Inhibition of interleukin-6 synthesis in an animal model of septic shock by anti-C5a monoclonal antibodies

The complement activation fragment C5a was recently shown to induce interleukin (IL)-6 synthesis by peripheral blood mononuclear cells. To understand better the role of C5a in cytokine regulation in vivo, we investigated the effects of complement depletion by cobra venom factor (CVF) or of anti-C5a monoclonal antibodies (mAb) on IL-6 generation in an animal model of septic shock. Complement-depleted pigs which were subsequently challenged with Escherichia coli generated significantly (p < 0.05) less IL-6 during the 6-h observation period than complement-sufficient controls. To address specifically the role of C5a in IL-6 regulation, we produced a C5a(57–74) peptide-specific mAb (T13/9) which neutralizes the bioactivity of porcine C5a. The mAb T13/9 does not crossreact with the precursor protein C5. The pretreatment of pigs with anti-C5a mAb T13/9 prior to the induction of sepsis resulted in a decrease of over 75 % in serum IL-6 bioactivity compared to control animals (p < 0.0001). These results indicate a role for C5a in the modulation of IL-6 synthesis in Gram-negative bacteremia.     

Analysis of a terminal Xp22.3 deletion in a patient with six monogenic disorders: implications for the mapping of X linked ocular albinism.

The molecular characterisation of chromosomal aberrations in Xp22.3 has established the map position of several genes with mutations resulting in diverse phenotypes such as short stature (SS), chondrodysplasia punctata (CDPX), mental retardation (MRX), ichthyosis (XLI), and Kallmann syndrome (KAL). We describe the clinical symptoms of a patient with a complex syndrome compatible with all these conditions plus ocular albinism (OA1). He has a terminal Xp deletion of at least 10 Mb of DNA. Both the mother and sister of the patient are carriers of the deletion and show a number of traits seen in Turner's syndrome. The diagnosis of ocular albinism was confirmed in the patient and his mother, who shows iris translucency, patches and streaks of hypopigmentation in the fundus, and macromelanosomes in epidermal melanocytes. By comparative deletion mapping we can define a deletion interval, which locates the OA1 gene proximal to DXS143 and distal to DXS85, with the breakpoints providing valuable starting points for cloning strategies.     

Swelling,Elasticity and Spatial Inhomogeneity of Poly(N,N‐dimethylacrylamide) Hydrogels Formed at Various Polymer Concentrations

Summary: The equilibrium swelling degree, modulus of elasticity and the spatial inhomogeneity of poly(N,N‐dimethylacrylamide) (PDMAAm) hydrogels were investigated over the entire range of the initial monomer concentration. The degree of dilution of the networks after their preparation was denoted by ν, the volume fraction of crosslinked polymer after the gel preparation. The linear swelling ratio of the gels increased linearly with increasing ν. Depending on the value of ν, three different gel regimes were observed: (1) For ν < 0.3, increasing ν decreases the extent of cyclization during crosslinking so that the effective crosslink density of gels increases with rising ν. (2) For 0.3 < ν < 0.7, increasing ν reduces the accessibility of the pendant vinyl groups during crosslinking due to steric hindrance at high polymer concentrations. As a result, the effective crosslink density of gels decreases with increasing ν. (3) For ν > 0.7, the modulus of elasticity increases sharply with increasing ν due to the increasing extent of chain entanglements in this high concentration regime. Static light scattering measurements on the gels show that the degree of spatial gel inhomogeneity in PDMAAm gels attains a maximum value at ν = 0.06. The appearance of a maximum as well as the ν‐dependence of scattered light intensities from gels was successfully reproduced by the theory proposed by Panyukov and Rabin.

Effective crosslink density ν_e of the hydrogels shown as a function of ν.     

