Faculty Bibliography

Reduced litter sizes in mice missing pentraxin 3 (Ptx3) have been attributed to fertilization failure. However, our global gene expression studies showed high uterine Ptx3 expression at the implantation site in mice, suggesting its role in blastocyst implantation. We initiated molecular and genetic studies in mice to explore the importance of uterine Ptx3 in this process. We found that Ptx3 is expressed in a unique and transient fashion at implantation sites. With the initiation of implantation on midnight of Day 4 of pregnancy, Ptx3 is expressed exclusively in stromal cells at the site of blastocysts. On Day 5, its expression is more intense in decidualizing stromal cells, but it disappears on Day 6. The expression again becomes evident in the deciduum on Day 7, followed by a more robust expression on Day 8, particularly at the antimesometrial pole. From Day 9, with the initiation of placentation, Ptx3 expression becomes undetectable. These results suggest a role for PTX3 in implantation and decidualization. Indeed, deletion of Ptx3 results in both compromised implantation and decidualization. Interleukin 1B (IL1B), a known inducer of Ptx3, is also transiently expressed in stromal cells at the implantation site, suggesting that IL1B is an inducer of uterine Ptx3 expression. In fact, uterine Ptx3 expression follows that of Il1b induced by lipopolysaccharide treatment on Day 7 of pregnancy. Collectively, these findings provide evidence for an important role for PTX3 in implantation and decidualization. This study has clinical implications, since PTX3 is expressed in the receptive endometrium, and trophoblast cells influence decidual Ptx3 expression in humans.

BACKGROUND: Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by ventricular arrhythmias, sudden death, and fatty or fibrofatty replacement of right ventricular myocytes. Recent studies have noted an association between human ARVD/C and molecular remodeling of intercalated disc structures. However, progress has been constrained by limitations inherent to human studies. OBJECTIVE: We studied the molecular composition of the intercalated disc structure in a naturally occurring animal model of ARVD/C (Boxer dogs). METHODS: We studied hearts from 12 Boxers with confirmed ARVD/C and 2 controls. Ventricular sections from 4 animals were examined by immunofluorescent microscopy. Frozen tissue samples were used for Western blot analysis. Proteins investigated were N-cadherin, plakophilin 2, desmoplakin, plakoglobin, desmin, and connexin 43 (Cx43). RESULTS: In control dogs, all proteins tested by immunofluorescence analysis yielded intense localized signals at sites of end-to-end cell apposition. In contrast, myocardial tissues from ARVD/C-afflicted Boxers showed preservation of N-cadherin staining but loss of detectable signal for Cx43 at the intercalated disc location. Western blots indicated that the Cx43 protein was still present in the samples. Gene sequencing analysis showed no mutations in desmoplakin, plakoglobin, Cx43, or plakophilin 2. CONCLUSION: Mutation(s) responsible for ARVD/C in Boxers lead, directly or indirectly, to severe modifications of mechanical and electrical cell-cell interactions. Furthermore, significant reduction in gap junction formation may promote a substrate for malignant ventricular arrhythmias. This model may help to advance our understanding of the molecular basis, pathophysiology, and potential therapeutic approach to patients with ARVD/C

Among 25 patients of mean age 91.5 years (range, 90-96 years) who received a total hip or knee arthroplasty at the authors' institution, 8% experienced surgical complications, 56% experienced at least 1 medical complication, and 80% received perioperative blood transfusions. At a mean follow-up of 4.1 years, patients were experiencing pain reduction and somewhat higher functional capacity and had slightly better survival characteristics than age-matched controls. Total hip and knee arthroplasty patients in this cohort should be told that they have a higher likelihood of experiencing perioperative medical complications and of receiving a blood transfusion than younger individuals; at the same time, they can expect pain relief as well as equal or better survival than their age-matched peers

The vulnerability or destabilization of atherosclerotic plaques has been directly linked to plaque composition. Imaging modalities, such as magnetic resonance (MR) imaging, that allow for evaluation of plaque composition at a cellular and molecular level, could further improve the detection of vulnerable plaque and may allow for monitoring the efficacy of antiatherosclerotic therapies. In this review we focus on MR imaging strategies for the detection and evaluation of atherosclerotic plaques and their composition. We highlight recent advancements in the development of MR pulse sequences, computer image analysis, and the use of commercially available MR contrast agents, such as gadopentic acid (Gd-DTPA), for plaque characterization. We also discuss molecular imaging strategies that are currently being used to design specific imaging probes targeted to biochemical and cellular markers of atherosclerotic plaque vulnerability.

