Carbon nanotubes as nanovectors for intracellular delivery of laronidase in Mucopolysaccharidosis type I.

The immobilization of proteins on carbon nanotubes (CNTs) has been widely reported mainly for the preparation of sensors while the conjugation of enzymes for therapeutic purposes has scarcely been considered. Herein we report, to the best of our knowledge, the first example of intracellular delivery of a therapeutic enzyme by means of CNTs, retaining its activity. Mucopolysaccharidosis I is a rare genetic disease characterized by the deficiency or absence of the activity of the α-l-iduronidase (IDUA) enzyme. We evaluated the capacity of the recombinant form of the human IDUA enzyme, laronidase (Aldurazyme®), conjugated with CNTs to be internalized by fibroblasts from subjects affected with Mucopolysaccharidosis type I and the capacity of the enzyme to retain its activity after internalization. The enzyme was successfully delivered into the lysosomal space and the enzymatic activity of the conjugate was preserved after internalization up to 48 hours. This paves the way towards the use of such a kind of construct for therapeutic applications.

Proinflammatory effects of diesel exhaust nanoparticles on scleroderma skin cells.

Autoimmune diseases are complex disorders of unknown etiology thought to result from interactions between genetic and environmental factors. We aimed to verify whether environmental pollution from diesel engine exhaust nanoparticulate (DEP) of actually operating vehicles could play a role in the development of a rare immune-mediated disease, systemic sclerosis (SSc), in which the pathogenetic role of environment has been highlighted. The effects of carbon-based nanoparticulate collected at the exhaust of newer (Euro 5) and older (Euro 4) diesel engines on SSc skin keratinocytes and fibroblasts were evaluated in vitro by assessing the mRNA expression of inflammatory cytokines (IL-1 α , IL-6, IL-8, and TNF-α) and fibroblast chemical mediators (metalloproteases 2, 3, 7, 9, and 12; collagen types I and III; VEGF). DEP was shown to stimulate cytokine gene expression at a higher extent in SSc keratinocytes versus normal cells. Moreover, the mRNA gene expression of all MMPs, collagen types, and VEGF genes was significantly higher in untreated SSc fibroblasts versus controls. Euro 5 particle exposure increased the mRNA expression of MMP-2, -7, and -9 in SSc fibroblasts in a dose dependent manner and only at the highest concentration in normal cells. We suggest that environmental DEP could trigger the development of SSc acting on genetically hyperreactive cell systems.

[Nanotechnologies for future applications in the biomedical field]. / Le nanotecnologie: potenziali applicazioni in campo biomedico.

Engineered nanomaterials are increasingly being used for commercial purposes and especially for biomedical and biotechnological applications. Carbon nanotubes in particular are viewed as a class of nanomaterials with high potential for biological applications due to their unique mechanical, physical and chemical properties. Carbon nanotubes are cylindrical molecules composed solely of carbon atoms. They can be thought of as a seamless cylinder formed from a graphitic sheet with a hexagonal lattice structure. The CNT ends resemble hemispherical buckyballs connected by a graphene cylinder. The properties of individual CNTs vary depending on their atomic structure. Upon their discovery, the extraordinary properties have made CNTs the focus of a wide spectrum of research topics with potential for development into viable commercial applications. Carbon nanotubes are the most promising new nanoparticles that can be used for drug and gene delivery. Among numerous potential applications, including DNA and protein sensors, in vitro cell markers, diagnostic imaging contrast agents, the use of these carbon-nanostructures as multifunctional biological transporters, agents for selective cancer destruction and drug and gene delivery systems has been explored. Moreover, various cell types have been shown to grow extremely well on C-nanotubes, giving a potential for applications such as scaffolds and structures/coatings for tissue regeneration/repair.

Toxicity and biocompatibility of carbon nanoparticles.

