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Preimplantation genetic diagnosis in a family at risk for recurrence of Herlitz junctional epidermolysis bullosa
ESDR I JSID I SID Abstracts
0076
0073 IDENTIFICATION AND STRUCTURE OF THE E6BP PEF’IIDE THAT BlNDS THE es Baleia. and PAPULOMAVIRUS E6 PROTEINS. Jasonam Elliot J. An&&y. Department of Dermatology, New England Medical Center and Tufts University School of Medicine, and Department of Biochemistry, Tufts University School of Medicine, Boston, MA 02 I1 1 (USA) We recently identified a cellular protein (E6BP, also known as ERC55) that binds cancer associated human (H) and bovine papillomavirus (BPV) E6 proteins. E6BP contains six EF hand calcium biding helix-loophelix motifs. Using a series of deletion mutants, a 25 amino acid peptide from the fourth F.F hand of E6BP was found to mediate complex fornx?tion with HPV and BPV E6. This region was necessary and sufficient for E6 association. Alanine replacement mutagenesis of residues in the second alpha helix of this EF hand greatly reduced E6 binding, whereas mutation of amino acids necesszuyfor chelation of calcium did not abrogate E6 association. Further deletions indicated that a 13 amino acid peptide containing this putative helix was sufficient for E6 binding. E6 also hinds E6-AP, an ubiquitin ligase, and this complex targets ~53 for ubiquitination and degradation. Previous studies defined an 18 amino acid region of E6-AP that is necessary and sufficient for E6 binding. Comparison of the E6-binding peptides in E6BP and E6AP revealed a consensus 7 amino acid sequence L- VQ E F/L L -G- D/E. This prediction is consistent with the mutagenesis results which showed these residues were critical for E6BP association. Using nuclear magnetic resonance (NMR) spectroscopy confirmed the alpha helical structure of the E6BP peptide and determined the position of the critical amino acids. Computer modeling predicts both E6 binding motifs fold similarly. Overexpression of the E6BP E6 binding peptide specifically inhibited growth of papillomavirus transformed cells. These results imply that E6AP and E6BP present a similar surface to E6 and an likely to interact with the same region of E6. Truncated forms of this E6BP domain interfere with E6 transforming functions.
HUMAN
HERPESVIRUS
6 (HHVL)
INFECTS
DENDRITIC
CELLS
(DC)
AND SUPPRESSES HIV REPLICATION IN CO-INFECTED _ * HGoldlne.# * and A Blauvelt.” CULTURES. H_,&ja.* V Kla,&Covtun. *Dermatology Branch, NC1 and #Division of Viral Products. FDA, Bethesda, MD. Primary infection wrtb HI-IV6causes rose&, and viral reactivation has been purported to be a co-factor ia the pmgressive loss of CD4* T cells (TC) observed in AIDS patients. Thus, recent studies have focused on HHV6-HIV interactions in TC, as well as other cells involved in immune responses, e.g., macrophages. Although DC play important roles in the generation of normal immune responses and in the immunapathogenesis of HIV disease, infection of DC by HHV6 and co-infection of DC by HHV6 and HIV have not been previously studied. We prepared DC from plastic-adherent peripherat blood leukocytes in the presence of GM-CSF and IL-4. and exposed purified DC to HHV6 alone (229 or U1102 strains), to HIV alone @Ill3or BaI. strains), or to HHV6 and HIV (i.e.. co-infection). Both strains of HHV6 could infect DC (as determined by semiquantitative PCR analyses of viral DNA, intraceIhdar monodonal antibody staining of viral proteins, and production of infectious virus in culture supematants). HHV6infected DC showed no marked cytopathic changes and were not impaired in their ability to stimulate allogeneic TC. Interestingly, in co-infected DC, HHV6 suppressed replication of both strains of HIV; similarly, HIV-induced syncytia blocked in these co-infected cultures. By contrast, HIV replicated well
formation
was
in co-infected TC and macrophages used as controls. This HHVb-mediated anti-HIV effect in DC was dependent on HHV6 infection. occurred either when HHV6 was added before or after HIV infection, and was not due to decreased cell surface expression or function of the HN co-receptors CD4, CCR5, and CXCR4. In summary. we show that HHV6 can infect DC, and that HHV6 blocks HIV replication in co-infected DC cultures. HHV6 may protect DC from HIV-induced cytopathicity in AIDS patients. In addition, these findings suggest that interactions between HIV and herpesviruses are.complex, and that observable outcome of dual infection is dependent on target cell type.
