Oligonucleotide PIK3CA/Chromosome 3 Dual in Situ Hybridization Automated Assay with Improved Signals, One-Hour Hybridization, and No Use of Blocking DNA.

The PIK3CA gene at chromosome 3q26.32 was found to be amplified in up to 45% of patients with squamous cell carcinoma of the lung. The strong correlation between PIK3CA amplification and increased phosphatidylinositol 3-kinase (PI3K) pathway activities suggested that PIK3CA gene copy number is a potential predictive biomarker for PI3K inhibitors. Currently, all microscopic assessments of PIK3CA and chromosome 3 (CHR3) copy numbers use fluorescence in situ hybridization. PIK3CA probes are derived from bacterial artificial chromosomes whereas CHR3 probes are derived mainly from the plasmid pHS05. These manual fluorescence in situ hybridization assays mandate 12- to 18-hour hybridization and use of blocking DNA from human sources. Moreover, fluorescence in situ hybridization studies provide limited morphologic assessment and suffer from signal decay. We developed an oligonucleotide-based bright-field in situ hybridization assay that overcomes these shortcomings. This assay requires only a 1-hour hybridization with no need for blocking DNA followed by indirect chromogenic detection. Oligonucleotide probes produced discrete and uniform CHR3 stains superior to those from the pHS05 plasmid. This assay achieved successful staining in 100% of the 195 lung squamous cell carcinoma resections and in 94% of the 33 fine-needle aspirates. This robust automated bright-field dual in situ hybridization assay for the simultaneous detection of PIK3CA and CHR3 centromere provides a potential clinical diagnostic method to assess PIK3CA gene abnormality in lung tumors.

Adjunctive HPV in-situ hybridization (ISH) assay as an aid in the diagnosis of cervical intraepithelial neoplasia in cervical tissue specimens: an analytical and functional characterization.

The purpose of this study was to develop and analytically and functionally validate a new human papillomavirus (HPV) in-situ hybridization (ISH) assay and to determine whether the use of this assay combined with hemotoxylin and eosin (H&E) staining could potentially improve the diagnostic accuracy of interpreting cervical intraepithelial neoplasia (CIN) in human cervical tissue specimens. An automated HPV ISH assay was developed using probes that targeted the broad spectrum of HPV genotypes most commonly associated with CIN. In an exploratory study, tissue sections (n=118) were stained with H&E alone and H&E with HPV ISH and evaluated by 6 general surgical pathologists. Results were compared with diagnoses established by expert pathologists on H&E alone. The change in specificity (diagnosis of no-CIN) and sensitivity (diagnosis of CIN) using H&E plus HPV versus H&E alone was determined. The HPV ISH assay detected 21 HPV genotypes and demonstrated no cross-reactivity to Epstein-Barr virus, cytomegalovirus, herpes simplex virus (HSV)-1, HSV-2, or human placental DNA. The assay detected HPV in a range of 1 to 600 copies on CaSki, HeLa, and SiHa xenografts. Use of this assay with H&E staining improved the average diagnostic specificity of the surgical pathologists from 68.5% to 89.9% (P<0.001), with fewer false-positive CIN 1 results (122 vs. 39). The diagnostic sensitivity was similar for assessments made with H&E alone and those made with HPV plus H&E (93.1% vs. 93.6%). In conclusion, a new automated broad-spectrum HPV ISH assay combined with H&E-stained slides contributed to better ascertainment of CIN than H&E staining alone.

Development of a method for the concentration and recovery of microsporidia from tap water.

Microsporidia are obligate intracellular parasites. Microsporidian spores infect a wide variety of hosts, including humans. The spores may be found in infected hosts' urine and feces, thus waterborne transmission is possible. This study details method development for the detection of microsporidia in tap water. In this study, filtration, centrifugation, purification, and detection parameters were optimized for the detection of microsporidia. The Pall-Gelman Envirocheck sampling capsule (Pall Gelman, Ann Arbor, MI) was chosen as the filter element. Optimal centrifugal force for spore recovery was 1500 x g. Additionally, it was determined that eluting microsporidia spores in a detergent elution buffer solution had a detrimental effect on spore recovery. A direct examination of the concentrate resulted in a greater recovery with less variability than subjecting the sample concentrate to a Percoll-sucrose density gradient purification step. The staining method employed for the detection spores was Calcofluor white (Sigma, St. Louis, MO). Percent recoveries for 10 L tap water samples (n = 5) using the Envirocheck sampling capsule without a density gradient purification step were 26.1+/-13.4 compared to 25+/-13.8 for samples subjected to a density gradient purification step.

IL-13 R130Q, a common variant associated with allergy and asthma, enhances effector mechanisms essential for human allergic inflammation.

Genetic factors are known to strongly influence susceptibility to allergic inflammation. The Th2 cytokine IL-13 is a central mediator of allergy and asthma, and common single-nucleotide polymorphisms in IL13 are associated with allergic phenotypes in several ethnically diverse populations. In particular, IL13+2044GA is expected to result in the nonconservative replacement of arginine 130 (R130) with glutamine (Q). We examined the impact of IL13+2044GA on the functional properties of IL-13 by directly comparing the activity of WT IL-13 and IL-13 R130Q on primary human cells involved in the effector mechanisms of allergic inflammation. Our results show that IL-13 R130Q was significantly more active than WT IL-13 in inducing STAT6 phosphorylation and CD23 expression in monocytes and hydrocortisone-dependent IgE switching in B cells. Notably, IL-13 R130Q was neutralized less effectively than WT IL-13 by an IL-13R2 decoy. Decreased neutralization of the minor variant could contribute to its enhanced in vivo activity. Neither IL-13 variant was able to engage T cells, which suggests that increased allergic inflammation in carriers of IL13+2044A depends on enhanced IL-13-mediated Th2 effector functions rather than increased Th2 differentiation. Collectively, our data indicate that natural variation in the coding region of IL13 may be an important genetic determinant of susceptibility to allergy.