A review is presented of the literature data concerning the effects induced by carbon nanoparticles on the biological environment and the importance of these effects in human and animal health. The discovery in 1985 of fullerenes, a novel carbon allotrope with a polygonal structure made up solely by 60 carbon atoms, and in 1991 of carbon nanotubes, thin carbon filaments (1-3 microm in length and 1-3 nm in diameter) with extraordinary mechanical properties, opened a wide field of activity in carbon research. During the last few years, practical applications of fullerenes as biological as well as pharmacological agents have been investigated. Various fullerene-based compounds were tested for biological activity, including antiviral, antioxidant, and chemiotactic activities. Nanotubes consist of carbon atoms arranged spirally to form concentric cylinders, that are perfect crystals and thinner than graphite whiskers. They are stronger than steel but very flexible and lightweight and transfer heat better than any other known material. These characteristics make them suitable for various potential applications such as super strong cables and tips for scanning probe microscopes, as well as biomedical devices for drug delivery, medical diagnostic, and therapeutic applications. The effects induced by these nanostructures on rat lung tissues, as well as on human skin and human macrophage and keratinocyte cells are presented.

Inflammatory status and cartilage regenerative potential of synovial fibroblasts from patients with osteoarthritis and chondropathy.

OBJECTIVES: To evaluate the inflammatory status and the cartilage regenerative potential of pathological synovial fibroblasts from patients with osteoarthritis (OA) compared with non-inflamed synovium (NS)-derived cells from patients with chondropathy. METHODS: The inflammatory cell phenotype was investigated based on the constitutive and inducible surface expression and secretion of various effector molecules using flow cytometry or ELISA assays. The capacity of cells to produce cartilage-like extracellular matrix was assessed using acid Alcian blue staining and type II collagen immunostaining after treatment with transforming growth factor beta1 (TGF-beta1). RESULTS: OA and NS fibroblasts consistently expressed CD29, CD44, CD49e, CD54, CD90 and CD106. Expression of high-affinity receptors for IL-4, IL-15, CXCL8 and CXCL12 was also detected but only intracellularly. All types of fibroblasts spontaneously released abundant amounts of CXCL12, CCL2, IL-6 and tissue inhibitor of metalloproteinase 1, while the production of IL-11, TGF-beta1, matrix metalloproteinase 1 (MMP-1) and MMP-9 was detected at moderate levels. Several other secreted factors remained undetectable. No statistically significant differences were noted between the two groups of fibroblasts. Treatment with the proinflammatory cytokine tumour necrosis factor alpha (TNF-alpha) up-regulated the same set of surface and secreted molecules, including CD54, CD106, membrane IL-15, CCL2 and CCL5. Under TGF-beta1 treatment and adipogenic culture conditions, both OA and NS fibroblasts displayed chondrogenic and adipocytic activities that were reduced in OA compared with NS cells. CONCLUSIONS: OA synovial fibroblasts did not display a distinct activated inflammatory phenotype compared with NS cells. However, they did differ in their reduced ability to produce cartilage-like matrix. This difference may be an additional important factor contributing to OA pathogenesis.

Pro-inflammatory and anti-inflammatory circulating cytokines and periprosthetic osteolysis.

We investigated the circulating levels of the main cytokines involved in bone resorption (IL-1beta, IL-6, TNF-alpha), prostaglandins (PGE2) and metalloproteases (MMP-1), as possible early markers of osteolysis, in the serum of eight patients with periprosthetic osteolysis and ten patients without osteolysis. All had received a cementless hip prosthesis (ABG-1). We also assessed the serum levels of IL-1 and TGF-beta anti-inflammatory cytokines exerting protective effect on bone resorption. The mean serum levels of IL-1beta, IL-6, TNF-alpha, TGF-beta, MMP-1, and PGE2 in patients with periprosthetic osteolysis did not differ significantly from those of patients without osteolysis or from those of normal controls. IL-11 serum levels were not detectable at all in any of the patients, while they were detected within normal reference values in the control subjects (significant inverse correlation). We believe that circulating cytokines cannot be regarded as markers of osteolysis, a condition characterised by a local inflammation without systemic signs of inflammation. On the contrary, the undetectable levels of IL-11 in implanted patients could provide evidence for a lack of balance between pro- and anti-inflammatory cytokines in these patients.

