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Publications (40)142.79 Total impact
Article: Mitochondrial DNA Variants Can Mediate Methylation Status of Inflammation, Angiogenesis and Signaling Genes[Show abstract] [Hide abstract]
ABSTRACT: Mitochondrial (mt) DNA can be classified into haplogroups representing different geographic and/or racial origins of populations. The H haplogroup is protective against age-related macular degeneration (AMD), while the J haplogroup is high risk for AMD. In the present study, we performed comparison analyses of human retinal cell cybrids, which possess identical nuclei, but mtDNA from subjects with either the H or J haplogroups, and demonstrate differences in total global methylation, expression patterns for two genes related to acetylation and five genes related to methylation. Analyses revealed that untreated-H and -J cybrids have different expression levels for nuclear genes (CFH, EFEMP1, VEGFA and NFkB2). However, expression levels for these genes become equivalent after treatment with a methylation inhibitor, 5-aza-2'-deoxycytidine. Moreover, sequencing of the entire mtDNA suggests that differences in epigenetic status found in cybrids are likely due to single nucleotide polymorphisms (SNPs) within the haplogroup profiles rather than rare variants or private SNPs. In conclusion, our findings indicate that mtDNA variants can mediate methylation profiles and transcription for inflammation, angiogenesis and various signaling pathways, which are important in several common diseases. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: email@example.com Molecular Genetics 05/2015; 24(16). DOI:10.1093/hmg/ddv173 · 6.39 Impact Factor
Article: Human Retinal Transmitochondrial Cybrids with J or H mtDNA Haplogroups Respond Differently to Ultraviolet Radiation: Implications for Retinal Diseases[Show abstract] [Hide abstract]
ABSTRACT: BACKGROUND: It has been recognized that cells do not respond equally to ultraviolet (UV) radiation but it is not clear whether this is due to genetic, biochemical or structural differences of the cells. We have a novel cybrid (cytoplasmic hybrids) model that allows us to analyze the contribution of mitochondrial DNA (mtDNA) to cellular response after exposure to sub-lethal dose of UV. mtDNA can be classified into haplogroups as defined by accumulations of specific single nucleotide polymorphisms (SNPs). Recent studies have shown that J haplogroup is high risk for age-related macular degeneration while the H haplogroup is protective. This study investigates gene expression responses in J cybrids versus H cybrids after exposure to sub-lethal doses of UV-radiation. METHODOLOGY/PRINCIPAL FINDINGS: Cybrids were created by fusing platelets isolated from subjects with either H (n = 3) or J (n = 3) haplogroups with mitochondria-free (Rho0) ARPE-19 cells. The H and J cybrids were cultured for 24 hours, treated with 10 mJ of UV-radiation and cultured for an additional 120 hours. Untreated and treated cybrids were analyzed for growth rates and gene expression profiles. The UV-treated and untreated J cybrids had higher growth rates compared to H cybrids. Before treatment, J cybrids showed lower expression levels for CFH, CD55, IL-33, TGF-A, EFEMP-1, RARA, BCL2L13 and BBC3. At 120 hours after UV-treatment, the J cybrids had decreased CFH, RARA and BBC3 levels but increased CD55, IL-33 and EFEMP-1 compared to UV-treated H cybrids. CONCLUSION/SIGNIFICANCE: In cells with identical nuclei, the cellular response to sub-lethal UV-radiation is mediated in part by the mtDNA haplogroup. This supports the hypothesis that differences in growth rates and expression levels of complement, inflammation and apoptosis genes may result from population-specific, hereditary SNP variations in mtDNA. Therefore, when analyzing UV-induced damage in tissues, the mtDNA haplogroup background may be important to consider. FAU - Malik, DeepikaPLoS ONE 06/2014; · 3.23 Impact Factor
Article: Human Retinal Transmitochondrial Cybrids with J or H mtDNA Haplogroups Respond Differently to Ultraviolet Radiation: Implications for Retinal Diseases[Show abstract] [Hide abstract]
ABSTRACT: Background It has been recognized that cells do not respond equally to ultraviolet (UV) radiation but it is not clear whether this is due to genetic, biochemical or structural differences of the cells. We have a novel cybrid (cytoplasmic hybrids) model that allows us to analyze the contribution of mitochondrial DNA (mtDNA) to cellular response after exposure to sub-lethal dose of UV. mtDNA can be classified into haplogroups as defined by accumulations of specific single nucleotide polymorphisms (SNPs). Recent studies have shown that J haplogroup is high risk for age-related macular degeneration while the H haplogroup is protective. This study investigates gene expression responses in J cybrids versus H cybrids after exposure to sub-lethal doses of UV-radiation. Methodology/Principal Findings Cybrids were created by fusing platelets isolated from subjects with either H (n = 3) or J (n = 3) haplogroups with mitochondria-free (Rho0) ARPE-19 cells. The H and J cybrids were cultured for 24 hours, treated with 10 mJ of UV-radiation and cultured for an additional 120 hours. Untreated and treated cybrids were analyzed for growth rates and gene expression profiles. The UV-treated and untreated J cybrids had higher growth rates compared to H cybrids. Before treatment, J cybrids showed lower expression levels for CFH, CD55, IL-33, TGF-A, EFEMP-1, RARA, BCL2L13 and BBC3. At 120 hours after UV-treatment, the J cybrids had decreased CFH, RARA and BBC3 levels but increased CD55, IL-33 and EFEMP-1 compared to UV-treated H cybrids. Conclusion/Significance In cells with identical nuclei, the cellular response to sub-lethal UV-radiation is mediated in part by the mtDNA haplogroup. This supports the hypothesis that differences in growth rates and expression levels of complement, inflammation and apoptosis genes may result from population-specific, hereditary SNP variations in mtDNA. Therefore, when analyzing UV-induced damage in tissues, the mtDNA haplogroup background may be important to consider.PLoS ONE 06/2014; 9(6):e99003. DOI:10.1371/journal.pone.0099003 · 3.23 Impact Factor
Article: Inherited Mitochondrial DNA Variants Can Affect Complement, Inflammation, and Apoptosis Pathways: Insights into Mitochondrial-Nuclear Interactions.[Show abstract] [Hide abstract]
ABSTRACT: Background Age-related macular degeneration (AMD) is the leading cause of vision loss in developed countries. While linked to genetic polymorphisms in the complement pathway, there are many individuals with high risk alleles that do not develop AMD, suggesting that other "modifiers" may be involved. Mitochondrial (mt) haplogroups, defined by accumulations of specific mtDNA single nucleotide polymorphisms (SNPs) which represent population origins, may be one such modifier. J haplogroup has been associated with high risk for AMD while the H haplogroup is protective. It has been difficult to assign biological consequences for haplogroups so we created human ARPE-19 cybrids (cytoplasmic hybrids), which have identical nuclei but mitochondria of either J or H haplogroups, to investigate their effects upon bioenergetics and molecular pathways.Methodology/Principal FindingsJ cybrids have altered bioenergetic profiles compared to H cybrids. Q-PCR analyses, show significantly lower expression levels for seven respiratory complex genes encoded by mtDNA. J and H cybrids have significantly altered expression of eight nuclear genes of the alternative complement, inflammation and apoptosis pathways. Sequencing of the entire mtDNA DNA was carried out for all the cybrids to identify haplogroup and non haplogroup defining SNPs.Conclusion/SignificancemtDNA can mediate cellular bioenergetics and expression levels of nuclear genes related to complement, inflammation and apoptosis. Sequencing data suggest that observed effects are not due to rare mtDNA variants but rather the combination of SNPs representing the J versus H haplogroups. These findings represent a paradigm shift in our concepts of mitochondrial-nuclear interactions.Human Molecular Genetics 02/2014; 23(13). DOI:10.1093/hmg/ddu065 · 6.39 Impact Factor
Article: Molecular and Bioenergetic Differences between Cells with African versus European Inherited Mitochondrial DNA Haplogroups: Implications for Population Susceptibility to Diseases.[Show abstract] [Hide abstract]
ABSTRACT: The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP turnover rates and lower levels of ROS production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.Biochimica et Biophysica Acta 11/2013; 1842(2). DOI:10.1016/j.bbadis.2013.10.016 · 4.66 Impact Factor
Article: Therapeutic Potential of the Poly(ADP-ribose) Polymerase Inhibitor Rucaparib for the Treatment of Sporadic Human Ovarian Cancer[Show abstract] [Hide abstract]
ABSTRACT: Here, we investigate the potential role of the PARP inhibitor rucaparib (CO-338, formerly known as AG014699 and PF-01367338) for the treatment of sporadic ovarian cancer. We studied the growth inhibitory effects of rucaparib in a panel of 39 ovarian cancer cell lines that were each characterized for mutation and methylation status of BRCA1/2, baseline gene expression signatures, copy number variations of selected genes, PTEN status, and sensitivity to platinum-based chemotherapy. To study interactions with chemotherapy, we used multiple drug effect analyses and assessed apoptosis, DNA fragmentation, and γH2AX formation. Concentration-dependent antiproliferative effects of rucaparib were seen in 26 of 39 (67%) cell lines and were not restricted to cell lines with BRCA1/2 mutations. Low expression of other genes involved in homologous repair (e.g., BCCIP, BRCC3, ATM, RAD51L1), amplification of AURKA or EMSY, and response to platinum-based chemotherapy was associated with sensitivity to rucaparib. Drug interactions with rucaparib were synergistic for topotecan, synergistic, or additive for carboplatin, doxorubicin or paclitaxel, and additive for gemcitabine. Synergy was most pronounced when rucaparib was combined with topotecan, which resulted in enhanced apoptosis, DNA fragmentation, and γH2AX formation. Importantly, rucaparib potentiated chemotherapy independent of its activity as a single agent. PARP inhibition may be a useful therapeutic strategy for a wider range of ovarian cancers bearing deficiencies in the homologous recombination pathway other than just BRCA1/2 mutations. These results support further clinical evaluation of rucaparib either as a single agent or as an adjunct to chemotherapy for the treatment of sporadic ovarian cancer. Mol Cancer Ther; 12(6); 1-14. ©2013 AACR.Molecular Cancer Therapeutics 05/2013; 12(6). DOI:10.1158/1535-7163.MCT-12-0813 · 5.68 Impact Factor
