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Biomedical subjects

M Zanetti

Publications and source records attributed to M Zanetti.

At least 91 records · Page 5Linked to original sources
PubMed1998

Cytotoxicity and apoptosis mediated by two peptides of innate immunity.

Antimicrobial peptides are present in a wide range of species, from protozoa to man, as effector molecules of innate immunity. Several bovine precursors of antimicrobial peptides have recently been identified, as deduced from cDNA, and assigned to the cathelicidin family. Two of these are the proforms of the antimicrobial peptides BMAP-27 and BMAP-28, which share a similar amino acid sequence, structural conformation, and toxic activity toward several bacterial and fungal strains. Here we report that they are cytotoxic to human tumor cells and normal proliferating, but not resting, lymphocytes at concentrations comparable to those microbiocidal. This effect is primarily due to damage of plasma membrane integrity. A more detailed investigation of the U937 cell line revealed that a Ca2+ influx into the cytosol occurs in the early steps of permeabilization. The perturbation of the membrane structure and the Ca2+ influx are followed by programmed death. A similar apoptosis inducing effect is also observed on in vitro activated human lymphocytes.

Amino Acid Sequence↗
PubMed1998

Biological characterization of a novel mammalian antimicrobial peptide.

A putative antimicrobial peptide of 34 residues was recently deduced from a bovine cathelicidin gene sequence and named BMAP-34. A peptide based on the deduced sequence was chemically synthesized and used to study the localization, structure and biological activities of BMAP-34. A Western blot analysis using antibodies raised to the synthetic peptide showed that BMAP-34 is stored as proform in the cytoplasmic granules of bovine neutrophils. CD spectroscopy indicates that the peptide assumes an amphipathic alpha-helical conformation, as also predicted by secondary structure analysis. The peptide exerts a broad spectrum antimicrobial activity against both Gram-negative and Gram-positive organisms, and is not active against eukaryotic cells. When tested on Escherichia coli ML-35, the kinetics of bacterial killing and of inner membrane permeabilization are slower than those observed for other alpha-helical peptides derived from cathelicidins.

Amino Acid Sequence↗
PubMed1998

Cloning and analysis of a transcript derived from two contiguous genes of the cathelicidin family.

Bac4 is a bovine cathelicidin gene contiguous to another member of this family named Bac7. Although mutations in the sequence suggested that Bac4 gene might be non-functional, primers based on Bac4 specific sequences allowed amplification of a 900 bp cDNA. The transcript comprises the sequences of exons 1, 2 and 3 of Bac7, and of exons 2, 3 and 4 of Bac4 gene and may result from a weak termination control of the transcription of the upstream Bac7 gene.

Amino Acid Sequence↗
PubMed1998

DNA immunization in relB-deficient mice discloses a role for dendritic cells in IgM-->IgG1 switch in vivo.

A single intraspleen inoculation of plasmid DNA coding for an immunoglobulin heavy chain gene initiates immunity and establishes immunologic memory against the antigenic determinants of transgenic immunoglobulins, somatic transgene immunization. During priming mice produce IgM but not IgG1 antibodies. Since IgM --> IgG1 class switch occurs spontaneously during the primary immune response to protein antigens we investigated possible mechanisms for failure of spontaneous isotype switch in vivo in this model of immunity. We found that inoculation of plasmid DNA in the form of a chimeric gene coding for granulocyte-macrophage colony-stimulating factor (GM-CSF) was able to drive IgG1 class switch readily after priming. Since GM-CSF activates cells of the dendritic lineage we tested the possibility that dendritic cells (DC) may be involved in regulating IgM --> IgG1 switch. To this end we used bone marrow chimeras constructed from mice carrying the null mutation for the relB member of the NF-kappaB/Rel family as these mice lack bone marrow-derived mature DC. RelB (-/-) mice and (-/-) bone marrow chimeras inoculated with DNA/GM-CSF did not produce IgG1 antibodies during the primary immune response. Since relB (-/-) bone marrow chimeras lack DC of donor origin but possess resident follicular dendritic cells we conclude that Ig class switch in vivo is regulated by the function of interdigitating dendritic cells (IDC). Thus, IDC may contribute to the qualitative aspects of the emerging immune response.

