation of the true picture of the genetic architecture of Sjögren's syndrome.

Narcoleptic-like Episodes in a Patient Receiving Pegylated Interferon-alpha 2b: A Case Report and Review of Literature.

A 26-year-old male developed narcoleptic-like episodes while on pegylated interferon-alpha 2b (peg-IFN-α2b) therapy for stage IIIB melanoma. Once peg-IFN-α2b was discontinued, the narcoleptic-like episodes eradicated. A case report and review of the literature are presented in this article.

Identification of material with paternal HLA antigen immunoreactivity from purported circulating immune complexes in patients with gestational trophoblastic neoplasia.

Circulating immune complex(es) (CIC) have been shown to rise progressively only when patients with hydatidiform molar pregnancy enter gonadotropin-documented remission. The CIC in 3 patients with gestational trophoblastic neoplasia (GTN)--1 with hydatidiform mole and 2 with choriocarcinoma--were characterized. Their clinical course was monitored by serial antigen-nonspecific polyethylene glycol (PEG) 6000-CIC assay and simultaneous human chorionic gonadotropin (HCG) assay from presentation until sustained gonadotropin-documented remission. As serial HCG progressively decreased to normal following surgical or chemotherapeutic reduction in tumor burden, PEG-CIC concurrently rose. Serum obtained at or near peak PEG-CIC levels was precipitated by 3.75% PEG 6000 and fractionated by column chromatography on Sephadex G-200 (exclusion limit, greater than 600,000 mol wt) in glycine-HCl and 1 M NaCl buffer at pH 2.8. None of the 5 elution fractions obtained from the 3 patients contained HCG or anti-HCG activity. However, in the hydatidiform molar patient, fractions 1 through 3 (mol wt greater than 67,000--and containing immunoglobulin) were shown to competitively inhibit complement-dependent antibody lysis on 1 of 4 paternal HLA haplotype (AW32) targets. In 2 of the 3 patients studied, low-molecular-weight fractions (not containing immunoglobulin) significantly inhibited reference anti-HLA binding of antisera directed against only 1 of 4 paternal HLA haplotypes. The immunospecificity of this inhibition was confirmed by criss-cross control assays in which elution fractions obtained from both of these patients were tested for inhibition of lymphocytolysis of both sets of paternal lymphocytes. None of these fractions were immunoreactive to maternal HLA haplotypes. Further analysis of serum from the hydatidiform molar patient revealed that no free complement-fixing antibody against paternal antigens could be found by conventional screening assays in unfractionated patient sera. Three of 4 paternal HLA antigens or non-complement-fixing anti-HLA immunoglobulin was detected in unfractionated pretreatment, treatment, and remission sera of the hydatidiform molar patient. Only in this patient's remission sera was unbound AW32 antigen or non-complement-fixing anti-AW32 antibody detected. These data demonstrate the successful characterization of at least 1 specific antigen fractionated from a tumor-associated immune complex. The implication that some patients with GTN may recognize and react to immunogenic paternal HLA antigens as part of their successful response to therapy for trophoblastic tumor is discussed.

In vivo hepatogenic capacity and therapeutic potential of stem cells from human exfoliated deciduous teeth in liver fibrosis in mice.

INTRODUCTION: Liver transplantation is a gold standard treatment for intractable liver diseases. Because of the shortage of donor organs, alternative therapies have been required. Due to their potential to differentiate into a variety of mature cells, stem cells are considered feasible cell sources for liver regeneration. Stem cells from human exfoliated deciduous teeth (SHED) exhibit hepatogenic capability in vitro. In this study, we investigated their in vivo capabilities of homing and hepatocyte differentiation and therapeutic efficacy for liver disorders in carbon tetrachloride (CCl4)-induced liver fibrosis model mice. METHODS: We transplanted SHED into CCl4-induced liver fibrosis model mice through the spleen, and analyzed the in vivo homing and therapeutic effects by optical, biochemical, histological, immunological and molecular biological assays. We then sorted human leukocyte antigen-ABC (HLA-ABC)-positive cells from primary CCl4-damaged recipient livers, and analyzed their fusogenicity and hepatic characteristics by flow cytometric, genomic DNA, hepatocyte-specific gene assays. Furthermore, we examined the treatment effects of HLA-positive cells to a hepatic dysfunction by a secondary transplantation into CCl4-treated mice. RESULTS: Transplanted SHED homed to recipient livers, and expressed HLA-ABC, human hepatocyte specific antigen hepatocyte paraffin 1 and human albumin. SHED transplantation markedly recovered liver dysfunction and led to anti-fibrotic and anti-inflammatory effects in the recipient livers. SHED-derived HLA-ABC-positive cells that were sorted from the primary recipient liver tissues with CCl4 damage did not fuse with the host mouse liver cells. Sorted HLA-positive cells not only expressed human hepatocyte-specific genes including albumin, cytochrome P450 1A1, fumarylacetoacetase, tyrosine aminotransferase, uridine 5'-diphospho-glucuronosyltransferase, transferrin and transthyretin, but also secreted human albumin, urea and blood urea nitrogen. Furthermore, SHED-derived HLA-ABC-positive cells were secondary transplanted into CCl4-treated mice. The donor cells homed into secondary recipient livers, and expressed hepatocyte paraffin 1 and human albumin, as well as HLA-ABC. The secondary transplantation recovered a liver dysfunction in secondary recipients. CONCLUSIONS: This study indicates that transplanted SHED improve hepatic dysfunction and directly transform into hepatocytes without cell fusion in CCl4-treated mice, suggesting that SHED may provide a feasible cell source for liver regeneration.