0077 PREIMPLANTATION GENETIC DIAGNOSIS IN A FAMILY AT RISK FOR RECURRENCE OF HERLITZ JUNCTIONAL EPIDERMOLYSIS BULLOSA. Peter B. Csetilmi-Friedman, Yaxu Tanq’. Jamie A. Grffo’ and Angela M. Christians. m 1D tol ColumblaUnivers N Y rk NY ;* Department ot Ubstetn’s and ~yn%&y%v York Universitv, &v !kk!Nk. The Herlit? type of juwtional 6pidenolysis bullosa (HJEB) is a severe inherited bullous disease which usually leads to the demise of the affected newborn dutfn the first mptis of life. Mutabons in genes enwding the three polypeptides o B the anchonng filament protein laminin 5 have been reported to underly this m. The @eaku bash of JEB provides the neces~a infwmstion for the development of sInglecell prelmplantation genetic diagnosis (P% D) far famik at dsk for mcwence. We studied a family wfth one healthy and one previously affected child which died from JEB. Mutation screening using hetercduplex analysis and direct sequencing of the PCR products revealed a previously described hot t meation in LAMB3 in tijtir, and a novel delayed @nk&x codon in i&3 in the father (TG& ;i$G For PGD, we ,developed a simultaneous four-exon. doubly nested multiplex assay, amphtyfng the two mutabons and two informah% intragenic polymorphisms, which were detxtible using the appropriate restriction enzyme. Single emblypnic cells were biopsied firm B-call embryos using standard micminanipukhon techniques, and subjected to the multiplex PCR assay and restriction enzyme digestion. The emb OS which proved not to can-y either of the mutations were mimptsnted in the m x er, and we am awaiting the result of the pregnancy test. This study demonstrates the sensitivity of multiilex PCR fwn single ~4; and illustrates the feasibility of POD for an iniwited skin disorder, for the first
0075
0078
UVB RADIATION ACTIVATES HIV IN HUMAN SKIN. Eric Slmoson. Brian Dswson and Poociano Cruz. Jr., Departments of Dermatology and Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA. Concern about a detrimental relntkmship behveen UV radiation and HIV infection has been fueled by the high prevalence in seropositive patients of photoresponsive chronic skin diseases (eosbmphilie foUlcolitis, psoriasis, eczema, pruritus) and by studies that have shown UVC, UVB, sunlight, or PUVA to act&ate HIV lo cultured cells and tranrgenic animals. However, none of the latter experiments were conducted in humans, and studies that examined the safety of phototbcnpy in HIV+ patients have been inconclusive. Thus, we were compelled to determine whether UVB radiation can activate HIV In human skin. We first developed a quantitative assay for measuring HIV In skin. RNA was extracted from biopsy specimens of and nonksioaal skln from HIV+ patients with psoriasis. To control for sample variation, sawn1 paired specimens were taken from each palent, with the least amount of extracted total RNAispeetmen serving IS the limiting factor for each experiment A PCR-based assay amplified and detected a target sequence in the conserved region of the HIV-1 gag gene, using established primers and a built-in qusntitation standard. Lesional skin contained 20-M HIV copies& RNA, whereas nonlesional skin had very low numbers. Similar findings were noted in Iesiooal vs. nonlesionn1 skin of HIV+ patients with moIluscum contaglosum. We next examined the elfect of in viw UVB ndiatton FSZO Sunlamps) on cutzmeeus virst load. Adjacent paired specimens were taken from psoriatic skin (one from an u&radiated site; the other from skin exposed to 1 single dose of 100300 J/m*). HIV copy number was increased (S- to IO-fold) in UVB-treated skin in comparison to the paired unirradiated control. This study provides tbe tlrst quantitative method for measuring HIV in skin. It documents the presence of a signiticant number of vlrnl copies In lesional siter. Finally, we demonstrate that a single low dox olUVB radiation does activate HIV in human akin.
