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Characterization of novel antigens in membranous nephropathyColes, Paige 17 June 2016 (has links)
INTRODUCTION: Membranous nephropathy is an autoimmune disease that targets glomeruli of the kidney. Previous discoveries in membranous nephropathy include the discovery of megalin as an antigen in the proximal tubular brush border fraction (Fx1A) and glomeruli of Heymann nephritis rats, identification of neutral endopeptidase in alloimmune neonatal nephropathy, and discovery of PLA2R and THSD7A as causal antigens in approximately 80-85% of primary membranous nephropathy cases. It was then recognized that there must be other antigens responsible for the remaining 15-20% of cases.
OBJECTIVES: The current study aims to screen membranous nephropathy patient serum samples via Western blotting for reactivity with potential antigens in protein extracts of normal human glomeruli, purify potential membranous nephropathy antigens, identify them with mass spectrometry, and validate these identifications with immunoprecipitation and immunohistochemical analysis. Previously, work had been done to identify one novel, 58 kDa antigen. A second novel antigen had been shown in the proximal tubule brush border. Finally, a third protein, CR1, was shown to contain corresponding antibodies in the antibody preparation used in the rat model of membranous nephropathy, making the antigen a protein of interest in human primary membranous nephropathy.
METHODS: Using human glomeruli obtained by detergent extraction, we isolated extracellular domains and identified two novel antigens, called 58-kDa and brush-border, with patient serum. We attempted to further purify the 58-kDa antigen with lectin binding columns and partition phase separation. Upon the identification of a small cohort of cases associated with autoimmune tubulointerstitial nephritis, we set out to determine if these sera recognized a novel antigen. Prior to screening human glomerular extract with these sera, we exposed it to partial proteolysis with trypsin, reducing agent β-mercaptoethanol, and tubular elements to further characterize the antigen before it was pulled down with anti-brush-border antigen+ and control IgG4 and analyzed by mass spectrometry. The third and final antigen we investigated was CR1, which we screened with membranous nephropathy sera and immunoblotted its antibody against different protein preparations.
RESULTS: Labeling of the extracellular portions of the 58-kDa and brush-border antigens with biotin was successful. It was determined that the 58-kDa antigen was not glycosylated due to its inability to bind lectin columns. The 58-kDa antigen was present in the hydrophilic layer when separated with tritonX-114 detergent. Partial proteolysis of the brush-border antigen with trypsin yielded bands at 140 kDa, 120 kDa and 95 kDa. The brush-border antigen was destroyed under reducing conditions. Candidate proteins for the brush-border antigen as determined by mass spectrometry include megalin and SVEP1. Membranous nephropathy sera were shown to be negative for anti-CR1+ antibody, and anti-CR1+ antibody was reactive with glomeruli and the TBS supernatant fraction.
CONCLUSIONS: This study suggests that the 58-kDa antigen which has antibodies in some human primary membranous nephropathy sera contains extracellular portion(s), is not glycosylated, but is membrane-associated. The data indicate that there is also potential for a membranous nephropathy antigen in the tubular brush border with an immunoreactive element around 95 kDa in size, that is sensitive to reducing conditions. Preliminary mass spectrometry information points toward megalin as the identification of this antigen. CR1 does not appear to be a causal antigen in human primary membranous nephropathy.
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Distribution of circulating and glomerular IgG subclasses against full-length anti-phospholipase A2 receptor and its epitopes in primary membranous nephropathyLi, Lee Chuan 08 April 2016 (has links)
OBJECTIVE: Research towards understanding PLA2R, the human antigen of primary membranous nephropathy has steadily gained ground since its discovery in 2009. This autoimmune kidney disease features a unique immunological character of high IgG4 prevalence, both in circulation and as immune complexes deposited in tissue. We seek to characterize and better understand the distribution of all IgG subclasses between serum and glomerular deposits, as well as characterize IgG reactivity directed against both full-length PLA2R and its immunogenic components.
METHODS: Using biopsy data obtained from renal pathology centers, we identified 13 patients with primary membranous nephropathy as well as biopsy immunofluorescence data for all IgG subclasses. We compared anti-PLA2R staining in glomeruli to serum anti-PLA2R using western blot, and analyzed concordance of subclass distribution between the two sets of data using Cohen's kappa score. We also studied and similarly analyzed, using western blot, subclass distribution of IgG against PLA2R epitopes CysR, CTLD1, and CTLD4-8.