Animals↗
PubMed1998

Immunological memory after somatic transgene immunization is positively affected by priming with GM-CSF and does not require bone marrow-derived dendritic cells.

Inoculation of plasmid DNA is a promising vaccination approach but optimal regimes and ways to enhance immunogenicity remain to be established. Among natural immunological adjuvants, granulocyte-macrophage colony-stimulating factor (GM-CSF) was shown to increase the potency of immunization against tumor cells and protein antigens. Here we studied the effect of GM-CSF on memory responses against a 12-mer B cell epitope in mice primed with a single DNA inoculation. The results show that GM-CSF given at priming as a DNA/GM-CSF chimeric vaccine enhances the magnitude of the anamnestic response irrespective of the form of antigen used subsequently in the booster immunization. Using mice lacking bone marrow-derived dendritic cells we also determined that the enhancing effect is not strictly dependent on these cells. These results expand our understanding of the activity of GM-CSF in vivo as a modulator of the immune response including immunological memory.

Animals↗
PubMed1998

Relationships between phenylalanine hydroxylation and plasma aromatic amino acid concentrations in humans.

We investigated the relationships between phenylalanine hydroxylation (Phe Hy) and plasma concentrations of phenylalanine, tyrosine, and glucagon in healthy male volunteers (N = 13; age, 29 +/- 3 years). Phe Hy, as well as the Phe and Tyr rate of appearance (Ra), were measured during L-[2H5]-Phe and L-[2H2]-Tyr continuous intravenous (i.v.) infusions both under basal postabsorptive conditions (N = 13) and following divergent changes of plasma aromatic amino acids (AAA) concentrations. Namely, AAA were increased by administration of a balanced synthetic mixed meal (n = 6) or selectively decreased by i.v. infusion of insulin along with a Phe-deficient, Tyr and tryptophan-deprived amino acid mixture ([IAA] n = 7). Following the meal, plasma Phe (54 +/- 3 to 81 +/- 12 micromol/L), plasma Tyr (54 +/- 4 to 91 +/- 7), Phe Hy (0.09 +/- 0.01 to 0.15 +/- 0.02 micromol/kg x min), Phe Ra (0.65 +/- 0.04 to 0.96 +/- 0.07), and Tyr Ra (0.51 +/- 0.03 to 0.93 +/- 0.11) all significantly increased (P < or = .05 v basal). IAA infusion significantly decreased plasma Phe (to 47 +/- 3 micromol/L), plasma Tyr (to 25 +/- 4), Phe Hy (to 0.07 +/- 0.004 micromol/kg x min), and Tyr Ra (to 0.29 +/- 0.02; all P < or = .05 v sal), while Phe Ra did not change (0.64 +/- 0.04, NS). Plasma glucagon did not change in the three experimental periods (basal, 85 +/- 7; meal, 72 +/- 10; IAA, 92 +/- 14 pg/mL; NS). Using linear regression analysis, plasma Phe was positively related to both Phe Hy (R2 = .76, P < .001) and plasma Tyr (R2 = .80, P < .001); Phe Hy and plasma Tyr were also significantly correlated (R2 = .60, P < .001). No correlation was found between Phe Hy and basal plasma glucagon (R2 = .04, NS). Using multiple regression analysis with plasma Tyr as the dependent variable, plasma Phe was still correlation with plasma Tyr (t = 4.29, P = .0002), while the relationship between Phe Hy and plasma Tyr was no longer significant (t = 0.69, P = .49). These data indicate that plasma Phe is closely associated with its own hydroxylative disposal in humans, and confirm that Phe conversion to Tyr may play a physiological role in maintaining balanced plasma phenylalanine and tyrosine concentrations.

