Affinity purification of a major and a minor allergen from dog extract: serologic activity of affinity-purified Can f I and of Can f I-depleted extract

Most dog-allergic patients react to a major 25 kd component on sodium dodecyl sulfate blots, Can f I (Ag 13). We initially raised monoclonal antibodies (Cf-3 and Cf-2) reactive with IgE-binding components distinct from Can f I. After a slight modification, we immunized other strains of mice and produced monoclonal antibodies coded Cf-1a and Cf-1b reactive with Can f 1. We affinity purified the allergens, Can f I and "dog allergen 2" with Cf-1a and Cf-2 ascites, respectively, and house dust-rich dog dander. Comparison of purified Can f I with dog saliva in RAST demonstrated that Can f I is a potent allergen for most dog-allergic patients (average response, 70%). After depletion of dog saliva of Can f I, a slightly lower contribution for Can f I was found, but the overall results supported the conclusion that Can f I is a major allergen in dog saliva. Comparison of purified dog allergen 2 with dog dander in RAST demonstrated that dog allergen 2 is less important for dog-allergic patients (average response, 23%). We radiolabeled the purified allergens and developed assays to measure Can f I and dog allergen 2 in allergen extracts and dust samples. Dog saliva was a strong allergen source, dog urine and feces contained very little of the allergens, and both allergens were found to a variable degree in the nine dog breeds tested.     

Usefulness of repeated N-terminal pro-B-type natriuretic peptide measurements as incremental predictor for long-term cardiovascular outcome after vascular surgery

Plasma N-terminal pro-B-type natriuretic peptide (NT-pro-BNP) levels improve preoperative cardiac risk stratification in vascular surgery patients. However, single preoperative measurements of NT-pro-BNP cannot take into account the hemodynamic stress caused by anesthesia and surgery. Therefore, the aim of the present study was to assess the incremental predictive value of changes in NT-pro-BNP during the perioperative period for long-term cardiac mortality. Detailed cardiac histories, rest left ventricular echocardiography, and NT-pro-BNP levels were obtained in 144 patients before vascular surgery and before discharge. The study end point was the occurrence of cardiovascular death during a median follow-up period of 13 months (interquartile range 5 to 20). Preoperatively, the median NT-pro-BNP level in the study population was 314 pg/ml (interquartile range 136 to 1,351), which increased to a median level of 1,505 pg/ml (interquartile range 404 to 6,453) before discharge. During the follow-up period, 29 patients (20%) died, 27 (93%) from cardiovascular causes. The median difference in NT-pro-BNP in the survivors was 665 pg/ml, compared to 5,336 pg/ml in the patients who died (p = 0.01). Multivariate Cox regression analyses, adjusted for cardiac history and cardiovascular risk factors (age, angina pectoris, myocardial infarction, stroke, diabetes mellitus, renal dysfunction, body mass index, type of surgery and the left ventricular ejection fraction), demonstrated that the difference in NT-pro-BNP level between pre- and postoperative measurement was the strongest independent predictor of cardiac outcome (hazard ratio 3.06, 95% confidence interval 1.36 to 6.91). In conclusion, the change in NT-pro-BNP, indicated by repeated measurements before surgery and before discharge is the strongest predictor of cardiac outcomes in patients who undergo vascular surgery.     

An integrated approach for identifying and mapping human genes.

We have developed a method for generating expressed-sequence maps of human chromosomes. The method involves several steps that begin with libraries of highly representative short cDNAs prepared by using random oligomers as primers. The cDNA inserts are amplified by PCR with flanking vector primers. Chromosomal region-specific cDNA packets are prepared by hybridization of the cDNA inserts to DNA derived from yeast artificial chromosomes (YACs) assigned to defined regions of human chromosomes. The cDNA packets are cloned into yeast chromosome fragmentation vectors and used for transformation of yeast bearing the YAC used for affinity purification. Sequences in the cDNAs undergo homologous recombination with the corresponding exons in the genomic DNA yielding a set of truncated YACs. Each unique truncation specifies the location of an exon in the YAC. Since all of the truncation events end with the same vector sequence, it is possible to rescue and sequence these ends to generate expressed sequence tags. The method couples rapid purification of region-specific cDNAs with precise mapping of their genes on YACs. Appropriately truncated YACs also provide easy access to gene regulatory sequences. We describe the feasibility of individual steps of the method using the factor IX (F9) gene as a model system and we present the mapping of several expressed sequences corresponding to a 330-kb YAC containing DNA from human chromosome 6p21. In addition, we obtained the sequence, including an intron-exon junction, flanking a particular truncation event.