Article: Mitochondrial DNA Variants Mediate Energy Production and Expression Levels for CFH, C3 and EFEMP1 Genes: Implications for Age-Related Macular Degeneration[Show abstract] [Hide abstract]
ABSTRACT: Mitochondrial dysfunction is associated with the development and progression of age-related macular degeneration (AMD). Recent studies using populations from the United States and Australia have demonstrated that AMD is associated with mitochondrial (mt) DNA haplogroups (as defined by combinations of mtDNA polymorphisms) that represent Northern European Caucasians. The aim of this study was to use the cytoplasmic hybrid (cybrid) model to investigate the molecular and biological functional consequences that occur when comparing the mtDNA H haplogroup (protective for AMD) versus J haplogroup (high risk for AMD). Cybrids were created by introducing mitochondria from individuals with either H or J haplogroups into a human retinal epithelial cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). In cybrid lines, all of the cells carry the same nuclear genes but vary in mtDNA content. The J cybrids had significantly lower levels of ATP and reactive oxygen/nitrogen species production, but increased lactate levels and rates of growth. Q-PCR analyses showed J cybrids had decreased expressions for CFH, C3, and EFEMP1 genes, high risk genes for AMD, and higher expression for MYO7A, a gene associated with retinal degeneration in Usher type IB syndrome. The H and J cybrids also have comparatively altered expression of nuclear genes involved in pathways for cell signaling, inflammation, and metabolism. Our findings demonstrate that mtDNA haplogroup variants mediate not only energy production and cell growth, but also cell signaling for major molecular pathways. These data support the hypothesis that mtDNA variants play important roles in numerous cellular functions and disease processes, including AMD.PLoS ONE 01/2013; 8(1):e54339. DOI:10.1371/journal.pone.0054339 · 3.23 Impact Factor
Article: Mitochondrial DNA haplogroups confer differences in risk for age-related macular degeneration: A case control study[Show abstract] [Hide abstract]
ABSTRACT: Background Age-related macular degeneration (AMD) is the leading cause of vision loss in elderly, Caucasian populations. There is strong evidence that mitochondrial dysfunction and oxidative stress play a role in the cell death found in AMD retinas. The purpose of this study was to examine the association of the Caucasian mitochondrial JTU haplogroup cluster with AMD. We also assessed for gender bias and additive risk with known high risk nuclear gene SNPs, ARMS2/LOC387715 (G > T; Ala69Ser, rs10490924) and CFH (T > C; Try402His, rs1061170). Methods Total DNA was isolated from 162 AMD subjects and 164 age-matched control subjects located in Los Angeles, California, USA. Polymerase chain reaction (PCR) and restriction enzyme digestion were used to identify the J, U, T, and H mitochondrial haplogroups and the ARMS2-rs10490924 and CFH-rs1061170 SNPs. PCR amplified products were sequenced to verify the nucleotide substitutions for the haplogroups and ARMS2 gene. Results The JTU haplogroup cluster occurred in 34% (55/162) of AMD subjects versus 15% (24/164) of normal (OR = 2.99; p = 0.0001). This association was slightly greater in males (OR = 3.98, p = 0.005) than the female population (OR = 3.02, p = 0.001). Assuming a dominant effect, the risk alleles for the ARMS2 (rs10490924; p = 0.00001) and CFH (rs1061170; p = 0.027) SNPs were significantly associated with total AMD populations. We found there was no additive risk for the ARMS2 (rs10490924) or CFH (rs1061170) SNPs on the JTU haplogroup background. Conclusions There is a strong association of the JTU haplogroup cluster with AMD. In our Southern California population, the ARMS2 (rs10490924) and CFH (rs1061170) genes were significantly but independently associated with AMD. SNPs defining the JTU mitochondrial haplogroup cluster may change the retinal bioenergetics and play a significant role in the pathogenesis of AMD.BMC Medical Genetics 01/2013; 14(1):4. DOI:10.1186/1471-2350-14-4 · 2.08 Impact Factor
Article: Developmental or degenerative – NR2E3 gene mutations in two patients with enhanced S cone syndrome[Show abstract] [Hide abstract]
ABSTRACT: Enhanced S Cone Syndrome is a rare autosomal recessive disorder characterized clinically by an absence of rod function, a replacement of most L and M cone function by S cone activity (Goldmann-Favre Syndrome) and by variable degrees of retinal degeneration in different families. The causative gene, nuclear receptor subfamily 2, group E, member 3 (NR2E3), controls the developmental sequence for rods and cones. The purpose of this study was to compare the nature and implications of mutations in two subjects with Enhanced S Cone Syndrome who have significantly different degrees of degenerative damage. A direct sequencing approach was used to identify the mutations. Genomic DNA was amplified from all the exons of NR2E3 and used as a template for sequencing. Of the two families studied, Case 1 is of Persian ethnicity while Case 2 is Brazilian. A total of six individuals within the two families were studied. Case 1 (original propositus of the syndrome) has the characteristic developmental rod/cone abnormality with large amplitude electroretinogram responses and no retinal degeneration. She was homozygous for a novel mutation, c.[del196-201del6] (p.G66-C67del), which lies entirely within the P-box for this gene. By comparison, Case 2 had Goldmann-Favre Syndrome with retinal degeneration and low electroretinogram signals. She was a compound heterozygote for c.[119-2A>C]+[del194-202del9] (p.N65-C67del), mutations that have been reported previously. Her second mutation overlaps that of Case 1 within the P-box. The novel in-frame homozygous deletion of Case 1, within the P-box motif of the DNA binding domain, caused a developmental abnormality without retinal degeneration. Case 2, with more traditional Goldmann-Favre Syndrome with retinal degeneration, was a compound heterozygote where one allele had a similar P-box deletion but the other was a splicing defect. Case 1 is the first reported homozygous deletion within the P-box. This is the first report of NR2E3 mutations in a Persian and a Brazilian family.Molecular vision 02/2011; 17:519-25. · 1.99 Impact Factor
Article: Characterization of Retinal and Blood Mitochondrial DNA from Age-Related Macular Degeneration Patients[Show abstract] [Hide abstract]
ABSTRACT: To determine mitochondrial (mt)DNA variants in AMD and age-matched normal retinas. Total DNA was isolated from retinas (AMD, n = 13; age-matched normal, n = 13), choroid (AMD, n = 3), and blood (AMD, n = 138; normal, n = 133). Long-extension-polymerase chain reaction amplified the full-length ( approximately 16.2 kb) mtDNA genome. Retinal mtDNA was sequenced for nucleotide variants and length heteroplasmy. Pyrosequencing was performed on heteroplasmic mtDNA. PCR amplification and enzyme digestions were used to analyze for nucleotide changes. Retinal mtDNA had a greater number of rearrangements and deletions than did blood mtDNA in normal samples (9.3 +/- 1.78 vs. 3 +/- 1.18, P = 0.019), and AMD samples (14.33 +/- 1.96 vs. 5.2 +/- 0.80, P = 0.0031. Five (55%) of 9 AMD patients had unreported SNPs, and 2 (16.6%) of 12 of the normal group did. The mtDNA coding region had 20 SNPs that produced amino acid changes. The noncoding MT-Dloop region had nucleotide heteroplasmy and length heteroplasmy. There were more SNPs per person in the AMD population than in the older (P = 0.003) and younger (P = 0.05) normal subjects. The C12557T (T-I) in the MT-ND5 gene was present in two AMD subjects (2/138) but was absent in the normal (0/133). Common mutations for Leber's hereditary optic neuropathy (LHON: G11778A; T14484C; and G3460A) were not present in AMD samples. AMD subjects have high levels of large mtDNA deletions/rearrangements in the retinas, unreported and amino acid-changing SNPs in the coding genome, and a greater number of SNPs per person in the noncoding MT-Dloop region. These mtDNA variants could diminish energy production efficiency, alter the mtDNA copy numbers and/or impact transcription in AMD retinas.Investigative ophthalmology & visual science 03/2010; 51(8):4289-97. DOI:10.1167/iovs.09-4778 · 3.40 Impact Factor
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ABSTRACT: We reported previously a 7-base intronic deletion close to the intron/exon junction of the SOD1 gene in 2 separate families with an autosomal-dominant form of keratoconus. The goal of this study was to determine if the 2 families (families A and H) shared a common haplotype by identifying closely linked new microsatellite markers flanking the SOD1 gene. Total genomic DNA was extracted from the blood of available members of families A and H. The DNA was amplified by polymerase chain reaction and digested with HpyCH4 III. A genomic contig was first constructed flanking the human SOD1 gene on chromosome 21q22.1-21q22.11. New polymorphic microsatellite markers were identified. All available individuals from the 2 families were genotyped using a set of 7 different markers (SOD1NU10, SOD1NU1, SOD1NU2, SOD1NU3, SOD1NU13, SOD1NU8, and SOD1NU9) to identify phase and the disease haplotype was constructed. Five of the 7 markers are novel (SOD1NU1, SOD1NU2, SOD1NU3, SOD1NU8, and SOD1NU9). Family A is a 3-generation family and the disease haplotype was inferred based on segregation data for 7 different markers. Family H shared only 3 of the disease-associated alleles (SOD1NU1, SOD1NU2, and SOD1NU13) compared with family A. Based on the dissimilarity of disease-associated alleles, the 2 families do not appear to share the same haplotype and therefore are not closely related. This strongly supports the uniqueness of the 7-base deletion in intron 2 of the SOD1 gene to the keratoconus phenotype.Cornea 09/2009; 28(8):902-7. DOI:10.1097/ICO.0b013e3181983a0c · 2.04 Impact Factor