Adult↗
PubMed1998

MR abnormalities of the intervertebral disks and adjacent bone marrow as predictors of segmental instability of the lumbar spine.

PURPOSE: To assess whether MR abnormalities of the intervertebral disks and adjacent bone marrow can predict segmental instability of the lumbar spine as diagnosed on functional radiographs. MATERIAL AND METHODS: A consecutive review was made of 60 patients examined with MR imaging and with lateral flexion and extension views of the lumbar spine. Sagittal T1- and T2-weighted images were evaluated blindly with regard to abnormalities of the intervertebral disk and the adjacent bone marrow. Segmental instability was diagnosed when a.p. translation of 3 mm or more was present on the functional radiographs. Moreover, the presence of osteophytes was evaluated on lateral standard radiographs. RESULTS: Of a total of 300 segments, 32 (10.7%) were unstable. Anular tears were the most relevant MR finding. Their sensitivity, specificity, and positive and negative predictive values for segmental instability were 18.8%, 97.0%, 42.9% and 90.9%. The corresponding values for traction osteophytes were 12.5%, 98.1%, 44.4% and 90.4%. Abnormalities of bone marrow were not significantly related to segmental instability (p=0.35). CONCLUSION: Functional radiographs should be considered in patients with anular tears or traction osteophytes. No correlation was found between segmental instability and abnormalities of bone marrow adjacent to the endplates.

Adult↗
PubMed1998

Radiographically negative stress related bone injury. MR imaging versus two-phase bone scintigraphy.

PURPOSE: To assess the diagnostic value of MR imaging relative to two-phase bone scintigraphy in radiographically negative stress-related injuries. MATERIAL AND METHODS: Sixteen consecutive patients presenting with stress-related bone injuries and normal standard radiographs were examined with two-phase bone scans and MR imaging. MR imaging protocols varied according to the region to be examined. The MR and scintigraphic images were evaluated blindly and separately by respectively two radiologists and two nuclear medicine physicians. The standard of reference was represented by a combination of clinical and scintigraphic findings as well as clinical follow-up. RESULTS: For MR imaging, sensitivity, specificity, and positive and negative predictive values for the presence of stress-related injuries for the two readers were 69/63%, 100/80%, 100/91%, and 50/40%, respectively. Interobserver agreement was good (kappa = 0.62). For scintigraphy, all abnormal and normal findings were correctly identified. CONCLUSION: For patients with clinically suspected stress-related injuries and a low probability of other active bone diseases (such as infection or neoplasm), bone scintigraphy should be the initial imaging modality.

Adolescent↗
PubMed1998

Collagen VI deficiency induces early onset myopathy in the mouse: an animal model for Bethlem myopathy.

To gain insight into the function of type VI collagen, the col6a1 gene was inactivated by targeted gene disruption in the mouse. The homozygous mutants lacked collagen VI in the tissues and showed histological features of myopathy such as fiber necrosis and phagocytosis and a pronounced variation in the fiber diameter. Muscles also showed signs of stimulated regeneration of fibers. Necrotic fibers were particularly frequent in the diaphragm at all ages examined. Similar, although milder, alterations were detected in heterozygous mutant mice, indicating haploinsufficiency of the col6a1 gene function. The data led us to conclude that collagen VI is necessary for maintenance of the integrity of muscle fibers and that the col6a1 -deficient mouse can be considered an animal model of Bethlem myopathy.

Animals↗
PubMed1998

Quantitative assessment of the muscles of the rotator cuff with magnetic resonance imaging.