RESULTS: All 13/13 (100%) patient samples were positive for circulating anti-PLA2R IgG4 in western blot, with 11/13 (84.6%) positive for IgG3, 6/13 (46.2%) for IgG2, and 11/13 (84.6%) for IgG1. When compared with biopsy immunofluorescence these results exhibited fair agreement for IgG4, IgG3, and IgG1; IgG2 was discordant with corresponding biopsies. For reactivity against PLA2R epitopes, 11/12 (91.7%) samples were positive for anti-CysR IgG4, 5/12 (41.7%) positive for IgG3, 0/11 (0.0%) for IgG2, and 8/11 (72.7%) for IgG1. Reactivity against epitopes CTLD1 and CTLD4-8 was detected less frequently than against CysR, though IgG4 was still the predominant subclass in almost all cases.
CONCLUSION: In general, levels of circulating IgG subclasses directed against PLA2R is concordant between serum and in biopsy, and as such serum anti-PLA2R can act as a good proxy for all IgG subclasses found in glomerular deposits. Furthermore, both full-length PLA2R and its extracellular domain containing the CysR epitope exhibit concordance between IgG3 and IgG4 levels, demonstrating potential for anti-CysR autoantibodies to be a good indicator for primary MN in addition to anti-PLA2R.
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Biochemical analysis of the factors controlling the process of membrane tubule formation from the Golgi complexWeigert, Roberto January 2000 (has links)
Membranous tubules are very abundant structures in living cells and form or are part of most intracellular organelles. The Golgi apparatus is mainly formed by tubules, which adopt different geometries and conformations. However, their physiological role has not yet been established and this is mainly due to the almost absolute lack of knowledge about the biochemical mechanisms regulating their formation, maintenance and disruption. The aim of this thesis was to investigate in a systematic way these mechanisms. The first step has been to set up an in vitro morphological assay suitable for the visualisation of Golgi-associated tubules in isolated Golgi stacks. This assay was based on electron microscopy and specifically on negative staining of whole-mount preparations. It allowed both qualitative and quantitative analysis of the morphological changes of Golgiassociated tubules after in vitro incubations. This assay was then used for screening several molecules or experimental conditions for their effect on tubular homeostasis. Among them, the most significant was BARS (BFA-dependent ADP-Ribosylation Substrate), a protein previously implicated in the maintenance of Golgi architecture. BARS has been found to cause the selective breakdown of the tubular part of the Golgi complex promoting fission events which convert the tubular structures into clusters of vesicles. This effect correlated with the enzymatic activity of BARS, which acts as an acyl-CoA dependent lysophosphatidic acid acyl transferase (LPAAT), increasing phosphatidic acid (PA) levels in Golgi membranes. This suggests that local modifications of the composition of the lipid bilayer is a possible mechanism for the fission of membranous tubules.
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A retrospective study characterizing the complete s open reading frame of hepatitis B virus from black children with membranous nephropathy treated with interferon alpha-2bGous, Natasha Myrna 06 August 2008 (has links)
ABSTRACT
In sub-Saharan Africa a causal relationship has been established between hepatitis B
virus (HBV) infection and membranous nephropathy (MN), especially in Black children.
The most common method of treatment is interferon therapy, which is however, only
effective in 30-40% of patients. The reason for this is unclear. The objective of this pilot
study was to determine whether mutations in the complete surface gene of HBV isolated
from Black children with HBV-associated MN before, during and after treatment with
interferon, had any effect on treatment response and vice versa. HBV DNA was extracted
from the serum of a responder, reverter and non-responder patient before, during (4 and
16 weeks) and after (40 weeks) IFN treatment. The preS1/preS2/S region was amplified
and cloned, and the clones sequenced. Sequence analyses revealed the preS2 region to be
the most variable in the reverter and non-responder and HBsAg was the most variable in
the non-responder. Phylogenetic analysis showed that the viral population dynamics
between the responder strains and the reverter/non-responder strains differed as a result
of various mutations found within the surface gene. Thus the presence of mutations in
preS2 and HBsAg of the non-responding patients may carry predictive markers for nonresponse
but further investigation would be needed to conclusively prove this.