Familial granulomatous synovitis, uveitis, and cranial neuropathies.

A family is presented that had what is believed to be a previously undescribed syndrome of granulomatous synovitis, bilateral recurrent uveitis, and cranial neuropathies. Affected members included the proband, his brother, father, and probably the decreased paternal grandmother. Disease onset was in childhood. Each had symmetric, boggy polysynovitis of the hands and wrists, resulting in nearly identical boutonniere deformities. Hand radiography in the proband and his brother revealed no erosions or joint destruction despite more than 20 years of disease. Synovectomy specimens in the proband and his brother showed granulomatous inflammation with giant cells. Recurrent, nongranulomatous, acute iridocyclitis with visual impairment afflicted the proband, brother, and father. Apparently corticosteroid-responsive bilateral neurosensory hearing loss occurred in the proband, and a transient sixth cranial nerve palsy in his brother. All members of the family were antinuclear antibody-, rheumatoid factor-, and HLA-B27-negative. Serum angiotensin-converting enzyme levels were within normal limits in all family members. The inheritance pattern of this syndrome is most consistent with an autosomal dominant mode.

Transfer of the IL-6 gene into a human colorectal carcinoma cell line and consequent enhancement of tumor antigen expression.

cDNA encoding the human IL gene (580 bp), inserted into a retroviral expression vector carrying neomycin resistance selective marker, was introduced into HT-29 human colon carcinoma cells by lipofection. Interleukin-6 activity was measured by ELISA and bioassay using B9 cells. Interleukin-6 secreted by transfected HT-29 cells was shown to be biologically active. The expression of the human tumor associated antigen CEA (carcinoembryonic antigen), HLA classes I and II, and ICAM-1 antigens in the transfected HT-29 cells were also analyzed by flow cytometry. Significant enhancement in the expression of CEA but not in the expression of HLA class I, HLA class II and ICAM-1 antigens, was observed in the transfected HT-29 cells as compared to the parental HT-29 cells. These results provide experimental evidence that enhancement of tumor antigen expression on tumor cells can be induced by IL-6 gene transfection, and suggest another potential role for the use of IL-6 gene transfer in the immunotherapy of human cancers.

Genetic control of HLA-linked immune responsiveness to (H,G)-A-L.

Fifty-three donors belonging to seven families were tested for their immune response potential to (H,G)-A-L. Most of these donors had been previously tested for their ability to respond to (T,G)-A-L and were all HLA typed as well. The heredity of the ability to respond to (H,G)-A-L by the production of an antigen-specific helper T cell factor is compatible with an autosomal dominant trait linked to HLA. The genotype of an HLA-A/B recombinant individual suggested that a gene controlling the immune response to (H,G)-A-L is linked to HLA-A. Lod scores also suggested a linkage between immune response potential to (H,G)-A-L and HLA-A. The different patterns of responses to (T,G)-A-L and (H,G)-A-L observed in many individuals are compatible with the notion that separate loci are governing the immune responses to the two synthetic polypeptides.

Use of DNA probes from the 5' flanking region of the HLA-B gene to examine polymorphism at the HLA-B locus.

Two DNA probes isolated from the region 5' to the HLA-B gene are described. One of these, pCH6, detects a TaqI polymorphism which is correlated with serological alleles of HLA-B. Both probes are used to show a high level of DNA sequence homology between the 5' flanking region of HLA-B and HLA-C.

Molecular analysis of HLA class I and class II antigen loss mutants reveals a homozygous deletion of the DR, DQ, and part of the DP region: implications for class II gene order.

The mutant human B-lymphoblastoid cell lines, 721.174 and 721.180, previously reported to exhibit greatly reduced expression of human HLA class I and II antigens (DeMars et al., Hum Immunol 11:77, 1984), were analyzed by Southern blotting using class II cDNA and genomic clones as hybridization probes. All genomic sequences complementary to DR alpha, DR beta, DQ alpha, and DQ beta probes were absent from these mutants. DZ alpha genomic sequences were deleted as were the DP alpha 1 and DP beta 1 loci but the DP beta 2 and most, if not all, of the DP alpha 2 locus were retained. However, no RNA transcripts for either DP alpha 2 or DP beta 2 could be detected. The mapping of the deletion breakpoint within the DP cluster allows the orientation of the loci in the DP region with respect to the centromere as follows: centromere, DP beta 2, DP beta 1, DP alpha 1, (DQ, DR). In addition, the analysis of a set of DR-, DQ-, DP+ homozygous deletion mutants (721.82, 721.84, and 721.101) reveals a deletion breakpoint between the DQ alpha 1/DQ beta 1 loci and the DQ alpha 2/DQ beta 2 loci. These mutants retain DZ alpha genomic sequences, tentatively mapping the DZ alpha locus between the DQ and the DP region. The residual ability of the DR-, DQ-, DP- mutants (174 and 180)* to stimulate allogeneic and autologous lymphoproliferative responses must be attributed to expression of as yet unidentified class II antigens, or to non-class II antigens.