IMPORTANCE Retinoblastoma (Rb) is one of the first tumors to have a known genetic etiology. However, because biopsy of this tumor is contraindicated, it has not been possible to define the effects of secondary genetic changes on the disease course. OBJECTIVE To investigate whether the aqueous humor (AH) of Rb eyes has sufficient tumor-derived DNA to perform genetic analysis of the tumor, including DNA copy number alterations. DESIGN, SETTING, AND PARTICIPANTS This investigation was a case series study at a tertiary care hospital (Children's Hospital Los Angeles) with a large Rb treatment center. Cell-free DNA (cfDNA) was isolated from 6 AH samples from 3 children with Rb, including 2 after primary enucleation and 1 undergoing multiple intravitreous injections of melphalan for vitreous seeding. Samples were taken between December 2014 and September 2015. MAIN OUTCOMES AND MEASURES Measurable levels of nucleic acids in the AH and identification of tumor-derived DNA copy number variation in the AH. The AH was analyzed for DNA, RNA, and micro-RNA using Qubit high-sensitivity kits. Cell-free DNA was isolated from the AH, and sequencing library protocols were optimized. Shallow whole-genome sequencing was performed on an Illumina platform, followed by genome-wide chromosomal copy number variation profiling to assess the presence of tumor DNA fractions in the AH cfDNA of the 3 patients. One child's cfDNA from the AH and tumor DNA were subjected to Sanger sequencing to isolate the RB1 mutation. RESULTS Six AH samples were obtained from 3 Rb eyes in 3 children (2 male and 1 female; diagnosed at ages 7, 20, and 28 months). A corroborative pattern between the chromosomal copy number variation profiles of the AH cfDNA and tumor-derived DNA from the enucleated samples was identified. In addition, a nonsense RB1 mutation (Lys→STOP) from 1 child was also identified from the AH samples obtained during intravitreous injection of melphalan, which matched the tumor sample postsecondary enucleation. Sanger sequencing of the AH cfDNA and tumor DNA with polymerase chain reaction primers targeting RB1 gene c.1075A demonstrated this same RB1 mutation. CONCLUSIONS AND RELEVANCE In this study evaluating nucleic acids in the AH from Rb eyes undergoing salvage therapy with intravitreous injection of melphalan, the results suggest that the AH can serve as a surrogate tumor biopsy when Rb tumor tissue is not available. This novel method will allow for analyses of tumor-derived DNA in Rb eyes undergoing salvage therapy that have not been enucleated.

Outer membrane protein A (OmpA) is an abundant protein of Escherichia coli and other enterobacteria with a multitude of functions. Although the structural features and porin function of OmpA were well studied, its role in the pathogenesis of various bacterial infections has been emerging for the past decade. The four extracellular loops of OmpA interact with a variety of host tissues for adhesion, invasion and evasion of host-defense mechanisms. This review describes how various regions present in the extracellular loops of OmpA contribute to the pathogenesis of neonatal meningitis induced by E. coli K1 and for many other functions. In addition, the function of OmpA like proteins such as OprF of Pseudomonas aeruginosa is also discussed herein.

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Outer membrane protein A (OmpA) has been implicated as an important virulence factor in several Gram-negative bacterial infections such as Escherichia coli K1, a leading cause of neonatal meningitis associated with significant mortality and morbidity. In this study, we generated E. coli K1 mutants that express OmpA in which three or four amino acids from various extracellular loops were changed to alanines, and we examined their ability to survive in several immune cells. We observed that loop regions 1 and 2 play an important role in the survival of E. coli K1 inside neutrophils and dendritic cells, and loop regions 1 and 3 are needed for survival in macrophages. Concomitantly, E. coli K1 mutants expressing loop 1 and 2 mutations were unable to cause meningitis in a newborn mouse model. Of note, mutations in loop 4 of OmpA enhance the severity of the pathogenesis by allowing the pathogen to survive better in circulation and to produce high bacteremia levels. These results demonstrate, for the first time, the roles played by different regions of extracellular loops of OmpA of E. coli K1 in the pathogenesis of meningitis and may help in designing effective preventive strategies against this deadly disease.Escherichia coli K1 is a prominent Gram-negative bacterium that causes meningitis in neonates with case fatality rates ranging from 5 to 30% of infected infants (1-4). Those who survive are often left with permanent neurological dysfunction such as hearing loss, mental retardation, and cortical blindness (5, 6). Despite the use of advanced antibiotics, the morbidity and mortality rates associated with E. coli K1 meningitis remain unchanged over the last few decades (7,8). In addition, because of a recent surge in antibiotic-resistant E. coli K1 strains, the mortality rates will further increase significantly (9, 10). Therefore, new modes of prevention are warranted for which the understanding disease pathophysiology is clearly necessary. Studies from this laboratory have demonstrated that E. coli K1 interacts with human brain microvascular endothelial cells (HBMEC) 2 to enter the central nervous system (11,12). The interaction of the bacterium with HBMEC is mediated by outer membrane protein A (OmpA) of E. coli K1 and a glycoprotein receptor, Ecgp96, on HBMEC (13, 14). OmpA initially binds to GlcNAc1-4GlcNAc epitopes of Ecgp96, followed by the peptide portion of the receptor (15). Of note, synthetic peptides that represent loops 1 and 2 of OmpA prevented the E. coli K1 invasion of HB-MEC (15). OmpA has been shown to be responsible for conferring serum resistance by binding to a complement regulator protein, C4b-binding protein (C4bp) (16, 17). Neonates having lower than the threshold levels of C4bp may be at a higher risk to E. coli K1 meningitis as evidenced when the bacterium treated with adult serum, which contained higher amounts of C4bp, could not invade HBMEC compared with newborn serum treatment (18). Nonetheless, neither of the sera prevents the entry of E. coli K1 into macrophages or dendritic cells (...