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Hepatitis-B-associated glomerular disease : a clinicopathological study of Hepatitis B virus associated Membranous Glomerulonephritis in Namibian and South African children 1974 – 2005 and a comparison with hepatitis B associated Membranous Glomerulonephritis as well as Idiopathic Membranous Glomerulonephritis in adultsBates, William D. 12 1900 (has links)
Thesis (PhD (Med))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Background and Objective: The most common cause of severe
proteinuria/nephrotic syndrome (NS) in children worldwide is minimal change disease
(MCD). This is also the pattern observed in white and Indian children in South Africa
(SA). By contrast, black and mixed race/coloured children of Southern Africa in the
1960s to 1990s were shown to have a different pattern of NS. One of the main
differences was the frequency of hepatitis B virus (HBV) associated
glomerulonephritis, usually membranous glomerulonephritis (MGN). The objective of
this project was a clinicopathological study of this subgroup of nephrotic children to
document the disease further and in particular to seek correlations between
pathological and clinical features including prognosis. A central focus was to
document the detailed ultrastructural examination of the renal biopsies of these
children and to correlate the spectrum of pathological features with demographic,
clinical, laboratory and prognostic features.
The hypothesis was that the clinicopathological features of HBV MGN in
children differed substantially from idiopathic MGN in general (children and
adults) and also from HBV MGN in adults and that HBV MGN in children should
be viewed as a distinct disease.
Patients and methods: The childhood (12 years and younger) patient cohort was
309 children with severe proteinuria/nephrotic syndrome who presented at Tygerberg
Hospital (TBH) over a 21 year period from 1974-1995, including 67 children from
Namibia. The study group was 71 children with HBV MGN who were followed up to
2005. The comparative adult group was 45 adults with MGN of whom 12 had HBV MGN and 33 idiopathic MGN. (A comparison could not be made with idiopathic MGN
in childhood as this centre only had 2 such patients during the study period.)
Demographic, clinical, laboratory and renal pathology data were collected, compared
and correlated.
Results: HBV associated MGN was the most frequent cause of NS in the Namibian
subgroup, 25/67 (37%) and the third most frequent, 71/309 (23%) in the childhood
cohort as a whole. The MGN group was 86% (71/83) of the total HBV childhood
nephrotic cohort, by far the dominant subgroup.
The average age of the 71 children with HBV MGN was 6.0 years (range 2-12 years)
at presentation and boys comprised 80% of the group. Hepatitis B envelope antigen
(HBeAg) was identified in the serum of 87% of children tested. Laboratory features
different from idiopathic MGN included more prominent haematuria, mildly raised
serum transaminases and more frequently lowered serum C3 and C4 levels. Light
microscopic examination of renal biopsies showed mesangial proliferation in all
patients but with minimal glomerular sclerosis and interstitial disease. On
ultrastructural examination mesangial and subendothelial deposits were common and
prominent as was mesangial interposition. The MGN of HBV in children therefore
frequently showed mesangiocapillary glomerulonephritis (MCGN) features in addition
to the subepithelial deposits of MGN. The subgroup of 23 whose renal biopsies
displayed severe mesangial interposition in addition to the subepithelial deposits of
MGN were termed the mixed HBV MGN-mesangiocapillary GN group. Virus like
bodies and tubuloreticular inclusion bodies were both found in more than 80% of
biopsies of childhood HBV MGN. HBeAg was identified in the subepithelial deposits
in the glomeruli. This was the first time this feature was demonstrated in Africa. The
46 South African children with HBV MGN showed a cumulative remission rate of 25% at 2 years and 52% at 4 years. Seven of the children (10%) of the total cohort
developed chronic renal failure (CRF). Age of 6 years and above at presentation and
severe mesangial deposits on biopsy correlated with fewer remissions and poorer
outcome. In 3 patients the interval between the diagnosis of HBV MGN and the onset
of CRF was more than 19 years with the longest being 23 years. The 358 cases of
childhood HBV MGN from Southern Africa constitute 37% of the reported childhood
patients.
Comparative data
A comparison was made between the 71 children with HBV MGN, 12 adults with
HBV MGN and 33 adults with idiopathic MGN. The main differences were that both
HBV MGN groups included only coloured and black patients and were more
predominantly male while the idiopathic MGN group included all races. In the HBV
patients, haematuria was more frequent and severe, liver enzymes were frequently
raised and C3 more frequently reduced than in the idiopathic cohort. Both groups of
adult MGN patients had normal C4 levels while the childhood HBV MGN group had
reduced C4 levels.
