The Digestive Diverticula in the Carnivorous Nudibranch, Melibe leonina, Do Not Contain Photosynthetic Symbionts.

A number of nudibranchs, including Melibe engeli and Melibe pilosa, harbor symbiotic photosynthetic zooxanthellae. Melibe leonina spends most of its adult life on seagrass or kelp, capturing planktonic organisms in the water column with a large, tentacle-lined oral hood that brings food to its mouth. M. leonina also has an extensive network of digestive diverticula, located just beneath its translucent integument, that are typically filled with pigmented material likely derived from ingested food. Therefore, the focus of this project was to test the hypothesis that M. leonina accumulates symbiotic photosynthetic dinoflagellates in these diverticula. First, we conducted experiments to determine if M. leonina exhibits a preference for light, which would allow chloroplasts that it might be harboring to carry out photosynthesis. We found that most M. leonina preferred shaded areas and spent less time in direct sunlight. Second, we examined the small green circular structures in cells lining the digestive diverticula. Like chlorophyll, they exhibited autofluorescence when illuminated at 480 nm, and they were also about the same size as chloroplasts and symbiotic zooxanthellae. However, subsequent electron microscopy found no evidence of chloroplasts in the digestive diverticula of M. leonina; the structures exhibiting autofluorescence at 480 nm were most likely heterolysosomes, consistent with normal molluscan digestion. Third, we did not find evidence of altered oxygen consumption or production in M. leonina housed in different light conditions, suggesting the lack of any significant photosynthetic activity in sunlight. Fourth, we examined the contents of the diverticula, using HPLC, thin layer chromatography, and spectroscopy. The results of these studies indicate that the diverticula did not contain any chlorophyll, but rather harbored other pigments, such as astaxanthin, which likely came from crustaceans in their diet. Together, all of these data suggest that M. leonina does sequester pigments from its diet, but not for the purpose of symbiosis with photosynthetic zooxanthellae. Considering the translucent skin of M. leonina, the pigmented diverticula may instead provide camouflage.

Varios nudibranquios, incluidos Melibe engeli y Melibe pilosa, albergan zooxantelas fotosintéticas simbióticas. Melibe leonina pasa la mayoría de su vida adulta en pastos marinos o quelpo, donde captura organismos planctónicos en la columna de agua con una gran capucha oral forrada por tentáculos que llevan comida a su boca. Melibe leonina también tiene una extensa red de divertículos digestivos, ubicados justo debajo de su tegumento translúcido, que generalmente están llenos de material pigmentado probablemente derivado de alimentos ingeridos. Por lo tanto, el objetivo de este proyecto fue evaluar la hipótesis de que M. leonina acumula dinoflagelados fotosintéticos simbióticos en estos divertículos. Primero, realizamos experimentos para determinar si M. leonina se orienta hacia la luz, lo cual permitiría a los cloroplastos que podría albergar el realizar la fotosíntesis. Descubrimos que la mayoría de M. leonina prefería las áreas sombreadas y pasaba menos tiempo bajo la luz solar directa. En segundo lugar, examinamos las pequeñas estructuras circulares verdes en las células que recubren los divertículos digestivos. Al igual que la clorofila, exhibieron autofluorescencia cuando se iluminaban a 480 nm, y también tenían aproximadamente el mismo tamaño que los cloroplastos y las zooxantelas simbióticas. No obstante, la microscopía electrónica no produjo evidencia de cloroplastos en los divertículos digestivos de M. leonina. Es probable que las estructuras que exhibieron autofluorescencia en 480 nm fuesen heterolisosomas, lo cual es consistente con la digestión normal de moluscos. En tercer lugar, no encontramos evidencia de un consumo o producción de oxígeno alterado en M. leonina alojadas varias condiciones lumínicas, lo cual sugiere la ausencia de actividad fotosintética significativa en la presencia de luz solar. En cuarto lugar, examinamos el contenido de los divertículos mediante HPLC, cromatografía en capa fina, y espectroscopia. Los resultados de estos estudios indican que los divertículos no contenían clorofila, pero si otros pigmentos como la astaxantina que probablemente provenía de crustáceos en su dieta. Nuestros datos sugieren que M. leonina secuestra pigmentos de su dieta, pero no con el propósito de la simbiosis con zooxantelas fotosintéticas. Teniendo en cuenta la piel translúcida de M. leonina, los divertículos pigmentados podrían quizás proporcionar camuflaje.

