CYP1A Expression in Caged Rainbow Trout Discriminates Among Sites with Various Degrees of Polychlorinated Biphenyl Contamination

It has become increasingly apparent that resident fish can develop resistance to chemicals in their environment, thus compromising their usefulness as sentinels of site-specific pollution. By using a stream system whose resident fish appear to have developed pollutant resistance (Brammell et al., Mar Environ Res 58:251–255, 2005), we tested the hypothesis that the pollutant-inducible biomarker, cytochrome P4501A (CYP1A), as measured in field-caged juvenile rainbow trout (Oncorhynchus mykiss), would reflect relative pollution differences between reference and polychlorinated biphenyl (PCB)-contaminated sites. Trout were caged in the Town Branch/Mud River system (Logan County, KY), a stream system undergoing remediation for PCBs. Fish were held in remediated (Town Branch), unremeditated (Mud River), and reference sites for 2 weeks during spring 2002. At the end of this period, gill and hepatic CYP1A expression were measured. To evaluate the relative PCB exposure of caged trout and provide a reference point against which to calibrate CYP1A response, PCB levels were quantified in sediments from each site. Hepatic CYP1A expression in caged trout clearly detected the presence of PCBs in the Town Branch/Mud River stream system. Sediment PCB levels and hepatic CYP1A expression in caged trout produced identical pollution rankings for the study sites. Gill CYP1A expression, although suggestive of site differences, was not statistically different among sites. Unlike resident fish, which failed to show site differences in hepatic CYP1A expression in this waterway (Brammell et al. 2005), caged fish proved to be a sensitive discriminator of relative PCB contamination in this system. In summary, we determined that CYP1A expression in caged fish reflected relative in situ pollutant exposure. The exposure paradigm confirmed that 2 weeks was a sufficient caging period for evaluating CYP1A response in this species at these temperatures (13–19°C). In addition, these studies demonstrate that tissue-specific CYP1A expression can provide insights into likely routes of exposure. We conclude that CYP1A expression in caged trout is a reliable and inexpensive first-pass determination of relative environmental pollutant exposure and bioavailability in aqueous systems.     

The chemotherapy of posterior Fossa tumors in childhood

Conventional therapy for brain tumors, consisting of neurosurgical intervention and radiotherapy, has not resulted in the successes achievable in other childhood malignancies. The role of adjuvant chemotherapy, well defined in many childhood cancers, has not yet contributed significantly to the treatment of children with brain tumors. Chemotherapy of recurrent tumors has produced regressions but no cures. The most active agents identified to date in the treatment of recurrent posterior fossa tumors include cisplatinum, cyclophosphamide and methotrexate. Future efforts will need to focus on the rational selection of drugs for study in limited agent histology-stratified phase II trials, with advancement of active agents into large randomized phase III adjuvant therapy trials.     

Effect of electric and chemical stimulation of the raphe obscurus on phrenic nerve activity in the cat

The effect of electrical and chemical (l-glutamate) stimulation of the raphe obscurus on phrenic nerve activity was examined in the cat. Phrenic nerve activity was recorded from a C5 nerve root in anesthetized, paralyzed and artificially ventilated cats. Neural discharge was quantitated by integrating the phrenic nerve activity. The respiratory frequency was determined from the integrated nerve signal. Focal electrical stimulation (18–144 μA; 5–40 Hz; 100 μs pulse duration) resulted in significant (P < 0.05) increases in both integrated phrenic nerve (IPN) amplitude and respiratory frequency. These changes were dependent upon current intensity and frequency of stimulation. The largest increases in IPN amplitude and respiratory frequency were47 ± 17%and146 ± 8%, respectively. To insure that the changes in integrated phrenic nerve activity (IPNA) were the result of stimulation of cell bodies and not axons of passage,l-glutamate (100, 200 nmol) was microinjected (100 nl) into the raphe obscurus. Significant (P < 0.05) dose-related changes occurred in integrated phrenic nerve amplitude with an increase of44 ± 13% at 100 nmol and80 ± 13% at 200 nmoll-glutamate. No significant increase in respiratory frequency was observed withl-glutamate microinjection. The results suggest that the raphe obscurus may be involved in respiratory control.     

Modification of peanut allergen Ara h 3: effects on IgE binding and T cell stimulation

BACKGROUND: Peanut allergy is a major health concern due to the increased prevalence, potential severity, and chronicity of the reaction. The cDNA encoding a third peanut allergen, Ara h 3, has been previously cloned and characterized. Mutational analysis of the Ara h 3 IgE-binding epitopes with synthetic peptides revealed that single amino acid changes at critical residues could diminish IgE binding. METHODS: Specific oligonucleotides were used in polymerase chain reactions to modify the cDNA encoding Ara h 3 at critical IgE binding sites. Four point mutations were introduced into the Ara h 3 cDNA at codons encoding critical amino acids in epitopes 1, 2, 3 and 4. Recombinant modified proteins were used in SDS-PAGE/Western IgE immunoblot, SDS-PAGE/Western IgE immunoblot inhibition and T cell proliferation assays to determine the effects of these changes on in vitro clinical indicators of peanut hypersensitivity. RESULTS: Higher amounts of modified Ara h 3 were required to compete with the wild-type allergen for peanut-specific serum IgE. Immunoblot analysis with individual serum IgE from Ara-h-3-allergic patients showed that IgE binding to the modified protein decreased approximately 35-85% in comparison to IgE binding to wild-type Ara h 3. Also, the modified Ara h 3 retained the ability to stimulate T cell activation in PBMCs donated by Ara-h-3-allergic patients. CONCLUSIONS: The engineered hypoallergenic Ara h 3 variant displays two characteristics essential for recombinant allergen immunotherapy; it has a reduced binding capacity for serum IgE from peanut-hypersensitive patients and it can stimulate T-cell proliferation and activation.     