The immune complex pattern in both of the HBV MGN adult and childhood groups on
biopsy was similar with more mesangial and subendothelial deposits as well as
mesangial interposition than the idiopathic group. Despite this similarity between the
two HBV groups, both adult groups showed more glomerular sclerosis and interstitial
disease than the childhood group. The clinical outcome of the children’s cohort was
better than the other 2 groups with remission (52%) more frequent at 4 years (p<
0.01) and better renal and patient survival.
Including the 83 cases from this series, at least 1243 renal biopsy proven cases of
HBV MGN have been reported in the English literature; children (80%) and adults (20%). The male gender predominance in both age groups for HBV MGN is similar
(children 79%; adults 84%) and significantly greater than for idiopathic MGN.
Conclusions: The findings confirm that HBV MGN in children is a distinct form of
GN which broadens the classical morphologic description of MGN by often including
a number of mesangiocapillary GN features. The subgroup of renal biopsies with the
most severe mesangiocapillary GN features was classified as the mixed HBV MGNmesangiocapillary
GN group. The MGN spectrum as a whole comprised 86% of the
HBV positive childhood group. HBV MGN was the most frequent association with
NS/severe proteinuria in the Namibian subgroup (37%) and the third largest group
(19%) in the SA children. It showed a relatively high spontaneous remission rate but
at least 10% of the children developed renal failure. Age of 6 years and above at
presentation and severe mesangial deposits on biopsy correlated with fewer
remissions and poorer outcome. Extended follow up (more than 15 years) was
required to demonstrate renal failure in some patients in the poor outcome group.
Urbanisation, associated with lower HBV carrier rates, and HBV vaccination (initiated
routinely in 1995 in SA), have already lead to a sharply decreasing incidence of this
disease in SA. HBV MGN has been a valuable and possibly unique model of human
GN and MGN in particular in that the HBeAg has been identified in both the serum
and glomeruli enabling confirmation of the aetiological role of HBeAg. / AFRIKAANSE OPSOMMING: Agtergrond en Doelwit: Die algemeenste oorsaak van erge proteïenurie/nefrotiese
sindroom (NS) in kinders wêreldwyd is minimale veranderingsiekte. Hierdie patroon
kom ook voor in blanke- en Indiër kinders in Suid-Afrika. In teenstelling hiermee is
aangetoon dat swart en kleurling/gemengde ras kinders in Suider Afrika tussen die
jare 1960s tot 1990s ’n ander patroon van nefrotiese sindroom gehad het. Een van
die hoof verskille was die algemene voorkoms van hepatitis B virus (HBV)
geassosieerde glomerulonefritis, gewoonlik membraneuse glomerulonefritis (MGN).
Die doelwit van hierdie projek was ’n klinies-patologiese studie van hierdie subgroep
van nefrotiese kinders ten einde die siekte verder te beskryf en veral om korrelasies
te tref tussen patologiese en kliniese kenmerke insluitende prognose. Die
gedetaileerde ultrastrukturele ondersoek van die kinders se nierbiopsies en die
korrelasie van die spektrum patologiese kenmerke met demografiese, kliniese,
laboratorium en prognostiese kenmerke was ‘n sentrale fokusarea.
Die hipotese was dat die klinies-patologiese kenmerke van HBV MGN in
kinders wesenlik van idiopatiese MGN in die algemeen verskil (in kinders en
volwassenes) en ook van HBV MGN in volwassenes, en dat die beeld in kinders
as ’n afsonderlike siekte beskou behoort te word.
Pasiënte en metodes: Die kinder kohort (12 jaar en jonger) was 309 kinders met
erge proteïenurie/nefrotiese sindroom wie in Tygerberg Hospitaal (TBH) behandel
was oor ‘n 21 jarige periode vanaf 1974 tot 1995, insluitende 67 kinders van Namibië.
Die studiegroep was 71 kinders met HBV MGN wie waar moontlik tot 2005 opgevolg was. Die vergelykende volwasse groep was 45 volwassenes met MGN van wie 12
HBV MGN gehad het en 33 idiopatiese MGN. (’n Vergelyking met idiopatiese MGN
in kinders kon nie gedoen word nie omdat hierdie sentrum net twee sulke pasiënte
tydens die studietyd behandel het.) Demografiese, kliniese, laboratorium en
nierpatologie inligting is versamel, vergelyk en gekorreleer.
