Maternal LAMP/p55gagHIV-1 DNA Immunization Induces In Utero Priming and a Long-Lasting Immune Response in Vaccinated Neonates

Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+ CD25+ Foxp3+ T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-gamma-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods.

SARS coronavirus nucleocapsid immunodominant T-cell epitope cluster is common to both exogenous recombinant and endogenous DNA-encoded immunogens

Correspondence between the T-cell epitope responses of vaccine immunogens and those of pathogen antigens is critical to vaccine efficacy. In the present study, we analyzed the spectrum of immune responses of mice to three different forms of the SARS coronavirus nucleocapsid (N): (1) exogenous recombinant protein (N-GST) with Freund's adjuvant; (2) DNA encoding unmodified N as an endogenous cytoplasmic protein (pN); and (3) DNA encoding N as a LAMP-I chimera targeted to the lysosomal MHC II compartment (p-LAMP-N). Lysosomal trafficking of the LAMP/N chimera in transfected cells was documented by both confocal and immunoelectron microscopy. The responses of the immunized mice differed markedly. The strongest T-cell IFN-gamma and CTL responses were to the LAMP-N chimera followed by the pN immunogen. In contrast, N-GST elicited strong T cell IL-4 but minimal IFN-gamma responses and a much greater antibody response. Despite these differences, however, the immunodominant T-cell ELISpot responses to each of the three immunogens were elicited by the same N peptides, with the greatest responses being generated by a cluster of five overlapping peptides, N76-114, each of which contained nonameric H2(d) binding domains with high binding scores for both class I and, except for N76-93, class II alleles. These results demonstrate that processing and presentation of N, whether exogenously or endogenously derived, resulted in common immunodominant epitopes, supporting the usefulness of modified antigen delivery and trafficking forms and, in particular, LAMP chimeras as vaccine candidates. Nevertheless, the profiles of T-cell responses were distinctly different. The pronounced Th-2 and humoral response to N protein plus adjuvant are in contrast to the balanced IFN-gamma and IL-4 responses and strong memory CTL responses to the LAMP-N chimera. (C) 2005 Elsevier Inc. All rights reserved.

Analysis of the peripheral T-cell compartment in the MHC class II deficiency syndrome.

To analyse the impact of lack of MHC class II expression on the composition of the peripheral T-cell compartment in man, the expression characteristics of several membrane antigens were examined on peripheral blood lymphocytes (PBL) and cultured T cells derived from an MHC-class-II-deficient patient. No MHC class II expression could be detected on either PBL or activated T cells. Moreover, the expression of MHC class I was reduced both on PBL and in vitro activated T cells compared to the healthy control. However, the reduced expression of CD26 observed on the PBL of the patient was restored after in vitro expansion. Despite the presumably class-II-deficient thymic environment, a distinct but reduced single CD4+ T-cell population was observed in the PBL of the patient. After in vitro expansion, the percentage of CD4+ cells dropped even further, most likely due to a proliferative disadvantage, compared to the single CD8+ T-cell population. However, proliferation analysis showed that T-cell activation via the TcR/CD3 pathway is not affected by the MHC class II deficiency.

TCR V alpha- and V beta-gene segment use in T-cell subcultures derived from a type-III bare lymphocyte syndrome patient deficient in MHC class-II expression.

Previously, we and others have shown that MHC class-II deficient humans have greatly reduced numbers of CD4+CD8- peripheral T cells. These type-III Bare Lymphocyte Syndrome patients lack MHC class-II and have an impaired MHC class-I antigen expression. In this study, we analyzed the impact of the MHC class-II deficient environment on the TCR V-gene segment usage in this reduced CD4+CD8- T-cell subset. For these studies, we employed TcR V-region-specific monoclonal antibodies (mAbs) and a semiquantitative PCR technique with V alpha and V beta amplimers, specific for each of the most known V alpha- and V beta-gene region families. The results of our studies demonstrate that some of the V alpha-gene segments are used less frequent in the CD4+CD8- T-cell subset of the patient, whereas the majority of the TCR V alpha- and V beta-gene segments investigated were used with similar frequencies in both subsets in the type-III Bare Lymphocyte Syndrome patient compared to healthy control family members. Interestingly, the frequency of TcR V alpha 12 transcripts was greatly diminished in the patient, both in the CD4+CD8- as well as in the CD4-CD8+ compartment, whereas this gene segment could easily be detected in the healthy family controls. On the basis of the results obtained in this study, it is concluded that within the reduced CD4+CD8- T-cell subset of this patient, most of the TCR V-gene segments tested for are employed. However, a skewing in the usage frequency of some of the V alpha-gene segments toward the CD4-CD8+ T-cell subset was noticeable in the MHC class-II deficient patient that differed from those observed in the healthy family controls.

