An ELISA serum assay for autoantibodies to HSP70 in immune-mediated hearing loss

A Western blot to detect anti-HSP70 autoantibodies has been reported to be of diagnostic value for immune-mediated hearing loss patients. While setting up this Western blot in our lab, we detected two main problems. First, some patients were positive for antibodies to a 70-kDa protein when tested against a whole cell lysate, but negative if the antigen used was purified HSP70. Second, if high amounts of purified HSP70 were loaded on the gel, both patients and healthy controls were positive. We have developed and optimized an ELISA as an alternative to the Western blot. This assay is more appropriate to identify positive and negative individuals because it is semi-quantitative. The ELISA is also more sensitive, requiring very low concentrations of the antigen and thus minimizing false positives. Finally, we demonstrated that immune-mediated hearing loss patients recognize mainly the native form of HSP70, a fact that potentially leads to false negatives when a denaturing Western blot assay is used for diagnosis. To test the diagnostic value of the ELISA, we performed a blind test with 70 hearing loss patients, as well as 30 healthy controls. A sensitivity of 84% and a specificity of 93% were obtained, superior to what has been reported so far for the Western blot.     

Identification of a 7S globulin as a novel coconut allergen.

BACKGROUND: Coconut (Cocos nucifera) is a monocotyledonous plant of the Arecaceae family. Allergy to coconut is infrequent, with only 5 cases reported so far in the medical literature. OBJECTIVE: To identify coconut allergens in 2 patients allergic to this food. METHODS: We describe 2 patients allergic to coconut: an adult pollen-allergic patient monosensitized to coconut who presented with severe oropharyngeal symptoms and a child with a previous allergy to walnut, not allergic to pollen, who developed anaphylaxis on coconut ingestion. Both patients had positive skin prick test results and serum specific IgE (CAP) to coconut. IgE sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting was performed to identify the allergens involved, and a strong IgE binding band detected in both patients was further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS). Stability to pepsin digestion of the coconut extract and its cross-reactivity with tree nuts were studied. RESULTS: An immunoblot showed an almost identical profile of IgE binding proteins in the coconut extract in both patients who reacted strongly to a band of approximately 29 kDa. The peptide analysis by MALDI-TOF MS of this band obtained the sequence GHGKREDPEKR. The protein with the highest correlation with this peptide was found to be a 7S globulin from Elaeis guineensis, another oil palm species also belonging to the Arecaceae family. The 29-kDa band was digested by pepsin in less than 1 minute. Cross-reactivity among coconut, walnut, and hazelnut was demonstrated by CAP inhibition in patient 2. CONCLUSION: We have identified a 7S storage protein as a novel coconut allergen.     

An animal model of human-type memory loss based on aging, lesion, forebrain ischemia, and drug studies with the rat

The goals of this research were to develop a within-subject test of spatial working memory and performance for the rat in a T-maze, based on a delayed alternation, or "win-shift" foraging strategy. Using this model, specific aims were to compare the effects of: (1) age, (2) basal forebrain, medial septal, and amygdala lesions, (3) four vessel occlusion (4-VO), forebrain ischemia, and (4) physostigmine, scopolamine, arecoline, piracetam, and clonidine on memory and performance of young middle-aged, and old rats. Aging significantly impaired working memory and performance of Long-Evans rats. Memory of septal and basal forebrain, but not of amygdala lesioned rats was significantly impaired without effects on performance. Transient, 4-VO forebrain ischemia produced significant memory impairment, without effects on performance, and highly selective CA1 cell loss in the hippocampus. Physostigmine enhanced working memory in middle-aged and old rats. Scopolamine impaired memory in young, middle-aged, and old rats. Physostigmine reversed the scopolamine impairments of working memory. Arecoline enhanced memory in old rats without effects on performance. Piracetam and clonidine had no direct effects on memory, but piracetam increased and clonidine decreased speed of performance. From the aging, lesion, ischemia, and drug studies it was concluded that there was a convergence of evidence from 4 different approaches for a critical role for the hippocampus, particularly the CA1 fields, in spatial working memory.     