Resultate: HBV geassosieerde MGN was die algemeenste oorsaak van NS in die
Namibiese subgroep, 25/67 (37%) en die derde mees algemeen, 71/309 (23%) in die
kinder kohort as geheel. Die MGN groep was 86% (71/83) van die totale HBV kinder
nefrotiese kohort en verreweg die oorheersende subgroep.
Die gemiddelde ouderdom van die 71 kinders met HBV MGN by presentering was
6.0 jaar (reikwydte 2-12 jaar) en seuns het 80% van die groep behels. Hepatitis B
omhullingsantigeen (envelope antigen- HBeAg) is aangetoon in die serum van 87%
van die kinders wie daarvoor getoets is. Laboratoriumkenmerke wat van idiopatiese
MGN verskil het, het ingesluit meer prominente hematurie, gering verhoogde serum
transaminases en meer dikwels verlaagde serum C3 en C4 vlakke. Ligmikroskopiese
ondersoek van die nierbiopsies het mesangiale proliferasie in elke pasiënt getoon,
maar met minimale glomerulêre sklerose en interstisiële siekte. Met ultrastrukturele
ondersoek was mesangiale en subendoteliële neerslae asook mesangiale
interposisie algemeen. Die MGN van HBV in kinders het dus dikwels kenmerke van
mesangiokapillêre glomerulonefritis getoon bo en behalwe die subepiteliële neerslae
van MGN. Die ondergroep van 23 van wie die nierbiopsies erge mesangiale
interposisie aangetoon het asook die subepiteliale neerslae van MGN is die
gemengde HBV MGN-mesangiokapillêre GN groep genoem. Virustipe liggaampies
en tubuloretikulêre insluitingsliggaampies is in meer as 80% van die biopsies
bevestig. HBeAg was in die subepiteliële neerslae identifiseer. Dit was die eerste
keer dat hierdie kenmerk in Afrika identifiseer is. Die 46 Suid-Afrikaanse kinders het ’n kumulatiewe remissie koers van 25% teen 2 jaar en van 52% teen 4 jaar
getoon. Sewe van die kinders (10%) van die hele kohort het kroniese nierversaking
(KNV) ontwikkel. Ouderdom van 6 jaar en meer by presentasie en erge mesangiale
neerslae in ‘n biopsie het met minder remissies en ’n swakker uitkoms gekorreleer.
Drie pasiënte het meer as 19 jaar na aanvanklike voordoening ooglopende KNV
ontwikkel, waarvan 23 jaar die langste interval was. Die 358 gevalle van kinderjare
HBV MGN van Suidelike-Afrika maak 37% uit van die gerapporteerde kinder
pasiënte.
Vergelykende data
’n Vergelyking is getref tussen die 71 kinders met HBV MGN, 12 volwassenes met
HBV MGN en 33 volwassenes met idiopatiese MGN. Die hoof verskille was dat beide
HBV groepe net kleurling en swart pasiënte ingesluit het en meer oorwegend manlik
was, terwyl die idiopatiese groep alle rasse ingesluit het. In die HBV pasiënte was
hematurie meer algemeen en erg, lewer ensieme meer dikwels verhoog en C3 meer
dikwels verlaag as in die idiopatiese kohort. Beide groepe van volwasse MGN
pasiënte het normale C4 vlakke getoon terwyl die kindergroep met HBV MGN
verlaagde C4 vlakke bewys het. Die immuunkompleks patroon in biopsies van die
HBV MGN volwasse en kindergroepe was soortgelyk met meer mesangiale en
subendoteliële neerslae asook meer mesangiale interposisie as in die idiopatiese
groep. Ten spyte van hierdie ooreenkoms tussen die twee HBV groepe, het die twee
volwasse groepe meer glomerulêre sklerose en interstisiële siekte as die kindergroep
vertoon. Die kliniese uitkoms van die kinderkohort was beter as die ander twee
groepe met remissie (52%) wat meer algemeen was teen 4 jaar (p< 0.01) en met
beter nier- en pasïent oorlewing. Ingeslote die 83 gevalle van hierdie reeks, is ten minste 1243 nierbiopsie bewysde
gevalle van HBV MGN in kinders (80%) en volwassenes (20%) in die Engelse
literatuur gerapporteer. Die manlike oorheersing in beide ouderdomsgroepe van HBV
MGN is soortgelyk (kinders 79%; volwassenes 84%) en betekenisvol meer as vir
idiopatiese MGN.