Nerve growth factor release by human synovial fibroblasts prior to and following exposure to tumor necrosis factor-alpha, interleukin-1 beta and cholecystokinin-8: the possible role of NGF in the inflammatory response.

OBJECTIVE: Aim of this study was to investigate the synthesis, release and effects of nerve growth factor (NGF) in human synovial cells isolated from synovial tissue specimen from healthy and osteoarthritis (OA) patients. METHODS: Human synovial fibroblasts cultures were established starting from healthy and osteoarthritis patients. NGF protein levels in the culture medium, NGFmRNA and high-affinity NGF receptor (Tyrosine kinase A: TrkA) expression in the cells were evaluated in basal conditions and after stimulation with pro-inflammatory cytokines or with the neuropeptide cholecystokinin-8 (CCK-8). The effect of NGF supplement to culture medium on cell proliferation, TrkA expression, and tumour necrosis factor-alpha (TNF-alpha) and inducible-nitric oxide synthase (iNOS) production was investigated. RESULTS: Under basal conditions human synovial cells produce and release NGF. Both interleukin-1-beta (IL-1 beta) and TNF-alpha, but not CCK-8 promote NGF synthesis and release from OA cells. TrkA NGF receptors are also expressed in both normal and OA synovial cells. NGF, but not IL-1 beta, TNF-alpha and CCK-8, enhances the expression of TrkA in isolated synovial cells. NGF down-regulates IL-1 beta-induced TNF-alpha and iNOS production by OA synovial fibroblasts. CONCLUSIONS: NGF is produced and released and TrkA receptors are expressed in synovial inflammation. Overexpression of NGF in inflammed joints might be involved in the modulation rather than in the induction of the joint inflammatory response.

Increase in free radicals on UHMWPE hip prostheses components due to inflamed synovial cell products.

Oxidative degradation of artificial UHMWPE joint implants caused by gamma-ray sterilization is thought to be responsible for the production of wear debris resulting in adverse tissue responses. On the other hand, it is well known that inflammation is associated with generation, by inflammatory cells, of free radicals (H(2)O(2) and NO) and destructive proteolytic enzymes (collagenases), which creates a strong oxidative environment. We hypothesized that when an UHMWPE implantation was performed in an inflammatory joint environment, the oxidative substances produced by inflamed synoviocytes could increase oxidative degradation of the polyethylene insert. We measured the amount of free radicals on conventional and on Duration-treated polyethylene samples by the electron spin resonance (ESR) technique before and after exposure of the samples to (1) inflamed synovial cell cultures; (2) normal synovial cell cultures; and (3) medium alone. We observed an increase in the number of free radicals on polyethylene samples after their immersion in cell cultures. Furthermore, it was observed that the increase of free radicals on polyethylene correlated with the degree of inflammation of synovial cells in culture.

An unusual transmission of Neisseria meningitidis: neonatal conjunctivitis acquired at delivery from the mother's endocervical infection.

BACKGROUND: Primary meningococcal conjunctivitis is assumed to be due to the direct inoculation of Neisseria meningitidis into the conjunctival sac from an exogenous source. According to a literature review, no case of neonatal conjunctivitis infection acquired at delivery from maternal endocervicitis has been published. GOAL: To report a case of meningococcal neonatal conjunctivitis acquired at delivery because of the mother's endocervical infection and cross-transmission of the strain with her partner. STUDY DESIGN: Strains were characterized by bacteriologic and serologic methods including grouping (agglutination), typing, and subtyping (enzyme-linked immunoabsorbent assay). Molecular analysis was done by pulsed-field gel electrophoresis. RESULTS: The three strains (newborn infant, mother, partner) were of the same antigenic formula (C:NT:P1.NT) and exhibited similar NheI and SpeI pulsed-field gel electrophoresis patterns. CONCLUSION: The identical phenotypic and genomic analysis of strains is the evidence for N meningitidis transmission at delivery from a maternal endocervical infection to the newborn infant and cross transmission between sexual partners.

Intracytoplasmic Th1 and Th2 cytokines in rheumatoid arthritis blood and synovial tissue.

In rheumatoid arthritis (RA), T cells have been proposed either as a main actor or as an epiphenomenon in such a primarily synoviocyte-driven disease. A major issue remains the remarkable paradox between the T cell infiltrate and the relative failure to detect definite markers of their activity. To determine the Th1/Th2 cytokine profile in RA synovium, we used a single cell flow cytometric assay for interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-4 and IL-10 in paired peripheral blood (PB) and synovial tissue (ST) lymphocytes from RA and osteoarthritis (OA) patients and PB lymphocytes from healthy controls. Cytokines were undetectable in unstimulated PB and ST lymphocytes. More stimulated PB and ST CD4(+)lymphocytes produced IFN-gamma than IL-4, for all individuals tested. RA PB CD4(+)lymphocytes showed the same Th1 cytokine pattern as normal controls. No increase of such a Th1 profile was observed for ST lymphocytes. A specific recruitment of T CD4(+)lymphocytes in the rheumatoid inflamed synovium could not be concluded on the basis of these results.

Characterization of an outbreak of tetM-containing Neisseria gonorrhoeae in Argentina.

Phenotypic and molecular characterization of an outbreak of 9 Neisseria gonorrhoeae (NG) isolates exhibiting high-level plasmid mediated resistance to penicillin and tetracycline (PP-TRNG) that took place in Tandil, Argentina between February and April 1995. Comparison with the patterns of the 3 PP-TRNG strains previously isolated were made. We determined the following markers for each strain: antimicrobial susceptibility, serogroup, auxotype, plasmid profile, presence of tetM determinant and restriction pattern of the tetM-containing plasmid. Antimicrobial tests values were: tetracycline disk diameter 12-14 mm, minimum inhibitory concentration (MIC) 32 micrograms/ml; penicillin disk diameter 6 mm, MIC 32 micrograms/ml and sensitive by both methods to spectinomycin, cefuroxime, ceftriaxone and ciprofloxacin. All isolates were of the same serogroup (WI). Ten of the strains, including the 9 from Tandil outbreak, were arginine-requiring, while the other 2 were methionine and arginine-requiring. All of them demonstrate the same plasmid profile (2.6, 3.2, 25.2 MDa). They were positive for the tetM determinant and the restriction analysis identified it is a Dutch-type plasmid. In spite of the temporal and geographical dispersion, PP-TRNG strains in Argentina seem to be highly homogeneous in terms of antimicrobial susceptibility, serogroup, plasmid profiles and even auxotype.

PIP: This study determines the phenotypic and molecular characterization of an outbreak of 9 Neisseria gonorrhea isolates exhibiting high-level plasmid-mediated resistance to penicillin and tetracycline (PP-TRNG) that took place in Tandil, Argentina. Subjects included patients attending Tandil's Sanatamarina Hospital. Individuals infected with sexually transmitted disease were examined using a standardized questionnaire. Results show that all isolates produced B-lactamase, were penicillin resistant (disk zone diameter 6 mm, MIC 32 mcg/ml) and high-level tetracycline resistant (disk zone diameter 12-14 mm, MIC 32 mcg/ml). Moreover, all strains were fully sensitive to cefuroxime, ceftriaxone, ciprofloxacin, and spectinomycin; while autotype and serogroup results showed that all the strains were arginine-requiring and of serogroup WI. In spite of the temporal and geographical dispersion, PP-TRNG strains in Argentina seem to be highly homogeneous in terms of antimicrobial susceptibility, serogroup, plasmid profiles, and even auxotype.

Markers of connective tissue activation in aseptic hip prosthetic loosening.

The fundamental role played by macrophages and fibroblasts of the synovial-like membrane in aseptic hip prosthesis loosening (AHPL) has recently been confirmed by numerous studies. In this study, the activity of these cells in patients with prosthetic loosening was analyzed by evaluating the main markers of fibroblast and macrophage activation in sera and in supernatants of cultured fibroblasts obtained from AHPL patients who underwent revision of a loose total hip arthroplasty implant. In these patients interleukin-1, hyaluronic acid (HA), and type III procollagen peptide were evaluated. The results were compared with those obtained in 13 patients with firmly fixed implants and 13 patients with osteoarthritis. Serum HA levels were significantly higher (779.3 +/- 951.6 micrograms/L) in patients with AHPL as compared with patients with firmly fixed implants (112.9 +/- 84.9 micrograms/L) and osteoarthritis (115.3 +/- 107.8 micrograms/L). Type III procollagen peptide levels were elevated in only 33.3% of patients with AHPL, whereas interleukin-1-beta (IL-1 beta) was detectable in 4 patients with AHPL but not in patients with firmly fixed implants or osteoarthritis. In supernatants, IL-1 beta was measurable in 4 of 6 fibroblast cultures, whereas type III procollagen peptide and HA were measurable in all cultures. The data confirm the existence of an inflammatory process in AHPL patients in which macrophages and fibroblasts play a key role. The detection, in these patients, of high circulating levels of IL-1 beta and HA is very important from a clinical point of view because they could be considered specific markers of inflammation.

[Physiopathological reasoning in the clinical diagnosis]. / Il ragionamento fisiopatologico nella diagnosi clinica.

It is generally acknowledged that the causal approach is used very seldom and only in the late stages of the diagnostic process, when hypotheses are refined and verified. The hypotheses generation is supposed to take place in the very first stage of the medical process, and the approach most frequently used is the probabilistic one. It is also believed that owing to its explanatory function, causal reasoning should be used only--and only sparingly--when solving intricate metabolic and endocrinological cases. The Authors refute this restrictive view of the causal approach's field of application, and examine the reasons that prevent its broader use, especially in the range of such pathologies. Amongst such reasons, the Authors highlight some present didactic trends: namely the fact that the basic teachings--which are also the necessary premise to the causal approach--are given too early, in the early years of the University syllabus, and remain therefore cut off from the actual medical teaching. On the basis of this analysis some alternative didactic methods are put forward.

[The presentation of clinical cases: the map is not the territory]. / La presentazione dei casi clinici: la mappa non è il territorio.

To establish their effectiveness for teaching purposes two different expository methods employed for the presentation of clinical cases are compared: the "traditional or conventional" expository method and the more recently introduced non-conventional expository method. The "traditional" expository method is characterized by its schematization and rigidity, which are features that misrepresent the real clinical situation. The non-conventional expository method is distinguished by its fluidity, by its different cognitive approach (iterative approach) and by its ability to make the cognitive processes underlying all clinical reasoning explicit. This is an element of fundamental importance for teaching. In addition, the authors point out the teaching limits implicit in the presentation of cases whatever their expository method. The authors conclude by maintaining that tutorials at the patient's bedside must form the basis of clinical teaching.

Primer informe en la República Argentina de resistencia a penicilina en Neisseria gonorrhoeae mediada por el plásmido de 3,2 MDal (africano) / First report in Argentine of resistance to penicillin in Neisseria gonorrhoeae madiated by the 3,2 MDal African plasmid

En la República Argentina, Buenos Aires (INM), detectamos la primer cepa productora de ß-lactamasa en 1980, incrementándose la prevalencia de estas cepas resistentes de 1,9 por ciento (1980-1984) a 30 por ciento (1984-1988) y a 37 por ciento (1989-1991). El objetivo de este trabajo es la caracterización de los plásmidos responsables de esta resistencia, en un estudio retrospectivo que abarca de 1985 a 1991. Se estudiaron 28 cepas de Neisseria gonorrhoeae productoras de penicilinasa (PPNG), enviadas para su estudio o aisladas en nuestro Servicio de exudados genitales. El 100 por ciento de las cepas presentó el plásmido críptico de 2,6 MDal. El plásmido de 3,2 MDal se encontró en 13 cepas (46,4 por ciento) y en el 92,3 por ciento de ellas acompañado por el de 24,4 MDal. En 15 cepas (53,60 por ciento) se detectó la presencia del plásmido de 4,4 MDal, sólo en 9 de ellas (60 por ciento) junto con el plásmido conjugativo. Este es el primer informe sobre la presencia del plásmido de 3,2 MDal (africano) en las cepas PPNG aisladas en la Argentina. Nuestro primer hallazgo de este plásmido data de 1988

Primer informe en la República Argentina de resistencia a penicilina en Neisseria gonorrhoeae mediada por el plásmido de 3,2 MDal (africano) / First report in Argentine of resistance to penicillin in Neisseria gonorrhoeae madiated by the 3,2 MDal African plasmid

En la República Argentina, Buenos Aires (INM), detectamos la primer cepa productora de ß-lactamasa en 1980, incrementándose la prevalencia de estas cepas resistentes de 1,9 por ciento (1980-1984) a 30 por ciento (1984-1988) y a 37 por ciento (1989-1991). El objetivo de este trabajo es la caracterización de los plásmidos responsables de esta resistencia, en un estudio retrospectivo que abarca de 1985 a 1991. Se estudiaron 28 cepas de Neisseria gonorrhoeae productoras de penicilinasa (PPNG), enviadas para su estudio o aisladas en nuestro Servicio de exudados genitales. El 100 por ciento de las cepas presentó el plásmido críptico de 2,6 MDal. El plásmido de 3,2 MDal se encontró en 13 cepas (46,4 por ciento) y en el 92,3 por ciento de ellas acompañado por el de 24,4 MDal. En 15 cepas (53,60 por ciento) se detectó la presencia del plásmido de 4,4 MDal, sólo en 9 de ellas (60 por ciento) junto con el plásmido conjugativo. Este es el primer informe sobre la presencia del plásmido de 3,2 MDal (africano) en las cepas PPNG aisladas en la Argentina. Nuestro primer hallazgo de este plásmido data de 1988 (AU)

Autoimmune features in atherosclerotic ischemic cardiomyopathy.

An high frequency of antimitochondrial autoantibodies has been reported in subjects affected with primary cardiomyopathies and it has been hypothesized that they could be involved in the pathogenesis of these diseases. In order to find out whether such autoantibodies could on the contrary represent an epiphenomenon of myocardial cell damage, we searched for antimitochondrial (AMA), antinuclear (ANA) and antismooth-muscle (SMA) non-organ specific autoantibodies in a group of 50 subjects (47 females and 3 males), over 65 years of age, affected with ischemic cardiomyopathy (ICM) due to atherosclerosis, a condition resembling other cardiomyopathies as it concerns ultrastructural aspects of myocardial tissue. The frequency of the autoantibodies tested in our patients resulted quite similar to that occurring in our healthy elderly control subjects (AMA: 14% vs 5.7%; ANA: 28% vs 23%; SMA: 12% vs 11.4%) and in normal aged population. On the basis of our data, the myocardial cell damage "per se" does not seem to influence significatively the production of non-organ specific autoantibodies.

Evaluation of the biological activity of unmodified synthetic eel calcitonin rectal capsules. Comparison with intramuscular administration and placebo.

Biological activity of eel calcitonin 100 IU administered by the rectal route was evaluated in healthy volunteers by measuring plasmatic variations of cyclic adenosine monophosphate (cAMP) after a single dose and during a 21-day treatment. This formulation appears to allow a bioavailability of the drug which is half of the same dose administered intramuscularly, and to cause a prompt and significant increase of plasma cAMP, with minimal variations of calcaemia. Repeated administration shows the persistence in time of this effect. The overall conclusion is that the amount of calcitonin absorbed through the rectal mucosa seems sufficient to induce a typical biological response related to the interaction of the hormone with its specific receptors in bone.

[Anamnesis: a diagnostic passe-partout]. / L'anamnesi: un passe-partout diagnostico.

There is universal consensus that the anamnesis is a fundamental and irreplaceable phase both for the formulation of the diagnosis and for establishing the correct physician-patient relation. In a high percentage of cases the diagnostic hypothesis formulated during the history-taking is successively confirmed. The analysis of the structure of the anamnesis has demonstrated to realize a cognitive approach of phenomenological type. This kind of approach fits with the structure of illness because of its dynamic aspect. That is the reason why it represents an indispensable diagnostic key.