CLONING AND EXPRESSION ANALYSIS OF AMAC-1,
lesional
ASSOCIATED
CC-CHEMOKINE
OF ANTIGEN-PRESENTING
A NOVEL TH2CELLS. y,
dt Dept. of Damtatology, Benjamin Kz&&CJUer. 0. Politz N. Hakii. S. C FraddiaMedialCaatex,TheFreeUaiwmityofBerlia,Be&,Gamtany we have closed a novel human cc-chemo~ae. aliematiw rlwop@e actionaswc~ated CC-&m&n+ (MCI-l. The isolated cDNA dose (803 bp) shows a single open reading frame of 261 bp coding for 89 amino acid residw; mature AMA&I protein consists of 69 amino acids with a moleadar weight of 7855 AMAC-I homology
is most closely related to MIP-la of 55% and 59%, respectively
with B cDNA
and protein
sequence
However, the expression pattern of AMAC-1 ia directly opposite to that of MI&la. While MI&la is iadwed by da&al macrophage axdiatora, MC-1 is induced in maorophages by altamative nwrophage
mediators
U.4.
IL13,
and K-10.
Expression
of AMAC-1
is inhibii
by IFN-y while GC met a slightly positive synargi& &Tactin combination with IL4. Peripheral blood moaocytes da sot express AMAC-1; tima couw experimntu
show that moaocvte-to-macrooh diientiation is a arareauisite for Ah&w-I expreasicat Expre&on of Ah&cl by GM-CSF/U-&du& mowxyta-duiwd dendtitic cells is compkx; mature adherant dead& cells, however, only express minor amouats of AMAC-1 mRNA In viw, AMAC-I is expressed by slvwlru macrophage8 f+om healthy parson, smokers, and asthmatic patieats. In conohtsioa, MC-1 is a novel Thz-awciat ad cc-chamokina possibly iavolved ia the antigen presenting cdl-dapaadent regulation of th-e Thlfl’h2 bahuw ia iaflammatory aad immune reactions.
0076
0073 IDENTIFICATION AND STRUCTURE OF THE E6BP PEF’IIDE THAT BlNDS THE es Baleia. and PAPULOMAVIRUS E6 PROTEINS. Jasonam Elliot J. An&&y. Department of Dermatology, New England Medical Center and Tufts University School of Medicine, and Department of Biochemistry, Tufts University School of Medicine, Boston, MA 02 I1 1 (USA) We recently identified a cellular protein (E6BP, also known as ERC55) that binds cancer associated human (H) and bovine papillomavirus (BPV) E6 proteins. E6BP contains six EF hand calcium biding helix-loophelix motifs. Using a series of deletion mutants, a 25 amino acid peptide from the fourth F.F hand of E6BP was found to mediate complex fornx?tion with HPV and BPV E6. This region was necessary and sufficient for E6 association. Alanine replacement mutagenesis of residues in the second alpha helix of this EF hand greatly reduced E6 binding, whereas mutation of amino acids necesszuyfor chelation of calcium did not abrogate E6 association. Further deletions indicated that a 13 amino acid peptide containing this putative helix was sufficient for E6 binding. E6 also hinds E6-AP, an ubiquitin ligase, and this complex targets ~53 for ubiquitination and degradation. Previous studies defined an 18 amino acid region of E6-AP that is necessary and sufficient for E6 binding. Comparison of the E6-binding peptides in E6BP and E6AP revealed a consensus 7 amino acid sequence L- VQ E F/L L -G- D/E. This prediction is consistent with the mutagenesis results which showed these residues were critical for E6BP association. Using nuclear magnetic resonance (NMR) spectroscopy confirmed the alpha helical structure of the E6BP peptide and determined the position of the critical amino acids. Computer modeling predicts both E6 binding motifs fold similarly. Overexpression of the E6BP E6 binding peptide specifically inhibited growth of papillomavirus transformed cells. These results imply that E6AP and E6BP present a similar surface to E6 and an likely to interact with the same region of E6. Truncated forms of this E6BP domain interfere with E6 transforming functions.