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ABSTRACT: To study the effects of hydrogen peroxide exposure on mitochondrial DNA (mtDNA) in cultured human corneal epithelial cells. In addition, we compared the integrity of mtDNA found in epithelial cells isolated from keratoconus (KC) and normal (NL) corneas. Telomerase immortalized human corneal epithelial cell line (hTCEpi) were cultured at pH 7.0 or pH 5.0 with or without 200 microM hydrogen peroxide (H2O2). Immunohistochemistry with a marker for oxidative damage, 8-hydroxy-2'-deoxyguanosine (8-OH-dG), was performed on KC and NL corneas (n = 10). Epithelial cells were isolated from KC corneas (n = 5) and NL corneas (n = 7). Total DNA was extracted, and the mtDNA was analyzed by long extension polymerase chain reaction (LX-PCR). The ratios of mtDNA to nuclear DNA were measured by PCR. The mtDNA control regions were PCR amplified and sequenced. In the epithelial cell cultures, the full-length LX-PCR mtDNA decreased 54% and 44% in the H2O2 + pH7 cultures and H2O2 + pH5 cultures, respectively. 8-OH-dG was present in all layers of KC epithelial cells but only in superficial layers of NL epithelial cells. The isolated KC and NL epithelial cells had comparable levels of full-length LX-PCR mtDNA (16.2 kb) and smaller sized mtDNA bands (4.3 +/- 0.99 vs 4.0 +/- 0.83 bands per individual, respectively). There were no significant differences in the control region nucleotide sequences in KC and NL epithelia. Hydrogen peroxide can significantly degrade LX-PCR mtDNA in vitro. Although the KC epithelium showed a higher degree of oxidative damage, the levels of mtDNA damage in NL and KC epithelial cells were similar to each other.Cornea 06/2009; 28(4):426-33. DOI:10.1097/ICO.0b013e31818c2c55 · 2.04 Impact Factor
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ABSTRACT: To examine the mtDNA control regions in normal and age-related macular degeneration (AMD) retinas. To identify the mtDNA variations associated with AMD. Retinas from 10 normal and 11 AMD globes were isolated and analyzed for mtDNA rearrangements by long extension-polymerase chain reaction (LX-PCR) and for the nature and frequency of single-nucleotide polymorphisms (SNPs) in the mtDNA control region by direct sequencing. Blood DNA was extracted from 99 AMD and 92 age-matched control subjects. The sequence variations that define haplogroups H, I, J, K, T, V, X, and U were characterized by PCR, restriction enzyme digestion, and/or sequencing. LX-PCR of retinal mtDNAs revealed high levels of rearrangements in the patients with AMD and the control subjects, consistent with the decline in mitochondrial function with age. However, the AMD retinas had higher oxidized DNA levels and a higher number of SNPs than controls (P = 0.02). The control region SNPs T16126C and A73G, commonly found in haplogroups J and T, were more frequent in the AMD retinas than in normal retinas. The associations between AMD and haplogroups J and T were confirmed and extended by analysis of blood DNA. SNPs at position a T16126C (J; odds ratio [OR] = 3.66), T16126C+G13368A (JT; OR = 10.27), A4917G+A73G (T4; OR = 5), and T3197C+A12308G (U5; OR = infinity), were all strongly associated with AMD. AMD retinas exhibited increased mtDNA control region SNPs compared to normal retinas. This correlated with an increased frequency of mtDNA SNPs associated with haplogroups J, T and U in patients with AMD. These results implicate mitochondrial alterations in the etiology of AMD.Investigative ophthalmology & visual science 02/2009; 50(6):2966-74. DOI:10.1167/iovs.08-2646 · 3.40 Impact Factor
Article: New mutation, P575L, in the GUCY2D gene in a family with autosomal dominant progressive cone degeneration[Show abstract] [Hide abstract]
ABSTRACT: To clinically characterize the retinal abnormalities and identify the mutation causing an autosomal dominant cone degeneration in an African American family. Clinical characterization of family members using fundus photography, fluorescein angiography, and electrophysiological testing. Standard molecular genetic methods were used, including segregation analysis and DNA sequencing of candidate genes. Genetic mutation screening was performed in 20 individuals: 10 clinically unaffected and 10 affected. The affected family members had findings consistent with a primary cone degeneration. A novel mutation, P575L, was found in exon 8 of the GUCY2D gene in 12 members of this family. In addition to finding a previously undescribed mutation in GUCY2D, 2 of the family members who were thought to be unaffected through routine clinical examinations also had this mutation. These findings suggest that autosomal dominant cone degeneration in this family demonstrated age-dependent penetrance, which appears incomplete. This is the first African American family reported with a mutation in GUCY2D. Because the disease in this family and the one we previously described is primarily a cone degeneration, this disease should be more properly classified as cone degeneration and be called cone degeneration 2. This study helps to expand the phenotype of the disease and help clinicians identify patients with cone degenerations.Archives of Ophthalmology 04/2008; 126(3):397-403. DOI:10.1001/archopht.126.3.397 · 4.40 Impact Factor
Article: of a case. Am J Ophthalmol 1939; 22 : 744–749. 6 Salas D, Murthy S, Champ C, Hawksworth N. Primary A 26-year-old woman was referred to the glaucoma tuberculosis of the conjunctiva. Eye 2001; 15 (Part 5): clinic because of elevated intraocular pressure (IOP) 674–676. found in a routine eye examination. The patient was 7 Cameron JA, Nasr AM, Chavis P. Epibulbar and otherwise healthy with no significant family history, ocular tuberculosis. Arch Ophthalmol 1996; 114 (6): 770–771. no visual complaints, or any history of recurrent 8 Flinnoff WC. Ocular tuberculosis, experimental and clinical. erosions. Arch Ophthalmol 1924; 53 : 130–136. Her best-corrected visual acuity was 20/20 in both 9 Grunert C. A contribution to the subject of tuber- eyes. Corneal sensitivity was normal. Slit-lamp culosis of the conjunctiva. Arch Ophthalmol 1899; 28 : examination revealed refractile, radial filamentary lines 540–556. 10 Eyre JWH. Tuberculosis of the conjunctiva: its in the anterior corneal stroma creating a lattice-work etiology, pathology and diagnosis. Lancet 1912; 1 : pattern, with sparing of the central cornea (Figure 1). 1319–1328. No opacification or corneal haze was noted. 11 Aclimandos WA, Kerr-Muir M. Tuberculous Gonioscopy showed high iris insertion and prominent keratoconjunctivitis. Br J Ophthalmol 1992; 76 : iris processes in both eyes (Figure 2). IOP was 34 mmHg 175–176. in the right eye and 29 mmHg in the left eye. Fundus AG Zaborowski 1 , BN Gundry 1 , ME Masenya 2 and L Visser 1 examination revealed an arcuate nerve fibre loss between the first-order inferior temporal vessels of the left eye. 1 Department of Ophthalmology, Nelson R Visual fields were normal. No change in the findings of the cornea, fundus, and visual fields was noted within Mandela School of Medicine, Durban, the 5 years of follow-up. IOP remained controlled under South Africa antiglaucoma treatment. 2 Department of Anatomical Pathology, Nelson R Familial and systemic amyloidosis were ruled out by Mandela School of Medicine, 52 Umbilo Road, extensive physical examination and laboratory work-up. Congella, Durban, KwaZulu-Natal 4013, The patient did not present any skin lesions, South Africa macroglossia, arrhythmias or megalocardy, hepatosplenomegaly, arthritis, and cranial or peripheral Correspondence: A Zaborowski, neuropathy. There was no evidence of pulmonary, Tel: þ 27 31 3603450; haematopoetic, or renal involvement. ESR, CRP, and Fax: þ 27 31 5727634. immunoglobulins levels were within normal limits. E-mail: email@example.com Echocardiography was normal. Skin biopsy was negativeEye 09/2006; 20(8):979-80. DOI:10.1038/sj.eye.6702092 · 2.08 Impact Factor
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ABSTRACT: To screen superoxide dismutase 1 (SOD1) on chromosome 21 as a possible candidate gene for familial keratoconus (KC). Total genomic DNA was extracted from the blood of 15 different KC families and 156 unaffected subjects. All five exons of the SOD1 gene were sequenced. For a rapid screening test, DNA was amplified by polymerase chain reaction (PCR), digested with HpyCH4 III or analyzed by radioactively end-labeled exon PCR. RNA was extracted from leukocytes and reverse transcribed to cDNA, and the PCR was amplified for splice variants. Some samples were cloned and sequenced. A heterozygous genomic 7-base deletion in intron 2 of the SOD1 gene was identified in two KC families (pedigrees 1 and 6). The deletion segregated within pedigree 1 and was absent in 312 chromosomes from normal individuals. RNA from the proband of pedigree 1 showed that in addition to the wild-type transcript, two other transcripts were expressed for the CuZn SOD (SOD1) gene: lacking entire exon 2 (LE2) and lacking entire exon 2 and entire exon 3 (LE2E3). A unique genomic deletion within intron 2 close to the 5' splice junction of the SOD1 gene was identified in three patients with KC. Moreover, mRNA from one affected individual also had two transcript splice variants (LE2 and LE2E3) that others have shown to code for proteins lacking the active site of the SOD1 enzyme. Further studies should be conducted to determine whether a causal relationship exists between these two events that may increase oxidative stress and be associated with KC.Investigative Ophthalmology & Visual Science 09/2006; 47(8):3345-51. DOI:10.1167/iovs.05-1500 · 3.40 Impact Factor
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ABSTRACT: To perform candidate gene screening for posterior polymorphous corneal dystrophy (PPCD). The initial 3 genes chosen, ID1, BCL2L1, and VSX1, lie within the region on chromosome 20 to which the PPCD gene has been linked, and mutations in VSX1 have previously been identified in patients with PPCD. DNA extraction, PCR amplification, and direct sequencing of the VSX1, BCL2L1, and ID1 genes were performed in 14 affected patients (12 families) as well as in unaffected family members and healthy control subjects. No coding region mutations in the BCL2L1 or ID1 genes were identified in affected patients. In the VSX1 gene, the previously identified Gly160Asp missense change was not present in any of our 12 probands, and the Asp144Glu mutation was identified in 1 affected patient as well as 1 unaffected control individual. Additionally, 2 synonymous substitutions were identified, Ala182Ala (8 affected patients from 8 families) and Gly239Gly (1 affected patient and 1 unaffected patient from the same family). In the ID1 gene, the synonymous substitution Gly216Gly was observed in 2 affected patients (2 families) who also demonstrated a single nucleotide change in both the 5'UTR (2129T>C) and 3'UTR (3267A>G). Another 5'UTR change, 2177T>C, was identified in 1 affected patient and his unaffected parent, both of whom also demonstrated the 2129T>C and 3267A>G changes. None of the 12 probands with PPCD demonstrated the previously described Gly160Asp mutation within the VSX1 gene. The Asp144Glu missense change, present in an affected patient as well as an unaffected control individual, appears to be a rare polymorphism, not a disease-causing mutation. No coding region changes were identified in the ID1 or BCL2L1 genes. Therefore, although we report a number of novel polymorphisms in the VSX1 and ID1 genes, the failure to identify any sequence variants that sort with the disease phenotype suggests that other genetic factors are involved in PPCD.Cornea 04/2005; 24(2):151-5. DOI:10.1097/01.ico.0000141235.26096.1d · 2.04 Impact Factor
