UNLABELLED: HIV-1 assembles at the plasma membrane of virus-producing cells as an immature, noninfectious particle. Processing of the Gag and Gag-Pol polyproteins by the viral protease (PR) activates the viral enzymes and results in dramatic structural rearrangements within the virion--termed maturation--that are a prerequisite for infectivity. Despite its fundamental importance for viral replication, little is currently known about the regulation of proteolysis and about the dynamics and structural intermediates of maturation. This is due mainly to the fact that HIV-1 release and maturation occur asynchronously both at the level of individual cells and at the level of particle release from a single cell. Here, we report a method to synchronize HIV-1 proteolysis in vitro based on protease inhibitor (PI) washout from purified immature virions, thereby temporally uncoupling virus assembly and maturation. Drug washout resulted in the induction of proteolysis with cleavage efficiencies correlating with the off-rate of the respective PR-PI complex. Proteolysis of Gag was nearly complete and yielded the correct products with an optimal half-life (t(1/2)) of ~5 h, but viral infectivity was not recovered. Failure to gain infectivity following PI washout may be explained by the observed formation of aberrant viral capsids and/or by pronounced defects in processing of the reverse transcriptase (RT) heterodimer associated with a lack of RT activity. Based on our results, we hypothesize that both the polyprotein processing dynamics and the tight temporal coupling of immature particle assembly and PR activation are essential for correct polyprotein processing and morphological maturation and thus for HIV-1 infectivity. IMPORTANCE: Cleavage of the Gag and Gag-Pol HIV-1 polyproteins into their functional subunits by the viral protease activates the viral enzymes and causes major structural rearrangements essential for HIV-1 infectivity. This proteolytic maturation occurs concomitant with virus release, and investigation of its dynamics is hampered by the fact that virus populations in tissue culture contain particles at all stages of assembly and maturation. Here, we developed an inhibitor washout strategy to synchronize activation of protease in wild-type virus. We demonstrated that nearly complete Gag processing and resolution of the immature virus architecture are accomplished under optimized conditions. Nevertheless, most of the resulting particles displayed irregular morphologies, Gag-Pol processing was not faithfully reconstituted, and infectivity was not recovered. These data show that HIV-1 maturation is sensitive to the dynamics of processing and also that a tight temporal link between virus assembly and PR activation is required for correct polyprotein processing.

• MeSH
• HIV-1 fyziologie   MeSH
• lidé MeSH
• posttranslační úpravy proteinů * MeSH
• proteiny viru HIV metabolismus   MeSH
• proteolýza MeSH
• sestavení viru * MeSH
• uvolnění viru z buňky * MeSH
• virologie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The critical requirements in developing clinical-grade human-induced pluripotent stem cells-derived neural precursors (hiPSCs-NPCs) are defined by expandability, genetic stability, predictable in vivo post-grafting differentiation, and acceptable safety profile. Here, we report on the use of manual-selection protocol for generating expandable and stable human NPCs from induced pluripotent stem cells. The hiPSCs were generated by the reprogramming of peripheral blood mononuclear cells with Sendai-virus (SeV) vector encoding Yamanaka factors. After induction of neural rosettes, morphologically defined NPC colonies were manually harvested, re-plated, and expanded for up to 20 passages. Established NPCs showed normal karyotype, expression of typical NPCs markers at the proliferative stage, and ability to generate functional, calcium oscillating GABAergic or glutamatergic neurons after in vitro differentiation. Grafted NPCs into the striatum or spinal cord of immunodeficient rats showed progressive maturation and expression of early and late human-specific neuronal and glial markers at 2 or 6 months post-grafting. No tumor formation was seen in NPCs-grafted brain or spinal cord samples. These data demonstrate the effective use of in vitro manual-selection protocol to generate safe and expandable NPCs from hiPSCs cells. This protocol has the potential to be used to generate GMP (Good Manufacturing Practice)-grade NPCs from hiPSCs for future clinical use.

