Development of an MHC class I L(d)-restricted PSA peptide-loaded tetramer for detection of PSA-specific CD8+ T cells in the mouse.

We set out to develop a PSA peptide-loaded tetramer for enumeration of PSA-specific CD8(+) T cells in the Balb/c mouse model. A candidate major histocompatibility complex (MHC) class I PSA peptide (HPQKVTKFML(188-197)) was selected on the basis of its ability to restimulate PSA-specific CD8(+) T cells to secrete interferon-γ in our assays. Next, H-2L(d)-restricted peptide-loaded and fluorescently labeled tetramers were produced in conjunction with the NIH Tetramer Core Facility, Atlanta, GA, USA. This tetramer was then tested for staining specificity and optimized for detection of PSA-specific CD8(+) T cells induced by our PSA-encoding adenovirus tumor vaccine. The MHC class I PSA peptide demonstrated successful restimulation of CD8(+) T cells isolated from mice previously vaccinated with a PSA-encoding adenovirus tumor vaccine, with no restimulation observed in control-vaccinated mice. The peptide-loaded H-2L(d) tetramer exhibited the desired binding specificity and allowed for detection and frequency determination of PSA-specific CD8(+) T cells by flow cytometry. We have successfully designed and validated a PSA peptide tetramer for use in the Balb/c mouse model that can be used to test PSA-based prostate cancer vaccines. Until now, PSA-specific CD8(+) T cells in the mouse have only been detectable via cytotoxic T-lymphocyte assays or intracellular cytokine staining, which primarily assess antigen-specific functional activity and not their absolute number. This research tool provides laboratories the ability to directly quantitate CD8(+) T cells elicited by PSA-specific immunotherapies and cancer vaccines that are tested in mouse models.

The red muscle morphology of the thresher sharks (family Alopiidae).

A more medial and anterior position of the red aerobic myotomal muscle (RM) and the presence of a vascular counter-current heat exchange system provide the functional elements that facilitate regional RM endothermy in tunas, lamnid sharks and the common thresher shark (Alopias vulpinus). The convergent RM morphology among all species capable of RM endothermy suggests that RM position is a strong predictor of fish endothermic capacity. The present study investigated the comparative RM morphology of the other two thresher shark species (bigeye thresher, Alopias superciliosus, and the pelagic thresher, Alopias pelagicus), for which there is no information regarding their capacity for RM endothermy, and compared these data with published works on A. vulpinus. The digitization of transverse sections along the body of A. superciliosus and A. pelagicus enabled quantification of the relative amount of RM and the position and placement of the RM along the body. The RM in both A. superciliosus and A. pelagicus is positioned subcutaneously, along the lateral edges of the myotomes, and is distributed relatively evenly over the trunk of the body. The position of maximum RM area is at 50% fork length (FL) for A. superciliosus and at 75% FL for A. pelagicus. The amount of RM (mean +/- S.E.M.) is 2.31+/-0.11% and 3.01+/-0.10% in A. superciliosus and A. pelagicus, respectively. When compared with A. vulpinus, all three alopiid sharks have a similar amount of RM. However, A. superciliosus and A. pelagicus differ from A. vulpinus in that they do not possess the medial and anterior RM arrangement that would likely facilitate metabolic heat conservation (RM endothermy).

Swimming performance studies on the eastern Pacific bonito Sarda chiliensis, a close relative of the tunas (family Scombridae) I. Energetics.

A large swim tunnel respirometer was used to quantify the swimming energetics of the eastern Pacific bonito Sarda chiliensis (tribe Sardini) (45-50 cm fork length, FL) at speeds between 50 and 120 cm s(-1) and at 18+/-2 degrees C. The bonito rate of oxygen uptake ((O(2)))-speed function is U-shaped with a minimum (O(2)) at 60 cm s(-1), an exponential increase in (O(2)) with increased speed, and an elevated increase in (O(2)) at 50 cm s(-1) where bonito swimming is unstable. The onset of unstable swimming occurs at speeds predicted by calculation of the minimum speed for bonito hydrostatic equilibrium (1.2 FL s(-1)). The optimum swimming speed (U(opt)) for the bonito at 18+/-2 degrees C is approximately 70 cm s(-1) (1.4 FL s(-1)) and the gross cost of transport at U(opt) is 0.27 J N(-1) m(-1). The mean standard metabolic rate (SMR), determined by extrapolating swimming (O(2)) to zero speed, is 107+/-22 mg O(2) kg(-1) h(-1). Plasma lactate determinations at different phases of the experiment showed that capture and handling increased anaerobic metabolism, but plasma lactate concentration returned to pre-experiment levels over the course of the swimming tests. When adjustments are made for differences in temperature, bonito net swimming costs are similar to those of similar-sized yellowfin tuna Thunnus albacares (tribe Thunnini), but the bonito has a significantly lower SMR. Because bonitos are the sister group to tunas, this finding suggests that the elevated SMR of the tunas is an autapomorphic trait of the Thunnini.