Article: Lattice cornea dystrophy associated with the Ala546Asp and Pro551Gln missense changes in the TCFBI gene[Show abstract] [Hide abstract]
ABSTRACT: To report a phenotypic variant of lattice corneal dystrophy associated with two missense changes, Ala546Asp and Pro551Gln, in the transforming growth factor-beta-induced gene (TGFBI). Experimental study. Genomic DNA was obtained from the proband as well as affected and unaffected family members. Exons 4, 11, 12, and 14 of the TGFBI gene were amplified and sequenced. Additionally, a corneal button excised from the proband was examined by light and transmission electron microscopy. Haplotype analysis was performed on the proband's family and members of a previously identified pedigree with the same TGFBI gene missense changes. Bilateral, symmetric, radially arranged, branching refractile lines within and surrounding an area of central anterior stromal haze were noted in the proband. Multiple polymorphic, refractile deposits were noted in the mid and posterior stroma in both the proband and her daughter. Light and electron microscopic analyses demonstrated amyloid and excluded the presence of deposits characteristic of granular corneal dystrophy. Screening of TGFBI exon 12 in the proband and her affected daughter revealed two missense changes, Ala546Asp and Pro551Gln (both absent in 250 control chromosomes). Haplotype analysis suggested that the mutations in this family and in a previously identified pedigree reflect a founder effect, rather than an independent occurrence. We present a phenotypic variant of lattice corneal dystrophy associated with the Ala546Asp and Pro551Gln missense changes in exon 12 of the TGFBI gene. A common ancestor appears to account for the missense mutations observed in this pedigree and in a previously reported family.American Journal of Ophthalmology 12/2004; 138(5):772-81. DOI:10.1016/j.ajo.2004.06.021 · 3.87 Impact Factor
Article: Novel mutations in the carbohydrate sulfotransferase gene (CHST6) in American patients with macular corneal dystrophy[Show abstract] [Hide abstract]
ABSTRACT: To further characterize the mutations within the CHST6 gene responsible for causing macular corneal dystrophy in a cohort of affected patients from the United States. Experimental study. Genomic DNA was extracted from buccal epithelium of 16 affected patients (14 families), 17 unaffected relatives, and 127 controls, followed by polymerase chain reaction amplification and direct sequencing of the CHST6 coding region. Subtyping of affected patients into type I and II macular corneal dystrophy was performed by measuring antigenic keratan sulfate (AgKS) serum levels. Haplotype analysis was performed in families that demonstrated common mutations. CHST6 coding region analysis in 10 patients identified as having type I macular corneal dystrophy revealed 10 sequence changes: eight missense mutations, four of which are novel (Met104Val, Tyr110Cys, Gln122Pro, and Leu276Pro) and four of which have been reported previously (Ser51Leu, Pro72Ser, Cys102Gly, and Leu200Arg); one novel homozygous nonsense mutation in two patients from a single family (c. 1683C>T, Gln331X); and one frameshift mutation in a heterozygous state in a single patient (c.1744_1751dupGTGCGCTG). Mutation analysis in the four patients identified as having type II macular corneal dystrophy (serum samples were not obtained from two affected patients) revealed three patients heterozygous for either the c.923G>C, c.969C>A, or c.1519T>C sequence changes. The fourth patient was compound heterozygous for c.969C>A and c.1291T>G. None of these changes was observed in 127 control individuals. Haplotype analysis using microsatellite markers flanking the CHST6 gene did not reveal a common founder for the Leu200Arg (1291T>G) missense mutation, present in five families, identifying this position as a mutation hot-spot. A variety of previously unreported mutations in the coding region of the CHST6 gene are associated with type I macular corneal dystrophy in a cohort of patients from the United States.American Journal of Ophthalmology 03/2004; 137(3):465-73. DOI:10.1016/j.ajo.2003.09.036 · 3.87 Impact Factor
Article: Keratoconus--no association with the transforming growth factor beta-induced gene in a cohort of American patients.[Show abstract] [Hide abstract]
ABSTRACT: Keratoconus is a noninflammatory, corneal thinning disorder leading to mixed myopic and irregular astigmatism and implicated as a major reason for cornea transplantations in the Western world. Genetic factors have been suggested as a cause of keratoconus. The levels of transforming growth factor beta-induced (TGFBI) protein have been reported to be altered in keratoconus tissues. Mutations in this gene are responsible for causing various corneal dystrophies. Given this strong evidence of the involvement of this gene in corneal dystrophies, we investigated possible mutations within this gene in 15 probands of families with keratoconus. All patients and control individuals had complete ophthalmological examination by a corneal specialist to determine their affectation status. The entire transcript of the TGFBI gene was analyzed by direct sequencing from patient DNA. We found 8 sequence variations within the gene, none of which was protein-altering changes. These changes were also observed in control individuals, and 4 are previously known polymorphisms. We concluded that the TGFBI gene is not responsible for causing keratoconus in these patients.Cornea 02/2004; 23(1):13-7. · 2.04 Impact Factor
Top Journals
- American Journal of Ophthalmology (5)
- Human Molecular Genetics (3)
- Cornea (3)
- PLoS ONE (3)
- Cornea (2)
University of California, Irvine
Irvine, California, United States
- Department of Ophthalmology
Jules Stein Eye InstituteMaryland, United States
University of California, Los Angeles
Los Angeles, California, United States
- Jules Stein Eye Institute
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical CenterTorrance, California, United States


text: Charming and beautiful hostel with wonderful hot water and a good breakfast! Not meticously clean nut close to the plaza, good restaurants and shopping. We had fun at the Para-ti tour, Sucre Sombero tour and eating the best chicken in town. we had a great time and hope to return!

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Leonardo Royal Hotel Düsseldorf Königsallee
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... a crazy concept for NZers but public drinking is perfectly legal in the oh so modern and rule-abiding German lands.
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Cobalt 336: More than your Average Bowrider
Tired of walking the Fort Lauderdale International Boat Show, Lenny Rudow found a comfy place to relax on board the Cobalt 336.
Eds Note: Want to see more 336 video? Check out Cobalt's YouTube tour of the 336.
Video transcript: I finally found somewhere comfortable to sit down, on this Cobalt 336. Now from this look, you probably think I'm sitting on a bowrider. Yeah, sort of...
Looks can be a little bit deceiving, particularly on a boat like this. Check out this cabin! Here underneath the helm station, there's plenty of room to kick back, relax, maybe turn on the AC.
And underneath the passenger's console, there's a full-sized head. An average bowrider? I don't think so. Find out more in the full review.
—Lenny Rudow
Watch this Cobalt 336 Short Take on YouTube.


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Great lake view. Cabin is rustic with no running water or sewer hookup. Boat dock in need of repair. Simple one room, 20 by 20 cabin. Not suitable for year around living in current condition. Mostly wooded 5.11 acres just minutes from Warrenton & I-70. Mobile homes are welcome. Very Private. Warrenton R-3 School District.
Sep 29 -
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3 beds 2 baths 1,440 sqft
For Sale by Owner
14.8 Acres of Woods, Custom Oak Log Home + Mark Twain Natl. Forest $169,000
What else could you want? Read on??
The photos paint the perfect picture, but the peace and serenity of the setting here can?t be expressed with words. This amazing seasoned Oak Log cabin offers 1440 square feet of living space: 2 bedrooms ( 11-1/2 by 15, and 9 by 12 feet) ...
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Price: $174,800
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More Info: http://listings.realbird.com/Real_Estate/29-381-ACRES--PAVED-ROADS/Troy/MO/K8O6H8N4/415598.aspx
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Page 1
Immunopathology and Infectious Disease
Pemphigus IgG Causes Skin Splitting in the
Presence of Both Desmoglein 1 and Desmoglein 3
Volker Spindler,* Detlev Drenckhahn,*
Detlef Zillikens,†and Jens Waschke*
From the University of Wu ¨rzburg,* Institute of Anatomy and Cell
Biology, Wu ¨rzburg; and the University of Lu ¨beck,†Department of
Dermatology, Lu ¨beck, Germany
According to the desmoglein (Dsg) compensation
concept, different epidermal cleavage planes ob-
served in pemphigus vulgaris and pemphigus folia-
ceus have been proposed to be caused by different
autoantibody profiles against the desmosomal pro-
teins Dsg 1 and Dsg 3. According to this model, Dsg 1
autoantibodies would only lead to epidermal splitting
in those epidermal layers in which no Dsg 3 is present
to compensate for the functional loss of Dsg 1. We
provide evidence that both pemphigus foliaceus-IgG
containing Dsg 1- but not Dsg 3-specific antibodies
and pemphigus vulgaris-IgG with antibodies to Dsg 1
and Dsg 3 were equally effective in causing epidermal
splitting in human skin and keratinocyte dissociation
in vitro. These effects were present where keratino-
cytes expressed both Dsg 1 and Dsg 3, demonstrating
that Dsg 3 does not compensate for Dsg 1 inactivation.
