Biomedical subjects

G Grandi

Publications and source records attributed to G Grandi.

At least 19 recordsLinked to original sources

PubMed2006

Immunopathology of Dirofilaria immitis infection.

Heartworm disease caused by Dirofilaria immitis affects canine and feline hosts, with infections occasionally being reported in humans. Studies have shown that both dirofilarial antigens and those derived from its bacterial endosymbiont Wolbachia, interact with the host organism during canine, feline and human infections and participate in the development of the pathology and in the regulation of the host's immune response. Both innate and acquired immune responses are observed and the development of the acquired response may depend on the host and, or on its parasitological status. This review aims at illustrating current research on the role of both D. immitis and Wolbachia, in the immunology and immunopathology of dirofilariosis.

Animals↗

PubMed2006

iNOs expression is stimulated by the major surface protein (rWSP) from Wolbachia bacterial endosymbiont of Dirofilaria immitis following subcutaneous injection in mice.

The bacterial endosymbiont Wolbachia of several species of filarial nematodes plays an important role in the inflammatory pathology of filariasis. Nitric oxide (NO) production has also been implicated in the immune response during filarial infections. Here we present data indicating that a recombinant Wolbachia surface protein (rWSP) induces iNOs mRNA expression and NO production, as well as IFN-gamma and a Th1-type antibody response, in inoculated BALB/c mice. This effect is not observed when mice are inoculated with a recombinant heat shock protein from Wolbachia (GroEL).

Animals↗

PubMed2006

Dogs with patent Dirofilaria immitis infection have higher expression of circulating IL-4, IL-10 and iNOS mRNA than those with occult infection.

Dirofilaria immitis is the agent of canine heartworm disease, in which adult worms reside in the pulmonary arteries, producing first stage larvae (microfilariae) that are released into the bloodstream. The present work describes the cytokine and iNOS mRNA expression in the peripheral blood of naturally infected dogs classified as either microfilariemic or amicrofilariemic. Results show that microfilariemic dogs had higher expression of IL-4 and iNOS mRNA than amicrofilariemic dogs. Furthermore, IL-10 mRNA expression was strongly expressed in dogs with circulating microfilariae, compared to only negligible expression in amicrofilariemic dogs. Finally, mf+ status was associated with a predominance in IgG1 production against worm antigens. These results would suggest that circulating mf may stimulate, like in other filarial infections, an immune bias towards unresponsiveness in D. immitis-infected dogs, consenting long-term adult worm survival.

Animals↗

PubMed2006

The intradermal Leishmanin reaction induces antigen-specific maturation of canine dendritic cells with up-regulation of MHCII synthesis and expression.

Dendritic cells (DCs) are professional antigen-presenting cells that reside in many tissues, including the skin. This study showed that intradermal injection of leishmanin in Leishmania infantum-infected dogs induced the "up-regulation" of surface MHCII expression, associated with progressive ultrastrucutural changes characteristic of DC maturation, including the formation of multilaminar MHC class II-containing compartments and arrays of tubulo-vesicular structures. These changes were not observed in control dogs from L. infantum non-endemic areas. The results indicated that canine DCs were effector cells in delayed-type hypersensitivity, that the leishmanin reaction was specific for a cell-mediated reaction to L. infantum in infected dogs, and that canine DCs possessed ultrastructural organelles reminiscent of those in activated human DCs.

Animals↗

PubMed2005

Aelurostrongylus abstrusus (cat lungworm) infection in five cats from Italy.

Infection by the cat lungworm Aelurostrongylus abstrusus is considered uncommon. Here, the authors report the clinical, diagnostic and therapeutic features of five infections recently observed in Italy. All cats were under 12 months of age. All except one cat had symptomatic infections, with cough, dyspnea, and weight loss with radiographic signs of broncopneumonia. All cats were eosinophilic. Larvae were present in fresh fecal smears and on flotation exam in all cats. Baermann larval recovery permitted definitive identification and, in one case, larvae per gram of feces (lpg) counts. One dose of ivermectin (400 microg/kg) was not effective in one cat, while one dose of selamectin (6 mg/kg) was effective in one of three cases and fenbendazole at 50 mg/kg given daily for 15 days was effective in four of four cases.

Animals↗

PubMed2004

Early development of the pituitary gland in Acipenser naccarii (Chondrostei, Acipenseriformes): an immunocytochemical study.

The distribution and appearance of secretory cells in the pituitary gland were investigated for the first time in a chondrostean species, Acipenser naccarii, from embryos to juveniles, by immunohistochemistry with mammalian and teleost hormone antisera. On 5.5 day post-fertilization (2.5 days pre-hatching), the pituitary of embryos appears as an oval cell mass with a narrow central cavity (hypophysial cleft), close to the ventral border of diencephalon under the third ventricle. At that time no neurohypophysis is observed, the adenohypophysis is not yet structurally divided into pars intermedia (PI) and pars distalis (PD) and only immunoreactive growth hormone cells are detectable. Seven days post-fertilization (1 day pre-hatching) the immunoreactive thyrotropic cells appear in the ventral region and the immunoreactive adrenocorticotropic cells in the posterior dorsal one. At hatching, some immunoreactive melanotropic (ir-MSH) cells are visible in the posterior dorsal region and some immunoreactive prolactin cells in the anterior one. Eight days later the immunoreactive somatolactin cells appear along the posterior dorsal border and the immunoreactive gonadotropic I (ir-GtH I) cells in the ventral region. Here, a few ir-GtH II cells finally appear in 76-86 day old juveniles. The gland elongates after hatching and in 8-day-old larvae two adenohypophysial regions are identified: a posterior (the presumptive PI) and an anterior one (the presumptive PD). In 156-166-day-old juveniles three regions (rostral and proximal pars distalis and pars intermedia) appear and a high number of ir-MSH cells are visible in the rostral region. The first protrusion of neurohypophysis into adenohypophysis is observed in 76-86-day-old juveniles and increases with age, branching into PI. The rostro-caudal distribution of the immunoreactive cells follows the spatial expression of the corresponding hormone gene families observed in zebra fish, suggesting similar differentiating mechanisms in teleosts and chondrosteans.

Adrenocorticotropic Hormone↗

PubMed2002

Gene expression profile in Neisseria meningitidis and Neisseria lactamica upon host-cell contact: from basic research to vaccine development.

Differential gene regulation in the human pathogen Neisseria meningitidis group B (MenB) and in Neisseria lactamica, a human commensal species, was studied by whole genome microarray after bacterial interaction with epithelial cells. Host-cell contact induced changes in the expression of 347 and 285 genes in MenB and N. lactamica, respectively. Of these, only 167 were common to MenB and N. lactamica, suggesting that a different subset of genes is activated by pathogens and commensals. Change in gene expression was stable over time in N. lactamica, but short-lived in MenB. A large part (greater than 30%) of the regulated genes encoded proteins with unknown function. Among the known genes, those coding for pili, capsule, protein synthesis, nucleotide synthesis, cell wall metabolism, ATP synthesis, and protein folding were down-regulated in MenB. Transporters for iron, chloride and sulfate, some known virulence factors, GAPDH and the entire pathway of selenocysteine biosynthesis were upregulated. Gene expression profiling indicates that approximately 40% of the regulated genes encode putative surface-associated proteins, suggesting that upon cell contact Neisseria undergoes substantial surface remodeling. This was confirmed by FACS analysis of adhering bacteria using mouse sera against a subset of recombinant proteins. Finally, a few surface-located, adhesion-activated antigens were capable of inducing bactericidal antibodies, indicating that microarray technology can be exploited for the identification of new vaccine candidates.

Bacterial Adhesion↗

PubMed2001

CD81 extracellular domain 3D structure: insight into the tetraspanin superfamily structural motifs.

Human CD81, a known receptor for hepatitis C virus envelope E2 glycoprotein, is a transmembrane protein belonging to the tetraspanin family. The crystal structure of human CD81 large extracellular domain is reported here at 1.6 A resolution. Each subunit within the homodimeric protein displays a mushroom-like structure, composed of five alpha-helices arranged in 'stalk' and 'head' subdomains. Residues known to be involved in virus binding can be mapped onto the head subdomain, providing a basis for the design of antiviral drugs and vaccines. Sequence analysis of 160 tetraspanins indicates that key structural features and the new protein fold observed in the CD81 large extracellular domain are conserved within the family. On these bases, it is proposed that tetraspanins may assemble at the cell surface into homo- and/or hetero-dimers through a conserved hydrophobic interface located in the stalk subdomain, while interacting with other liganding proteins, including hepatitis C virus E2, through the head subdomain. The topology of such interactions provides a rationale for the assembly of the so-called tetraspan-web.

