Biomedical subjects

D Soldati

Publications and source records attributed to D Soldati.

At least 19 recordsLinked to original sources

PubMed2001

Modulation of myosin A expression by a newly established tetracycline repressor-based inducible system in Toxoplasma gondii.

We have developed a control system for regulating gene activation in Toxoplasma gondii. The elements of this system are derived from the Escherichia coli tetracycline resistance operon, which has been widely used to tightly control gene expression in eukaryotes. The tetracycline repressor (tetR) interferes with transcription initiation while the chimeric transactivator, composed of the tetR fused to the activating domain of VP16 transcriptional factor, allows tet-dependent transcription. Accordingly, tetracycline derivatives such as anhydrotetracycline, which we found to be well tolerated by T.gondii, can serve as effector molecules, allowing control of gene expression in a reversible manner. As a prerequisite to functionally express the tetR in T.gondii, we used a synthetic gene with change of codon frequency. Whereas no activation of transcription was achieved using the synthetic tetracycline-controlled transactivator, tTA2(s), the TetR(s )modulates parasite transcription over a range of approximately 15-fold as measured for several reporter genes. We show here that the tetR-dependent induction of the T.gondii myosin A transgene expression drastically down-regulates the level of endogenous MyoA. This myosin is under the control of a tight feedback mechanism, which occurs at the protein level.

Animals↗

PubMed2001

Attenuated Toxoplasma gondii ts-4 mutants engineered to express the Leishmania antigen KMP-11 elicit a specific immune response in BALB/c mice.

In order to test recombinant Toxoplasma as adjuvant and live vaccine carrier in the infectious disease model of murine experimental leishmaniasis, we engineered the attenuated, temperature-sensitive Toxoplasma gondii strain ts-4 to express the heterologous Leishmania antigen kinetoplastid membrane protein-11 (KMP-11). Transgenic ts-4 clones were obtained which express KMP-11 as cytoplasmatic protein or target it to the secretory pathway of the tachyzoites. Immunization of BALB/c mice with these stably transformed parasites elicited proliferative responses to both T. gondii antigen and recombinant KMP-11. When challenged with Leishmania major, we observed significant protection in animals that had been vaccinated with the KMP-11-expressing ts-4 mutants. The adjuvant attenuated only the onset of the Leishmania infection, but animals were ultimately not able to control the disease. Thus, our findings demonstrate that recombinant Toxoplasma has the potential to serve as an efficient vaccine carrier for cutaneous leishmaniasis. Furthermore, they establish a protective role for the antigen KMP-11 when given in such a vaccine formulation.

Animals↗

PubMed2001

Toxoplasma gondii myosins B/C: one gene, two tails, two localizations, and a role in parasite division.

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

Alternative Splicing↗

PubMed2001

Identification and characterization of an escorter for two secretory adhesins in Toxoplasma gondii.

The intracellular protozoan parasite Toxoplasma gondii shares with other members of the Apicomplexa a common set of apical structures involved in host cell invasion. Micronemes are apical secretory organelles releasing their contents upon contact with host cells. We have identified a transmembrane micronemal protein MIC6, which functions as an escorter for the accurate targeting of two soluble proteins MIC1 and MIC4 to the micronemes. Disruption of MIC1, MIC4, and MIC6 genes allowed us to precisely dissect their contribution in sorting processes. We have mapped domains on these proteins that determine complex formation and targeting to the organelle. MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. In contrast, MIC1 is linked to a quality control system and is absolutely required for the complex to leave the early compartments of the secretory pathway. MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act. We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite.

Animals↗

PubMed2001

Treatment of early stage squamous-cell carcinoma of the glottic larynx: endoscopic surgery or cricohyoidoepiglottopexy versus radiotherapy.

BACKGROUND: Both surgery and radiotherapy are recognized treatments of T1-T2 squamous cell carcinoma of the larynx. We retrospectively analyze and compare the oncological outcome of patients treated in a single institution, either by endoscopic surgery or partial supracricoid laryngectomy versus radiation therapy. METHODS: The medical records of 156 patients treated between 1983 and 1996 with either surgery (n = 75) or radiotherapy (n = 81) were reviewed. Male to female ratio, median age, and T-stage distribution were comparable. RESULTS: With a median follow-up time of 59 months, the 5-year cause-specific survival rate of 93% was identical for both groups. The actuarial incidence of metachronous second primaries was 7% at 5 years. Local control at 5 years remained 84% after surgery and 77% after radiotherapy. Anterior commissure infiltration was shown to represent a negative predictive factor of local control for radiotherapy (p =.01). Salvage treatment brought ultimate local control to 96% of patients after surgery and 94% after radiation therapy with long-term laryngeal preservation rate altered significantly (p =.05) in the group of patients who received radiotherapy (90.1% vs 97.4%). CONCLUSION: The treatment of laryngeal cancer is always a compromise between oncological efficiency and preservation of function. Our data suggest that, assuming proper selection of patients, radiation therapy and surgery yield similar local control and survival rates. The functional disadvantages after surgery are moderate and clearly counterbalanced by a significant decrease in long-term laryngeal preservation rate after radiotherapeutic treatment.