Development of DNA vaccines against hemolytic-uremic syndrome in a murine model

Shiga toxin type 2 (Stx2) produced by Escherichia coli O:157H7 can cause hemolytic-uremic syndrome in children, a disease for which there is neither a vaccine nor an effective treatment. This toxin consists of an enzymatically active A subunit and a pentameric B subunit responsible for the toxin binding to host cells, and also found to be immunogenic in rabbits. In this study we developed eukaryotic plasmids expressing the B subunit gene of Stx2 (pStx2B) and the B subunit plus the gene coding for the A subunit with an active-site deletion (pStx2 Delta A). Transfection of eukaryotic cells with these plasmids produced proteins of the expected molecular weight which reacted with specific monoclonal antibodies. Newborn and adult BALB/c mice immunized with two intramuscular injections of each plasmid, either alone or together with the same vector expressing the granulocyte and monocyte colony-stimulating factor (pGM-CSF), elicited a specific Th1-biased humoral response. The effect of pGM-CSF as an adjuvant plasmid was particularly notable in newborn mice and in pStx2B-vaccinated adult mice. Stx2-neutralizing activity, evaluated in vitro on VERO cell monolayers, correlated with in vivo protection. This is the first report using plasmids to induce a neutralizing humoral immune response against the Stx2.     

Genetic relationship between Streptococcus pneumoniae isolates from nasopharyngeal and cerebrospinal fluid of two infants with Pneumococcal Meningitis

The molecular epidemiology of Streptococcus pneumoniae isolates from carriage and cerebrospinal fluid (CSF) concurrently recovered from the same individual has not yet been reported. By using pulsed-field gel electrophoresis, we demonstrated the genetic linkage among strains from CSF and nasopharynges of two children with pneumococcal meningitis.     

Reducing the time of sperm-oocyte interaction in human in-vitro fertilization improves the implantation rate

Human oocyte development was evaluated after a reduced timeexposure to spermatozoa in vitro. A total of 119 patients wereassigned to two study groups in a randomized prospective studyin which each patient‘s oocytes were exposed to spermatozoafor either 1 h (group 1 – 58 patients) or the standard16 h incubation period (group 2 – 61 patients). The fertilizationrate obtained in group 1 was higher than in group 2 (285/393,73%, and 272/410, 66% respectively), suggesting that the spermatozoa-oocyteinteraction occurs within 1 h. This was confirmed in a studyin vitro using fluorescently labelled spermatozoa and normaloocyte-cumulus complexes. Spermatozoa enter the cumulus complexwithin 15 min, traverse the cumulus layer within 3 h, and firstappear in the oocyte cortex at 4 h post-insemination. The incidenceof polyspermy was higher in oocytes exposed to spermatozoa for16 h (3%) than for 1 h (1%). There was no difference in thecleavage rate or morphological characteristics of embryos fromboth study groups. However, when evaluating the timing of embryodevelopment, group 1 generated a significantly higher percentageof four to five cell embryos when compared to group 2 (55 versus39%; P < 0.001), documented at 40 h post-insemination. Theimplantation and pregnancy rates for group 1 were 11 and 28%,while the corresponding rates for group 2 were 8 and 15%. Thissuggests that a reduced exposure of oocyte to spermatozoa favoursembryo viability, possibly due to a decrease in potential damagefrom sperm metabolic waste products.     

Classification and subtype prediction of adult soft tissue sarcoma by functional genomics

Adult soft tissue sarcomas are a heterogeneous group of tumors, including well-described subtypes by histological and genotypic criteria, and pleomorphic tumors typically characterized by non-recurrent genetic aberrations and karyotypic heterogeneity. The latter pose a diagnostic challenge, even to experienced pathologists. We proposed that gene expression profiling in soft tissue sarcoma would identify a genomic-based classification scheme that is useful in diagnosis. RNA samples from 51 pathologically confirmed cases, representing nine different histological subtypes of adult soft tissue sarcoma, were examined using the Affymetrix U95A GeneChip. Statistical tests were performed on experimental groups identified by cluster analysis, to find discriminating genes that could subsequently be applied in a support vector machine algorithm. Synovial sarcomas, round-cell/myxoid liposarcomas, clear-cell sarcomas and gastrointestinal stromal tumors displayed remarkably distinct and homogenous gene expression profiles. Pleomorphic tumors were heterogeneous. Notably, a subset of malignant fibrous histiocytomas, a controversialhistological subtype, was identified as a distinct genomic group. The support vector machine algorithm supported a genomic basis for diagnosis, with both high sensitivity and specificity. In conclusion, we showed gene expression profiling to be useful in classification and diagnosis, providing insights into pathogenesis and pointing to potential new therapeutic targets of soft tissue sarcoma.