Antibacterial Activity of Ethanolic Extract of Cinnamon Bark, Honey, and Their Combination Effects against Acne-Causing Bacteria.

Propionibacterium acnes and Staphylococcus epidermidis are the major skin bacteria that cause the formation of acne. The present study was conducted to investigate antibacterial activity of ethanolic extract of cinnamon bark, honey, and their combination against acne bacteria. The antibacterial activity of extract of cinnamon bark and honey were investigated against P. acnes and S. epidermidis using disc diffusion. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were attained using Clinical and Laboratory Standard Institute (CLSI) methods. The interaction between cinnamon bark extract and honey was determined using a checkerboards method. The results showed that the MICs of cinnamon bark extract and honey against P. acne were 256 µg/mL and 50% v/v, respectively, while those against S. epidermidis were 1024 µg/mL and 50% v/v, respectively. The MBC of cinnamon bark extract against P. acnes and S. epidermidis were more than 2048 µg/mL, whereas the MBC for honey against P. acnes and S. epidermidis were 100%. The combination of cinnamon bark extract and honey against P. acnes and S. epidermidis showed additive activity with a fractional inhibitory concentration index (FICI) value of 0.625. Therefore, the combination of cinnamon bark extract and honey has potential activity against acne-causing bacteria.

Serum levels of metal ions in female patients with breast cancer.

BACKGROUND: Breast cancer is the second commonest form of cancer among women. Several studies have been conducted to identify potential risk factors. However, role of trace elements or metals in causing breast cancer has not been studied to great extent. AIMS: To estimate the serum levels of calcium, copper, magnesium, iron, phosphorus and zinc and determine their role in causing breast cancer in female patients. SETTINGS AND DESIGN: A case-control study on female patients with breast cancer was conducted in a private superspecialty hospital and Cancer centre situated in Southern part of India. MATERIALS AND METHODS: Newly diagnosed female patients with breast cancer in the age group of 30-60 y attending Oncology clinic were included in the study. These cases were selected irrespective of type and stage of the disease. The age matched control subjects were drawn from apparently healthy women attending master health check at superspecialty hospital. The patients or controls suffering from co-morbid conditions which affect serum levels of metal ions and other malignancies, and those undergoing treatment for breast cancer were excluded from the study. Serum was separated and tests were performed according to standard procedure for each metal ion on the same day. The estimation of metal ions was done by UV-Visible Spectrophotometer-CHEM 7. STATISTICAL ANALYSIS: Independent Samples T-test was used to calculate difference between the two means. The p-value of <0.05 was considered as significant. RESULTS: The study was conducted on 54 female patients with breast cancer and 54 female controls with mean age of 47.2±8.14 y and 46.8±8.4 y respectively. There was statistically significant increase in serum levels of calcium, copper, iron and phosphorus in patients with breast cancer when compared to controls. The increase in serum levels of magnesium was insignificant. A statistically significant decrease in serum zinc levels was observed in patient with breast cancer when compared to controls. CONCLUSION: The present study highlights the role of calcium, copper, iron, phosphorus, magnesium and zinc in the pathogenesis of breast cancer. The estimation of serum levels of these metal ions has a potential role in early detection and monitoring of breast cancer.

Energy Sources for Ablation in the Pericardial Space.

Catheter ablation has been widely used for the management of cardiac arrhythmias. Transvenous endocardial catheter ablation successfully eliminates or modifies the critical substrate for most arrhythmias. Most arrhythmias can be eliminated with conventional endocardial mapping and radiofrequency energy delivery, but some critical arrhythmic substrates are not accessible via endocardial access and this has led to epicardial mapping and ablation in addition to traditional endocardial mapping techniques. This article reviews current approaches to epicardial ablation and discusses the specialized tools that increase ablation efficacy and safety.

A nationwide survey on the prevalence of atrioesophageal fistula after left atrial radiofrequency catheter ablation.