• MeSH
• buněčná diferenciace MeSH
• indukované pluripotentní kmenové buňky * MeSH
• krysa rodu rattus MeSH
• leukocyty mononukleární MeSH
• lidé MeSH
• nervové kmenové buňky * MeSH
• neurony metabolismus   MeSH
• virus Sendai genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Retroviruses assemble and bud from infected cells in an immature form and require proteolytic maturation for infectivity. The CA (capsid) domains of the Gag polyproteins assemble a protein lattice as a truncated sphere in the immature virion. Proteolytic cleavage of Gag induces dramatic structural rearrangements; a subset of cleaved CA subsequently assembles into the mature core, whose architecture varies among retroviruses. Murine leukemia virus (MLV) is the prototypical γ-retrovirus and serves as the basis of retroviral vectors, but the structure of the MLV CA layer is unknown. Here we have combined X-ray crystallography with cryoelectron tomography to determine the structures of immature and mature MLV CA layers within authentic viral particles. This reveals the structural changes associated with maturation, and, by comparison with HIV-1, uncovers conserved and variable features. In contrast to HIV-1, most MLV CA is used for assembly of the mature core, which adopts variable, multilayered morphologies and does not form a closed structure. Unlike in HIV-1, there is similarity between protein-protein interfaces in the immature MLV CA layer and those in the mature CA layer, and structural maturation of MLV could be achieved through domain rotations that largely maintain hexameric interactions. Nevertheless, the dramatic architectural change on maturation indicates that extensive disassembly and reassembly are required for mature core growth. The core morphology suggests that wrapping of the genome in CA sheets may be sufficient to protect the MLV ribonucleoprotein during cell entry.

• MeSH
• elektronová kryomikroskopie MeSH
• genové produkty gag chemie   genetika   ultrastruktura   MeSH
• HEK293 buňky MeSH
• HIV-1 chemie   genetika   ultrastruktura   MeSH
• kapsida chemie   ultrastruktura   MeSH
• krystalografie rentgenová MeSH
• kvarterní struktura proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• myši MeSH
• proteinové domény MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• tomografie elektronová MeSH
• virion chemie   genetika   ultrastruktura   MeSH
• virové plášťové proteiny chemie   genetika   ultrastruktura   MeSH
• virus myší leukemie chemie   genetika   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• srovnávací studie MeSH

Defective FAS (CD95/Apo-1/TNFRSF6) signaling causes autoimmune lymphoproliferative syndrome (ALPS). Hypergammaglobulinemia is a common feature in ALPS with FAS mutations (ALPS-FAS), but paradoxically, fewer conventional memory cells differentiate from FAS-expressing germinal center (GC) B cells. Resistance to FAS-induced apoptosis does not explain this phenotype. We tested the hypothesis that defective non-apoptotic FAS signaling may contribute to impaired B cell differentiation in ALPS. We analyzed secondary lymphoid organs of patients with ALPS-FAS and found low numbers of memory B cells, fewer GC B cells, and an expanded extrafollicular (EF) B cell response. Enhanced mTOR activity has been shown to favor EF versus GC fate decision, and we found enhanced PI3K/mTOR and BCR signaling in ALPS-FAS splenic B cells. Modeling initial T-dependent B cell activation with CD40L in vitro, we showed that FAS competent cells with transient FAS ligation showed specifically decreased mTOR axis activation without apoptosis. Mechanistically, transient FAS engagement with involvement of caspase-8 induced nuclear exclusion of PTEN, leading to mTOR inhibition. In addition, FASL-dependent PTEN nuclear exclusion and mTOR modulation were defective in patients with ALPS-FAS. In the early phase of activation, FAS stimulation promoted expression of genes related to GC initiation at the expense of processes related to the EF response. Hence, our data suggest that non-apoptotic FAS signaling acts as molecular switch between EF versus GC fate decisions via regulation of the mTOR axis and transcription. The defect of this modulatory circuit may explain the observed hypergammaglobulinemia and low memory B cell numbers in ALPS.