Carcinoma, Squamous Cell↗

PubMed2001

Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii.

Host-cell invasion by apicomplexan parasites is extremely rapid and relies on a sequence of events that are tightly controlled in time and space. In most Apicomplexa, the gliding motility and host-cell invasion are tightly coupled to the release of microneme proteins at the apical tip of the parasites and their redistribution toward the posterior pole. This movement is dependent on an intact parasite actomyosin system. Micronemes are involved in the trafficking and storage of ligands (MICs) for host-cell receptors that are not only structurally related but also functionally conserved among the Apicomplexa. In Toxoplasma gondii, the repertoire of membrane-spanning microneme proteins includes adhesins such as TgMIC2 and escorters such as TgMIC6. The latter forms a complex with the soluble adhesins, TgMIC1 and TgMIC4 and assures their proper sorting to the mironemes. Escorters are also anticipated to bridge host-cell receptors to the parasite membrane during invasion. Most TgMICs are proteolytically cleaved either during their transport along the secretory pathway and/or after exocytosis. The biological significance of these processing events is largely unknown. One of these processing events targets a conserved motif close to the membrane-spanning domain causing the release of the processed form of the micronemes from the parasite surface. The cleavages occurring after release might contribute to the disassembly of the complexes and thus to fission between the parasitophorous vacuole and the host plasma membrane at the end of the invasion process. Gliding motility and host-cell penetration involve the redistribution of the micronemes toward the posterior pole of the parasites. This capping process involves actin polymerisation, myosin adenosine triphosphatase activation and the establishment of a connection between the MICs-receptor complexes and the actomyosin system of the parasite. The most carboxy-terminal end of the MICs cytoplasmic tails is implicated in this process, but the precise nature of the connection with the actomyosin system remains to be elucidated.

Actomyosin↗

PubMed2001

Identification of a human immunodominant B-cell epitope within the GRA1 antigen of Toxoplasma gondii by phage display of cDNA libraries.

Excreted secreted antigens of the protozoan parasite Toxoplasma gondii play a key role in stimulating the host immune system during acute and chronic infection. With the aim of identifying the immunodominant epitopes of T. gondii antigens involved in the human B-cell response against the parasite, we employed a novel immunological approach. A library of cDNA fragments from T. gondii tachyzoites was displayed as fusion proteins to the amino-terminus of lambda bacteriophage capsid protein D. The lambda D-tachyzoite library was then affinity-selected by using a panel of sera of pregnant women, all infected with the parasite. Some of the clones identified through this procedure matched the sequence of the dense granule GRA1 protein (p24), allowing us to identify its antigenic regions. In particular, the analysis of human antibody response against the recombinant GRA1 antigen fragments revealed the existence of an immunodominant epitope (epi-24 peptide).

Amino Acid Sequence↗

PubMed2001

Targeting of soluble proteins to the rhoptries and micronemes in Toxoplasma gondii.

Rhoptry and microneme organelles of the protozoan parasite Toxoplasma gondii are closely associated with host cell adhesion/invasion and establishment of the intracellular parasitophorous vacuole. In order to study the targeting of proteins to these specialized secretory organelles, we have engineered green fluorescent protein (GFP) fusions to the rhoptry protein ROP1 and the microneme protein MIC3. Both chimeras are correctly targeted to the appropriate organelles, permitting deletion analysis to map protein subdomains critical for targeting. The propeptide and a central 146 amino acid region of ROP1 are sufficient to target GFP to the rhoptries. More extensive deletions result in a loss of rhoptry targeting; the GFP reporter is diverted into the parasitophorous vacuole via dense granules. Certain MIC3 deletion mutants were also secreted into the parasitophorous vacuole via dense granules, supporting the view that this route constitutes the default pathway in T. gondii, and that specific signals are required for sorting to rhoptries and micronemes. Deletions within the cysteine-rich central region of MIC3 cause this protein to be arrested at various locations within the secretory pathway, presumably due to improper folding. Although correctly targeted to the appropriate organelles in living parasites, ROP1-GFP and MIC3-GFP fusion proteins were not secreted during invasion. GFP fusion proteins were readily secreted from dense granules, however, suggesting that protein secretion from rhoptries and micronemes might involve more than a simple release of organellar contents.