BACKGROUND: There are limited data on the prevalence of atrioesophageal fistula (AEF) after left atrial radiofrequency catheter ablation for atrial fibrillation (AF). The purpose of this study was to determine the prevalence and factors associated with AEF using a nationwide anonymous survey. METHODS AND RESULTS: The information solicited included the practice setting, number of left atrial ablations performed for AF, prevalence of AEF, clinical presentation and outcome of these patients, ablation strategy, type of ablation catheter, and energy settings used to deliver radiofrequency energy. The survey was mailed to 1,874 members of the Heart Rhythm Society within the US and 585 physicians responded (31%). AEF was reported in six of the 20,425 patients who underwent a left atrial ablation procedure (0.03%). All six patients suffered from major cerebrovascular events. Five of the six patients died (83%). The patient who survived had residual hemiparesis. There was no association between the risk of AEF and the case volume. In five patients, wide area circumferential ablation was performed. In the remaining patient, pulmonary vein isolation by ostial ablation was employed. In all cases an 8-mm tip ablation catheter was used. The power in patients who did and did not develop AEF were 58 +/- 13 and 41 +/- 9 W, respectively (P = 0.03). In one patient AEF occurred despite <1 degrees C rise recorded from an esophageal temperature probe. In the remaining patients no specific method was used to visualize the location of the esophagus. CONCLUSIONS: Based on the responses to the survey, the risk of AEF appears to be <1%. However, AEF is associated major cerebrovascular events and leads to death in >80% of the patients.

Fine specificity of anti-fibrillin-1 autoantibodies in primary pulmonary hypertension syndrome.

Autoantibodies to fibrillin-1 (Fbn-1) have been found in systemic sclerosis (SSc), calcinosis, Raynaud's esophagael dysmotility, sclerodectyly, and telaengectasia (CREST) and mixed connective tissue disease (MCTD) diseases. The purpose of this study was to determine whether patients with primary pulmonary hypertension (PPH) and appetite-suppressant-associated PPH have anti-Fbn-1 autoantibodies. In addition we assessed the human leucocyte antigen (HLA) class II alleles (DRB1, 3, 4, 5 and DQB1) in these patients in order to determine whether the response is genetically restricted. The frequency of anti-Fbn-1 autoantibodies in patient groups was compared with that of a control group of 88 healthy patients, and HLA was correlated similarly with a group of 51 healthy subjects. Anti-Fbn-1 autoantibodies were found at high frequency in PPH: in 70 of 75 adults with PPH (93%), in 28 of 33 children with PPH (84.8) and in 12 of 18 (67%) patients with appetite-suppressant-associated PPH. Utilization of two Fbn-1 fusion proteins allowed us to determine the dominant determinant region, recognized by anti-Fbn-1 autoantibodies, which may be located on the N-terminal fragment of the Fbn-1 protein. No significant immunogenetic correlations were found when the PPH patient groups were compared with normal controls. This novel category of autoantibodies is found in diseases characterized by endothelial and extracellular matrix protein alterations and fibrosis.

Genetic and immunologic features associated with scleroderma-like syndrome of TSK mice.

Tight-skin (TSK) mouse, the experimental model for scleroderma, develops cutaneous hyperplasia and autoantibodies to scleroderma specific autoantigens. TSK syndrome is caused by a mutation on chromosome 2. Induction of cutaneous hyperplasia is due to intragenic duplication of exons 17 to 40 of fibrillin-1 gene, mapping close to TSK locus. The mutant mouse expresses a 14kb Fbn transcript in addition to 11kb wild-type transcript. Immunoprecipation analysis confirms that the mutant transcript is functional and codes for a 420kD fibrillin. The occurrence of TSK syndrome is independent of the presence of mature lymphocytes although splenic/bone marrow cells appear to be capable of transferring the disease in normal animals. Transgenic mice expressing mutant transgene develop mild skin thickness with associate biochemical changes but do not develop anti-topo I antibodies. Among the other factors that may contribute to the develop- ment of hyperplasia, collagen V seems to play an important role.

Spontaneous occurrence of anti-fibrillin-1 autoantibodies in tight-skin mice.