An Approach for Widening the Bioequivalence Acceptance Limits in the Case of Highly Variable Drugs

Purpose. Highly variable drugs pose a problem in bioequivalence assessment because they often fail to meet current regulatory acceptance criteria for average bioequivalence (80–125%). This paper examines alternative approaches to establishing bioequivalence. Methods. Suggested solutions have included alternate study designs, e.g., replicate and multiple dose studies, reducing the level of the confidence interval, and widening the acceptance limits. We focus on the latter approach. Results. A rationale is presented for defining wider acceptance limits for highly variable drugs. Two previously described methods are evaluated, and a new method having more desirable properties is proposed. Conclusions. We challenge the one size fits all current definition of bioequivalence acceptance limits for highly variable drugs, proposing alternative limits or goal posts which vary in accordance with the intrasubject variability of the reference product.     

Breast milk studies in the Bitterfeld district

Human milk samples were tested for pesticide residues (particularly chlorinated hydrocarbons) in 1996 in continuation of the environmental medical tests conducted in 1990 and 1993 in Bitterfeld county. Although the overall human milk contamination due to chlorinated hydrocarbons was reduced, there were still higher concentrations of DDT and beta-HCH detected compared to the concentrations found in what was formerly West Germany. The objective of this follow-up testing of 106 human milk samples was to observe overall trends as well as the specific effects of sanitary and environmental protection measures after three years. Sample preparation included fat extraction and chromatographic column studies. The extracts were analyzed by means of two different gas chromatography systems based on electron-catching detectors. Overall, the contamination was reduced compared to the years of 1990 and 1993. Contaminant concentrations did not exceed the "tolerable concentration" while using a safety factor of 10. However, few beta-HCH (2% of the samples), HCB (22%), total DDT (20%), and more than half of total PCB samples exceeded the "tolerable concentrations" while using a safety factor of 100. A major decrease in the average contamination was noticed for total PCB (63% decrease for 0.3 mg/kg of milk fat), beta-HCH (77% decrease for 0.056 mg/kg of milk fat), and HCB (57% decrease for 0.091 mg/kg of milk fat). The concentration of the heptachlorepoxide was even reduced to 1%. Whereas the DDT and DDD concentrations were also reduced compared to 1993 (83% decrease for 0.033 mg/kg of milk fat, and 84% decrease of 0.005 mg/kg of milk fat), the DDE concentration increased (32% increase for 0.755 mg/kg of milk fat). This can be explained by the decomposition of enzymes from DDT to DDE and the extremely high accumulation tendency of this metabolite. The results show that the contamination of women over time was influenced by two contrary factors. On one hand, the internal exposure increases with age; on the other hand lactation eliminates contaminants from the body of the mother. Comparison to other states' data shows that the results match the overall picture of the residue situation in Germany. Results of the environmental-medical investigations from 1996 demonstrate a predominant decrease of contamination with chlorinated hydrocarbons within the human body. This tendency was possibly influenced by two developments. The global usage of persistent chlorinated hydrocarbons was critically observed and regulated more strictly in recent years, which decreased their concentration tremendously. The other reason for the reduction of contamination by chlorinated hydrocarbons in the county of Bitterfeld could be the improving environmental situation in this region.     

Epidemiology of periodontal diseases in adults from Latin America

A decade has passed since we first reviewed the epidemiology of periodontal diseases in Latin America. At that time, lack of population‐based studies was the norm and our conclusions were based on very limited evidence. The aim of the present comprehensive review was to update and expand our previous work by providing a broad overview of Latin America and its current social, economic and demographic status and by focusing on the epidemiology of periodontal diseases in Latin American adults published in the last 15 years. The amount of periodontal epidemiological data available has increased but is still restricted to a few countries only. The scope of the literature available has also broadened to include oral health‐related quality of life and systemic interactions; however, most studies had methodological limitations that might have biased their results. In general, periodontitis was very prevalent, but severe periodontal destruction was localized. Besides being associated with well‐established risk factors, periodontitis was associated with demographics and socio‐economic factors in Latin American populations. To advance epidemiological knowledge, population‐based cross‐sectional and longitudinal studies, using appropriate methodologies, should be the future focus of the research agenda of researchers and public health planners in Latin American countries.     

Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.