Leishmania major promastigote entry of an autophagy-like compartment and amastigote escape from the parasitophorous vacuole

In this thesis, we investigated the interaction of the obligate intracellular parasite Leishmania (L.) major with two phenotypes of human monocyte derived macrophages (hMDMs). Thereby we focused on the development and maturation of the parasitophorous vacuole (PV) and could show that compartment development is dependent on the parasite stage.rnFocusing on the ultrastructure of PVs containing axenic amastigotes, we demonstrated that the parasites are partially located in damaged PVs or in the cytoplasm of the host. Moreover, we visualized multiple amastigotes in a common PV 144 h p.i. in pro-inflammatory hMDM I but not in anti-inflammatory hMDM II indicating different PV development. rnRegarding the promastigote form, we demonstrated a different uptake of viable and apoptotic L. major promastigotes by hMDMs. Viable promastigotes are predominantly taken up via the flagellum tip whereas apoptotic promastigotes enter the cells via the parasite body. Analyzing compartment maturation, we found that 20-30% of the PVs get positive for the early maturation markers PI3P and EEA1 independent of the viability of the parasites and unaffected by the human macrophage type. Subsequently, 25-40% of the parasites acquire the autophagy marker LC3 on their PV, what is independent of the viability of the parasites as well. We quantified this and in hMDM II less LC3-positive compartments formed compared to hMDM I. Analyzing the ultrastructure, we investigated that the compartments consist of a single-membrane PV characteristic for LC3-associated phagocytosis (LAP). Involvement of LAP was confirmed by demonstrating that the protein kinase ULK1 is dispensable for LC3-compartment formation around Leishmania PVs. Visualizing compartment dynamics in real time showed that apoptotic promastigotes are degraded in LC3-positve compartments, whereas viable promastigotes are able to get rid of LC3-protein on their PV suggesting an involvement in parasite development and survival. In this thesis, we established a lentiviral based fluorescent imaging technique that we combined with High-Pressure-Freezing (HPF) and high-resolution 3D electron microscopy. We visualized a promastigote in a LC3-compartment whose ultrastructure showed an opening of the PV to the outside. To identify new LAP markers involved in Leishmania infection, we established an immuno-magnetic isolation protocol for the purification of Leishmania containing compartments.rnIn conclusion, this study suggests that L. major compartment biogenesis and maturation in pro- and anti-inflammatory human macrophages is dependent on the parasite stage and is different between axenic amastigotes, viable promastigotes and apoptotic promastigotes. Understanding the development and maturation of Leishmania parasites in human host cells is important to control and combat the neglected disease leishmaniasis in the future.rn

Investigación sobre las causas que pudieron originar el hundimiento de la cubierta del tercer depósito del Canal de Isabel II en 1905