HeartMate III: pump design for a centrifugal LVAD with a magnetically levitated rotor.

A long-term, compact left ventricular assist device (LVAD), the HeartMate III, has been designed and fabricated, featuring a centrifugal pump with a magnetically levitated rotor. The pump has been optimized by in vitro testing to achieve a design point of 7 L/min against 135 mm Hg at high hydrodynamic efficiency (30%) and to be capable of up to 10 L/min under such a load. Furthermore, the pump has demonstrated no mechanical failures, low hemolysis (4-10 mg/dl plasma free Hb), and low thrombogenicity during six (40, 27, 59, 42, 27, and 49-day) in vivo bovine studies.

Investigation of the 'switch-epitope' concept with random peptide libraries displayed as thioredoxin loop fusions.

The 'FLITRX' random peptide library, consisting of dodecamer loop peptides displayed on a thioredoxin-flagellin scaffold on Escherichia coli, was used to select peptide sequences with affinity for a monoclonal antibody. These peptides were further screened for pH- and metal-sensitive antibody binding. Several zinc-sensitive peptides were identified, termed 'switch epitopes'. A soluble, monomeric thioredoxin loop ('Trxloop') insertion analog of a FLITRX switch epitope was constructed and its antibody binding properties were characterized by Western blots. Zinc-dependent antibody recognition was maintained in the Trxloop protein although the apparent antibody affinity was lower. This Trxloop protein bound to an immobilized metal affinity chromatography matrix, similar to a 'histidine-patch' thioredoxin variant, and was reversibly precipitated by 1 mM Zn(2+) or Cu(2+) ions. Residues important for zinc and antibody binding were determined by site-directed mutagenesis. The Trxloop antibody affinity was increased by saturation mutagenesis. Biotinylated Trxloop ('Biotrxloop') variants of the original and improved affinity Trxloop proteins were constructed and characterized by surface plasmon resonance measurements. Increased antibody affinity was partially due to a slower antibody desorption rate, although the relative adsorption rates were dependent on the amount of immobilized Biotrxloop protein, indicating an influence of avidity on the apparent affinity.

The Heartmate III: design and in vivo studies of a maglev centrifugal left ventricular assist device.

A compact implantable centrifugal left ventricular assist device (LVAD) (HeartMate III) featuring a magnetically levitated impeller is under development. The goal of our ongoing work is to demonstrate feasibility, low hemolysis, and low thrombogenicity of the titanium pump in chronic bovine in vivo studies. The LVAD is based on so-called bearingless motor technology and combines pump rotor, drive, and magnetic bearing functions in a single unit. The impeller is rotated (theta z) and levitated with both active (X, Y) and passive (Z, theta x, theta y) suspension. Six prototype systems have been built featuring an implantable titanium pump (69 mm diameter, 30 mm height) with textured blood contacting surfaces and extracorporeal electronics. The pumps were implanted in 9 calves (< or = 100 kg at implant) that were anticoagulated with Coumadin (2.5 < or = INR < or = 4.0) throughout the studies. Six studies were electively terminated (at 27-61 days), 1 study was terminated after the development of severe pneumonia and lung atelectasis (at 27 days) another study was terminated after cardiac arrest (at 2 days) while a final study is ongoing (at approximately 100 days). Mean pump flows ranged from 2 to 7 L/min, except for brief periods of exercise at 6 to 9 L/min. Plasma free hemoglobin ranged from 4 to 10 mg/dl. All measured biochemical indicators of end organ function remained within normal range. The pumps have met performance requirements in all 9 implants with acceptable hemolysis and no mechanical failures.

