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Biomedical subjects

G H Giambartolomei

Publications and source records attributed to G H Giambartolomei.

15 recordsLinked to original sources
PubMed2005

Mutation in the P36 gene of Mycobacterium bovis provokes attenuation of the bacillus in a mouse model.

P36 is a member of a family of secreted proteins distributed throughout the genus Mycobacterium. The central domain of these proteins contains several amino acid PGLTS repeats, which differ considerably between species. P36, also called exported repetitive protein (Erp) in M. tuberculosis, has been shown to be associated with virulence since the disruption of its gene impaired multiplication of both virulent M. tuberculosis and M. bovis BCG in cultured macrophages and immunocompetent mice. In order to demonstrate that P36 is a putative virulence factor of wild-type Mycobacterium bovis we generated a P36 mutant by gene disruption and we evaluated its replication in spleen and lungs of infected mice. In this study, the mutant strain displays low levels of multiplication in mice, indicating that the P36 gene is important for in vivo growth of M. bovis.

Animals↗
PubMed2001

Limited diagnostic usefulness of antibodies to cytoplasmic proteins of Brucella in early-treated human brucellosis.

Antibodies to cytoplasmic proteins (CP) of Brucella have been shown to be useful for the diagnosis of human brucellosis; however, some early-diagnosed patients lack such an antibody response while having high titers of antibodies to lipopolysaccharide (LPS). To address which factors determine this serological discrepancy in the early stages of brucellosis we examined the antibody response to CP and LPS of 21 patients involved in an outbreak of B. melitensis infection who had a short duration of clinical illness at diagnosis (3-40 d). At diagnosis, antibodies to LPS (IgM and/or IgG) were found in all patients, while anti-CP antibodies were detected in 16 subjects (76%). At 6 weeks post-diagnosis IgG to CP (with or without IgM) had been detected in 13 patients and IgM alone had been found in 4; however, 4 other patients (19%) had no response to CP. No significant differences were found between these 3 groups in terms of age, gender, antimicrobial agents or factors that could hamper the immune response. Notably, however, the 4 non-responders and 3 of the 4 patients having only IgM to CP had started antibiotic therapy within 14 d post-symptoms, while treatment was started later in 9 of 13 patients who developed anti-CP IgG. In addition, maximum titers of IgG to CP tended to be lower in early-treated patients. These results suggest that very early antibiotic therapy hampers the antibody response to Brucella CP but has little impact on the anti-LPS response. Given the higher specificity of the former and the higher sensitivity of the latter, both reactivities should be measured in order to diagnose human brucellosis.

Antibodies, Bacterial↗
PubMed2000

Structural, functional and immunological studies on a polymeric bacterial protein.

The characterization of proteins from Brucella spp, the causative agent of brucellosis, has been the subject of intensive research. We have described an 18-kDa cytoplasmic protein of Brucella abortus and shown the potential usefulness of this protein as an antigen for the serologic diagnosis of brucellosis. The amino acid sequence of the protein showed a low but significant homology with that of lumazine synthases. Lumazine is an intermediate product in bacterial riboflavin biosynthesis. The recombinant form of the 18-kDa protein (expressed in E. coli) folds like the native Brucella protein and has lumazine-synthase enzymatic activity. Three-dimensional analysis by X-ray crystallography of the homolog Bacillus subtilis lumazine synthase has revealed that the enzyme forms an icosahedral capsid. Recombinant lumazine synthase from B. abortus was crystallized, diffracted X rays to 2.7-A resolution at room temperature, and the structure successfully solved by molecular replacement procedures. The macromolecular assembly of the enzyme differs from that of the enzyme from B. subtilis. The Brucella enzyme remains pentameric (90 kDa) in its crystallographic form. Nonetheless, the active sites of the two enzymes are virtually identical at the structural level, indicating that inhibitors of these enzymes could be viable pharmaceuticals across a broad species range. We describe the structural reasons for the differences in their quaternary arrangement and also discuss the potential use of this protein as a target for the development of acellular vaccines.

Animals↗
PubMed1999

Internalization and resistance to degradation of Alzheimer's A beta 1-42 at nanomolar concentrations in THP-1 human monocytic cell line.