En la presente investigación se analiza la causa del hundimiento del cuarto compartimento del Tercer Depósito del Canal de Isabel II el 8 de abril de 1905, uno de los más graves de la historia de la construcción en España: fallecieron 30 personas y quedaron heridas otras 60. El Proyecto y Construcción de esta estructura era de D. José Eugenio Ribera, una de las grandes figuras de la ingeniería civil en nuestro país, cuya carrera pudo haber quedado truncada como consecuencia del siniestro. Dado el tiempo transcurrido desde la ocurrencia de este accidente, la investigación ha partido de la recopilación de la información relativa al Proyecto y a la propia construcción de la estructura, para revisar a continuación la información disponible sobre el hundimiento. De la construcción de la cubierta es interesante destacar la atrevida configuración estructural, cubriéndose una inmensa superficie de 74.000 m2 mediante una sucesión de bóvedas de hormigón armado de tan sólo 5 cm de espesor y un rebajamiento de 1/10 para salvar una luz de 6 m, que apoyaban en pórticos del mismo material, con pilares también muy esbeltos: 0,25 m de lado para 8 m de altura. Y todo ello en una época en la que la tecnología y conocimiento de las estructuras con este "nuevo" material se basaban en buena medida en el desarrollo de patentes. En cuanto a la información sobre el hundimiento, llama la atención en primer lugar la relevancia de los técnicos, peritos y letrados que intervinieron en el juicio y en el procedimiento administrativo posterior, poniéndose de manifiesto la trascendencia que el accidente tuvo en su momento y que, sin embargo, no ha trascendido hasta nuestros días. Ejemplo de ello es el papel de Echegaray -primera figura intelectual de la época- como perito en la defensa de Ribera, de D. Melquiades Álvarez -futuro presidente del Congreso- como abogado defensor, el General Marvá -uno de los máximos exponentes del papel de los ingenieros militares en la introducción del hormigón armado en nuestro país-, que presidiría la Comisión encargada del peritaje por parte del juzgado, o las opiniones de reconocidas personalidades internacionales del "nuevo" material como el Dr. von Emperger o Hennebique. Pero lo más relevante de dicha información es la falta de uniformidad sobre lo que pudo ocasionar el hundimiento: fallos en los materiales, durante la construcción, defectos en el diseño de la estructura, la realización de unas pruebas de carga cuando se concluyó ésta, etc. Pero la que durante el juicio y en los Informes posteriores se impuso como causa del fallo de la estructura fue su dilatación como consecuencia de las altas temperaturas que se produjeron aquella primavera. Y ello a pesar de que el hundimiento ocurrió a las 7 de la mañana... Con base en esta información se ha analizado el comportamiento estructural de la cubierta, permitiendo evaluar el papel que diversos factores pudieron tener en el inicio del hundimiento y en su extensión a toda la superficie construida, concluyéndose así cuáles fueron las causas del siniestro. De los resultados obtenidos se presta especial atención a las enseñanzas que se desprenden de la ocurrencia del hundimiento, enfatizándose en la relevancia de la historia -y en particular de los casos históricos de error- para la formación continua que debe existir en la Ingeniería. En el caso del hundimiento del Tercer Depósito algunas de estas "enseñanzas" son de plena actualidad, tales como la importancia de los detalles constructivos en la "robustez" de la estructuras, el diseño de estructuras "integrales" o la vigilancia del proceso constructivo. Por último, la investigación ha servido para recuperar, una vez más, la figura de D. José Eugenio Ribera, cuyo papel en la introducción del hormigón armado en España fue decisivo. En la obra del Tercer Depósito se arriesgó demasiado, y provocó un desastre que aceleró la transición hacia una nueva etapa en el hormigón estructural al abrigo de un mayor conocimiento científico y de las primeras normativas. También en esta etapa sería protagonista. This dissertation analyses the cause of the collapse of the 4th compartment of the 3th Reservoir of Canal de Isabel II in Madrid. It happened in 1905, on April 8th, being one of the most disastrous accidents occurred in the history of Spanish construction: 30 people died and 60 were injured. The design and construction supervision were carried out by D. José Eugenio Ribera, one of the main figures in Civil Engineering of our country, whose career could have been destroyed as a result of this accident. Since it occurred more than 100 years ago, the investigation started by compiling information about the structure`s design and construction, followed by reviewing the available information about the accident. With regard to the construction, it is interesting to point out its daring structural configuration. It covered a huge area of 74.000 m2 with a series of reinforced concrete vaults with a thickness of not more than 5 cm, a 6 m span and a rise of 1/10th. In turn, these vaults were supported by frames composed of very slender 0,25 m x 0,25 m columns with a height of 8 m. It is noteworthy that this took place in a time when the technology and knowledge about this "new" material was largely based on patents. In relation to the information about the collapse, its significance is shown by the important experts and lawyers that were involved in the trial and the subsequent administrative procedure. For example, Echegaray -the most important intellectual of that time- defended Ribera, Melquiades Álvarez –the future president of the Congress- was his lawyer, and General Marvá -who represented the important role of the military engineers in the introduction of reinforced concrete in our country-, led the Commission that was put in charge by the judge of the root cause analysis. In addition, the matter caught the interest of renowned foreigners like Dr. von Emperger or Hennebique and their opinions had a great influence. Nonetheless, this structural failure is unknown to most of today’s engineers. However, what is most surprising are the different causes that were claimed to lie at the root of the disaster: material defects, construction flaws, errors in the design, load tests performed after the structure was finished, etc. The final cause that was put forth during the trial and in the following reports was attributed to the dilatation of the roof due to the high temperatures that spring, albeit the collapse occurred at 7 AM... Based on this information the structural behaviour of the roof has been analysed, which allowed identifying the causes that could have provoked the initial failure and those that could have led to the global collapse. Lessons have been learned from these results, which points out the relevance of history -and in particular, of examples gone wrong- for the continuous education that should exist in engineering. In the case of the 3th Reservoir some of these lessons are still relevant during the present time, like the importance of detailing in "robustness", the design of "integral" structures or the due consideration of construction methods. Finally, the investigation has revived, once again, the figure of D. José Eugenio Ribera, whose role in the introduction of reinforced concrete in Spain was crucial. With the construction of the 3th Reservoir he took too much risk and caused a disaster that accelerated the transition to a new era in structural concrete based on greater scientific knowledge and the first codes. In this new period he would also play a major role.

