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.     

Chlamydia trachomatis major outer membrane protein variants escape neutralization by both monoclonal antibodies and human immune sera.

We have identified two families of novel Chlamydia trachomatis isolates with amino acid changes within the major outer membrane protein (MOMP) variable domains: one family of Da, D*, and D- and one family of Ia and I-. In order to determine whether these MOMP variants can escape antibody neutralization of infectivity, we tested both the D and I prototype strains and the variants in a complement-independent in vitro neutralization assay. We found that variants can indeed escape neutralization by both monoclonal antibodies and polyclonal human immune sera that neutralize the prototype strain.     

Applications of a rat multiple tissue gene expression data set

With the sequencing and assembly of the rat genome comes the difficult task of assigning functions to genes. Tissue localization of gene expression gives some information about the potential role of a gene in physiology. Various examples of the utility of multiple tissue gene expression data sets are illustrated here. First, we highlight their use in finding genes that might play an important role in a particular tissue on the basis of exclusive expression in that tissue or coexpression with a gene or genes with known function. Second, we show how this data might be used to explain known phenotypic differences between strains. Third, we show how expression patterns of genes in a genomic interval might identify candidate genes in quantitative trait loci (QTL) mapping studies. Lastly, we show how multiple tissue and species data can help researchers prioritize follow up studies to microarray experiments. All of these applications of multiple tissue gene expression data sets will play a role in functionally annotating the rat genome.     

Corneal crystals in nephropathic cystinosis: natural history and treatment with cysteamine eyedrops

Although renal disease is the most prominent feature of the lysosomal storage disease cystinosis, corneal cystine crystal formation remains a major complication, leading to photophobia, corneal erosions, and keratopathies. Moreover, the extent of corneal crystal accumulation reflects the course and severity of the disease itself, and the cornea is accessible to direct examination. Therefore, we employed a scoring system, based on a library of slit-lamp photographs of corneas with increasing crystal densities (0.00-3.00), to assess the degree of crystal accumulation in 170 patients with nephropathic cystinosis examined at the National Institutes of Health between 1976 and 2000. None of the patients had received topical cystine-depleting therapy at the time of the evaluation. In this natural history study, infants in the first year of life had absent or minimal corneal crystals, i.e., a corneal cystine crystal score (CCCS) of 0 or 0.25. However, the CCCS increased linearly with age, such that every patient had visible crystals by 16 months of age, and plateaued at approximately 3.00 by early adolescence. Longitudinal studies in representative patients support the cross-sectional results. Individuals homozygous for the common 57-kb deletion involving the cystinosis gene (CTNS) displayed the same course of corneal crystal accumulation as did individuals not bearing the large deletion. Patients with ocular or nonnephropathic cystinosis had CCCSs that were, in general, half those expected for patients with nephropathic cystinosis of the same age. Administration of 0.55% cysteamine eyedrops, given 6 to 12 times per day, dissolved corneal cystine crystals in 10 representative patients with nephropathic cystinosis aged 1 to 32 years within 8 to 41 months.     

Regional distribution of fiber types in developing baboon diaphragm muscles

Fiber type distribution and mean fiber area were determined for seven sites in diaphragm muscles of premature (140 days gestation), full-term (180 days gestation), and adult baboons. Within a group, data did not differ significantly amongst the seven sites. The diaphragm of premature animals had a large proportion [56(+/- 2)%] of type IIc fibers, smaller proportions of type I, IIo, and IIh fibers [16(+/- 2), 21(+/- 1), and 7(+/- 2)%, respectively], and no type IIg fibers. Full-term animals had fewer type IIc [2(+/- 1)%] fibers, greater proportions of type I [46(+/- 2)%], IIh [23(+/- 1)%], and IIg [11(+/- 1)%] fibers, and a similar proportion of type IIo fibers [17(+/- 1)%]. Diaphragm from adult baboons had similar proportions of type IIh, IIg, and IIc fibers in females [39(+/- 4), 20(+/- 2), 1(+/- 1), 41(+/- 5), and 1(+/- 1)%] and males [48(+/- 2), 16 (+/- 1), 0(+/- 0), 36(+/- 2), and 3(+/- 2)%]. Fiber area for premature [143(+/- 9), 210(+/- 15), 231(+/- 15), and 156(+/- 16) microns2 for type I, IIo, IIh, and IIc fibers], newborn [317(+/- 32), 374(+/- 36), 468(+/- 42), 498(+/- 43), and 322(+/- 37) microns2 for type I, IIo, IIh, IIg, and IIc fibers], and for type I, IIo, IIg, and IIc fibers from adult female [1,759(+/- 130), 2,365(+/- 284), 5,026(+/- 742), and 1,843(+/- 111) microns2] and adult male [2,513(+/- 221), 3,987(+/- 267), 6,102(+/- 376), and 2,833(+/- 151) microns2] baboons indicated growth which correlated with body weight. Our results also show that metabolic and contractile enzymes develop normally, but growth of respiratory muscle fibers is arrested, during 10 days following premature birth.     