Rather, the cleavage plane in intact human skin
caused by pemphigus autoantibodies was similar to
the plane of keratinocyte dissociation in response to
toxin B-mediated inactivation of Rho GTPases. Be-
cause we recently demonstrated that pemphigus-IgG
causes epidermal splitting by inhibition of Rho A, we
propose that Rho GTPase inactivation contributes to the
mechanisms accounting for the cleavage plane in pem-
phigus skin splitting.
(Am J Pathol 2007, 171:906–916;
DOI: 10.2353/ajpath.2007.070028)
Pemphigus is an autoimmune blistering skin disease
caused by autoantibodies directed to the cadherin-type
adhesion molecules desmoglein (Dsg) 1 and 3.1–4Anti-
bodies against other antigens, including cholinergic re-
ceptors, have also been implicated in the pathogene-
sis.5–9Several theories have been proposed to explain
the mechanisms underlying blister formation by Dsg au-
toantibodies. However, the different clinical phenotypes
of pemphigus vulgaris (PV) and pemphigus foliaceus
(PF) are still not fully understood.10–12It is well estab-
lished that PV is characterized by deep (suprabasal)
epidermal blistering, whereas PF exhibits superficial blis-
tering (involving granular or spinous cell layers). In PF,
mucous membranes are not involved; whereas in PV,
mucosal disease is a regular finding, and skin involve-
ment may occur in addition.4Interestingly, these pheno-
types usually correlate with different autoantibody pro-
files. PV patients with only mucosal lesions show
autoantibodies to Dsg 3 but not to Dsg 1. PF patients
(skin involvement only) reveal autoantibodies to Dsg 1
but not to Dsg 3. In PV patients with both mucous mem-
brane and skin involvement, autoantibodies to Dsg 3 and
Dsg 1 may be detected.4,13,14These observations, to-
gether with studies reporting that Dsg 1 is predominantly
expressed in superficial epidermal layers but absent in
the suprabasal layer, whereas Dsg 3 is restricted to deep
epidermal layers, and that Dsg 1 is less abundantly ex-
pressed in mucosal epithelia, have led to the desmoglein
compensation hypothesis as an explanation for the dif-
ferent epidermal cleavage planes in PV and PF.3,4,15–17
According to this model, in the deep epidermis, Dsg 3
compensates for the functional loss of Dsg 1 induced by
Dsg 1-specific autoantibodies, resulting in superficial
blistering in PF.
However, more recently, this model has been repeat-
edly questioned.2,10,11i) The model does not explain why
blistering in patients with mucocutaneous PV, where Dsg
1- and Dsg 3-specific autoantibodies are present, occurs
suprabasally and not throughout the epidermis. ii) The
desmoglein compensation hypothesis postulates that the
different forms of pemphigus and the clinical stages of PV
are distinguishable by antibody profiles and histology.
This view has recently been challenged.2,18iii) To vali-
date the desmoglein compensation theory, expression
patterns of desmogleins have been evaluated in mouse
Supported by Deutsche Forschungsgemeinschaft (grant SFB 487 TP B5)
and the Interdisziplina ¨res Zentrum fu ¨r Klinische Forschung Wu ¨rzburg
(grant TP A-51).
Accepted for publication May 30, 2007.
Address reprint requests to Dr. Jens Waschke, Institute of Anatomy and
Cell Biology, Julius-Maximilians-University, Koellikerstrasse 6, D-97070
Wu ¨rzburg, Germany. E-mail: email@example.com.
The American Journal of Pathology, Vol. 171, No. 3, September 2007
Copyright © American Society for Investigative Pathology
DOI: 10.2353/ajpath.2007.070028
Page 2
models.4,16However, recent studies indicate that the
expression patterns of Dsg 1 and Dsg 3 in mice pro-
foundly differ from those in humans.19iv) To visualize the
desmoglein localization pattern in human epidermis, au-
toantibodies from patients with PV and PF have been
applied.15,20In these studies, either whole sera of PV and
PF patients were used without characterization of the
autoantibody profile20or IgG fractions were used after
immunosabsorption with recombinant Dsg extracellular
domains fused to the Fc portion of human IgG.15More
recently, it has been suggested that immunoabsorption
using this fusion protein containing the extracellular do-
main of desmogleins is not entirely specific. Therefore,
the specificity of autoantibodies used for immunolocal-
ization of desmogleins in human skin in previous studies
is not entirely clear.9v) Finally, for desmoglein compen-
sation to be a valid mechanism in human epidermis, only
autoantibodies against Dsg 1 and Dsg 3 should cause
epidermal splitting, and these antibodies should specifi-
cally block the adhesive function of the one desmoglein
subtype (Dsg 1 or Dsg 3) to which they are directed.4
Therefore, the desmoglein compensation model has
been used to support the hypothesis that autoantibodies
directlyaffect desmoglein
drance.16,17This has also become a matter of discussion
because other antibodies, including those antibodies to
cholinergic receptors have been found to be pathogen-
ic.2,9However, we have shown that PF-IgG causes ker-
atinocyte dissociation and inhibition of Dsg 1 binding by
mechanisms different from steric hindrance because PF-
IgG was not effective in reducing Dsg 1-mediated
transinteraction when probed by single-molecule atomic
force microscopy.21This observation supported the idea
that autoantibody-triggered signaling mechanisms are
involved in pemphigus pathogenesis. Among others,
catenin and Rho protein signaling have been shown to be
involved in pemphigus pathogenesis.22–24These as-
pects led us to reconsider the desmoglein compensation
concept and to evaluate the role of Rho GTPase inacti-
vation in pemphigus skin blistering.
binding bysterichin-
Materials and Methods
Cell Culture and Test Reagents
The immortalized human keratinocyte cell line HaCaT
was grown in Dulbecco's modified Eagle's medium (Life
Technologies, Karlsruhe, Germany) supplemented with
50 U/ml penicillin-G, 50 ?g of streptomycin, and 10%
fetal calf serum (Biochrom, Berlin, Germany) in a humid-
ified atmosphere (95% air/5% CO2) at 37°C. The cultures
were used for all experiments that used confluent mono-
layers and were grown in Dulbecco's modified Eagle's
medium at 1.8 mmol/L Ca2?. Dsg 1 expression was de-
tected by dot blot analysis and immunostaining (day 7
after plating). All experiments were performed using Dul-
becco's modified Eagle's medium containing 1.8 mmol/L
Ca2?. Cytotoxic necrotizing factor 1 (CNF-1) (a kind gift
from Gudula Schmidt, University of Freiburg, Freiburg,
Germany) was used at 300 ng/ml for 120 minutes as
described previously.23The cell-permeable C 3 fusion
toxin (C3FT) from Clostridium botulinum,23toxin B from
Clostridium difficile, and lethal toxin (LT) from Clostridium
sordellii were contributed by Holger Barth (University of
Ulm, Ulm, Germany) and used at 300 ng/ml for 180
minutes. For experiments using human epidermis, 800
ng/ml toxin B was required.
Purification of PF-IgG
Purification was performed as described previously.21Sera
from two PF patients and two patients suffering from a
clinically, histologically, and serologically and from a volun-
teer without any skin disease (control) were used for the
present study. Patients' sera were tested by enzyme-linked
immunosorbent assay for reactivity against Dsg 1 and Dsg
3, respectively. IgG fractions PF-IgG 1 and 2 contained Dsg
1 antibodies but no Dsg 3 antibodies, whereas PV-IgG 1
and 2 contained Dsg 1 and Dsg 3 antibodies (enzyme-
linked immunosorbent assay cut-off was 2025). IgG frac-
tions were purified by affinity chromatography using protein
A agarose as described previously.21Concentrations of all
for all experiments.
Skin Biopsies from Pemphigus Patients
Skin biopsies were taken from PV patient 1 and PF patient
2 when disease was initially diagnosed. After paraffin
embedding, semisectioning (5-?m thickness) was per-
formed, and sections were stained with hematoxylin and
eosin (H&E).
Ex Vivo Model of Human Skin Splitting
The model was used as described previously.23Skin
pieces were taken from fresh cadavers of individuals not
suffering from any skin disease who had donated their
bodies to the Institute of Anatomy and Cell Biology of
Wu ¨rzburg. Specimens were incubated with Dulbecco's
modified Eagle's medium containing 10% fetal calf serum
and 1.8 mmol/L Ca2?for 24 hours in the presence or
absence of PV-IgG, PF-IgG, or toxin B. After brief rinsing
with phosphate-buffered saline (PBS; consisting of 137
mmol/L NaCl, 2.7 mmol/L KCl, 8.1 mmol/L Na2HPO4, and
1.5 mmol/L KH2PO4, pH 7.4), skin specimens were
mounted on copper plates using Reichert-Jung mounting
medium (Cambridge Instruments, Nußloch, Germany)
and frozen in liquid nitrogen. Cryosections (5 ?m thick)
were obtained using a Reichert-Jung 2800 Frigocut
(Cambridge Instruments). For each condition, three to
five pieces of skin (2 ? 2 mm) from at least two different
cadavers were used and incubated in the presence or
absence of patient IgG separately. After immunostaining
as described below, serial sectioning was performed. In
three to five skin pieces incubated with PV-IgG, PF-IgG,
or control IgG, 39 to 63 different sections were evaluated.