RATIONALE AND OBJECTIVES: The purpose of this study was to establish a magnetic resonance (MR) imaging standard for quantification of the muscles of the rotator cuff. METHODS: Parasagittal T1-weighted turbo spin-echo images of the shoulder were obtained in 70 asymptomatic subjects (35 women, 35 men; age range: 21-70 years, mean: 45 years). Standardized cross-sectional areas (rotator cuff muscle areas divided by the area of the supraspinatus fossa) and standardized signal intensities (related to signal intensities of the teres major muscle) were measured and compared with 30 patients with different stages of rotator cuff tears and 10 patients with glenohumeral instability. In addition, a so-called tangent sign was evaluated with the hypothesis that a healthy supraspinatus muscle crosses a line (tangent) drawn through the superior borders of the scapular spine and the superior margin of the coracoid. RESULTS: Cross-sectional areas of the muscles of the rotator cuff were variable in asymptomatic subjects. Cross-sectional areas (but not signal intensities) did discriminate patients with different stages of rotator cuff tears from asymptomatic subjects. The tangent sign was negative in all asymptomatic subjects but positive in four and nine of 10 patients with medium and large rotator cuff tears, respectively. CONCLUSIONS: A method for quantification of the muscles of the rotator cuff using MR imaging is presented. Cross-sectional areas can be used for quantification of the muscles of the rotator cuff. The tangent sign is a useful MR sign for atrophy of the supraspinatus muscle.

Adult↗
PubMed1998

Assessment of dorsal or ventral intercalated segmental instability configurations of the wrist: reliability of sagittal MR images.

PURPOSE: To determine whether a dorsal intercalated segmental instability (DISI) or ventral intercalated segmental instability configuration can be mimicked on sagittal magnetic resonance (MR) images in asymptomatic subjects with unremarkable lateral radiographs. MATERIALS AND METHODS: In 10 asymptomatic adult volunteers, capitolunate, scapholunate, and radiolunate angles were measured on lateral radiographs and compared with those measured on T1-weighted spin-echo MR images obtained in neutrally positioned wrists with subtle variations in the sagittal imaging plane. MR measurements also were obtained in 15 degrees radially and 15 degrees ulnarly deviated wrists. RESULTS: In neutrally positioned wrists, the mean capitolunate, scapholunate, and radiolunate MR angles were 13.6 degrees-14.5 degrees, 4.0 degrees-9.9 degrees, and 18.0 degrees-20.3 degrees larger, respectively, than those on lateral radiographs. In 15 degrees radially deviated wrists, the mean MR angles were similar to those on lateral radiographs. In 15 degrees ulnarly deviated wrists, the mean MR angles were 32.3 degrees, 16.6 degrees, and 37.1 degrees larger than those on lateral radiographs. At MR imaging, a DISI configuration would have been diagnosed in four subjects with neutrally positioned wrists and in eight subjects with ulnarly deviated wrists. CONCLUSION: The lunate appears more dorsally tilted on sagittal MR images than on lateral radiographs, and a DISI configuration can be mimicked. Subtle errors in the selection of the imaging plane do not substantially influence measurements; however, wrist positioning is crucial.

Adult↗
PubMed1998

Lumbar spine: quantitative and qualitative assessment of positional (upright flexion and extension) MR imaging and myelography.

PURPOSE: To compare measurements of the sagittal diameter of the lumbar dural sac obtained at positional magnetic resonance (MR) imaging and at functional myelography and to assess the influence of various body positions on the dural sac and the intervertebral foramina. MATERIALS AND METHODS: Thirty consecutive patients referred for lumbar myelography were examined with an open 0.5-T MR imager, Sagittal T2-weighted fast spin-echo images were acquired with patients in the supine, upright flexion, and upright extension positions. The midsagittal diameter of the dural sac was measured at the level of the disks on MR images and myelograms. Foraminal sizes on the MR images were scored independently by two observers. RESULTS: Correlation between MR imaging and myelographic measurements was high (r = .81-.97). A small but statistically significant positional dependence of the dural sac diameter was found in the lower lumbar spine. Position-dependent differences in foraminal scores were uncommon. CONCLUSION: Quantitative assessment of sagittal dural sac diameters is comparable between lumbar myelography and positional MR imaging. In a selected patient population, only small changes in the sagittal diameter of the dural sac and foraminal size can be expected between various body positions, and the information gained in addition to that from standard MR imaging is limited [corrected].