A Randomized Clinical Trial of Topical Cysteamine Disulfide (Cystamine) versus Free Thiol (Cysteamine) in the Treatment of Corneal Cystine Crystals in Cystinosis

In nephropathic cystinosis, corneal cystine crystals cause severe photophobia and corneal erosions. Topical cysteamine dissolves these crystals, but cannot be marketed because it rapidly oxidizes to the disulfide form, cystamine, at room temperature. Since cystamine itself could be used commercially, we compared the efficacy of cystamine and cysteamine with respect to cystine crystal dissolution in a randomized, double-masked clinical trial. One eye each of 14 patients with cystinosis was randomized to either cystamine or cysteamine, 0.5%, with 0.01% benzalkonium chloride; the companion eye was treated with the alternate preparation. Corneal crystals were photographed and a density score was assigned to each slide based on 13 standard slides. After 8–20 months, 6 patients showed significant reduction of the corneal crystal score in only one eye. In each case, the improved eye was the cysteamine-treated eye. Theoretically, cysteamine should dissolve both intracellular and extracellular crystals, whereas cystamine should dissolve only intracellular crystals because it must first be reduced to the free thiol by the cytoplasmic-reducing environment. Hence, the lack of efficacy of the disulfide cystamine suggests that some corneal cystine crystals in cystinosis patients are extracellular, and that another form of stable, topical cysteamine must be developed for cystinosis patients.     

Maternal immunization modulates the primary immune response to 2-phenyl-oxazolone in BALB/c mice

The development of the antibody repertoire in newborn mice is greatly influenced by idiotypic network interactions. It has been demonstrated that anti-idiotypic antibodies either directly injected or transferred from the mother may alter the repertoire for life. For an elucidation of the underlying mechanisms we have analyzed the primary immune response to 2-phenyl-5-oxazolone (phOx) coupled to chicken serum albumin (CSA) in BALB/c mice after complete disappearance of maternal antibodies which originated from different stages of affinity maturation. Depending on the serum titers of the mothers after primary (1° mo), secondary (2° mo) or tertiary (3° mo) immunization, maternal anti-phOx IgG persisted in F1 mice for up to 9 months. In addition, F1 mice born to 2° mo developed – even without immunization – an anti-phOx IgM titer which reached levels similar to an antigen-induced primary response. An enhancement of the early primary anti-phOx as well as anti-CSA response was seen in F1 mice born from 1° mo, whereas the response was delayed when born to 2° mo and 3° mo. The antibody titers in the latter group of mice remained at a lower level for 3 months. In contrast, mice of the F2 generation which received a smaller amount of the same collection of maternal antibodies as F1 mice from 3° mo exhibited a quite different primary response: (i) They showed an earlier onset in their anti-CSA response. (ii) Whereas normally a plateau in antibody titer was reached by the 4th weak after immunization, in 55 % of the F2 mice a prolonged increase of the anti-phOx and anti-CSA antibody titers was observed. At 12 weeks after antigenic challenge, titers reached plateau levels of 6 × 10⁵ which were never before seen in a primary phOx or CSA response. Thus, depending on its own immunological experience, the maternal immune system induces a state of memory in the offspring which results in a faster and/or enhanced immune response in the F1 and F3 generations.     