• MeSH
• apoptóza genetika   MeSH
• hypergamaglobulinemie * MeSH
• lidé MeSH
• lymfoproliferativní nemoci * genetika   MeSH
• TOR serin-threoninkinasy MeSH
• zárodečné centrum lymfatické uzliny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Formation of a mature core is a crucial event for infectivity of retroviruses such as Mason-Pfizer monkey virus (M-PMV). The process is triggered by proteolytic cleavage of the polyprotein precursor Gag, which releases matrix, capsid (CA), and nucleocapsid proteins. Once released, CA assembles to form a mature core - a hexameric lattice protein shell that protects retroviral genomic RNA. Subtle conformational changes within CA induce the transition from the immature lattice to the mature lattice. Upon release from the precursor, the initially unstructured N-terminus of CA is refolded to form a β-hairpin stabilized by a salt bridge between the N-terminal proline and conserved aspartate. Although the crucial role of the β-hairpin in the mature core assembly has been confirmed, its precise structural function remains poorly understood. RESULTS: Based on a previous NMR analysis of the N-terminal part of M-PMV CA, which suggested the role of additional interactions besides the proline-aspartate salt bridge in stabilization of the β-hairpin, we introduced a series of mutations into the CA sequence. The effect of the mutations on virus assembly and infectivity was analyzed. In addition, the structural consequences of selected mutations were determined by NMR spectroscopy. We identified a network of interactions critical for proper formation of the M-PMV core. This network involves residue R14, located in the N-terminal β-hairpin; residue W52 in the loop connecting helices 2 and 3; and residues Q113, Q115, and Y116 in helix 5. CONCLUSION: Combining functional and structural analyses, we identified a network of supportive interactions that stabilize the β-hairpin in mature M-PMV CA.

• MeSH
• AIDS opičí genetika   metabolismus   MeSH
• buněčné linie MeSH
• HEK293 buňky MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• mutace genetika   MeSH
• sekundární struktura proteinů genetika   MeSH
• sekvence aminokyselin MeSH
• sestavení viru genetika   MeSH
• virion genetika   metabolismus   MeSH
• virové plášťové proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Rekombinantní humánní erytropoetin (rHuEPO) je používá při léčbě etiologicky rozdílných anémií (např. anémie při chronické renální insuficienci, anémie nedonošenců, anémie při určitých onkologických hematologických onemocněních, anémie vyvolané chemoterapií, anémie při HIV infekci a korekci anémie před chirurgických zákrokem). Působením na pluripotentní kmenové buňky kostní dřeně zrychluje erytropoézu 3–4 krát, což se projevuje vzrůstem počtu retikulocytů od třetího dne podávání. RHuEPO se může úspěšně použít jako součást postupů pro vyhýbání se transfuzím krve. Odpověď na léčbu se zlepšuje při podání perorálního nebo intravenózního železa. Erytropoetin se prokázal jako účinný v léčbě anémie při různých stavech a v kombinaci se železem, nebo bez něho může být alternativou transfuze erytrocytárního koncentrátu.

Recombinant human erythropoietin (rHuEPO) is used in the treatment of various types of anaemia (eg. in patients with chronic renal insufficiency, anaemia in premature babies, anaemia in certain onco-hematological illnesses, chemotherapy-induced anaemia, anaemia in human immunodeficiency virus (HIV) infection, and to redress anaemia before surgery). Erythropoietin stimulates the production of red blood cells from the bone marrow, speeds up red cell maturation 3–4 times and increases the reticulocyte count within 3–4 days. RHuEPO can be effectively used in strategies to avoid blood transfusion. The response is improved by oral or intravenous iron. Erythropoietin has proven efficacy in addressing anaemia associated with a variety of conditions and, with or without iron, can be an alternative to red blood cells transfusion.

• MeSH
• anemie farmakoterapie   terapie   MeSH
• erythropoetin aplikace a dávkování   farmakologie   terapeutické užití   MeSH
• erytropoéza fyziologie   účinky léků   MeSH
• kombinovaná farmakoterapie MeSH
• lékařská onkologie metody   MeSH
• lidé MeSH
• nádory farmakoterapie   komplikace   MeSH
• nežádoucí účinky léčiv komplikace   MeSH
• pooperační komplikace farmakoterapie   terapie   MeSH
• železo aplikace a dávkování   farmakologie   terapeutické užití   MeSH
• Check Tag
• lidé MeSH

Extrahepatální projevy infekce virem hepatitidy C (HCV) jsou velmi časté. Nejčastější z nich je smíšená kryoglobulinemie. Protilátky anti-HCV a ribonukleovou kyselinu viru, HCV RNA, lze nalézt v kryoprecipitátech, společně s revmatoidním faktorem. Kryoglobuliny se skládají z komplexu imunoglobulinů, které in vitro precipitují při ochlazení pod teplotu lidského těla. Vaskulitida je vyvolána ukládáním těchto imunokomplexů v malých cévách. Souvislost s infekcí HCV je považována za prokázanou i u membranoproliferativní glomerulonefritidy, leukocytoklastické vaskulitidy, lymfoproliferativních onemocnění (zejména B-buněčného lymfomu), Sjögrenova a sicca syndromu, lichen planus, porfyria cutanea tarda a diabetes mellitus. Velmi pravděpodobný je i vztah chronické infekce HCV k onemocněním štítné žlázy, artralgiím, jinak nevysvětlitelné únavě a autoimunitní hepatitidě.