Comparative studies of high performance swimming in sharks I. Red muscle morphometrics, vascularization and ultrastructure.

Tunas (family Scombridae) and sharks in the family Lamnidae are highly convergent for features commonly related to efficient and high-performance (i.e. sustained, aerobic) swimming. High-performance swimming by fishes requires adaptations augmenting the delivery, transfer and utilization of O(2) by the red myotomal muscle (RM), which powers continuous swimming. Tuna swimming performance is enhanced by a unique anterior and centrally positioned RM (i.e. closer to the vertebral column) and by structural features (relatively small fiber diameter, high capillary density and greater myoglobin concentration) increasing O(2) flux from RM capillaries to the mitochondria. A study of the structural and biochemical features of the mako shark (Isurus oxyrinchus) RM was undertaken to enable performance-capacity comparisons of tuna and lamnid RM. Similar to tunas, mako RM is positioned centrally and more anterior in the body. Another lamnid, the salmon shark (Lamna ditropis), also has this RM distribution, as does the closely related common thresher shark (Alopias vulpinus; family Alopiidae). However, in both the leopard shark (Triakis semifasciata) and the blue shark (Prionace glauca), RM occupies the position where it is typically found in most fishes; more posterior and along the lateral edge of the body. Comparisons among sharks in this study revealed no differences in the total RM quantity (approximately 2-3% of body mass) and, irrespective of position within the body, RM scaling is isometric in all species. Sharks thus have less RM than do tunas (4-13% of body mass). Relative to published data on other shark species, mako RM appears to have a higher capillary density, a greater capillary-to-fiber ratio and a higher myoglobin concentration. However, mako RM fiber size does not differ from that reported for other shark species and the total volume of mitochondria in mako RM is similar to that reported for other sharks and for tunas. Lamnid RM properties thus suggest a higher O(2) flux capacity than in other sharks; however, lamnid RM aerobic capacity appears to be less than that of tuna RM.

Comparative studies of high performance swimming in sharks II. Metabolic biochemistry of locomotor and myocardial muscle in endothermic and ectothermic sharks.

Metabolic enzyme activities in red (RM) and white (WM) myotomal muscle and in the heart ventricle (HV) were compared in two lamnid sharks (shortfin mako and salmon shark), the common thresher shark and several other actively swimming shark species. The metabolic enzymes measured were citrate synthase (CS), an index of aerobic capacity, and lactate dehydrogenase (LDH), an index of anaerobic capacity. WM creatine phosphokinase (CPK) activity, an index of rapid ATP production during burst swimming, was also quantified. Enzyme activities in RM, WM and HV were similar in the two lamnid species. Interspecific comparisons of enzyme activities at a common reference temperature (20 degrees C) show no significant differences in RM CS activity but higher CS activity in the WM and HV of the lamnid sharks compared with the other species. For the other enzymes, activities in lamnids overlapped with those of other shark species. Comparison of the HV spongy and compact myocardial layers in mako, salmon and thresher sharks reveals a significantly greater spongy CS activity in all three species but no differences in LDH activity. Adjustment of enzyme activities to in vivo RM and WM temperatures in the endothermic lamnids elevates CS and LDH in both tissues relative to the ectothermic sharks. Thus, through its enhancement of both RM and WM enzyme activity, endothermy may be an important determinant of energy supply for sustained and burst swimming in the lamnids. Although lamnid WM is differentially warmed as a result of RM endothermy, regional differences in WM CS and LDH activities and thermal sensitivities (Q(10) values) were not found. The general pattern of the endothermic myotomal and ectothermic HV muscle metabolic enzyme activities in the endothermic lamnids relative to other active, ectothermic sharks parallels the general pattern demonstrated for the endothermic tunas relative to their ectothermic sister species. However, the activities of all enzymes measured are lower in lamnids than in tunas. Relative to lamnids, the presence of lower WM enzyme activities in the thresher shark (which is in the same order as the lamnids, has an RM morphology similar to that of the mako and salmon sharks and may be endothermic) suggests that other factors, such as behavior and swimming pattern, also affect shark myotomal organization and metabolic function.

Review: Analysis of the evolutionary convergence for high performance swimming in lamnid sharks and tunas.