Morphological and Functional Association of Sec22b/ERS-24 with the pre-Golgi Intermediate Compartment

Yeast Sec22p participates in both anterograde and retrograde vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus by functioning as a v-SNARE (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein receptor) of transport vesicles. Three mammalian proteins homologous to Sec22p have been identified and are referred to as Sec22a, Sec22b/ERS-24, and Sec22c, respectively. The existence of three homologous proteins in mammalian cells calls for detailed cell biological and functional examinations of each individual protein. The epitope-tagged forms of all three proteins have been shown to be primarily associated with the ER, although functional examination has not been carefully performed for any one of them. In this study, using antibodies specific for Sec22b/ERS-24, it is revealed that endogenous Sec22b/ERS-24 is associated with vesicular structures in both the perinuclear Golgi and peripheral regions. Colabeling experiments for Sec22b/ERS-24 with Golgi mannosidase II, the KDEL receptor, and the envelope glycoprotein G (VSVG) of vesicular stomatitis virus (VSV) en route from the ER to the Golgi under normal, brefeldin A, or nocodazole-treated cells suggest that Sec22b/ERS-24 is enriched in the pre-Golgi intermediate compartment (IC). In a well-established semi-intact cell system that reconstitutes transport from the ER to the Golgi, transport of VSVG is inhibited by antibodies against Sec22b/ERS-24. EGTA is known to inhibit ER–Golgi transport at a stage after vesicle/transport intermediate docking but before the actual fusion event. Antibodies against Sec22b/ERS-24 inhibit ER–Golgi transport only when they are added before the EGTA-sensitive stage. Transport of VSVG accumulated in pre-Golgi IC by incubation at 15°C is also inhibited by Sec22b/ERS-24 antibodies. Morphologically, VSVG is transported from the ER to the Golgi apparatus via vesicular intermediates that scatter in the peripheral as well as the Golgi regions. In the presence of antibodies against Sec22b/ERS-24, VSVG is seen to accumulate in these intermediates, suggesting that Sec22b/ERS-24 functions at the level of the IC in ER–Golgi transport.

Interleaflet clear space is reduced in the membrane of COP I and COP II-coated buds/vesicles.

Intracellular transfers between membrane-bound compartments occur through vesicles that bud from a donor compartment to fuse subsequently with an acceptor membrane. We report that the membrane that delimits COP I or COP II-coated buds/vesicles from the endoplasmic reticulum and the Golgi complex has a thinner interleaflet clear space as compared with the surrounding, noncoated parental membrane. This change is compatible with a compositional change of the membrane bilayer during the budding process.

Spliceosomal small nuclear ribonucleoprotein particle trafficking and maturation in the nuclear compartment

We show for the first time that upon injection into the cytoplasm of the oocyte, fluorescein-labeled spliceosomal snRNAs, in the context of functional snRNPs, are targeted to elongating pre-mRNAs. This finding presents us with a novel assay with which to dissect the mechanism by which snRNPs are targeted to nascent pre-mRNA transcripts. Two critical advantages offered by this system are immediately evident. First, it allows us to investigate the mechanisms employed to recruit snRNPs as it actually transpires within the realm of the cell nucleus. Second, it allows a genome-wide analysis of snRNP recruitment to nascent transcripts, and, hence, the conclusions drawn from these studies do not depend on the sequence of any particular promoter or pre-mRNA. Indeed, it is with this assay that we have stumbled upon a most unanticipated discovery: Contrary to the current paradigm, the co-transcriptional recruitment of splicing snRNPs to nascent transcripts is not contingent on their role in splicing in vivo. Based on these and other data, we have constructed a two-step recruitment-loading model wherein snRNPs are first recruited to pre-mRNA transcripts and only then loaded directly onto cis-acting sequences on nascent pre-mRNA. While conducting studies on snRNP trafficking, a new discovery was made. We found that the lampbrush chromosomes could be visualized by light microscopy in vivo, and that these chromosomes have an architecture that is identical with those in formaldehyde treated nuclear spread preparations. Importantly, we now have the first system with which we can examine the dynamic interactions of macromolecules with specific RNA polymerase II transcriptional units in the live nucleus.