Extrahepatic manifestations of hepatitis C virus infection (HCV) are very common. The most common of these is mixed cryoglobulinaemia. Anti-HCV antibodies and viral ribonucleic acid, HCV RNA, can be found in the cryoprecipitates, together with the rheumatoid factor. Cryoglobulins consist of a complex of immunoglobulins that in vitro precipitate upon the cooling bellow the human body temperature. Vasculitis is caused by the deposition of such immune complexes in the small blood vessels. A link with the HCV infection is considered to be established with membranoproliferative glomerulonephritis, leukocytoclastic vasculitis, lymphoproliferative disorders (in particular B cell lymphoma), Sjögren and sicca syndrome, lichen planus, porfyria cutanea tarda and diabetes mellitus. Very probable is the relationship of chronic HCV infection and thyroid disease, arthralgias, otherwise unexplained fatigue and autoimmune hepatitis.

• Klíčová slova
• extrahepatální manifestace,
• MeSH
• antivirové látky aplikace a dávkování   terapeutické užití   MeSH
• chronická hepatitida C * diagnóza   farmakoterapie   komplikace   MeSH
• cyklofosfamid terapeutické užití   MeSH
• diabetes mellitus 2. typu farmakoterapie   komplikace   MeSH
• glukokortikoidy terapeutické užití   MeSH
• kryoglobulinemie diagnóza   farmakoterapie   komplikace   MeSH
• lichen planus diagnóza   farmakoterapie   MeSH
• lymfoproliferativní nemoci diagnóza   etiologie   MeSH
• nemoci ledvin diagnóza   farmakoterapie   komplikace   MeSH
• rituximab aplikace a dávkování   terapeutické užití   MeSH

Dosažené pokroky v protinádorové léčbě jsou do jisté míry vykoupeny výskytem jejích nežádoucích účinků. K nejzávažnějším patří tzv. s léčbou související akutní myeloidní leukemie. Nárůst jejich incidence je v současnosti jedním z nejrychlejších mezi nádorovými chorobami. Příčiny vzniku nejsou přesně objasněny, předpokládá se účast několika patogenetických mechanismů. Prognóza těchto onemocnění je obecně velmi nepříznivá, na čemž se podílí jak toxicita předchozí protinádorové léčby, tak i nepříznivý biologický charakter leukemie. Volba léčebného přístupu závisí na celkovém stavu nemocného a na genetické prognóze konkrétního onemocnění. Pro většinu nemocných je jedinou potenciálně kurativní metodou léčby alogenní transplantace krvetvorných buněk. Snížení rizika vzniku těchto komplikací a rozšíření jejich léčebných možností patří k důležitým aktuálním úkolům onkologie a hematologie.

Advances achieved in anticancer treatment are partly redeemed by adverse events. So called therapy-related acute myeloid leukemia belongs to the most serious ones. Their incidence growth is one of the most rapid among neoplastic disorders nowadays. Causes of developement of these complications are not clearly clarified. Several pathogenetic mechanisms involvement is anticipated. Generally speaking, prognosis of these disorders is very poor, partly due to toxicity of previous antitumour treatment, partly due to adverse biological character of this leukemia group. The therapeutic approach depends on patient`s performance status as well as genetic prognosis of the disease itself. Allogeneic hematopoietic cells transplantation is the only potentially curative option for majority of patients. Reduction of incidence of the disease and expansion of therapeutic possibilities are very important tasks of both oncology and heamatology.

• Klíčová slova
• sekundární leukemie,
• MeSH
• akutní myeloidní leukemie * etiologie   farmakoterapie   chemicky indukované   MeSH
• antitumorózní látky škodlivé účinky   MeSH
• cytostatické látky škodlivé účinky   MeSH
• lidé MeSH
• lymfoproliferativní nemoci komplikace   MeSH
• myelodysplastické syndromy chemicky indukované   MeSH
• nádory prsu komplikace   MeSH
• nádory vyvolané zářením MeSH
• radioterapie škodlivé účinky   MeSH
• sekundární malignity * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