Microglial cell involvement in Alzheimer's disease has been related to amyloid beta (A beta) internalization, the release of inflammatory cytokines and the development of neuritic plaques. The human monocyte/macrophage THP-1 cell line has been widely used as a model of human microglial cells. We used THP-1 cells to study the adsorption, internalization and resistance to degradation of A beta1-40 and A beta1-42 isoforms offered at nanomolar concentrations and free of large aggregates, conditions that may mimic a pre-fibrillar stage of A beta in the brain. Under these conditions, A betas did not induce THP-1 activation, as assessed by interleukin-1beta expression. A beta1-42 showed a preferential adsorption and intracellular accumulation as compared to A beta1-40, supporting that competent nuclei for A beta1-42 ordered aggregation may be formed inside microglial cells. In light of the possible neurotoxicity of soluble A beta1-42, we propose that amyloid formation within brain phagocytic cells may be a protective mechanism in early stages of the disease.

Amyloid beta-Peptides↗
PubMed1999

Induction of pro- and anti-inflammatory cytokines by Borrelia burgdorferi lipoproteins in monocytes is mediated by CD14.

We previously showed that heat-killed Borrelia burgdorferi spirochetes and lipidated outer surface protein A (L-OspA) stimulated the in vitro production of interleukin-10 (IL-10) in peripheral blood mononuclear cells (PBMC) from uninfected humans and rhesus monkeys (G. Giambartolomei et al., Infect. Immun. 66:2691-2697, 1998). Here we demonstrate that uninfected human peripheral blood monocytes, but not B or T cells, are the cells that transcribe the IL-10 cytokine gene in response to heat-killed B. burgdorferi. B. burgdorferi similarly induced an upregulation of the IL-1beta and IL-6 cytokine genes in monocytes and the production of IL-10 and IL-6 in culture supernatants of the human monocytic cell line THP-1. Purified L-OspA (but not unlipidated OspA [U-OspA] or U-OspC) also stimulated the production of both cytokines in THP-1 cells in a dose-dependent fashion, suggesting that acylation of the OspA protein molecule is required for the production of both anti- and pro-inflammatory cytokines in naive monocytes. A lipohexapeptide that contained the tripalmitoyl-modified cysteine motif (Pam3Cys-Hex) of B. burgdorferi lipoproteins but with an arbitrary peptide sequence had the same effect. Monoclonal antibodies (MAbs) MY4 and 60bca, both of which bind to CD14 and are known to block lipopolysaccharide (LPS)-mediated cytokine production, were able to block L-OspA-mediated IL-10 and IL-6 cytokine production. In contrast, MAb 26ic, which also binds to CD14 but does not block LPS function, failed to inhibit L-OspA-mediated cytokine production. These data suggest that activation of monocytes and production of both anti- and pro-inflammatory cytokines induced by lipoproteins proceeds via the CD14 receptor. LPS binding protein was not required for OspA-induced cytokine production. Our results demonstrate that pro- and anti-inflammatory cytokines induced by B. burgdorferi lipoproteins in PBMC are produced by monocytes and that lipoprotein and LPS signaling pathways share at least the initial signaling event that involves the CD14 receptor.

Acute-Phase Proteins↗
PubMed1998

Diminished production of T helper 1 cytokines and lack of induction of IL-2R+ T cells correlate with T-cell unresponsiveness in rhesus monkeys chronically infected with Brugia malayi.

The relationship between antigen-specific responsiveness, parasitic burden, and lymphatic pathology was investigated in nine rhesus monkeys with chronic Brugia malayi infections. Specifically, in vitro proliferation, cytokine gene expression and production, IL-2R expression on T cells, microfilaria (mf) densities, and lymphedema were evaluated. PBMC from three animals (two mf- one mf+) proliferated in response to filarial antigen (responder monkeys, RM) and cells from six animals (5 mf+; one mf-) did not (nonresponder monkeys, NRM). All RM showed lymphedema and none of the NRM did. Antigen-specific IL-2 and IFN-gamma (mRNA and protein) were induced in PBMC from all RM whereas PBMC from only one of six NRM responded with IL-2 and IFN-gamma expression. IL-4 transcripts were induced in PBMC from two of three RM and in cells from all six NRM. IL-10 mRNA expression and protein production were induced in PBMC from two of three RM and in cells from five of six NRM. A marked increase in the frequency of IL-2R+ T cells was observed in antigen-stimulated PBMC cultures of RM but not in those of NRM. The data show that diminished production of Th1 cytokines and lack of induction of IL-2R+T cells may contribute to the unresponsiveness of PBMC from NRM to filarial antigen. They also show that the polarization of immune responses and lymphatic pathology observed in rhesus monkeys is similar to that generally described in human lymphatic filariasis patients.

Animals↗
PubMed1998

Borrelia burgdorferi stimulates the production of interleukin-10 in peripheral blood mononuclear cells from uninfected humans and rhesus monkeys.