After each section was harvested, at least 50 ?m of tissue
was discarded. In the next section, it was verified by
Cleavage Plane in Pemphigus
AJP September 2007, Vol. 171, No. 3
Page 3
microscopic evaluation that no blistering was found to
ensure that each blister measured was counted not more
than once. For each blister, localization was analyzed
and described as deep splitting when suprabasal split-
ting or splitting within the lower spinous layer was de-
tected and described as superficial splitting when split-
ting was located in the upper spinous or granular layer.
For studies using toxin B, skin was fixed at room temper-
ature with 2% formaldehyde (freshly prepared from para-
formaldehyde) in PBS, dehydrated in ascending concen-
trations of ethanol (50, 70, and 96% and 3 ? 100%; 10
minutes each), equilibrated with propylene oxide (two
times for 15 minutes), and embedded in Epon 812. Semi-
thin sections (1-?m thick) were stained with toluidine blue
or prepared for immunostaining by incubation (5 minutes
each) with sodium-methanolate, sodium-methanolate
mixed with toluol (1:1), acetone (2?), and H2O, followed
by PBS before immunostaining was performed as de-
scribed below.
HaCaT cells were grown on coverslips to confluence as
described above (7 days) and incubated with PF-IgG or
PV-IgG for 24 hours at 37°C. After incubation with auto-
antibodies, culture medium was removed, and monolay-
ers were fixed for 10 minutes at room temperature with
2% formaldehyde (freshly prepared from paraformalde-
hyde) in PBS. Afterward, monolayers were treated with
0.1% Triton X-100 in PBS for 5 minutes at room temper-
ature. Cryosections of human skin were dried on a heat
plate for 30 minutes and fixed with ice-cold acetone
(?20°C) for 5 minutes. After rinsing with PBS at room
temperature, cryosections/semithin sections of human
skin or HaCaT cells were preincubated for 30 minutes
with 10% normal goat serum and 1% bovine serum albu-
min at room temperature and incubated for 16 hours at
4°C with mouse monoclonal antibody directed to the
ectodomain of human Dsg 1 (clone p124; Progen, Heidel-
berg, Germany) or Dsg 3 (Zytomed, Berlin, Germany) or
against Rho family GTPases Rho A or Rac1 (Upstate, Ham-
burg, Germany), respectively (dilution 1:100 in PBS each).
After several rinses with PBS (three times for 5 minutes),
monolayers were incubated for 60 minutes at room temper-
burg, Germany). For visualization of filamentous actin (F-
actin), ALEXA-phalloidin (diluted 1:60 in PBS; Mobitec,
Go ¨ttingen, Germany) was used (incubation for 1 hour at
room temperature). Cells incubated with antibodies or
ALEXA-phalloidin were rinsed with PBS (three times for
5 minutes). Immunostaining of cryosections from skin
specimens was performed after incubation with PF-IgG
and PV-IgG as described for cell culture monolayers.
Finally, coverslips were mounted on glass slides with
60% glycerol in PBS containing 1.5% n-propyl gallate
(Serva, Heidelberg, Germany) as antifading com-
pound. Monolayers and cryosections/semithin sections
were examined using an LSM 510 (Zeiss, Go ¨ttingen,
Germany). Images were processed using Adobe Pho-
toshop 7.0 software (Adobe Systems, San Jose, CA).
Transmission Electron Microscopy
As described previously,21HaCaT monolayers were
fixed in 2.5% glutaraldehyde containing 0.01% ruthenium
red in 0.1 mol/L sodium cacodylate, pH 7.35, for 1 hour at
4°C. After rinsing in 0.1 mol/L cacodylate (three times for
5 minutes) cells were postfixed in 2% OSO4in 0.1 mol/L
cacodylate for 1 hour at room temperature. Cells were
rinsed again in cacodylate and dehydrated in ascending
concentrations of methanol (25, 50, 70, and 80%; 2 ?
95%; and 2 ? 100%; 10 minutes each). After incubation
in 70% methanol, monolayers were placed for 1 hour in
saturated uranyl acetate in 70% methanol for 1 hour at
dark. After dehydration, cells were equilibrated in pro-
pylene oxide (two times for 10 minutes) and embedded in
Epon 812. Semithin sections (1 ?mol/L) were stained with
toluidine blue, and ultrathin sections were contrasted with
uranyl acetate and lead citrate and examined with a LEO
AB 912 electron microscope (Zeiss, Jena, Germany).
Laser Tweezer
Expression and purification of recombinant Dsg 1, coat-
ing of polystyrene beads, and the laser tweezer set-up
were described previously in detail.21Through all exper-
iments, the laser intensity was 42 mW in the focal plane.
Coated beads (10 ?l of stock solution) were suspended
in 200 ?l of culture medium and allowed to interact with
HaCaT monolayers for 30 minutes at 37°C before mea-
suring the number of bound beads (control values).
Beads were considered tightly bound when resisting la-
ser displacement at the 42-mW setting. For every condi-
tion, 100 beads were counted. Afterward, control IgG,
PV-IgG, or PF-IgG was applied for 120 minutes in the
absence or presence of CNF-1; or toxin B, C3FT, or LT
was added for 180 minutes before the number of bound
beads was counted again. Percentage of beads resisting
laser displacement under various experimental condi-
tions was normalized to control values.
Differences in bead adhesion and epidermal splitting
were assessed using Student's t-test. In text and bar
diagrams, values were expressed as mean ? SEM. Sta-
tistical significance was assumed for P ? 0.05.
PV-IgG and PF-IgG Caused Epidermal Splitting
of Layers Expressing Dsg 1 and Dsg 3 in Intact
Human Skin in Vitro
The cleavage planes in skin specimens from the PV and
PF patients who contributed IgG fractions for this study
clearly differed from each other and were typical for PV
and PF, respectively. Representative patterns are shown
in Figure 1, a and b. In contrast, in organ cultures of
human skin from individuals not suffering from pemphi-
gus disease, incubation for 24 hours with PV-IgG (con-
AJP September 2007, Vol. 171, No. 3
Spindler et al
Page 4
taining Dsg 1 and Dsg 3 antibodies; Table 1) and PF-IgG
(Dsg 1 antibodies only; Table 1) resulted in predomi-
nantly deep splitting (Figure 1, c and d; Table 2). For PV,
both IgG fractions tested (PV-IgG 1 and PV-IgG 2)
caused suprabasal epidermal splitting with frequencies
of 87% (n ? 23, PV-IgG 1) and 89% (n ? 90, PV-IgG 2),
respectively. For PF, the cleavage plane of 76% (PF-IgG
1, n ? 68) and 64% (PF-IgG 2, n ? 22) of splits was also
localized in lower epidermal layers. It has to be noted that
the mean split length induced by pemphigus IgG ranged
from 75.5 ? 14 (PV-IgG 1) to 105.5 ? 21 ?m/mm (PV-IgG
2) and from 36.7 ? 6 (PF-IgG 2) to 82.3 ? 11 ?m/mm
(PF-IgG 1). Thus, the split length induced by pemphigus
IgG in vitro in the short time period of 24 hours is sub-
stantially smaller than the size of blisters in pemphigus
In a second set of experiments, we characterized the
expression pattern of Dsg 1 and Dsg 3 in deep layers
of human epidermis in which pemphigus IgG-induced
splitting occurred in vitro. Immunostaining of cryosec-
tions (5 ?m) using specific monoclonal antibodies di-
rected against human Dsg 1 and Dsg 3 revealed that
these desmogleins display a broad overlapping distri-
bution pattern in human epidermis. Dsg 1 (Figures 2a
and 3a) was expressed throughout the entire epider-
mis with an apicobasally decreasing gradient. Expres-
sion was especially strong in the granular layer,
whereas in the basal layer, fluorescence was weak but
still present. This was even more obvious in semithin
sections from plastic-embedded epidermis (1 ?m),
which showed continuous Dsg 1 staining in the gran-
ular and upper spinous layer, whereas individual des-
mosomes could be detected in the basal layer (com-
pare Figure 5b). Dsg 3 was found in all epidermal
Table 1.
Antibody Profile of Patient's IgG
ELISA Dsg 1 (U/ml)Dsg 3 (U/ml)
PV-IgG 1
PV-IgG 2
PF-IgG 1
PF-IgG 2
ELISA, enzyme-linked immunosorbent assay.
Table 2.
Location of Splitting in Human Skin in Vitro
sections (n)
PV-IgG 1
PV-IgG 2
PF-IgG 1
PF-IgG 2
Figure 2. In cultured intact human epidermis, PV-IgG caused epidermal
splitting where both Dsg1 and Dsg3 were present. Skin was immunostained
for Dsg 1 (a, c, and e) and Dsg 3 (b, d, and f). In untreated skin, Dsg 1 and
Dsg 3 displayed broad overlapping distribution patterns (a and b). Dsg 1 was
expressed throughout the epidermis with a decreasing gradient from super-
ficial to basal layers. Despite its basal prominence, Dsg 3 was present in all
layers except the upper granular layer. Incubation with PV-IgG containing
antibodies against both Dsg 1 and Dsg 3 lead to suprabasal splitting (c–f).
Note that Dsg1 was present in the basal layer underneath the split (arrows
in c and e) and that Dsg 3 was found above the cleavage plane (arrows in
d and f). Bar ? 50 ?m.
Figure 1. Histology from pemphigus patients whose sera were used for this
study revealed typical blister localization (a and b). The H&E-stained paraffin
sections showed suprabasal epidermal cleavage in the PV histology and
superficial blistering in the PF lesion. In human skin in vitro (c and d),
PV-IgG-induced splitting was suprabasally located, whereas PF-IgG-induced
cleavage was found within the spinous layer. Bar ? 50 ?m in all panels.