Gevolgtrekkings: Die bevindinge bevestig dat HBV MGN in kinders ’n afsonderlike
vorm van GN is wat die klassieke beskrywing van MGN verbreed deur die algemene
insluiting van ’n aantal mesangiokapillêre GN kenmerke. Die ondergroep van nier
biopsies met erge mesangiokapillêre GN kenmerke is as die gemengde HBV MGNmesangiokapillêre
GN groep geklassifiseer. Die MGN spektrum in geheel het 86%
van die HBV positiewe kindergroep behels. HBV MGN was die mees algemene
assosiasie met NS/erge proteïenurie in die Namibiese subgroep (37%) en die derde
grootse groep (19%) onder die SA kinders. Die siekte het ’n relatiewe hoë spontane
remissiekoers getoon, maar ten minste 10% van die kinders het nierversaking
ontwikkel. Ouderdom van 6 jaar en meer by presentasie en erge mesangiale
neerslae in ‘n nierbiopsie het met minder remissies en ’n slegter uitkoms gekorreleer.
Uitgebreide opvolg (meer as 15 jaar) was nodig om nierversaking in sommige van
die swak uitkomsgroep aan te toon.
Verstedeliking is geassosieerd met laer HBV draersyfers en hierdie faktor saam met
algemene HBV inenting in die kinderjare (wat in 1995 in SA begin was), het ’n skerp
daling in die voorkoms van hierdie siekte in SA teweeg gebring. HBV MGN is ’n
waardevolle en moontlik unieke model van menslike GN en MGN, veral omdat die
HBeAg in beide die serum en glomeruli identifiseer kon word om die etiologiese rol
van HBeAg te bevestig.
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Regulation of Membrane Fusion Events During Caenorhabditis elegans SpermatogenesisWashington, Nicole Leanne January 2005 (has links)
FER-1 is required for fusion of specialized vesicles, called membranous organelles, with the sperm plasma membrane during Caenorhabditis elegans spermiogeneis. To investigate the role of FER-1 in membranous organelle fusion, I first examined ten fer-1 mutations and found that they all cause the same defect in membrane fusion. FER-1 and the ferlin protein family are membrane proteins with four to seven C2 domains which commonly mediate Ca2+-dependent lipid-processing events. Most of the fer-1 mutations fall within these C2 domains, showing that they have distinct, non-redundant functions. I found that membranous organelle fusion requires intracellular Ca2+ and that C2 domain mutations alter Ca2+ sensitivity. This suggests that the C2 domains are involved in Ca2+ sensing and further supports their independent function. Using two immunological approaches we found three FER-1 isoforms, two of which may arise from FER-1 by proteolysis. By both light and electron microscopy these FER-1 proteins are localized to membranous organelle membranes. Together, these results suggest that the ferlin family members may share a conserved mechanism to regulate cell-type specific membrane fusion.In Chapter III, I present additional results toward studying the function of FER-1 using several broad-based approaches. First, I present a bioinformatics analysis of FER-1 C2 domains and the preliminary results of their calcium-dependent phospholipid binding capabilities. Second, preliminary interactions found with individual FER-1 functional domains by a yeast-two hybrid screen are discussed. Lastly, I present results from a candidate-gene approach to identify additional regulators of MO fusion, the sperm-specific synaptobrevins.