Heat-killed Borrelia burgdorferi spirochetes stimulate in vitro production of interleukin-10 (IL-10) at both mRNA and protein levels in peripheral blood mononuclear cells (PBMC) of uninfected rhesus monkeys. A concomitant down-modulation of IL-2 gene transcription was observed. Neither IL-4 nor gamma interferon gene expression was ostensibly affected by B. burgdorferi spirochetes. These phenomena were observed regardless of whether the stimulating spirochetes belonged to the B. burgdorferi sensu stricto, Borrelia afzelii, or Borrelia garinii genospecies, the three main species that cause Lyme disease. B. burgdorferi also induced production of IL-10 in uninfected human PBMC, indicating that this effect might play a role in human Lyme disease. Purified lipidated outer surface protein A (OspA), but not its unlipidated form, induced the production of high levels of IL-10 in uninfected human PBMC. Thus, the lipid moiety is essential in the induction of IL-10 in these PBMC. B. burgdorferi M297, a mutant strain that lacks the plasmid that encodes OspA and OspB, also induced IL-10 gene transcription in PBMC, indicating that this phenomenon is not causally linked exclusively to OspA and its lipid moiety. These results demonstrate that B. burgdorferi can stimulate the production of an antiinflammatory, immunosuppressive cytokine in naive cells and suggest that IL-10 may play a role both in avoidance by the spirochete of deleterious immune responses and in limiting the inflammation that the spirochete is able to induce.

Animals↗
PubMed1996

Blocking anti-Trichinella spiralis antibodies in chronically infected rats.

Specific anti-newborn larva antibodies present in the serum of rats chronically infected with Trichinella spiralis were incapable of inducing killing of newborn larvae by activation of normal peritoneal cells. These late antibodies blocked the cytotoxic reaction induced by early antibodies produced a few weeks after infection. Passive transference of late serum to normal mice failed to induce protective immunity against infection by newborn T. spiralis larvae. When late immunoserum was fractionated by gel filtration, blocking activity was found only in the fraction containing IgG subclasses. By indirect immunofluorescence assay and cytotoxic reaction it was shown that blocking antibodies were specific for newborn larvae and could not be adsorbed with muscle larvae. It is concluded that the synthesis of anti-newborn larva antibodies is modulated in the course of a chronic infection: early antibodies developing shortly after infection are cytotoxic, whereas blocking antibodies predominate in the late population. Furthermore, the results suggest that during a chronic infection, resistance to reinfection may be modified.

Animals↗
PubMed1996

Humoral immune response against lipopolysaccharide and cytoplasmic proteins of Brucella abortus in cattle vaccinated with B. abortus S19 or experimentally infected with Yersinia enterocolitica serotype 0:9.

The humoral immune responses against three different antigens of Brucella abortus were monitored by enzyme-linked immunosorbent assay in cattle vaccinated with B. abortus S19 or experimentally infected with Yersinia enterocolitica serotype 0:9. Immunoglobulin G (IgG) and IgM responses against (i) B. abortus lipopolysaccharide (LPS), (ii) total cytoplasmic proteins depleted of LPS (LPS-free CYT), and (iii) B. abortus 18-kDa cytoplasmic protein were measured. Vaccinated animals and Yersinia-infected animals developed high anti-LPS IgM and IgG titers, which overlapped with those obtained with sera from B. abortus 544-infected animals used as positive controls. In contrast, only a slight or negative IgG and IgM response against LPS-free CYT and the 18-kDa protein was detected in vaccinated or Yersinia-infected cattle, although its levels were always significantly lower than those of B. abortus 544-infected animals. These data indicate that cytoplasmic proteins of B. abortus could be useful for the differential diagnosis of bovine brucellosis.

Animals↗
PubMed1996

Interspecies cross-reactivity of a monoclonal antibody directed against wheat chloroplast fructose-1,6-bisphosphatase.