Adhesins, Bacterial↗

PubMed2001

Toxoplasmosis: a persisting challenge.

The sixth biennial International Congress on Toxoplasmosis, organized by Uwe Gross (University of Göttingen, Germany), was held on 21-25 May 2001 in Freising, Germany. The first meeting of this kind in 1990 was attended by only 26 investigators and this year there were 115 participants covering various research topics including the immunology, epidemiology, cellular and molecular biology of Toxoplasma gondii.

Animals↗

PubMed2001

Knowledge about cholesteatoma, from the first description to the modern histopathology.

OBJECTIVE: The aim of this study was to understand the historical development of the knowledge of cholesteatoma. MATERIALS AND METHODS: Review of the literature from 1683 to 1999 concerning the date-related knowledge of cholesteatoma. RESULTS: In 1683, Duverney first described a temporal bone tumor probably corresponding to a cholesteatoma. Until 1838, when Müller coined the term cholesteatoma, nothing new about this condition appeared in medical publications. After 1838, three main theories about the pathogenesis of cholesteatoma were published. Virchow, in 1855, considered cholesteatoma to be a tumor arising from the metaplasia of mesenchymal cells to epidermal cells, growing then as tumoral cells. Gruber, Wendt, and von Troeltsch, in 1868, considered cholesteatoma to be the result of a metaplasia not of bone cells but of tympanic mucosa cells into a malpighian epithelium. Politzer, in 1869, assumed that cholesteatoma was a glandular neoplasm of middle ear mucosa. Bezold and Habermann, in 1889, considered cholesteatoma to be the result of migration of the external ear canal epidermis into the tympanic cavity via a marginal perforation after acute or chronic otitis. It took 40 years of discussions about these three theories to finally confirm that Habermann and Bezold were correct. CONCLUSION: The knowledge of cholesteatoma has evolved with other medical branches. As otologists began to monitor their patients in vivo, not limiting their observations to temporal bone dissections, the genesis of cholesteatoma became well understood. Today, with immunology and new histopathologic techniques, it is anticipated that we will learn much more about cholesteatoma.

Cholesteatoma, Middle Ear↗

PubMed2000

The toxoplasma micronemal protein MIC4 is an adhesin composed of six conserved apple domains.

The initial stage of invasion by apicomplexan parasites involves the exocytosis of the micronemes-containing molecules that contribute to host cell attachment and penetration. MIC4 was previously described as a protein secreted by Toxoplasma gondii tachyzoites upon stimulation of micronemes exocytosis. We have microsequenced the mature protein, purified after discharge from micronemes and cloned the corresponding gene. The deduced amino acid sequence of MIC4 predicts a 61-kDa protein that contains 6 conserved apple domains. Apple domains are composed of six spacely conserved cysteine residues which form disulfide bridges and are also present in micronemal proteins from two closely related apicomplexan parasites, Sarcocystis muris and Eimeria species, and several mammalian serum proteins, including kallikrein. Here we show that MIC4 localizes in the micronemes of all the invasive forms of T. gondii, tachyzoites, bradyzoites, sporozoites, and merozoites. The protein is proteolytically processed both at the N and the C terminus only upon release from the organelle. MIC4 binds efficiently to host cells, and the adhesive motif maps in the most C-terminal apple domain.

Amino Acid Sequence↗

PubMed2000

Cholesteatoma in children: techniques and results.

A retrospective analysis of the medical records of all cases of cholesteatoma in children treated between 1981 and 1996 was performed. The charts of 59 children with cholesteatoma were reviewed. A total of 62 ears received surgery over 15 years. The median follow-up period was 5 years. There were 132 operations. An intact canal wall (ICW) procedure was performed in 29% of the ears in the first stage, a canal wall down (CWD) procedure in 37%, a transmeatal atticotomy (TA) in 21%, a tympanoplasty (T) in 6.5% and a myringoplasty (M) in 6.5% of the ears. The ICW procedure had a higher rate of residual/recurrent cholesteatoma than did CWD (P=0.8), TA (P=0.4), T (P=0.5), and M (P=0.05). The CWD procedure had a lower rate than TA (P=0.7), but a higher rate than T (P=0.6) and M (P=0.09). Lastly, TA had a higher rate than T (P=0.7) and M (P=0.1). Auditory results were similar for type II and type III reconstructions (P=0.5). An air-bone gap of less than 20 dB was achieved in 51% of the ears, and 80% had a gap of less than 40 dB. We found a clear difference, although not statistically significant, in the personal rate of recurrent cholesteatoma. It was 26% for surgeons who had performed more than 350 otological surgeries and fell to 15% for the most-experienced surgeon (1715 operations). It rose to 34% for the less experienced surgeons (P=0.8). We recommend that surgery for children with cholesteatoma be reserved for experienced surgeons.