Cleavage Plane in Pemphigus
AJP September 2007, Vol. 171, No. 3
Page 5
layers except the uppermost granular layer, but stain-
ing was more prominent in the basal layer compared
with upper spinous layers (Figures 2b and 3b). Be-
cause both PV-IgG and PF-IgG caused skin splitting
primarily in deep epidermal layers in vitro, we next
performed immunostaining for Dsg 1 and Dsg 3 in
consecutive cryosections from skin incubated with PV-
IgG or PF-IgG. Moderate to strong Dsg 1 staining was
seen in the basal layer underneath the suprabasally
located splitting areas caused by PV-IgG 1 and 2
(arrows in Figure 2, c and e). Similarly, PF-IgG 1 and 2
caused epidermal splitting of spinous layers contain-
ing both Dsg 1 (arrows in Figure 3, c and e) and Dsg 3
(arrows in Figure 3, d and f). Taken together, these
data indicate that, at least in intact epidermis in vitro,
PV-IgG and PF-IgG are capable to induce epidermal
splitting where Dsg 1 and Dsg 3 are present at similar
immunoreactive levels.
PF-IgG Is Sufficient to Induce Dissociation of
Human Keratinocytes Expressing Both Dsg 1
and Dsg 3
To support this observation further, we performed immu-
nostaining of HaCaT cells after incubation with patients'
IgG. To detect sensitively cell dissociation and to dem-
onstrate that effects of pemphigus IgG on Dsg 1 local-
ization were confined to areas of cell dissociation, cortical
cytoplasm was labeled with ALEXA-phalloidin for F-actin,
and desmogleins were immunostained with specific
monolayers, even small intercellular gaps can be de-
tected by the lack of green fluorescence.21In untreated
cells, Dsg 1 and Dsg 3 were located along cell junctions
that were also labeled strongly for F-actin (Figure 4A,
a–c). These distribution patterns were not affected by
24-hour incubation with control IgG from a healthy volun-
teer (Figure 4A, d–f). However, incubation with PF-IgG 1
and 2 (shown for PF-IgG 1 in Figure 4A, g) as well as with
PV-IgG 1 and 2 (shown for PV-IgG 1 in Figure 4A, j)
induced profound alterations of the actin cytoskeleton
and keratinocyte dissociation, leading to formation of
large intercellular gaps (arrows). As revealed by double-
labeling, Dsg 1 immunoreactivity was drastically reduced
in areas exhibiting cell dissociation in response to treat-
ment with PF-IgG 1 (Figure 4A, h) and PV-IgG 1 (Figure
4A, k). In contrast, the effect of PF-IgG and PV-IgG on
Dsg 3 staining differed. After incubation with PF-IgG 1,
Dsg 3 immunoreactivity was weak only at margins facing
intercellular gaps but was strong elsewhere (Figure 4A, i).
These data support the findings from intact epidermis
that PF-IgG (containing Dsg 1-specific antibodies only) is
sufficient to induce loss of adhesion and keratinocyte
dissociation. In contrast, treatment with PV-IgG 1 resulted
in fragmentation of the continuous Dsg 3 staining
throughout the monolayer, indicating a generalized re-
duction of desmosomes. This result was confirmed by
transmission electron microscopy (Figure 4B). In con-
trols, neighboring cell borders were tightly aligned with
numerous desmosomes visible between adjacent cells
(Figure 4B, a and b). In contrast, after treatment with
PV-IgG 1 (Figure 4B, a and d), large gaps were present
(Figure 4B, c), and desmosomes were completely miss-
ing in many areas of the monolayers. Where desmo-
somes were present, they were localized on filopodial
processes connecting neighboring cells (Figure 4B, c
and d). Notably, these desmosomes were linked to thick
bundles of keratin filaments (arrows).
Inhibition of Rho GTPases Caused Keratinocyte
Dissociation in Deep Epidermal Layers
The observations described above indicate that the
localization of epidermal splitting induced in vitro by
autoantibodies from pemphigus patients is difficult to
explain by the desmoglein compensation concept that
is based on different expression patterns of Dsg 1 and
Dsg 3 throughout the epidermal layers. Recently, we
found that epidermal splitting and keratinocyte disso-
Figure 3. Similar to PV-IgG, PF-IgG also caused epidermal splitting
where both Dsg1 and Dsg3 were present. Skin was immunostained for
Dsg 1 (a, c, and e) and Dsg 3 (b, d, and f). Compared with untreated skin
(a and b), incubation with PF-IgG containing only Dsg 1-specific anti-
bodies resulted in splitting where expression of Dsg 1 as well as Dsg 3
was found (c–f). Dsg 1 was present underneath the cleavage plane
(arrows in c and e), and Dsg 3 immunoreactivity was detected above the
split (arrows in d and f). Bar ? 50 ?m.
AJP September 2007, Vol. 171, No. 3
Spindler et al


text:
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text:
Topic: Carat
Epic Wave of Media Reviews Comes at a Crucial Time for Media Buying
Negotiating upfronts while pitching clients' business
By Andrew McMains on May. 29, 2015 - 11:31 AM EDT
The seemingly endless fire hose of media reviews—20 in the past six months, representing some $17.3 billion in annual media spending—could not have come at a worse time. Just as agencies…
P&G, Largest Advertiser in the U.S., Is Putting Its North American Media Business in Review
Spending exceeds $2.6 billion
By Noreen O'Leary on May. 15, 2015 - 10:13 AM EDT
America's biggest ad spender is putting its North American media business into review, according to sources. Just five months after Procter & Gamble named Kristine Decker as its new U.S. media…
Only Roster Shops Will Get a Crack at Coke's U.S. Media Account
Incumbent MediaVest competes with 3 of the brand's global partners
By Andrew McMains on Apr. 22, 2015 - 12:27 PM EDT
Like the media reviews of SC Johnson and Wells Fargo, Coca-Cola's U.S. contest is a roster-shops-only affair. Incumbent MediaVest is competing against three agencies that handle Coke business overseas: MediaCom, Carat…
Videa's President Discusses the Future of Automated Sales
And what TV should embrace so it doesn't lose out
By Janet Stilson on Apr. 6, 2015 - 7:37 PM EDT
Specs Who Shereta Williams Current gig President, Videa Old gig Vp, corporate development, Cox Media Group Age 41 Services have launched in the past and been unable to make a go of it. Why will yours succeed? If…
California Healthcare Exchange Dangles Its $50 Million Account
Review is in its early stages
By Noreen O'Leary on Mar. 19, 2015 - 3:02 PM EDT
Covered California, the state's healthcare exchange, is looking for an agency to help expand the pool of residents enrolled in health insurance coverage. A request for proposals for exchange's advertising account…
Mindshare Fills a Role That's Redefining the Agency
Rolf Olsen succeeds Bob Ivins as North American data chief
By Andrew McMains on Mar. 16, 2015 - 7:01 AM EDT
In his ongoing bid to redefine what a media agency can be, Mindshare North American CEO Colin Kinsella has recast in a significant role. Kinsela has named iProspect's Rolf Olsen to succeed Bob Ivins…
GMC Touts Precision in New Ads from Leo Burnett
Brand will double its media spending this year
By Kristina Monllos on Mar. 2, 2015 - 4:19 PM EST
General Motors said it will doubling down on GMC's media spending for 2015 amid a long-term plan to grow the brand in the next 10 years. The first step in the…
Massage Envy Trades Hometown Agency for San Francisco Shop
Venables, Bell + Partners snaps up creative business
By Andrew McMains on Feb. 9, 2015 - 12:20 PM EST
Some wins are more satisfying than others. But you'd love to be the planner leading research on Venables, Bell + Partners' newest account. Why? Because it's a chain known as Massage…
Dentsu Aegis Creates New CEO Role in the U.S.
Northern Europe chief Rob Horler starts the new gig in April
By Noreen O'Leary on Jan. 29, 2015 - 12:54 PM EST
Rob Horler is the new CEO of Dentsu Aegis Network, USA, with oversight of strategy development and execution across the company's 20 offices in the region. London-based Horler, who joined Aegis…
Bloomin' Brands Shifts Its Media Account to a New Agency
Casual restaurant owner spends $150 million in media annually
By Andrew McMains on Dec. 22, 2014 - 5:42 PM EST
Bloomin' Brands is moving on. After seven years with Carat, the parent company of Outback Steakhouse, Bonefish Grill and Carrabba's Italian Grill has completed a review and shifted its media business…


text:
New Author Webpage
Meanderings and Muses isn't going anywhere.
BUT - I do have a new webpage which will focus mostly on my writing.
If you're interested in reading more about my books, anthologies, and events, please click over to
There are a few videos of me reading from "Whimsey: A Novel," and from other favorite authors' work.
With more videos still to come.
The new page is a work in progress with plans for still more to come - I'm just still trying to figure out exactly what that might be - so check back from time to time, please.
See you there!
Tuesday, February 22, 2011
Jill Harman Smith
6 comments:
- Julie D said...
Gorgeous! Wow, so talented.
- February 22, 2011 at 9:25 PM
- Cathy Webster (Olliffe) said...
Beautiful photos.. you look like a model!
- February 22, 2011 at 9:40 PM
- Mason Canyon said...
Beautiful photos. I agree with Cathy, you do look like a model. Is this a new career you're looking at now that you have some free time on your hands. :) The photo of Harley is cute.
Thoughts in Progress
- February 22, 2011 at 10:51 PM
- Vicki Lane said...
You introduced me to Jill a while back and I've been enjoying her blog right along.
- February 23, 2011 at 12:30 AM
- Jill said...
Wow Kaye!!! How sweet of you!!! Kaye is a beautiful woman and very easy to photograph. I am not a professional photographer just a friend and neighbor. Not hard when you have a pretty lady and a cute dog. Ha.
- February 23, 2011 at 1:14 AM
- Kaye Barley said...
Thanks, everyone for stopping by. And for the compliments - wow. See what a talented photographer can do?!?!