Elasmobranchs and bony fishes have evolved independently for more than 400 million years. However, two Recent groups, the lamnid sharks (Family Lamnidae) and tunas (Family Scombridae), display remarkable similarities in features related to swimming performance. Traits separating these two groups from other fishes include a higher degree of body streamlining, a shift in the position of the aerobic, red, locomotor muscle that powers sustained swimming to a more anterior location in the body and nearer to the vertebral column, the capacity to conserve metabolic heat (i.e. regional endothermy), an increased gill surface area with a decreased blood-water barrier thickness, a higher maximum blood oxygen carrying capacity, and greater muscle aerobic and anaerobic enzyme activities at in vivo temperatures. The suite of morphological, physiological, and biochemical specializations that define "high-performance fishes" have been extensively characterized in the tunas. This review examines the convergent features of lamnid sharks and tunas in order to gain insight into the extent that comparable environmental selection pressures have led to the independent origin of similar suites of functional characteristics in these two distinctly different taxa. We propose that, despite differences between teleost and elasmobranch fishes, lamnid sharks and tunas have evolved morphological and physiological specializations that enhance their swimming performance relative to other sharks and most other high performance pelagic fishes.

Water-tunnel studies of heat balance in swimming mako sharks.

The mako shark (Isurus oxyrinchus) has specialized vascular networks (retia mirabilia) forming counter-current heat exchangers that allow metabolic heat retention in certain regions of the body, including the aerobic, locomotor red muscle and the viscera. Red muscle, white muscle and stomach temperatures were measured in juvenile (5-13.6 kg) makos swimming steadily in a water tunnel and exposed to stepwise square-wave changes in ambient temperature (T(a)) to estimate the rates of heat transfer and to determine their capacity for the activity-independent control of heat balance. The rates of heat gain of red muscle during warming were significantly higher than the rates of heat loss during cooling, and neither the magnitude of the change in T(a) nor the direction of change in T(a) had a significant effect on red muscle latency time. Our findings for mako red muscle are similar to those recorded for tunas and suggest modulation of retial heat-exchange efficiency as the underlying mechanism controlling heat balance. However, the red muscle temperatures measured in swimming makos (0.3-3 degrees C above T(a)) are cooler than those measured previously in larger decked makos. Also, the finding of non-stable stomach temperatures contrasts with the predicted independence from T(a) recorded in telemetry studies of mako and white sharks. Our studies on live makos provide new evidence that, in addition to the unique convergent morphological properties between makos and tunas, there is a strong functional similarity in the mechanisms used to regulate heat transfer.

Echocardiographic and hemodynamic determinations of the ventricular filling pattern in some teleost fishes.

The current concept of ventricular filling in elasmobranch and teleost fishes is that atrial contraction is the primary, if not the exclusive, determinant of ventricular filling. Recent echocardiographic and on-line hemodynamic data for elasmobranchs, however, have demonstrated a biphasic ventricular filling pattern, characterized by an early phase that occurs during ventricular relaxation and a late phase that follows atrial systole. This study reports echocardiographic and hemodynamic analyses of ventricular filling in three teleost genera (Paralabrax, Channa, Monopterus) having markedly different heart morphologies. Both the profiles of the atrioventricular pressure gradient in Paralabrax and the ventricular inflow velocity in all three genera indicate a biphasic ventricular filling pattern. Although the relative contribution of the early and late filling phases differed among the species studied, interspecific differences in heart structure did not obscure the biphasic pattern. Also, pericardiectomy did not affect the biphasic ventricular filling pattern in Paralabrax. The presence of biphasic filling in teleosts establishes a functional similarity with the elasmobranchs and, because the biphasic ventricular filling pattern predominates in higher vertebrates, suggests that this ventricular filling mechanism may be present in the entire subphylum Vertebrata.

Oxygen transport and cardiovascular responses to exercise in the yellowfin tuna Thunnus albacares.

Yellowfin tuna Thunnus albacares (1400-2175 g) instrumented with electrocardiogram electrodes and pre- and post-branchial catheters were subjected to incremental swimming velocity tests. Increasing velocity, from a minimal speed of 1.0 FLs-1, where FL is fork length, resulted in a 1.4-fold increase in heart rate (from 61.4 to 84.6 beats min-1), an elevated ventral-aortic blood pressure (from 10.8 to 12.2 kPa) and a decreased systemic vascular resistance. Relative branchial vascular resistance at minimal speed ranged from 24.4 to 40.0% of total vascular resistance and tended to increase with velocity. Yellowfin blood has a high oxygen-carrying capacity (16-18 ml O2 dl-1), and a low in vivo oxygen affinity (P50 = 5.3 kPa). Exercise caused a rise in arterial saturation (from 74 to 88%) and a decline in venous saturation (from 48 to 44%), resulting in a 1.3-fold increase in tissue oxygen extraction from the blood (arterial-venous oxygen content difference). Whereas arterial oxygen partial pressure (PO2) tended to increase with exercise, venous PO2 remained unchanged (approximately 5.3 kPa). The observed decrease in venous oxygen content was brought about by a lowered blood pH (from 7.80 to 7.76) and a large Bohr shift. Cardiac output and the increased blood oxygen extraction are estimated to have contributed nearly equally to the increased oxygen consumption during exercise. The large venous oxygen reserve still available to yellowfin tuna at maximal prolonged velocities suggests that the maximal oxygen delivery potential of the cardiovascular system in this species is not fully utilized during aerobic swimming. This reserve may serve other aerobic metabolic processes in addition to continuous swimming.