Adolescent↗

PubMed2000

The microneme protein MIC4, or an MIC4-like protein, is expressed within the macrogamete and associated with oocyst wall formation in Toxoplasma gondii.

The expression and localisation of MIC4, or an immuno-cross reacting MIC4-like protein, was examined in the enteric forms of Toxoplasma gondii using immunocytochemistry. In addition to being located within the micronemes of the merozoites, MIC4 or the MIC4-like protein was present within the macrogamete and was associated with the developing oocyst wall. The macrogamete is characterised by two types of structurally distinct wall forming bodies (WFB1 and 2). However, by immuno-electron microscopy, it was possible to identify two populations of dense granules (WFB1) which appear to form sequentially during macrogamete development. The first granules to form (WFB1a) stained positively with anti-MIC4 and were followed by MIC4 negative granules (WFB1b). During oocyst wall formation, the WFB1a and b sequentially released their contents onto the surface with WFB1a material forming an anti-MIC4 positive outer veil, while the WFB1b forms the electron dense outer layer of the oocyst wall. The inner layer was formed by WFB2. Thus, for the first time, it was possible to identify two populations of dense granules (WFB1a and b) involved in the formation of different parts of the oocyst wall. It was not possible to analyse the contents of macrogametes by western blot to unequivocally identify the antigen recognised by the polyclonal antisera as MIC4.

Animals↗

PubMed2000

A dibasic motif in the tail of a class XIV apicomplexan myosin is an essential determinant of plasma membrane localization.

Obligate intracellular parasites of the phylum Apicomplexa exhibit gliding motility, a unique form of substrate-dependent locomotion essential for host cell invasion and shown to involve the parasite actin cytoskeleton and myosin motor(s). Toxoplasma gondii has been shown to express three class XIV myosins, TgM-A, -B, and -C. We identified an additional such myosin, TgM-D, and completed the sequences of a related Plasmodium falciparum myosin, PfM-A. Despite divergent structural features, TgM-A purified from parasites bound actin in an ATP-dependent manner. Isoform-specific antibodies revealed that TgM-A and recombinant mycTgM-A were localized right beneath the plasma membrane, and subcellular fractionation indicated a tight membrane association. Recombinant TgM-D also had a peripheral although not as sharply defined localization. Truncation of their respective tail domains abolished peripheral localization and tight membrane association. Conversely, fusion of the tails to green fluorescent protein (GFP) was sufficient to confer plasma membrane localization and sedimentability. The peripheral localization of TgM-A and of the GFP-tail fusion did not depend on an intact F-actin cytoskeleton, and the GFP chimera did not localize to the plasma membrane of HeLa cells. Finally, we showed that the specific localization determinants were in the very C terminus of the TgM-A tail, and site-directed mutagenesis revealed two essential arginine residues. We discuss the evidence for a proteinaceous plasma membrane receptor and the implications for the invasion process.

Actins↗

PubMed2000

Two conserved amino acid motifs mediate protein targeting to the micronemes of the apicomplexan parasite Toxoplasma gondii.

The micronemal protein 2 (MIC2) of Toxoplasma gondii shares sequence and structural similarities with a series of adhesive molecules of different apicomplexan parasites. These molecules accumulate, through a yet unknown mechanism, in secretory vesicles (micronemes), which together with tubular and membrane structures form the locomotion and invasion machinery of apicomplexan parasites. Our findings indicated that two conserved motifs placed within the cytoplasmic domain of MIC2 are both necessary and sufficient for targeting proteins to T. gondii micronemes. The first motif is based around the amino acid sequence SYHYY. Database analysis revealed that a similar sequence is present in the cytoplasmic tail of all transmembrane micronemal proteins identified so far in different apicomplexan species. The second signal consists of a stretch of acidic residues, EIEYE. The creation of an artificial tail containing only the two motifs SYHYY and EIEYE in a preserved spacing configuration is sufficient to target the surface protein SAG1 to the micronemes of T. gondii. These findings shed new light on the molecular mechanisms that control the formation of the microneme content and the functional relationship that links these organelles with the endoplasmic reticulum of the parasite.

Amino Acid Motifs↗

PubMed2000

Toxoplasma gondii catalase: are there peroxisomes in toxoplasma?