BACKGROUND: Chyothorax is an uncommon medical condition. To the best of our knowledge, there have been no detailed English-language report dealing with its cytopathologic findings and diagnostic pitfalls CASES: A 12-year-old boy, hemodialysis dependent, with congenital nephrotic syndrome due to focal segmental glomerular sclerosis and a failed renal transplant, developed shortness of breath. Physical and radiologic examinations revealed a left pleural effusion. A 7-year-old boy developed shortness of breath, with a subsequent finding of a left pleural effusion. Multiple osteolytic skeletal lesions were found in this patient. Both patients underwent thoracocentesis. Cytologically, both fluids contained many relatively uniform, large lymphoid cells with high nuclear/cytoplasmic (N/C) ratio, condensed chromatin and occasional nucleoli, resembling blasts. Some nuclei were convoluted. Mitotic figures were present. Foamy macrophages were present in both cases. The differential diagnosis of these populations of cells included a lymphoproliferative disorder. However, the mature T-lymphocytic nature of the cells was confirmed by immunohistochemistry performed on cell block preparations, confirming the clinical impression of chylothorax in both cases. The first patient had chylothorax as a result of trauma due to therapeutic interventions (subclavian vein cannulation), in the second patient the chylothorax was a part of Gorham-Stout syndrome. CONCLUSION: The large T-lymphocytes that are the major cellular component of chylothorax may arouse suspicion of a lymphoproliferative disorder. Attention to the clinical history and immunophenotyping confirm the benign nature of the pleural space fluid. Also, abundant foamy macrophages can be considered a low-power clue to this diagnosis.

• MeSH
• chylotorax diagnóza   metabolismus   patologie   MeSH
• diferenciální diagnóza MeSH
• dítě MeSH
• finanční podpora výzkumu jako téma MeSH
• imunohistochemie MeSH
• lidé MeSH
• lymfoproliferativní nemoci diagnóza   MeSH
• T-lymfocyty metabolismus   patologie   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH

Mechanisms responsible for natural control of human immunodeficiency type 1 (HIV) replication in elite controllers (EC) remain incompletely defined. To determine if EC generate high quality HIV-specific IgA responses, we used Western blotting to compare the specificities and frequencies of IgA to HIV antigens in serum of gender-, age- and race-matched EC and aviremic controllers (HC) and viremic noncontrollers (HN) on highly active antiretroviral therapy (HAART). Concentrations and avidity of IgA to HIV antigens were measured using ELISA or multiplex assays. Measurements for IgG were performed in parallel. EC were found to have stronger p24- and V1V2-specific IgG responses than HN, but there were no IgG differences for EC and HC. In contrast, IgA in EC serum bound more frequently to gp160 and gag proteins than IgA in HC or HN. The avidity of anti-gp41 IgA was also greater in EC, and these subjects had stronger IgA responses to the gp41 heptad repeat region 1 (HR1), a reported target of anti-bacterial RNA polymerase antibodies that cross react with gp41. However, EC did not demonstrate greater IgA responses to E. coli RNA polymerase or to peptides containing the shared LRAI sequence, suggesting that most of their HR1-specific IgA antibodies were not induced by intestinal microbiota. In both EC and HAART recipients, the concentrations of HIV-specific IgG were greater than HIV-specific IgA, but their avidities were comparable, implying that they could compete for antigen. Exceptions were C1 peptides and V1V2 loops. IgG and IgA responses to these antigens were discordant, with IgG reacting to V1V2, and IgA reacting to C1, especially in EC. Interestingly, EC with IgG hypergammaglobulinemia had greater HIV-specific IgA and IgG responses than EC with normal total IgG levels. Heterogeneity in EC antibody responses may therefore be due to a more focused HIV-specific B cell response in some of these individuals. Overall, these data suggest that development of HIV-specific IgA responses and affinity maturation of anti-gp41 IgA antibodies occurs to a greater extent in EC than in subjects on HAART. Future studies will be required to determine if IgA antibodies in EC may contribute in control of viral replication.

• MeSH
• afinita protilátek MeSH
• dospělí MeSH
• ELISA MeSH
• HIV infekce farmakoterapie   imunologie   MeSH
• HIV obalový protein gp41 imunologie   MeSH
• HIV protilátky biosyntéza   MeSH
• imunoglobulin A krev   MeSH
• imunoglobulin G krev   MeSH
• lidé středního věku MeSH
• lidé MeSH
• vysoce aktivní antiretrovirová terapie * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH