Antibodies to cell envelope proteins of Pseudomonas aeruginosa in cystic fibrosis patients.

Many vaccines containing somatic and secreted antigens of Pseudomonas aeruginosa have been reported. The vaccines containing lipopolysaccharide have been found to provide type-specific protection, but the endotoxin content of these vaccines does not make it feasible to use them in patients who are already debilitated. Outer membrane proteins could be effective as vaccines, as they can be purified free of lipopolysaccharide, and also because they are common to all serotypes of P. aeruginosa. To be effective as a vaccine, such proteins must be immunogenic and accessible from the outside of the intact bacterial cell. In this study, we showed that systemic antibodies were produced frequently to two cell envelope proteins with masses of 58,500 and 37,500 daltons and occasionally to 34,000-dalton protein of P. aeruginosa in cystic fibrosis patients with chronic lung infections. In rabbits immunized with whole, fixed cells of P. aeruginosa, antibodies were also produced against the 58,500-dalton proteins. Thus, the 58,500-dalton cell envelope protein of P. aeruginosa was the only immunogenic protein that was accessible to the immune system when whole, fixed cells were used for immunization. These serum antibodies did not protect the cystic fibrosis patients against further lung infection with P. aeruginosa.     

Deposition and passage of transthyretin through the blood-nerve barrier in recipients of familial amyloid polyneuropathy livers

Familial amyloid polyneuropathy (FAP) is characterized by deposition of mutated transthyretin (TTR) in the peripheral nervous system. Prior to amyloid fibrils, nonfibrillar TTR aggregates are deposited inducing oxidative stress with increased nitration (3-NT). As the major source of TTR is the liver, liver transplantation (LT) is used to halt FAP. Given the shortage of liver donors, domino LT (DLT) using FAP livers is performed. The correlation between TTR deposition in the skin and nerve was tested in biopsies from normal individuals, asymptomatic carriers (FAP 0) and FAP patients; in FAP 0, nonfibrillar TTR was observed both in the skin and nerve in the same individuals; in patients, amyloid was detected in both tissues. The occurrence of amyloidosis in recipients of FAP livers was evaluated 1-7 years after DLT: TTR deposition occurred in the skin 3 years after transplantation either as amyloid or aggregates; in one of the recipients, fibrillar TTR was present in the epineurium 6 years after DLT. Deposits were scarce and 3-NT immunostaining was irrelevant. Nerve biopsies from DLT recipients had no FAP-related neuropathy. Our findings suggest that TTR amyloid formation occurs faster than predicted and that TTR of liver origin can cross the blood-nerve barrier. Recipients of FAP livers should be under surveillance for TTR deposition and tissue damage.     

Antibiotic resistance in exopolysaccharide-forming Staphylococcus epidermidis clinical isolates from orthopaedic implant infections

The opportunistic pathogen Staphylococcus epidermidis is able to produce biofilm and to frequently cause implant infections. In recent years, it has also exhibited an increasing antimicrobial drug resistance. Here, the resistance to a panel of 16 different antibiotics in 342 clinical strains of S. epidermidis from orthopaedic implant infections has been investigated. The isolates were pheno- and genotyped for extracellular polysaccharide production, relevant to staphylococcal biofilm formation, in order to ascertain possible associations with antibiotic resistance. Approximately 10% of the isolates were found to be sensitive to all screened antibiotics. In all, 37-38% were resistant to beta-lactams such as oxacillin and imipenem, while the resistance to penicillin, ampicillin, cefazolin, cefamandole, was consistently observed in over 80% of the strains. Erythromycin- and clindamycin- resistant strains were approximately 41% and 16%, respectively. Of the isolates, 10% was resistant to chloramphenicol, 23% to sulfamethoxazole and 26% to ciprofloxacin. Resistance to vancomycin was never observed. Interestingly, exopolysaccharide-producing strains exhibited a significantly higher prevalence in the resistance to the four aminoglycosides (gentamicin, amikacin, netilmicin, tobramycin), to sulfamethoxazole and to ciprofloxacin with respect to non-producing isolates. Moreover, multiple resistance to antibiotics was more frequent among exopolysaccharide-forming strains.     

Biological activities of monoclonal antibodies to Mycoplasma pneumoniae membrane glycolipids.

A purified preparation of membranes was obtained by using a unique method of treating Mycoplasma pneumoniae with the ATPase inhibitor, diethylstilbestrol. This method was shown to yield highly purified membranes with little or no cytoplasmic contamination. These membranes were used to immunize mice for subsequent productions of monoclonal antibodies (MAbs). Hybridoma culture supernatants were screened by enzyme-linked immunosorbent assay with whole-cell M. pneumoniae and lipid extract antigens. Four stable MAbs were obtained and characterized. MAb CP3-46F5 reacted with a protein of a molecular weight of approximately 52,000 as determined by Western blot (immunoblot). MAbs CP3-50C2, CP3-53C5, and CP3-53C8 did not react with any antigens on Western blots but did bind to at least 10 distinct glycolipid bands as determined by orcinol staining on thin-layer chromatograms of M. pneumoniae lipid extracts. The MAbs did not react with similarly prepared lipid extracts from Mycoplasma genitalium, Mycoplasma neurolyticum, and Mycoplasma gallisepticum. These MAbs did not inhibit M. pneumoniae metabolism or attachment to WiDr cell cultures. The anti-glycolipid MAbs recognize determinants specific to M. pneumoniae, unlike polyclonal hyperimmune sera against M. pneumoniae, which cross-react with lipid extracts of M. genitalium.     