HUMAN
HERPESVIRUS
6 (HHVL)
INFECTS
DENDRITIC
CELLS
(DC)
AND SUPPRESSES HIV REPLICATION IN CO-INFECTED _ * HGoldlne.# * and A Blauvelt.” CULTURES. H_,&ja.* V Kla,&Covtun. *Dermatology Branch, NC1 and #Division of Viral Products. FDA, Bethesda, MD. Primary infection wrtb HI-IV6causes rose&, and viral reactivation has been purported to be a co-factor ia the pmgressive loss of CD4* T cells (TC) observed in AIDS patients. Thus, recent studies have focused on HHV6-HIV interactions in TC, as well as other cells involved in immune responses, e.g., macrophages. Although DC play important roles in the generation of normal immune responses and in the immunapathogenesis of HIV disease, infection of DC by HHV6 and co-infection of DC by HHV6 and HIV have not been previously studied. We prepared DC from plastic-adherent peripherat blood leukocytes in the presence of GM-CSF and IL-4. and exposed purified DC to HHV6 alone (229 or U1102 strains), to HIV alone @Ill3or BaI. strains), or to HHV6 and HIV (i.e.. co-infection). Both strains of HHV6 could infect DC (as determined by semiquantitative PCR analyses of viral DNA, intraceIhdar monodonal antibody staining of viral proteins, and production of infectious virus in culture supematants). HHV6infected DC showed no marked cytopathic changes and were not impaired in their ability to stimulate allogeneic TC. Interestingly, in co-infected DC, HHV6 suppressed replication of both strains of HIV; similarly, HIV-induced syncytia blocked in these co-infected cultures. By contrast, HIV replicated well
formation
was
in co-infected TC and macrophages used as controls. This HHVb-mediated anti-HIV effect in DC was dependent on HHV6 infection. occurred either when HHV6 was added before or after HIV infection, and was not due to decreased cell surface expression or function of the HN co-receptors CD4, CCR5, and CXCR4. In summary. we show that HHV6 can infect DC, and that HHV6 blocks HIV replication in co-infected DC cultures. HHV6 may protect DC from HIV-induced cytopathicity in AIDS patients. In addition, these findings suggest that interactions between HIV and herpesviruses are.complex, and that observable outcome of dual infection is dependent on target cell type.
0077 PREIMPLANTATION GENETIC DIAGNOSIS IN A FAMILY AT RISK FOR RECURRENCE OF HERLITZ JUNCTIONAL EPIDERMOLYSIS BULLOSA. Peter B. Csetilmi-Friedman, Yaxu Tanq’. Jamie A. Grffo’ and Angela M. Christians. m 1D tol ColumblaUnivers N Y rk NY ;* Department ot Ubstetn’s and ~yn%&y%v York Universitv, &v !kk!Nk. The Herlit? type of juwtional 6pidenolysis bullosa (HJEB) is a severe inherited bullous disease which usually leads to the demise of the affected newborn dutfn the first mptis of life. Mutabons in genes enwding the three polypeptides o B the anchonng filament protein laminin 5 have been reported to underly this m. The @eaku bash of JEB provides the neces~a infwmstion for the development of sInglecell prelmplantation genetic diagnosis (P% D) far famik at dsk for mcwence. We studied a family wfth one healthy and one previously affected child which died from JEB. Mutation screening using hetercduplex analysis and direct sequencing of the PCR products revealed a previously described hot t meation in LAMB3 in tijtir, and a novel delayed @nk&x codon in i&3 in the father (TG& ;i$G For PGD, we ,developed a simultaneous four-exon. doubly nested multiplex assay, amphtyfng the two mutabons and two informah% intragenic polymorphisms, which were detxtible using the appropriate restriction enzyme. Single emblypnic cells were biopsied firm B-call embryos using standard micminanipukhon techniques, and subjected to the multiplex PCR assay and restriction enzyme digestion. The emb OS which proved not to can-y either of the mutations were mimptsnted in the m x er, and we am awaiting the result of the pregnancy test. This study demonstrates the sensitivity of multiilex PCR fwn single ~4; and illustrates the feasibility of POD for an iniwited skin disorder, for the first