The intracellular protozoan parasite Toxoplasma gondii, like all members of the phylum Apicomplexa, is known to possess many organelles: in addition to mitochondria and the compartments of the secretory pathway, there is a reduced chloroplast (the apicoplast) and the phylum-specific components of the apical complex: dense granules, micronemes and rhoptries. Conspicuously missing so far are microbodies, organelles that can be found in nearly all eukaryotic organisms. Microbodies show a large variation with regard to their size, number and contents, depending on the organism and cell type. One marker enzyme of this single membrane-bound organelle is catalase, which is responsible for the degradation of hydrogen peroxide to water and oxygen. The EST project in T. gondii revealed the existence of two overlapping clones which showed similarity with catalase, and these were used to clone the corresponding gene. The predicted sequence of T. gondii catalase has -AKM at the C terminus, which falls within the consensus of the PTS1 peroxisomal targeting signal. Southern blot analysis confirmed the presence of a single copy gene. Northern and western blot analyses showed that the catalase gene is transcribed and translated. Immunofluorescence assays using an antibody raised against a catalase peptide identified a distinct structure towards the apical end, but other catalase-specific antibodies failed to confirm this localisation. Cell fractionations indicated that the majority of the enzyme was in the cytosol. The fusion of the C-terminal twelve amino acids, including AKM, or the canonical peroxisomal targeting signal, -SKL, to GFP resulted in predominantly cytosolic localization in T. gondii. There was therefore no evidence for membrane-bound peroxisomes in Toxoplasma.

Amino Acids↗

PubMed2000

[Treatment of laryngeal cancer: cordectomy or crico-hyoido-epiglottopexy versus radiotherapy].

INTRODUCTION: Both radiotherapy and endoscopic or open functional surgery are recognised treatments for laryngeal cancer stage I and II. A comparison between two groups of patients treated with either modality may clarify the indications for both treatments. METHODS: Over a period of 13 years two separate series of patients were treated for laryngeal cancer (stage I and II) by either surgery (n = 72) or radiotherapy (n = 81). We have analysed and compared the two groups. RESULTS: Statistical analyses show a better local control among patients treated with surgery, when the anterior commissure was involved (p < 0.01) or with extension of the tumour (T2). However, long-term survivals were not significantly different in the two groups. As postradiation recurrence was diagnosed at an early stage, salvage (requiring total laryngectomy in many cases) was efficient but contributed to an appreciable difference in the long-term laryngeal preservation rate between the two groups (91% after radiotherapy and 99% after surgery). CONCLUSION: The treatment of laryngeal cancer must always compromise between oncological efficiency and functional preservation. With anterior commissure involvement (T1b) or more extensive disease (T2), surgery appears to be better. Therefore, preservation of perfect laryngeal function should be subordinate to oncological safety.

Carcinoma, Squamous Cell↗

PubMed2000

[Socioeconomic aspects in the therapy of peritonsillar absscess].

OBJECTIVE: To determine which treatment for quinsy is the most cost-effective option. MATERIAL AND METHOD: 277 quinsies were diagnosed in 262 patients in our institution between 1.1.1991 and 31.12.1998. Median age was 29 years (1-89 years), and sex ratio was 3 males:2 females. 82 quinsy tonsillectomies (AC), 91 drainages with interval tonsillectomy (DAF) and 104 drainages of the abscess without tonsillectomy (D) were carried out. RESULTS: Median hospital stay was 3 (1-7) days for quinsy tonsillectomy, and 10 (4-18) days for drainages with interval tonsillectomy (hospital stay after drainage added to hospital stay after tonsillectomy). If interval tonsillectomy is performed as an outpatient procedure, median hospital stay decreases to 5 (2-8) days. For patients who refused tonsillectomy, median hospital stay was 3 (0-14) days. Median disability after treatment was 15 (7-30) days for quinsy tonsillectomy. For drainages with interval tonsillectomy (disability succeeding the drainage added to that following interval tonsillectomy), the median is 20 (15-52) days. For patients who refused tonsillectomy it is 6 (0-15) days. In the AC group we counted 9 late haemorrhages (11%). In the DAF group 14 patients (15%) presented a late haemorrhage; 6 patients (6%) presented a postoperative superinfection of the tonsillar fossae. DISCUSSION: On the basis of the tariffs of our institution (CHUV), and of statistical data obtained from the National Institute for Social Insurance (SUVA) with regard to the economic impact of each day of disability, the cost of the various treatment options is presented. CONCLUSION: It results from our study that in the absence of a significant difference in the rate of complications, and even considering the possibility of carrying out interval amygdalectomy on an outpatient basis, the most cost-effective treatment of peritonsillar abscess is quinsy tonsillectomy.

Adolescent↗