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Analyses ultrastructurales et biochimiques des membranes cellulaires associées aux complexes de réplication du virus de l'hépatite C / Ultrastructural and biochemical analyses of cellular membranes associated with the Hepatitis C virus replication complexFerraris, Pauline 16 December 2011 (has links)
Comme pour la plupart des virus à ARN+, le VHC induit des remaniements membranaires appelés membranous web. Les protéines non structurales virales formant le complexe de réplication du virus sont associées à ces membranes néosynthétisées. La compréhension de la mise en place de ces membranes cellulaire est encore actuellement mal connue. Afin d’étudier ce phénomène, nous avons dans un premier temps sélectionné des clones cellulaires Huh7.5 hébergeants un réplicon sous-génomiquedu virus. Nous avons ainsi pu mettre en évidence la présence d’un réseau multivésiculaire semblant provenir de l’induction de mécanismes d’autophagie. Plus récemment l’utilisation du modèle de propagation du virus complet nous a permis de mieux caractériser ce réseau multivésiculaire en déterminant trois sous réseaux vésiculaires structuralement différents. L’analyse de cette étude est effectuée principalement par microscopie électronique avec des techniques innovantes tels que la reconstruction tridimensionnelle et des immunogolds. / As other RNA viruses, HCV induces membrane alterations termed membranous web and its nonstructural proteins forming the viral replication complex are associated to these neo-synthesized membranes. The mechanism underlying these host cell membranes alterations is still currently unknown. To investigate this mechanism, we initially selected Huh7.5 cells clones harbouring a HCV subgenomic replicon. We were able to demonstrate the presence of a multivesicular network apparently linked to the autophagy induction mechanisms. More recently, using the cell culture-adapted HCVsystem, we better characterized this network by determining three multivesiculars vesicles structurally different subnets. This study was carried out mainly by performing electron microscopy observations,with using innovative techniques such as three-dimensional reconstruction and immunogold.
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Nouveaux marqueurs diagnostiques et pronostiques dans la glomérulonéphrite extra-membraneuse : suivi des anticorps anti-PLA2R1 chez le greffé rénal : caractérisation des épitopes reconnus par les anticorps anti-PLA2R1 : identification d’une nouvelle cible antigénique / New diagnostic and prognostic marker in membranous nephropathySeitz-Polski, Barbara 15 December 2014 (has links)
La Glomérulonéphrite extra-membraneuse est une maladie auto-immune rare mais grave qui conduit dans 30% des cas à une insuffisance rénale chronique terminale nécessitant le recours à la dialyse ou la greffe rénale. Dans les suites d’une greffe, la GEM récidive dans 30 à 40% des cas. En 2009, l’équipe du Pr. Salant en collaboration avec notre équipe a montré que 70% des patients présentant une GEM étaient porteurs d’anticorps (Ac) dirigés contre le récepteur des phospholipases A2 (PLA2R1). Le titre d’anticorps est corrélé à l’activité de la maladie. Il n’existe actuellement aucun biomarqueur permettant de prédire l’évolution de la fonction rénale d’un patient lors de sa prise en charge : dans 30% des cas les patients présentent une rémission spontanée sans traitement immunosuppresseur. Le traitement de la GEM repose sur un traitement symptomatique et une réévaluation après 6 mois. En cas de maladie active persistante, il faut débuter un traitement immunosuppresseur. Dans les formes graves, cette période d’observation de 6 mois peut être à l’origine de lésions irréversibles. Nous avons validé un test ELISA permettant de quantifier les Ac anti-PLA2R1 au cours du suivi de patients porteurs d’une GEM. Ce test nous a permis de montrer sur une cohorte de 15 patients greffés dans les suites d’une GEM qu’un titre d’Ac anti-PLA2R1 persistant après la greffe était associé à un risque de récidive de la maladie sur le greffon. Nous avons ensuite produit dans des cellules HEK les orthologues de PLA2R1 (les récepteurs humain, lapin et murin). / Membranous Nephropathy (MN) is a major cause of nephrotic syndrome in adults. It is a rare but severe kidney disease with different etiologies and outcomes. In most cases (85%), the disease is idiopathic (iMN) and has an autoimmune origin. One third of patients develop end-stage kidney disease and are on kidney transplant waiting list. MN recurred in 30% after transplantation. Another third enter in spontaneous remission under renin-angiotensin system blockade. The treatment of iMN is controversial. KDIGO guidelines recommend a supportive symptomatic treatment with RAS-blockade and diuretics in all patients with iMN, and immunosuppressive therapy in case of renal function deterioration or persistent nephrotic syndrome. Therefore, immunosuppressive treatments are often started only after significant and potentially irreversible complications. No biological markers can predict clinical outcome and orient therapy. A major breakthrough was the discovery of autoantibodies to the phospholipase A2 receptor (PLA2R1, 180 kDa) in 70% of iMN patients in 2009, which has now allowed to develop diagnosis and prognosis tests for better medical care. During my PhD, I have first participated to the development of an ELISA which is now commercially available. I then used this latter to demonstrate that persistent anti-PLA2R1 activity can predict iMN recurrence after transplantation in a retrospective cohort of 15 patients. We then screened 50 patients with iMN on native kidney for their cross-reactivity to human (h), rabbit (rb) and mouse (m) PLA2R1 by western blot (WB) and antigen-specific ELISAs.