Spectral karyotyping combined with locus-specific FISH simultaneously defines genes and chromosomes involved in chromosomal translocations

Genes that play roles in malignant transformation have often been found proximate to cancer-associated chromosomal breakpoints. Identifying genes that flank chromosomal reconfigurations is thus essential for cancer cytogenetics. To simplify and expedite this identification, we have developed a novel approach, based on simultaneous spectral karyotyping and fluorescence in situ hybridization (FISH) which, in a single step, can identify gross chromosomal aberrations as well as detect the involvement of specific loci in these rearrangements. Signals for specifically queried genes (FISH probe) were easily detectable in metaphase cells, together with the signals from painted chromosomes (spectral karyotyping probes). The concentration and size of the FISH probes could cover a wide range and still be used successfully. Some of the nucleotide-bound dyes used for the labeling, as Cy3, Spectrum Orange, Alexa 594, Texas Red, and Rhodamine 110, were particularly efficient. More than one gene can be queried in the same metaphase, because multiple FISH probes could be hybridized simultaneously. To demonstrate this technique, we applied it to the myeloma cell line Karpas 620, which has numerous chromosomal rearrangements. The approach that we present here will be particularly useful for the analysis of complex karyotypes and for testing hypotheses arising from cancer gene expression studies. Published 2000 Wiley-Liss, Inc.     

The Bioperl toolkit: Perl modules for the life sciences

The Bioperl project is an international open-source collaboration of biologists, bioinformaticians, and computer scientists that has evolved over the past 7 yr into the most comprehensive library of Perl modules available for managing and manipulating life-science information. Bioperl provides an easy-to-use, stable, and consistent programming interface for bioinformatics application programmers. The Bioperl modules have been successfully and repeatedly used to reduce otherwise complex tasks to only a few lines of code. The Bioperl object model has been proven to be flexible enough to support enterprise-level applications such as EnsEMBL, while maintaining an easy learning curve for novice Perl programmers. Bioperl is capable of executing analyses and processing results from programs such as BLAST, ClustalW, or the EMBOSS suite. Interoperation with modules written in Python and Java is supported through the evolving BioCORBA bridge. Bioperl provides access to data stores such as GenBank and SwissProt via a flexible series of sequence input/output modules, and to the emerging common sequence data storage format of the Open Bioinformatics Database Access project. This study describes the overall architecture of the toolkit, the problem domains that it addresses, and gives specific examples of how the toolkit can be used to solve common life-sciences problems. We conclude with a discussion of how the open-source nature of the project has contributed to the development effort.     

Organochlorine,organobromine, metal,and selenium residues in bottlenose dolphins (Tursiops truncatus) collected during an unusual mortality event in the Gulf of Mexico, 1990

Polychlorinated biphenyls (PCBs), cis-chlordane, oxychlordane, heptachlor epoxide, mirex, hexachlorobenzene (HCB), lindane, octachlorostyrene (OCS), p,p-DDE,p,p-DDT, dieldrin, triphenylphosphate (TPP), polybrominated biphenyls (PBBs), and polybrominated diphenyl ethers (PBDPEs) were measured in the blubber, and five metals (mercury, lead, cadmium, chromium, and manganese) and selenium were measured in the liver of bottlenose dolphins (Tursiops truncatus) obtained from the Gulf of Mexico during an unusual mortality event in 1990. The collection of animals included fetuses, sucklings (<1 year old), immature dolphins (2–5 years old), and adults of both sexes. PCBs, p,p-DDE, HCB, and PBDPEs were detected in the blubber of each animal. Mean concentrations of organic contaminants were generally highest in adult males. p,p-DDE was the single component analyte measured at the highest concentration. Immature females had greater concentrations of most chlorinated organics than adult females. Mercury and cadmium concentrations in liver increased with increasing age-class. The correlation between mercury and selenium in all animals was r=0.96, with a mole ratio of 0.90. Concentrations of lead, manganese, cadmium, and chromium did not follow any particular age-class trend.