Light scattering from aqueous solutions of poly(acrylamide-co-maleic anhydride)

Light scattering measurements from aqueous solutions for two samples of poly(acrylamide-co-maleic anhydride) in the presence of salt are conducted. One of the samples exhibits a negative initial slope and a minimum in the plot of the conventional reciprocal scattered intensity function of sin² (θ/2). The explanation for this anomaly is a large optical anisotropy of the segment. A correction for this effect of segmental anisotropy is made. The behaviour of the other sample of this copolymer is typical for a polyelectrolyte. By analysing the disymmetry of the scattered light, the influence of the salt concentration on the polyion is evidenced by the determination of the macromolecular dimensions. Light scattering data allow the evaluation of the Mark-Kuhn-Houwink exponent a for various degrees of dissociation of the polyion; a correlation between the variation of a and the shape of the macromolecule is observed.     

Dopamine receptors in human brain: autoradiographic distribution of D1 sites

The distribution of dopamine D1 receptors has been determined in post mortem human brain tissues using in vitro receptor autoradiography, with ([3H]N-methyl) SCH 23390 as ligand. The highest densities of dopamine D1 sites were seen in the nucleus caudatus, putamen, globus pallidus pars medialis and substantia nigra. Intermediate densities were associated with the amygdala, mammillary bodies, cerebral cortex and CA1. The remaining part of the hippocampus as well as the diencephalon, brainstem and cerebellum contained low levels of [3H]SCH 23390 binding sites. The distribution of D1 receptors in the human brain closely resembles that reported for the rat brain. In addition, there was a good correlation between the anatomical localization of D1 sites and the distribution of dopaminergic nerve terminals in the central nervous system. The densities of D1 receptors in the human brain were observed to markedly decrease with age during the first decades of life. However, no further modifications were found beyond the age of 40 years. We did not observe any significant influence of other parameters such as gender and post mortem delay in our samples.     

Quantitative light microscopic autoradiographic localization of cholinergic muscarinic receptors in the human brain: forebrain

The distribution of muscarinic cholinergic receptors in the human forebrain and cerebellum was studied in detail by quantitative autoradiography using N-[3H]methylscopolamine as a ligand. Only postmortem tissue from patients free of neurological diseases was used in this study. The highest densities of muscarinic cholinergic receptors were found in the striatum, olfactory tubercle and tuberal nuclei of the hypothalamus. Intermediate to high densities were observed in the amygdala, hippocampal formation and cerebral cortex. In the thalamus muscarinic cholinergic receptors were heterogeneously distributed, with densities ranging from very low to intermediate or high. N-[3H]Methylscopolamine binding was low in the hypothalamus, globus pallidus and basal forebrain nuclei, and very low in the cerebellum and white matter tracts. The localization of the putative muscarinic cholinergic receptors subtypes M1 and M2 was analysed in parallel using carbachol and pirenzepine at a single concentration to partially inhibit N-[3H]methylscopolamine binding. Mixed populations of both subtypes were found in all regions. M1 sites were largely predominant in the basal ganglia, amygdala and hippocampus, and constituted the majority of muscarinic cholinergic receptors in the cerebral cortex. M2 sites were preferentially localized in the diencephalon, basal forebrain and cerebellum. In some areas such as the striatum and substantia innominata there was a tendency to lower densities of muscarinic cholinergic receptors with increasing age. In general, we observed a slight decrease in M2 sites in elderly cases. Muscarinic cholinergic receptor concentrations seemed to be reduced following longer postmortem periods. The distribution of acetylcholinesterase was also studied using histochemical methods, and compared with the localization of muscarinic cholinergic receptors and other cholinergic markers. The correlation between the presence of muscarinic cholinergic receptors and the involvement of cholinergic mechanisms in the function of specific brain areas is discussed. Their implication in neurological diseases is also reviewed.     

Site-specific mutagenicity of stereochemically defined 1,N2-deoxyguanosine adducts of trans-4-hydroxynonenal in mammalian cells

Trans-4-hydroxynonenal (HNE) is a toxic compound produced endogenously during lipid peroxidation. HNE is a potent electrophile that is reactive with both proteins and nucleic acids. HNE preferentially reacts with deoxyguanosine to form four stereoisomeric HNE-deoxyguanosine (HNE-dG) adducts: (6R, 8S, 11R), (6S, 8R, 11S), (6R, 8S, 11S), and (6S, 8R, 11R). These adducts were synthesized into 12-mer oligodeoxynucleotides, inserted into a DNA shuttle vector and evaluated for the ability of each stereoisomer to induce mutagenesis when replicated through mammalian cells. The resultant mutagenicity of these adducts was related to their stereochemistry, in that two of the HNE-dG adducts, (6R, 8S, 11R) and (6S, 8R, 11S), were significantly more mutagenic than the (6R, 8S, 11S) and (6S, 8R, 11R) HNE-dG adducts. These data conclusively demonstrate that HNE-derived DNA adducts can be mutagenic in mammalian cells and their ability to cause mutations is dictated by their stereochemistry.     

Immune and autoimmune aspects of diabetes mellitus

The immune and autoimmune aspects of diabetes mellitus are reviewed. Emphasis is given to the clinical association of diabetes with other autoimmune disease; the increased incidence of organ-specific autoimmunity in diabetic patients; the occurrence of humoral and cell-mediated antipancreas (islet) autoimmunity in diabetes; the association of HLA with juvenile-onset, insulin-dependent diabetes mellitus and with certain specific subpopulations of diabetic patients; the possible role of viruses in the etiology of diabetes; and the occurrence of alterations in humoral and cell-mediated immunity, granulocyte function, and the host defense against infectious agents in human diabetics and in animals with experimental diabetes.