0075
0078
UVB RADIATION ACTIVATES HIV IN HUMAN SKIN. Eric Slmoson. Brian Dswson and Poociano Cruz. Jr., Departments of Dermatology and Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA. Concern about a detrimental relntkmship behveen UV radiation and HIV infection has been fueled by the high prevalence in seropositive patients of photoresponsive chronic skin diseases (eosbmphilie foUlcolitis, psoriasis, eczema, pruritus) and by studies that have shown UVC, UVB, sunlight, or PUVA to act&ate HIV lo cultured cells and tranrgenic animals. However, none of the latter experiments were conducted in humans, and studies that examined the safety of phototbcnpy in HIV+ patients have been inconclusive. Thus, we were compelled to determine whether UVB radiation can activate HIV In human skin. We first developed a quantitative assay for measuring HIV In skin. RNA was extracted from biopsy specimens of and nonksioaal skln from HIV+ patients with psoriasis. To control for sample variation, sawn1 paired specimens were taken from each palent, with the least amount of extracted total RNAispeetmen serving IS the limiting factor for each experiment A PCR-based assay amplified and detected a target sequence in the conserved region of the HIV-1 gag gene, using established primers and a built-in qusntitation standard. Lesional skin contained 20-M HIV copies& RNA, whereas nonlesional skin had very low numbers. Similar findings were noted in Iesiooal vs. nonlesionn1 skin of HIV+ patients with moIluscum contaglosum. We next examined the elfect of in viw UVB ndiatton FSZO Sunlamps) on cutzmeeus virst load. Adjacent paired specimens were taken from psoriatic skin (one from an u&radiated site; the other from skin exposed to 1 single dose of 100300 J/m*). HIV copy number was increased (S- to IO-fold) in UVB-treated skin in comparison to the paired unirradiated control. This study provides tbe tlrst quantitative method for measuring HIV in skin. It documents the presence of a signiticant number of vlrnl copies In lesional siter. Finally, we demonstrate that a single low dox olUVB radiation does activate HIV in human akin.
CLONING AND EXPRESSION ANALYSIS OF AMAC-1,
lesional
ASSOCIATED
CC-CHEMOKINE
OF ANTIGEN-PRESENTING
A NOVEL TH2CELLS. y,
dt Dept. of Damtatology, Benjamin Kz&&CJUer. 0. Politz N. Hakii. S. C FraddiaMedialCaatex,TheFreeUaiwmityofBerlia,Be&,Gamtany we have closed a novel human cc-chemo~ae. aliematiw rlwop@e actionaswc~ated CC-&m&n+ (MCI-l. The isolated cDNA dose (803 bp) shows a single open reading frame of 261 bp coding for 89 amino acid residw; mature AMA&I protein consists of 69 amino acids with a moleadar weight of 7855 AMAC-I homology
is most closely related to MIP-la of 55% and 59%, respectively
with B cDNA
and protein
sequence
However, the expression pattern of AMAC-1 ia directly opposite to that of MI&la. While MI&la is iadwed by da&al macrophage axdiatora, MC-1 is induced in maorophages by altamative nwrophage
mediators
U.4.
IL13,
and K-10.
Expression
of AMAC-1
is inhibii
by IFN-y while GC met a slightly positive synargi& &Tactin combination with IL4. Peripheral blood moaocytes da sot express AMAC-1; tima couw experimntu
show that moaocvte-to-macrooh diientiation is a arareauisite for Ah&w-I expreasicat Expre&on of Ah&cl by GM-CSF/U-&du& mowxyta-duiwd dendtitic cells is compkx; mature adherant dead& cells, however, only express minor amouats of AMAC-1 mRNA In viw, AMAC-I is expressed by slvwlru macrophage8 f+om healthy parson, smokers, and asthmatic patieats. In conohtsioa, MC-1 is a novel Thz-awciat ad cc-chamokina possibly iavolved ia the antigen presenting cdl-dapaadent regulation of th-e Thlfl’h2 bahuw ia iaflammatory aad immune reactions.