Heart rate and stroke volume contribution to cardiac output in swimming yellowfin tuna: response to exercise and temperature.

Cardiac performance in the yellowfin tuna (Thunnus albacares, 673-2470 g, 33-53 cm fork length, FL) was examined in unanesthetized fish swimming in a large water tunnel. Yellowfin tuna were fitted with either electrocardiogram electrodes or a transcutaneous Doppler blood-flow probe over the ventral aorta and exposed to changes in swimming velocity (range 0.8-2.9 FLs-1) or to an acute change in temperature (18-28 degrees C). Heart rates (fH) at +/-1 degree C (30-130 beats min-1) were lower on average than previous measurements with non-swimming (restrained) tunas and comparable with those for other active teleosts at similar relative swimming velocities. Although highly variable among individuals, fH increased with velocity (U, in FLs-1) in all fish (fH = 17.93U + 49.93, r2 = 0.14, P < 0.0001). Heart rate was rapidly and strongly affected by temperature (Q10 = 2.37). Blood flow measurements revealed a mean increase in relative cardiac output of 13.6 +/- 3.0% with exercise (mean velocities 1.23-2.10 FLs-1) caused by an 18.8 +/- 5.4% increase in fH and a 3.9 +/- 2.3% decrease in stroke volume. These results indicate that, unlike most other fishes, cardiac output in yellowfin tuna is regulated primarily through increases in fH. Acute reductions in ambient temperature at slow swimming velocities resulted in decreases in cardiac output (Q10 = 1.52) and fH (Q10 = 2.16), but increases in stroke volume (Q10 = 0.78). This observation suggests that the lack of an increase in stroke volume during exercise is not due to the tuna heart operating at maximal anatomical limits.

Mechanisms of venous return and ventricular filling in elasmobranch fish.

The current concept of ventricular filling in the elasmobranch fish (sharks and rays) is that a subambient pericardial pressure establishes a negative diastolic pressure gradient for the atrium and that ventricular end-diastolic volume is exclusively determined by atrial systole. In contrast, recent findings using echo-Doppler and digital imaging techniques have demonstrated two filling phases in the elasmobranch ventricle. In this study, simultaneous atrial and ventricular pressure measurements made on sharks with an open or intact pericardium establish that atrial pressure is above ventricular diastolic pressure until the onset of ventricular systole. A positive biphasic atrioventricular pressure gradient thus ensures ventricular filling during early diastole, as a result of ventricular relaxation, as well as during atrial systole. Although a reduction in pericardial pressure resulted in a decline in the atrial and ventricular pressure, a positive atrioventricular pressure gradient is conserved. The finding that atrial diastolic pressure is not lower than ventricular diastolic pressure, when combined with previous results showing that pericardial pressure is generally at or above ambient and that ventricular filling is biphasic, constitutes a strong body of evidence favoring the operation of a direct venous inflow as the mechanism by which the elasmobranch heart fills.

Percutaneous caliceal irrigation during extracorporeal shock wave lithotripsy for lower pole renal calculi.

A percutaneous nephrostomy tube coiled in the lower pole calix was used to irrigate stone fragments during extracorporeal shock wave lithotripsy in a small series of patients. This procedure is suggested as a possible means of improving lithotripsy results for larger, lower pole stones.

Lithotripsy of urinary calculi by tunable pulsed dye lasers: a randomized in vitro study.

Pulsed dye laser lithotripsy has been used in the treatment of urinary calculi. We compared the efficacy of 2 pulsed dye lasers with different pulse durations in lithotripsy. A total of 20 pairs of human kidney stones was matched by size and composition, and randomized for laser lithotripsy under identical in vitro conditions. Each stone received 150 shocks at sequential energy settings between 40 and 140 mJ. while in a laser fiber compatible stone basket until complete fragmentation occurred. Stone fragments were separated by size and weight. We found that the laser with the shorter pulse duration fragmented stones with less total energy and produced fewer fragments greater than 3.35 mm. The differences were statistically significant.