Adult↗
PubMed1998

MR imaging of the lumbar spine: prevalence of intervertebral disk extrusion and sequestration, nerve root compression, end plate abnormalities, and osteoarthritis of the facet joints in asymptomatic volunteers.

PURPOSE: To identify the magnetic resonance (MR) abnormalities of the lumbar spine that have a low prevalence in asymptomatic patients and thus determine the findings that are predictive of low back pain in symptomatic patients. MATERIALS AND METHODS: Sagittal T1-weighted and sagittal and axial T2-weighted MR images were obtained in 60 asymptomatic volunteers aged 20-50 years. The MR images were evaluated with regard to intervertebral disk abnormalities, end plate abnormalities, and osteoarthritis of the facet joints by two musculoskeletal radiologists independently. RESULTS: Disk bulging or disk protrusion was found in 42 (14%) and 48 (16%) of the intervertebral spaces in 37 (62%) and 40 (67%) subjects, respectively. High-signal-intensity zones were found commonly (in 23 [7.7%] and 25 [8.3%] of the intervertebral spaces in 19 (32%) and 20 (33%) subjects, respectively). Disk extrusions were less common (in 11 [3.7%] and 11 [3.7%] of the intervertebral spaces in 11 (18%) and 11 (18%) subjects, respectively). There were no disk sequestrations. A nerve root compression in a single intervertebral space was diagnosed by one reader. End plate abnormalities were found in two (0.7%) and six (1.9%) of the intervertebral spaces in two (3%) and six (10%) subjects, respectively. No severe osteoarthritis was diagnosed by either reader. CONCLUSION: In patients younger than 50 years, disk extrusion and sequestration, nerve root compression, end plate abnormalities, and osteoarthritis of the facet joints are rare and, therefore, may be predictive of low back pain in symptomatic patients.

Adult↗
PubMed1998

Tendinopathy and rupture of the tendon of the long head of the biceps brachii muscle: evaluation with MR arthrography.

OBJECTIVE: The purpose of this study was to assess the diagnostic role of MR arthrography in patients with tendinopathy or rupture of the long biceps tendon. MATERIALS AND METHODS: MR arthrograms of 42 consecutive patients with arthroscopic or surgical confirmation of diagnosis (16 normal biceps tendons, 19 with tendinopathy, and seven with ruptures) were analyzed independently by two radiologists. Visibility of the biceps tendon, caliber changes, contour irregularities, and signal intensities were assessed separately in the parasagittal and axial planes. In addition, the two radiologists made an overall evaluation of abnormalities of the biceps tendon using both MR imaging planes. RESULTS: The most reliable MR findings for tendinopathy were caliber changes (sensitivity was 59% for observer 1 and 82% for observer 2; specificity was 88% and 64%, respectively) and signal abnormalities (sensitivity, 77% and 88%, respectively; specificity, 75% and 43%, respectively) in the parasagittal plane. Absence of visualization of the tendon in the parasagittal plane was a reliable sign for rupture (sensitivity, 86% and 86%, respectively; specificity, 94% and 87%, respectively). The overall sensitivity for detecting abnormalities (tendinopathy or rupture) was 92% for observer 1 and 89% for observer 2. Specificity was 56% and 81%, respectively. CONCLUSION: MR findings of tendinopathy and rupture of the biceps tendon are subtle. However, the combination of several MR criteria in two imaging planes makes a reasonably accurate diagnosis possible. The biceps tendon should not only be assessed in the bicipital sulcus on axial images but also on parasagittal images.

Adult↗
PubMed1997

Structural organization of the bovine cathelicidin gene family and identification of a novel member.