Tight-skin (TSK) mouse represents an experimental for systemic sclerosis, displaying cutaneous hyperplasia, connective tissue alterations in the internal organs and developing autoantibodies against several scleroderma target autoantigens. TSK mouse syndrome is associated with a mutation in fibrillin-1 (Fbn-1), the major component of 10 nm microfibrils. Here, we have investigated whether TSK mouse develops autoimmunity to Fbn-1 similar to scleroderma target autoantigens. Our results show that anti-Fbn-1 IgG autoantibodies are present in high titer in many TSK mice. Specificity of these antibodies was confirmed by competitive inhibition assays and Western blotting analysis using recombinant human Fbn-1 protein. TSK mouse autoantibodies recognize a conserved epitope present in the C region of Fbn-1. These results indicate the presence of Fbn-1 specific T and B cells in TSK mouse repertoire.

Effect of targeted mutation in collagen V alpha 2 gene on development of cutaneous hyperplasia in tight skin mice.

Collagen V plays a major regulatory role in the formation of heterotypic fibers of the dermis and cartilaginous tissues as well as in the assembly of extracellular matrix. The pN/pN mouse, which is defective in collagen V alpha 2 gene, exhibits skeletal abnormalities, skin fragility, and alterations in the collagen fiber organization, whereas the TSK/+ mouse, which is defective in fibrillin-1, the major component of microfibrils present in the extracellular matrix, develops cutaneous hyperplasia and autoimmunity. We have studied the role of collagen V in the formation of heterotypic collagen fibers in F1 mice, which are obtained by breeding pN/pN with TSK/+ mice. Our results show that F1 progeny neither develop cutaneous hyperplasia nor produce anti-topoisomerase I autoantibodies, unlike TSK/+ mice. The diameter of the collagen fibrils in the skin is also comparable to that found in control mice. Thus, the phenotypic changes observed in the TSK mouse could be reversed by genetic complementation with a collagen V-defective mouse.

Structure of the mutant fibrillin-1 gene in the tight skin (TSK) mouse.

Mice carrying the tight skin (TSK) mutation harbors a 3.0-kb genomic duplication (exons 17-40) of the fibrillin-1 gene (Fbn-1) located on band F of chromosome 2 as TSK mutation. We cloned and sequenced the mutated Fbn-1 gene, since it is believed to be responsible for TSK syndrome. Sequence analysis showed numerous amino acid differences in the 5' and 3' segments between the TSK mutation and wild-type fbn-1 gene, but any amino acid difference between the TSK mutation and C57BL/6 mice. (TSK and C57B1/6 mice are genetically similar, differing only by TSK mutation.) Four amino acid differences were observed between two copies of TSK's fbn-1 gene encoded by exons 17-40. Our results suggest that the majority of structural differences occurred in the N and C termini segments during strain divergence and only a few after the duplication event.

B-cell deficiency does not abrogate development of cutaneous hyperplasia in mice inheriting the defective fibrillin-1 gene.

Tight-skin (TSK) mouse, the experimental model for scleroderma, develops cutaneous hyperplasia, cardiac hypertrophy, pulmonary emphysema and autoimmunity against scleroderma target autoantigens. The cutaneous hyperplasia is associated with the accumulation of microfibrils and elastic fibers in the middle and deep dermis. Fibrillin-1 (Fbn-1) is a major component of the 10-12 nm microfibrils found in the extracellular matrix. In this study we report the identification of a genetic marker in the Fbn-1 gene that can distinguish the mutant phenotype. TSK mice exhibit an unique polymorphism in the Fbn-1 gene. RNA analysis, PCR analysis and sequence determination of the mutant gene showed that the Fbn-1 gene polymorphism is due to intragenic duplication of a segment of the gene coding for 3.0 Kb of mRNA sequence (10 Kb of the genome). Histological analysis of skin samples from F1 progeny obtained by crossing TSK mice with JH-/-, RAG2-/- or vit/vit showed a significant correlation between the inheritance of the defective Fbn-1 gene and the development of cutaneous hyperplasia. Further, our results also show that in mice deficient in mature B cells inheriting the defective Fbn-1 gene, development of cutaneous hyperplasia is not abrogated. Thus, production of autoantibodies or the presence of mature B lymphocytes do not play an integral role in the pathogenesis of cutaneous hyperplasia.