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Functional Morphology of the Vestibular End Organs in the Red-eared Slider Turtle, Trachemys scripta elegans.Riddell, Clinton D. 21 May 2014 (has links)
No description available.
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Toxoplasma gondii : étude du réseau de nanotubes membranaires de la vacuole parasitophore et des protéines GRA associées / Toxoplasma gondii,parasitophorous vacuole,dense granules,PI(4,5) P2,membranous tubules , amphipathic alpha helicesBittame, Amina 14 January 2011 (has links)
Dans la cellule hôte, Toxoplasma gondii se développe dans une vacuole parasitophore (VP) caractérisée par un réseau de nanotubes membranaires (RNM) dont la composition, le mécanisme de formation et la fonction sont obscures. Quelques protéines GRA, dont GRA2 et GRA6, sont sécrétées dans la VP à partir des granules denses puis ciblées au RNM. Cette localisation s'accorde avec l'hélice alpha-hydrophobe de GRA6 et les hélices alpha-amphipathiques de GRA2. Avant et après sécrétion dans la VP, les protéines GRA sont partiellement solubles. Le phénotype de parasites délétés de leur(s) gène(s) GRA2 et/ou GRA6 révèle que ces 2 protéines sont indispensables à la formation du RNM. J'ai montré 1) qu'avant leur insertion dans les membranes de la VP, la solubilité des protéines GRA est préservée grâce à des interactions hydrophobes avec peut être, des micelles de l'espace vacuolaire ; 2) que GRA12, une nouvelle protéine du RNM, n'interagit pas avec GRA2 dans ces membranes. 3) que l'adressage spécifique de GRA6 au RNM est déterminé par son domaine N-terminal hydrophile. 4) J'ai montré que GRA2 recombinante a une affinité pour le phosphatidyl inositol (4, 5) diphosphate avec lequel elle interagit via ses hélices alpha-amphipathiques. GRA2 déforme des liposomes de courbure membranaire importante pour générer de courts tubules membranaires. La tubulation est accentuée par GRA6 qui s'associe aux liposomes, quelque soit leur diamètre. Ces résultats valident le rôle direct de GRA2 et GRA6 dans la formation du RNM et laissent envisager un modèle de sa formation, dans lequel GRA6 favoriserait l'assemblage de vésicules lipidiques que GRA2 fusionnerait en tubules membranaires. / Within the host cell, Toxoplasma gondii multiplies in a parasitophorous vacuole (PV) characterized by a membranous nanotubular network (MNN). Its components, the mechanism of its formation and its function remain unknown. A few GRA proteins, including GRA2 and GRA6, are secreted from the dense granules into the PV and are targeted to the MNN. This location is in agreement with the hydrophobic alpha-helix predicted in GRA6 and with the GRA2 amphipatic alpha-helices. However, before and after their secretion in the PV, the GRA proteins are partially soluble. The phenotypic analysis of parasites deleted from their GRA2 and/or GRA6 gene(s) had shown that both these proteins are indispensable for MNN formation. During my thesis, I showed that before their insertion into the PV membranes, the GRA proteins solubility is preserved by establishing hydrophobic interactions, likely with micelles in the PV space. I also showed that GRA12, a novel MNN-associated protein, does not interact with GRA2 within these membranes. Using GRA6 as a model of study, I contributed to demonstrate that the GRA6 specific targeting to the MNN relies on its N-terminal hydrophilic domain. I demonstrated that recombinant GRA2 recognizes inositol (4, 5) biphosphate with which it interacts via its amphipatic alpha-helices. GRA2 deforms liposomes of steep membrane curvature into short membranous tubules. The tubulation is increased by GRA6 which associates with liposomes independently of their diameter. These results validate the direct role of both GRA2 and GRA6 in MNN formation and led us to propose a model in which GRA6 would tether vesicles, the fusion of which would be induced by GRA2.
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