Cathelicidins are a group of myeloid antimicrobial peptide precursors found in a variety of mammalian species. Transcripts of this family show a highly conserved 5' region corresponding to the 5' untranslated region, signal peptide and propiece, and diverse 3' regions encoding structurally varied C-terminal sequences that correspond to mature antimicrobial peptides after proteolytic release from the precursors. To establish the size of the bovine gene family encoding these proteins, lambda genomic clones were isolated by screening a bovine library with a probe based on the conserved cDNA region of bovine members. Restriction mapping, hybridization studies and sequence analysis identified 11 distinct genes that based on the intergenic distances of contiguous genes appear to be in close physical proximity. Among these, a novel gene encoding the precursor of a putative alpha-helical antimicrobial peptide was recognized and sequenced. The novel gene appears to be expressed in bovine bone marrow myeloid cells, spleen and testis.

Amino Acid Sequence↗
PubMed1997

In vivo role of B lymphocytes in somatic transgene immunization.

Immunity generated by in vivo inoculation of plasmid DNA is a straightforward and potentially valuable new approach to immunization. Little is known about the type of cells involved, the various immunological aspects, and the destiny of the transgene. In this report, we describe a system in which immunity is the result of in vivo targeting of B lymphocytes. This was accomplished using plasmid DNA encoding an immunoglobulin heavy-chain gene under the control of immunoglobulin promoter and enhancer elements. We show persistence of the transgene in splenic B lymphocytes for at least 3 months, i.e., the average life span of long-lived B lymphocytes in the mouse. The transgene could not be detected in any other lymphoid or nonlymphoid organs over a period of 6 months. We also established that the transgene is integrated in the host DNA. These studies bring new understanding to the events underlying the in vivo use of plasmid DNA. Moreover, the characteristics of this new approach make somatic transgene immunization a model system to study the immunogenicity of endogenous antigens in adult animals.

Animals↗
PubMed1997

Identification of CRAMP, a cathelin-related antimicrobial peptide expressed in the embryonic and adult mouse.

Cathelicidins are the precursors of potent antimicrobial peptides that have been identified in several mammalian species. Prior work has suggested that members of this gene family can participate in host defense through their antimicrobial effects and activate mesenchymal cells during wound repair. To permit further study of these proteins a reverse transcriptase-polymerase chain reaction approach was used to identify potential mouse homologs. A full-length 562-base pair cDNA clone was obtained encoding an NH2-terminal prepro domain homologous to other cathelicidins and a unique COOH-terminal peptide. This gene, named Cramp for cathelin-related antimicrobial peptide, was mapped to chromosome 9 at a region of conserved synteny to which genes for cathelicidins have been mapped in pig and man. Northern blot analysis detected a 1-kilobase transcript that was expressed in adult bone marrow and during embryogenesis as early as E12, the earliest stage of blood development. Reverse transcriptase-polymerase chain reaction also detected CRAMP expression in adult testis, spleen, stomach, and intestine but not in brain, liver, heart, or skeletal muscle. To evaluate further the expression and function of CRAMP, a peptide corresponding to the predicted COOH-terminal region was synthesized. CD spectral analysis showed that CRAMP will form an amphipathic alpha-helix similar to other antimicrobial peptides. Functional studies showed CRAMP to be a potent antibiotic against Gram-negative bacteria by inhibiting growth of a variety of bacterial strains (minimum inhibitory concentrations 0.5-8.0 microM) and by permeabilizing the inner membrane of Escherichia coli directly at 1 microM. Antiserum against CRAMP revealed abundant expression in myeloid precursors and neutrophils. Thus, CRAMP represents the first antibiotic peptide found in cells of myeloid lineage in the mouse. These data suggest that inflammatory cells in the mouse can use a nonoxidative mechanism for microbial killing and permit use of the mouse to study the role such peptides play in host defense and wound repair.

Amino Acid Sequence↗