Correlation between the concentration of serum anti-topoisomerase I autoantibodies and histological and biochemical alterations in the skin of tight skin mice.

Cutaneous hyperplasia observed in tight skin mice is due to a mutation located on chromosome 2. While homozygous mice die in utero, the heterozygotes survive. TSK syndrome is associated with the presence of autoantibodies specific for scleroderma target autoantigens. The presence of autoantibodies specific for topoisomerase I is characteristic of both human and murine disease. We have generated two distinct genotypes of mice, TSK/+ and +/+ with respect to the TSK trait by breeding TSK mice with immunodeficient mouse strains. Since the mutated gene of TSK syndrome has not yet been cloned, only histological and biochemical criteria were used for defining TSK genotype. In the F1 mice derived by mating TSK/+ mice with RAG2-/-, JH-/-, or C57BLvit/vit mice, we have found a good correlation between the amount of serum anti-topoisomerase I autoantibodies present and the histopathological and biochemical alterations that are characteristic of TSK scleroderma-like syndrome.

Antifibrillarin autoantibodies present in systemic sclerosis and other connective tissue diseases interact with similar epitopes.

Autoantibodies specific against fibrillarin, a 34-kD nucleolar protein associated with U3-snRNP, are present in patients with systemic sclerosis (SSc). To understand the mechanisms involved in the induction of these autoantibodies, we prepared a series of human fibrillarin recombinant proteins covering the entire molecule and analyzed their interaction with the autoantibodies present in various connective tissue diseases. Our results showed that antifibrillarin autoantibodies are present not only in SSc, as previously reported, but also in a variety of other connective tissue diseases. Patients with SSc (58%), mixed connective tissue diseases (60%), CREST syndrome (calcinosis, Raynaud phenomenon, esophageal dismotility, sclerodactyly, and telangiectasia syndrome) (58%), systemic lupus erythematosus (39%), rheumatoid arthritis (60%), and Sjogern's syndrome (84%) showed presence of antifibrillarin autoantibodies. Results obtained from competitive inhibition radioimmunoassay and Western blot analyses with purified recombinant fusion proteins revealed that these autoantibodies react primarily with epitope(s) present in the NH2- (AA 1-80) and COOH-terminal (AA 276-321) domains of fibrillarin. Autoantibodies reacting with internal regions of fibrillarin are less frequent. Analysis of the hydrophilicity profiles of reactive peptides showed presence of three potential antigenic sites in the NH2- and two in the COOH-terminal regions. While a hexapeptide sequence NH2 terminus of fibrillarin is shared with an Epstein-Barr virus-encoded nuclear antigen, the COOH-terminal region shares sequence homology with P40, the capsid protein encoded by herpes virus type 1. Interestingly, these two regions of fibrillarin also contain the most immunodominant sequences, as predicted by surface probability and the Jameson and Wolf antigenic index. These observations suggest that molecular mimicry might play an important role in the induction of antifibrillarin autoantibodies.

Molecular characterization of J558 genes encoding tight-skin mouse autoantibodies: identical heavy-chain variable genes code for antibodies with different specificities.

Tight-skin mouse, a mutant strain with a single gene defect, develops cutaneous hyperplasia and specific autoantibodies, like humans affected by scleroderma. The autoantibodies produced in the tight-skin mouse are encoded primarily by heavy-chain variable (VH) genes from the J558 family. To understand the genetic basis of production of autoantibodies, we have analyzed the structure of J558 genes encoding these autoantibodies. The results showed that J558 genes encoding these antibodies were not derived from a selected germ-line gene(s) or a single subfamily but were derived from genes belonging to diverse J558 subfamilies. However, two prototype VH genes representing two new subfamilies were found to be repeatedly expressed in their germ-line form in eight independent clones. Autoantibodies with distinct specificities appear to be generated by pairing of similar/identical VH genes with different V kappa genes derived from the same or different families. Fourteen of 18 autoantibodies shared a conserved heptapeptide sequence motif, YNEKFKG, in the second complementarity-determining region of heavy chains. Usage of germ-line genes from diverse J558 subfamilies bearing a common motif to encode autoantibodies suggests a regulatory role for this motif. Thus, selection and expansion of the autoreactive B-cell repertoire in the tight-skin mouse appear to be VH-gene mediated. The frequency of N nucleotide addition at diversity-joining (D-JH) junctions was lower, whereas the frequency of usage of the DFL16 segment was higher. Finally, in contrast to normal and other autoimmune mouse strains, the frequencies of D-D fusions and D inversions were higher in tight-skin mouse total immunoglobulin as well as autoantibody repertoires.