Jill, you may not think of yourself as a professional photographer, but that wonderful artistic talent of yours shines right through.
- February 23, 2011 at 11:56 AM


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Pediatric urologist may be able to offer peace of mind
Q: My son (4 years old) has had trace (non-visible) blood in his urine for about a month (the doctor has been detecting it — has had him checked weekly the past month). I have looked it up, but I just wanted to see if you knew of anything we should be super concerned about. I am actually very stressed about it. — J.M., Sandy Springs
A: Blood in the urine is called hematuria. You have already noticed that blood in the urine is not always visible to the eye and is called microscopic hematuria. This means that it is detected in the laboratory on a sample of urine. The question is where is the source of the microscopic blood, and it could be anywhere within the urinary tract. The urinary tract is composed of the two kidneys, two ureters (small tube leading from the kidney to the bladder), bladder and urethra (the tube from the bladder to the outside of the body).
A common cause of microscopic blood in the urine is related to a urinary tract infection, and I assume that an infection has been ruled out, or been treated; the infection could be in the bladder or in the kidney. Trauma can also be associated with blood in the urine. So any recent accidents or trauma to the kidneys would be considered. A direct blow to the kidney can cause blood in the urine. Another common reason for microscopic blood in the urine is a kidney stone, and I assume that has been ruled out as well.
In adults, the doctors would have a longer list of conditions to consider from menstruation and sexual activity in women to enlarged prostate glands in men. Polycystic kidney disease and sickle cell disease are two inherited disorders that may cause hematuria. It is also important to rule out the presence of benign tumors or cancer of the kidney or bladder when a person presents with microscopic hematuria.
If it's just a trace amount, ask how it has been detected — did the doctor look at the urine under a microscope and count the RBC or did he use a dipstick (a strip of specially treated paper that changes color when dipped in the urine). Often a dipstick will be used, and if the paper changes color the doctor or lab technician will examine the urine specimen with a microscope and count the number of red blood cells. It only takes a single red blood cell change the dipstick to trace amount.
The way a urine specimen is collected is important as well. Most doctors will request a clean, mid-stream voided specimen, where you cleanse around the urethra, urinate a little and then begin to collect the sample of urine. With a young child, collecting the sample of urine is always easy. However, it is probably easier with boys than girls. In some situations, the doctor may collect the urine specimen by inserting a small catheter directly into the bladder.
If your son continues to have trace amounts of blood in the urine, and your pediatrician has not done all the testing that makes you feel comfortable, then see a pediatric urologist sooner rather than later for peace of mind. He will do a several tests to make sure that nothing is wrong and it's just an idiopathic thing (no known cause and no worrying). Diagnostic
procedures can include further testing of urine samples to detect infections or kidney disease. Blood tests may be ordered to assess how well the kidneys are working. There are several types of imaging tests that can be done as well to see the kidneys, ureters and bladder, and these can vary from an ultrasound to an intravenous pyelogram (IVP), in which a special dye is injected into a vein and pictures are made as it travels through the body to the kidneys. On occasion, the doctor may decide that he needs to see inside the urethra and bladder and will do a cystoscopy, in which a small tube is inserted into the bladder.
If his blood work (blood urea nitrogen, creatinine) and his urinalysis (protein in the urine, white blood cells, etc.) are normal (other than the hematuria) then the blood in the urine may not have any significant problem attached to it and the doctor just checks your son out once a year to make sure there have been no changes.
Send your health questions to Debbie Jackson, PhD, MN, Clemson University, 302 Sikes Hall, Clemson, SC 29634-5155 or email email@example.com.
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4.5 stars for Lethal Game from Night Owl Reviews!
Julie Rowe presents readers with a fantastic follow up in this sweet and sexy action filled suspense. This story is about two of my favourite characters in quite some time. I'm truly all about the people I am reading about and if there is no connection I can't enjoy the book, but that was definitely not an issue here. Sophia is a spitfire with a chip on her shoulder bigger than she is. Connor is almost too good to be true but once I caught a glimpse of his flaws I quickly fell for him. I loved how action-packed this story was and how compelling the situation is without being unnecessarily graphically violent. The situation is dire but the book remains realistic without being over the top gritty. Danger and desire mix in the perfect balance that kept me glued to my ereader all night. This is the second story in the series and I enjoyed catching up with some of the characters I met last time around and meeting so many interesting new ones. I cannot wait to see what is going to happen next and just how the danger will be handled. I'm hooked and I believe you will be too.


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Our Man in Arlington
Much ado of late in the Arlington theater scene. The creative-but-resented-by-some Artisphere, the county announced Dec. 17, will likely shut down June 30. It's a casualty of subsidies that failed to produce the envisioned self-sustaining, hip arts and drama institution that would help revitalize nighttime Rosslyn. That same day, the […]Read more ›


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Three dozen former U.S. officials are urging President Obama, who meets Friday with Iraq's prime minister, to demand that the prime minister release seven kidnapped Iranian dissidents and help relocate thousands of others guaranteed U.S. protection at a refugee camp in Baghdad.
"Only your intervention can save their lives," they said in a letter to Mr. Obama.
The former officials — who include ex-CIA directors, retired generals and former diplomats — accused Iraqi troops, acting on behalf of Iran, of executing 52 dissidents and kidnapping the seven Iranians in a Sept. 1 attack on Camp Ashraf.
The victims were among about 100 dissidents who remained in that refugee camp north of the Iraqi capital to protect property belonging to about 3,000 others who relocated to Camp Liberty, near Baghdad International Airport.
All carried U.S. identification cards classifying them as "protected persons" under international treaties.
"This was premeditated murder planned at the highest level [of the Iraqi government] and carried out by Iraqi forces and agents, using equipment and training provided by United States forces," the former U.S. officials said in their letter.
They also expressed fear the Iraqi Prime Minister Nouri al-Maliki intends to turn the dissidents over to Iran to face execution.
The dissidents are the formerly armed wing of the National Council of Resistance of Iran, which has fought for the overthrow of the Iranian theocratic regime for decades.
The armed rebels turned over their weapons to U.S. forces after the ouster of Iraqi dictator Saddam Hussein in 2003.
They were under U.S. protection until 2009, when Iraq took over the administration of the refugee camps.
The signers of the letter to Mr. Obama include several prominent Democrats such as former Vermont Gov. Howard Dean, a former party chairman; and Patrick J. Kennedy, a former congressman from Rhode Island.
Foreign visitors in Washington this week include:
• Defense Minister Artis Pabriks of Latvia, who briefs the Heritage Foundation on security challenges in the Baltics.


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British Fans opinions on Warren's Boxing Channel.
Warren launches new boxing channel
Posted on Sep 22, 2011
Veteran promoter Frank Warren has joined forces with Boxing Channel Media to launch an independent, groundbreaking new boxing subscription channel called BoxNation.
Set to launch on Friday, September 30 at 7pm on Sky channel 456, BoxNation is the first ever channel dedicated to serving the UK's live fight enthusiasts and sports fans, and will feature Warrens long list of fighters.
Viewers will be able to see the channel for free initially before it becomes a subscription channel at the end of the year where viewers will then have to purchase it for £10 per month.
The channel has committed to showing two live championship promotions from the UK and around the world each week, and has also done deals with top German promoters Sauerland Events and US heavy hitters Golden Boy Promotions.
In addition to championship boxing, BoxNation will also cover major amateur tournaments and Mixed Martial Arts events, as well as bringing back the cult show Bunceys Boxing Hour with hugely popular pundit Steve Bunce.
The programmers have also promised daily fight news updates and an extensive archive bringing back classic fights from home and abroad and featuring some of the world's greatest ever boxers including Muhammad Ali, Mike Tyson, Sugar Ray Leonard and many more.
Warren who is still contracted to Sky Sports till June next year confirmed he would see the contract out while voicing his excitement about taking his marquee fighters to BoxNation and having the opportunity to get them in the ring more often throughout the year.
This is what all boxing fans have been waiting for a dedicated channel, 100 per cent committed to showing the best fights from around the world. Its a big challenge in my career, but Im really excited by BoxNation and it takes me back to my early years when I was at ITV doing a show a week, said Warren.
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Just another money making scheme. They will show a couple of good bouts to draw people in and then everything will dry up a couple of months in.
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Its a good thing. But its useless to me because its not on Freeview. Plus i dont see it lasting very long.
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its free for te first month or so
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so on top of the sky sports package we now gotta fork out another 10 quid on top?
good luck with that cause i sure ain't paying for it
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wow! this is the best news all year
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I heard they will start off by showing 2 bums fighting
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I'm going to wait and see what his programming schedule looks like, but I don't mind forking out an extra tenner a month if i'm going to get some good fights out of it. 2 live fights a week he's saying are guaranteed, hopefully not *****y fights from Azerbaijan or Pakistan.
The channel will always be streamed no doubt, but it will be nice to be able to have a pure 100% dedicated boxing channel that i can flick on whatever time of day or night and always have a fight on my screen
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text:
NASA on March 4 released a draft request for proposal for the fifth iteration of its Solutions for Enterprise-Wide Procurement, or SEWP, IT contract. (Read the cover letter).
SEWP is a 10-year, indefinite-delivery-indefinite-quantity contract with delivery orders for IT and audio-visual products and services. The contract is open to any government agency.
Among several changes, officials are reducing the number of competition groups from 13 in SEWP IV to five in SEWP V. They expect this change to reduce the cost to both industry and the government. SEWP V has a set-aside for small firms located in Historically Underutilized Business Zones. And NASA plans to fully track orders as well. Officials want to provide agencies with detailed information of their buying patterns primarily to assist with strategic sourcing.
NASA is also holding an "industry day" on March 11 to allow contractors to ask questions about the procurement vehicle. Washington Technology reported that more than $9.4 billion in task orders have been awarded under SEWP IV.
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Matthew Weigelt is a freelance journalist who writes about acquisition and procurement.
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