Amino Acid Sequence↗

PubMed2001

Rationally designed strings of promiscuous CD4(+) T cell epitopes provide help to Haemophilus influenzae type b oligosaccharide: a model for new conjugate vaccines.

The age-related and T cell-independent immunological properties of most capsular polysaccharides limit their use as vaccines, especially in children under 2 years of age. To overcome these limitations, polysaccharide antigens have been successfully conjugated to a variety of carrier proteins, such as diphtheria toxoid or tetanus toxoid (TT) and the diphtheria mutant (CRM197) to produce very successful glycoconjugate vaccines. The increasing demand for new conjugate vaccines requires the availability of additional carriers providing high and long-lasting T helper cell immunity. Here we describe the design and construction of three recombinant carrier proteins (N6, N10, N19) constituted by strings of 6, 10 or 19 human CD4(+) T cell epitopes from various pathogen-derived antigens, including TT and proteins from Plasmodium falciparum, influenza virus and hepatitis B virus. Each of these epitopes is defined as universal in that it binds to many human MHC class II molecules. When conjugated to Haemophilus influenzae type b (Hib) oligosaccharide, these carriers elicit a potent anti-Hib antibody response in mice. In the case of the N19-Hib conjugate, this response is at least as good as that observed with CRM197-Hib, a conjugate vaccine currently used for mass immunization. We also show that some of the universal epitopes constituting the recombinant carriers are specifically recognized by two human in vitro systems, suggesting that T cell memory is provided by the selected epitopes. The data indicate that rationally designed recombinant polyepitope proteins represent excellent candidates for the development and clinical testing of new conjugate vaccines.

Amino Acid Sequence↗

PubMed2001

Combined automated PCR cloning, in vitro transcription/translation and two-dimensional electrophoresis for bacterial proteome analysis.

The most popular approach for proteomics analysis is based on the combination of two-dimensional gel electrophoresis and mass spectrometry (MS). Although very effective, the method suffers from a number of limitations, the most serious one being the necessity to have expensive and sophisticated instrumentation requiring handling by skilled personnel. Here we propose an alternative approach which may offer some advantages over the current methods, at least for some specific applications. The method is based on two-dimensional gel separation of radiolabeled synthetic proteins derived from transcription/translation reactions of linear polymerase chain reaction amplified genes. The gel is autoradiographed and this is superimposed on the sample gel whose protein spots have to be identified. Matching between autoradiographs and sample gel spots allows immediate protein identification. The method has been validated identifying six proteins from a membrane protein preparation of Neisseria meningitidis MC58 strain. All proteins were correctly identified as judged by confirmation analysis with MS. The approach is particularly useful when a specific subset of proteins needs to be identified in a complex protein mixture.

Animals↗

PubMed2001

Antibacterial vaccine design using genomics and proteomics.

After 200 years of practice, vaccinology has proved to be very effective in preventing infectious diseases. However, several human and animal pathogens exist for which vaccines have not yet been discovered. As for other fields of medical sciences, it is expected that vaccinology will greatly benefit from the emerging genomics technologies such as bioinformatics, proteomics and DNA microarrays. In this article the potential of these technologies applied to bacterial pathogens is analyzed, taking into account the few existing examples of their application in vaccine discovery.

Animals↗

PubMed2001

Iris alteration using mechanical iris retractors.

A 74-year-old woman had a 3-port pars plana vitrectomy for a dropped nucleus after complicated phacoemulsification. Flexible nylon iris retractors were positioned to dilate the small pupil. The patient died of a pulmonary embolus 1 day after surgery. The eyeball was removed at autopsy, and pathology of the iris was investigated. Localized damage to the iris where the retractor hooks were positioned was noted, but no other alterations to the iris were observed. These findings may explain the recovery of pupil function that is common after surgery with mechanical iris retractors.

Aged↗

PubMed2001

Crystallization and preliminary crystallographic studies on the large extracellular domain of human CD81, a tetraspanin receptor for hepatitis C virus.

The large extracellular domain of CD81, a member of the tetraspanin family and a receptor protein for hepatitis C virus envelope E2 glycoprotein, has been expressed, purified and subsequently crystallized using the sitting-drop vapour-diffusion technique. Native diffraction data to 1.6 A resolution were obtained at the ID14 beamline of the European Synchrotron Radiation Facility from a flash-frozen crystal at 100 K. The crystals belong to space group P2(1), with unit-cell parameters a = 31.5, b = 77.2, c = 38.5 A, beta = 107.4 degrees, and are likely to contain two extracellular domains (2 x 99 residues) per asymmetric unit.