PD-L2 is a second ligand for PD-1 and inhibits T cell activation.

Programmed death I (PD-I)-deficient mice develop a variety of autoimmune-like diseases, which suggests that this immunoinhibitory receptor plays an important role in tolerance. We identify here PD-1 ligand 2 (PD-L2) as a second ligand for PD-1 and compare the function and expression of PD-L1 and PD-L2. Engagement of PD-1 by PD-L2 dramatically inhibits T cell receptor (TCR)-mediated proliferation and cytokine production by CD4+ T cells. At low antigen concentrations, PD-L2-PD-1 interactions inhibit strong B7-CD28 signals. In contrast, at high antigen concentrations, PD-L2-PD-1 interactions reduce cytokine production but do not inhibit T cell proliferation. PD-L-PD-1 interactions lead to cell cycle arrest in G0/G1 but do not increase cell death. In addition, ligation of PD-1 + TCR leads to rapid phosphorylation of SHP-2, as compared to TCR ligation alone. PD-L expression was up-regulated on antigen-presenting cells by interferon gamma treatment and was also present on some normal tissues and tumor cell lines. Taken together, these studies show overlapping functions of PD-L1 and PD-L2 and indicate a key role for the PD-L-PD-1 pathway in regulatingT cell responses.

Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation.

PD-1 is an immunoinhibitory receptor expressed by activated T cells, B cells, and myeloid cells. Mice deficient in PD-1 exhibit a breakdown of peripheral tolerance and demonstrate multiple autoimmune features. We report here that the ligand of PD-1 (PD-L1) is a member of the B7 gene family. Engagement of PD-1 by PD-L1 leads to the inhibition of T cell receptor-mediated lymphocyte proliferation and cytokine secretion. In addition, PD-1 signaling can inhibit at least suboptimal levels of CD28-mediated costimulation. PD-L1 is expressed by antigen-presenting cells, including human peripheral blood monocytes stimulated with interferon gamma, and activated human and murine dendritic cells. In addition, PD-L1 is expressed in nonlymphoid tissues such as heart and lung. The relative levels of inhibitory PD-L1 and costimulatory B7-1/B7-2 signals on antigen-presenting cells may determine the extent of T cell activation and consequently the threshold between tolerance and autoimmunity. PD-L1 expression on nonlymphoid tissues and its potential interaction with PD-1 may subsequently determine the extent of immune responses at sites of inflammation.

Identification of a human follicular dendritic cell molecule that stimulates germinal center B cell growth.

The initial interaction between B cells and follicular dendritic cells (FDCs) appears to be essential for germinal center (GC) formation. To identify molecules regulating this interaction, we generated FDC-staining monoclonal antibodies (mAbs) and screened them for their ability to block FDC-mediated costimulation of growth and differentiation of CD40-stimulated B cells. Using one of the inhibitory mAbs, 8D6, we expression cloned the cDNA encoding the 8D6 antigen (Ag) from a human FDC line, HK. The 8D6 Ag is a novel protein of 282 amino acids that is expressed abundantly on FDCs. Monolayers of COS cells transiently transfected with the 8D6 Ag cDNA stimulate B cell growth. The mAb 8D6 blocks the costimulatory function completely. The inhibitory activity of the mAb 8D6 was demonstrated to be due to an inhibition of cell cycle progression of CD40 ligand-stimulated GC B cells. In addition, the mAb 8D6 inhibits the growth of a lymphoma of GC origin, L3055, which depends on FDCs or HK cells for its growth. These findings suggest that the primary function of FDCs in the GC is to stimulate B cell growth. An FDC signal molecule, 8D6 Ag, may be an important molecule to mediate this function.