A novel single chain I-A(b) molecule can stimulate and stain antigen-specific T cells

Multimers of soluble major histocompatibility complex class I and II molecules have proven to be useful reagents in quantifying and following specific T cell populations. This study describes the design, generation, and characterization of a novel, single chain I-A(b) molecule which utilizes a unique linker derived from the murine invariant chain. A fragment of the invariant chain, residues 58-85, binds to a region proximal to the class II peptide binding groove and stabilizes occupancy of the class II invariant chain-associated peptide. We have utilized this fragment, replacing CLIP with the Ealpha peptide sequence, to lock the attached peptide into the class II binding groove. The single chain I-A(b) molecule was recognized by a panel of conformation-sensitive, I-A(b)-specific, monoclonal antibodies. Membrane-bound and soluble forms of the single chain I-A(b) stimulated an antigen-specific T cell hybridoma, and tetramers made from soluble monomers stained these cells. The unique features of this molecule may be useful in the design of recombinant T cell receptor ligands containing peptides with low affinity for MHC.     

Clinical Importance of Identifying Coagulase-Negative Staphylococci Isolated from Blood Cultures: Evaluation of MicroScan Rapid and Dried Overnight Gram-Positive Panels versus a Conventional Reference Method

We evaluated the clinical usefulness of species identification of blood isolates of coagulase-negative staphylococci as a predictor of the clinical significance of the isolates. In addition, we compared results of species identification obtained with MicroScan Rapid Gram-Positive Identification panels and Dried Overnight (Conventional) Gram-Positive Identification panels with those obtained by a tube reference method. Two hundred eighty-five blood isolates were tested, including 92 judged to represent true bacteremia and 193 judged to represent contamination. The most common species detected were Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus haemolyticus. These three species accounted for nearly 98% of the clinically significant isolates and 89% of the contaminants. The isolation of other species almost always represented contamination. However, identification of the three most common species did not help distinguish pathogens from contaminants. Both the Rapid and the Dried Overnight Gram-Positive panels identified S. epidermidis strains accurately, but the panels performed less well for the other species. Analysis revealed that S. hominis was frequently misidentified due to the presence of a previously unknown subspecies. Based on the initial results, revised investigational Dried Overnight Gram-Positive Identification panels (CPID-2) were prepared and tested. The CPID-2 panels identified 85 to 95% of S. epidermidis strains, 76 to 86% of S. hominis strains, and 88 to 92% of S. haemolyticus strains with high probability (>85%) and, overall, represented a significant improvement over the other panels for identification of these staphylococcal species.     

Arteriovenous glomeruli of the human spinal cord and their possible functional implications

A previous investigation of the radiculomedullary vascular distribution of the human spinal cord showed an immediate filling of the peripheral spinal veins during arterial injections. Because the perfused medium, a mixture of latex and India ink, had a sufficient viscosity to preclude capillary passage, an extensive system of arteriovenous anastomoses (AVA) was indicated. The injection of five additional anatomic cadaver spinal cords was undertaken to specifically determine the intraparenchymal position and structure of these AVA. It was found that only cleared and transilluminated longitudinal parasagittal sections adequately showed that the AVA were formed by an extensive array of intricately coiled arteriovenous glomeruli (AVG) that could pass substantial amounts of blood from third order arterial branches to equivalently sized veins, bypassing the capillary bed. The epithelioid structure of these AVG channel walls, coupled with their highly innervated adventitial tissue indicated a possible functional relationship to similar structures found in the ends (digital pads) of mammalian extremities. Stained serial sections of injected cords showed the vascular relations with nerve fibers derived from the intraspinal parenchyma as well as a possible extraparenchymal autonomic fiber source. The numbers and extent of these AVG complexes suggested they might be involved in the remarkable autoregulation of spinal cord blood flow (SCBF) that is impervious to middle range changes in systemic blood pressure. Future research may more accurately determine the functional role of these previously unreported human spinal AVG, particularly in their possible relation to SCBF autoregulation in normal and injured spines.     

Towards immunotherapy for peanut allergy

PURPOSE OF REVIEW: Food allergy is a major cause of life-threatening hypersensitivity reactions. Peanut allergy is the most serious of the hypersensitivity reactions to foods due to its persistence and high risk of severe anaphylaxis. Currently, strict avoidance of the allergenic food and ready access to self-injectable epinephrine is the 'standard of care' for food allergy. Based on extensive characterization of food allergens and a better understanding of the immunological mechanisms underlying allergic disease, promising therapeutic modalities for food allergy treatment and prevention are being developed. RECENT FINDINGS: Immunotherapeutic strategies include peptide immunotherapy, mutated protein immunotherapy and DNA immunization, which all strive to decrease the deleterious Th2 response. Another approach already in clinical trials for peanut allergy is the anti-IgE therapy which prevents circulating IgE from binding to effector cells, consequently decreasing clinical symptoms after peanut ingestion. In order to be applicable, these strategies must be well tolerated, inexpensive and easily administered. Realistic treatment options would likely involve a combination of different approaches. SUMMARY: Food allergy affects approximately 4-6% of children and 3-4% of adults. Peanut allergy can be devastating as reactions range from urticaria to severe anaphylactic shock and death. The only preventive measure for peanut allergy is strict avoidance of the incriminating food. It is likely immunotherapy will be available in the near future as a well tolerated and effective therapy for treating peanut allergy. The use of the anti-IgE therapy in conjunction with other immunotherapy would possibly be the best treatment option in the future.