The primary structure of several chloroplast fructose-1,6-bisphosphatase (CFBPase) was deduced from DNA sequences, but only spinach, pea and rapeseed enzymes have been characterized structurally. We analyzed whether CFBPases from different phylogenetic origin contain a common epitope. To this end a DNA fragment of 1200 base pairs encoding 338 amino acid residues of wheat CFBPase (38 kDa) was cloned in the expression plasmid pGEX-1 in frame with the gene coding for glutathione S-transferase (GT) of Schistosoma japonicun (26.5 kDa). Upon transformation of Escherichia coli and induction with isopropyl-beta-D-thiogalactopyranoside, centrifugation of the lysate partitioned 10% of the fusion protein in the supernatant fraction and the remaining 90% in the precipitate. The expected 65 kDa protein was purified from both the soluble and the particulate fraction by affinity chromatography on columns of glutathione-agarose. This fusion protein was successfully used to produce a monoclonal antibody that specifically recognized the CFBPase region of the fusion protein but not the GT moiety. Moreover, the monoclonal antibody immunoreacted not only with polypeptides (ca. 40 kDa) present in leaf crude extracts of other varieties of wheat (Triticum spelta, T. aestivum and T. durum), but also with homogeneous preparations of the spinach (Spinacia oleracea) and rapeseed (Brassica napus) enzymes. Thus, the cross reaction of this monoclonal antibody with counterparts from different plant species indicates the persistency of a common epitope through biological evolution.

Amino Acid Sequence↗
PubMed1995

Immune cytotoxic activity of human eosinophils against Trichinella spiralis newborn larvae.

The ability of human eosinophils to kill the newborn larvae (NBL) of Trichinella spiralis of different maturation status, in the presence of antibody, was studied. A cytotoxic in vitro test was performed using NBL less than 2 h of age (NBL2) or NBL maintained in culture at 37 degrees C for 20 h (NBL20), peripheral blood eosinophils, anti-Trichinella serum and human fresh serum as source of complement. Under these experimental conditions eosinophils from normal individuals attached to NBL2 as well as to NBL20 but only the latter were killed. On the other hand, eosinophils from volunteers with eosinophilia killed NBL regardless of larval age. Neither adherence nor significant mortality was observed in the absence of immune serum. These results indicate that NBL maturation and eosinophil activation status are crucial for antibody-dependent cellular cytotoxic reaction (ADCC).

Animals↗
PubMed1993

Immune killing of newborn Trichinella larvae by human leucocytes.

The capacity of human leucocytes from normal donors to kill the newborn larvae of the nematode Trichinella spiralis in vitro, in the presence of serum from infected individuals, was studied using newborn larvae (NBL) less than 2 h of age or NBL that had been maintained in culture at 37 degrees C for 20 h. Neutrophils and monocytes attached to newborn Trichinella larvae and killed them, regardless of their age. When eosinophils were used, 20 h old NBL were killed whereas 2 h old NBL were not. Complement was essential in the cytotoxic effect of leucocytes. These results indicate that host defence against T. spiralis in humans may be a complex mechanism in which different cell types can be involved. They also show that the age of maturation of the NBL is of paramount importance in the antibody-dependent cellular cytotoxicity reaction.

Animals↗
PubMed1993

[Serological, clinical, and epidemiological aspects of an outbreak of trichinosis in Azul, province of Buenos Aires].

An outbreak of human trichinellosis that took place in Southern Buenos Aires Province, an endemic area for this zoonosis, was studied. Eighteen individuals were infected by eating swine sausages lacking veterinary control. Clinical symptoms, as well as serological studies related to antibody production and cytotoxic capacity of the sera are described. Eight interdicted pigs and their products were analysed. In order to determine the transmission network of this zoonosis, wild rats were hunted in the farm where the swine were bred. The clinical symptoms of the infected individuals were: myalgia, edema, fever and cephalea. Sixty-one percent of the patients showed antibodies against muscle larvae 15-20 days after ingestion of the infected meat. Five out of eighteen patients showed anti-newborn larvae antibodies 40-60 days postinfection. The sausage burden was 5.3 muscle larvae/g. Two out of eight swine were diagnosed employing serological techniques, while parasitic techniques detected only one. The rat is an important reservoir in the domestic cycle of this zoonosis.

Adolescent↗
PubMedDate not supplied

Study of immune defence against Trichinella spiralis in BCG treated mice.

In vivo and in vitro studies were performed to determine the effect of BCG- activated macrophages on 2-hour-old newborn larvae (NBL2) of Trichinella spiralis. In vitro studies were done by the technique of antibody-dependent cellular cytotoxicity (ADCC) using peritoneal cells of BALB/c mice which had been treated with BCG 4-6 days before. No significant differences were found between the percentage of mortality caused by cells from treated animals and that corresponding to the untreated ones (control mice). The same result was obtained when only glass-adherent cell populations only were used. Intravenous administration of NBL2 and anti-T. spiralis antibodies to BCG-treated mice resulted in the same percentage of infectivity as that corresponding to control animals. These results indicate that an increase in the activity of macrophages induced by BCG neither alters the cytotoxic activity of the effector cells nor plays a direct role in the destruction of NBL2 of T. spiralis.

Animals↗