Transcuprein is a high-affinity copper carrier in the plasma that is involved in the initial distribution of copper entering the blood from the digestive tract. To identify and obtain cDNA for this protein, it was purified from rat plasma by size exclusion and copper-chelate affinity chromatography, and amino acid sequences were obtained. These revealed a 190-kDa glycosylated protein identified as the macroglobulin alpha(1)-inhibitor III, the main macroglobulin of rodent blood plasma. Albumin (65 kDa) copurified in variable amounts and was concluded to be a contaminant (although it can transiently bind the macroglobulin). The main macroglobulin in human blood plasma (alpha(2)-macroglobulin), which is homologous to alpha(1)-inhibitor III, also bound copper tightly. Expression of alpha(1)I3 (transcuprein) mRNA by the liver was examined in rats with and without copper deficiency, using quantitative polymerase chain reaction methodology and Northern blot analysis. Protein expression was examined by Western blotting. Deficient rats with 40% less ceruloplasmin oxidase activity and liver copper concentrations expressed about twice as much alpha(1)I3 mRNA, but circulating levels of transcuprein did not differ. Iron deficiency, which increased liver copper concentrations by threefold, reduced transcuprein mRNA expression and circulating levels of transcuprein relative to what occurred in rats with normal or excess iron. We conclude that transcupreins are specific macroglobulins that not only carry zinc but also carry transport copper in the blood, and that their expression can be modulated by copper and iron availability.

Bis(monoacylglycero)phosphate (BMP) is a unique lipid enriched in the late endosomes participating in the trafficking of lipids and proteins through this organelle. The de novo biosynthesis of BMP has not been clearly demonstrated. We investigated whether phosphatidylglycerol (PG) and cardiolipin (CL) could serve as precursors of de novo BMP synthesis using two different cellular models: CHO cells deficient in phosphatidylglycerophosphate (PGP) synthase, the enzyme responsible for the first step of PG synthesis; and human lymphoblasts from patients with Barth syndrome (BTHS), characterized by mutations in tafazzin, an enzyme implicated in the deacylation-reacylation cycle of CL. The biosynthesis of both PG and BMP was reduced significantly in the PGP synthase-deficient CHO mutants. Furthermore, overexpression of PGP synthase in the deficient mutants induced an increase of BMP biosynthesis. In contrast to CHO mutants, BMP biosynthesis and its fatty acid composition were not altered in BTHS lymphoblasts. Our results thus suggest that in mammalian cells, PG, but not CL, is a precursor of the de novo biosynthesis of BMP. Despite the decrease of de novo synthesis, the cellular content of BMP remained unchanged in CHO mutants, suggesting that other pathway(s) than de novo biosynthesis are also used for BMP synthesis

Virology laboratories historically have used direct fluorescent-antibody assay (DFA) and culture to detect six or seven respiratory viruses. Following the discovery of five new human respiratory viruses since 2000, there is an increasing need for diagnostic tests to detect these emerging viruses. We have developed a new test that can detect 20 different respiratory virus types/subtypes in a single 5-h test. The assay employs multiplex PCR using 14 virus-specific primer pairs, followed by a multiplexed target-specific primer extension (TSPE) reaction using 21 primers for specific respiratory virus types and subtypes. TSPE products were sorted and identified by using a fluid microsphere-based array (Universal Array; TmBioscience Corporation, Toronto, Canada) and the Luminex x-MAP system. The assay detected influenza A and B viruses; influenza A virus subtypes H1, H3, and H5 (including subtype H5N1 of the Asian lineage); parainfluenza virus types 1, 2, 3, and 4; respiratory syncytial virus types A and B; adenovirus; metapneumovirus; rhinovirus; enterovirus; and coronaviruses OC43, 229E, severe acute respiratory syndrome coronavirus, NL63, and HKU1. In a prospective evaluation using 294 nasopharyngeal swab specimens, DFA/culture detected 119 positives and the respiratory virus panel (RVP) test detected 112 positives, for a sensitivity of 97%. The RVP test detected an additional 61 positive specimens that either were not detected by DFA/culture or were positive for viruses not tested for by DFA/culture. After resolution of discordant results by using a second unique PCR assay and by using a combined reference standard of positivity, the RVP test detected 180 of 183 true positives, for a sensitivity of 98.5%, whereas DFA and culture detected only 126 of 183 true positives, for a sensitivity of 68.8%. The RVP test should improve the capabilities of hospital and public health laboratories for diagnosing viral respiratory tract infections and should assist public health agencies in identifying etiologic agents in respiratory tract infection outbreaks.

Fibrillins are the structural components of extracellular microfibrils that impart physical properties to tissues, alone or together with elastin as elastic fibers. Genetic studies in mice have revealed that fibrillin-rich microfibrils are also involved in regulating developmental programs and homeostatic processes through the modulation of TGF-beta/BMP signaling events. A new paradigm has thus emerged whereby the spatiotemporal organization of microfibrils dictates both the cellular activities and physical properties of connective tissues. These observations have paved the way to novel therapeutic approaches aimed at counteracting the life-threatening complications in human conditions caused by dysfunctions of fibrillin-rich microfibrils.