Antigens, CD↗

PubMed2000

Complete genome sequence of Neisseria meningitidis serogroup B strain MC58.

The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.

Antigenic Variation↗

PubMed2000

Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing.

Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.

Amino Acid Sequence↗

PubMed2000

Structure-function analysis of hepatitis C virus envelope-CD81 binding.

Hepatitis C virus (HCV) is a major human pathogen causing chronic liver disease. We have recently found that the large extracellular loop (LEL) of human CD81 binds HCV. This finding prompted us to assess the structure-function features of HCV-CD81 interaction by using recombinant E2 protein and a recombinant soluble form of CD81 LEL. We have found that HCV-E2 binds CD81 LEL with a K(d) of 1.8 nM; CD81 can mediate attachment of E2 on hepatocytes; engagement of CD81 mediates internalization of only 30% of CD81 molecules even after 12 h; and the four cysteines of CD81 LEL form two disulfide bridges, the integrity of which is necessary for CD81-HCV interaction. Altogether our data suggest that neutralizing antibodies aimed at interfering with HCV binding to human cells should have an affinity higher than 10(-9) M, that HCV binding to hepatocytes may not entirely depend on CD81, that CD81 is an attachment receptor with poor capacity to mediate virus entry, and that reducing environments do not favor CD81-HCV interaction. These studies provide a better understanding of the CD81-HCV interaction and should thus help to elucidate the viral life cycle and to develop new strategies aimed at interfering with HCV binding to human cells.

Amino Acid Sequence↗

PubMed2000

Evaluation of hepatitis C virus glycoprotein E2 for vaccine design: an endoplasmic reticulum-retained recombinant protein is superior to secreted recombinant protein and DNA-based vaccine candidates.

Hepatitis C virus (HCV) is the leading causative agent of blood-borne chronic hepatitis and is the target of intensive vaccine research. The virus genome encodes a number of structural and nonstructural antigens which could be used in a subunit vaccine. The HCV envelope glycoprotein E2 has recently been shown to bind CD81 on human cells and therefore is a prime candidate for inclusion in any such vaccine. The experiments presented here assessed the optimal form of HCV E2 antigen from the perspective of antibody generation. The quality of recombinant E2 protein was evaluated by both the capacity to bind its putative receptor CD81 on human cells and the ability to elicit antibodies that inhibited this binding (NOB antibodies). We show that truncated E2 proteins expressed in mammalian cells bind with high efficiency to human cells and elicit NOB antibodies in guinea pigs only when purified from the core-glycosylated intracellular fraction, whereas the complex-glycosylated secreted fraction does not bind and elicits no NOB antibodies. We also show that carbohydrate moieties are not necessary for E2 binding to human cells and that only the monomeric nonaggregated fraction can bind to CD81. Moreover, comparing recombinant intracellular E2 protein to several E2-encoding DNA vaccines in mice, we found that protein immunization is superior to DNA in both the quantity and quality of the antibody response elicited. Together, our data suggest that to elicit antibodies aimed at blocking HCV binding to CD81 on human cells, the antigen of choice is a mammalian cell-expressed, monomeric E2 protein purified from the intracellular fraction.

Animals↗

PubMed1999

Recombinant and truncated tetanus neurotoxin light chain: cloning, expression, purification, and proteolytic activity.

Tetanus neurotoxin (TeNT) consists of two disulfide-linked polypeptide chains, heavy (H) and light (L). The L chain is a zinc endopeptidase protein highly specific for vesicle-associated membrane protein (VAMP), which is an essential component of the exocytosis apparatus. Here we describe the cloning of the L chain of TeNT from Clostridium tetani strain Y-IV-3 (WS 15) and its expression in Escherichia coli as a glutathione S-transferase fusion protein. The full-length recombinant L chain, corresponding to residues 1-457, was obtained as a mixture of proteins of slightly different mass with identical N-terminal ends. To obtain a product useful for structural analysis and crystallization, a COOH-terminally truncated L chain (residues 1-427) was cloned, expressed, and purified with high yield. This truncated L chain is more active than the full-length and wild-type proteins in the hydrolysis of VAMP. Preliminary experiments of crystallization of the truncated recombinant L chain gave encouraging results.

Cloning, Molecular↗