Immunochemical and molecular characterization of anti-RNA polymerase I autoantibodies produced by tight skin mouse.

Autoantibodies against nuclear proteins like RNA polymerase I (RNA pol I) are produced in a number of rheumatic autoimmune diseases. Production of antibodies specific for the 190-kD subunit of RNA pol I appears to be characteristic in the patients with systemic sclerosis. Previous investigations have shown that the tight skin (TSK) mouse is an experimental model for systemic sclerosis. In the present study we show that the TSK mice produce high titers of anti-RNA pol I antibodies, both of IgM and IgG classes. To characterize the immunochemical properties of these antibodies we obtained a large panel of hybridomas from these mice. Analysis of these hybridomas revealed that clonal frequency of autoreactive B cells specific for RNA pol I are higher in the TSK mice that in the controls. mAbs obtained from the TSK mice were specific for the 190-kD subunit and cross-reacted with Escherichia coli and phage T7 RNA polymerases (155-, 150-, and 107-kD polypeptides). We have also demonstrated that these antibodies bind better to the phosphorylated enzymes. The anti-RNA pol I mAbs were divided into three groups in terms of their functional property. The first group of antibodies increased the catalytic activity of the enzyme whereas the antibodies of the second group inhibited the enzymatic activity. Competitive inhibition RIAs showed that these two groups of antibodies bound to distinct epitopes. The third group of antibodies was neutral and had no activity on the enzyme function. These results suggest that TSK mouse anti-RNA pol I antibodies recognize three or more conserved epitopes. To understand the molecular basis of the generation of such autoreactive antibodies we analyzed their V gene repertoire. Northern analysis of RNAs of 14 TSK hybridomas showed that the VH genes encoding these antibodies were mainly from VH J558 family. It is possible that these genes were derived from a single germline gene or from a set of related genes of a single subgroup.

Tight-skin mouse autoantibody repertoire: analysis of VH and VK gene usage.

In the previous studies we have shown that tight-skin (TSK) mouse is an experimental model for systemic sclerosis. This mutant mouse develops autoantibodies specific for scleroderma target antigens. To determine whether the expansion of autoantibody repertoire in TSK mouse occurs by selective expansion of certain variable region gene families, and whether CD5+ B cells contribute significantly to the production of autoantibodies, we have analyzed a panel of 60 hybridomas producing autoantibodies specific for scleroderma target autoantigens. Northern analysis of RNAs from these hybridomas showed that 70% were expressing genes from VHJ558 family while genes from 36-09 and J606 families were not at all represented. In contrast, in the cDNA libraries derived from splenic B cells, the expression of VHJ558 and 36-09 gene families were at an expected frequency corresponding to their genomic complexity (44% and 11.6%, respectively). These results demonstrate that there is a strong bias toward the use of J558 genes in TSK mouse autoantibody repertoire. On the other hand the expression of VK gene families was mostly random and corresponded to their frequency in splenic C kappa cDNA library. The pairing of VH:VK genes was stochastic. Analysis of the expression of J segments, however, revealed that JH2 and JK2 were predominantly used in the autoantibodies. Analysis of the expression CD5 mRNA in these hybridomas indicate that CD5+ B cells do not contribute significantly to the autoimmunity in TSK mice. These findings suggest that the expansion of peripheral autoreactive B cells in TSK mouse is determined by their immunoglobulin variable region rather than the genetic properties linked to a particular B cell subset.