Development of a new peptide-bead coupling method for an all peptide-based Luminex multiplexing assay for detection of Plasmodium falciparum antibody responses.

Using a recombinant protein antigen for antibody testing shows a sum of antibody responses to multiple different immune epitopes existing in the protein antigen. In contrast, the antibody testing to an immunogenic peptide epitope reflects a singular antibody response to the individual peptide epitope. Therefore, using a panel of peptide epitopes provides an advantage for profiling multiple singular antibody responses with potential to estimate recent malaria exposure in human infections. However, transitioning from malaria immune epitope peptide-based ELISA to an all peptide bead-based multiplex Luminex assay presents some challenges including variation in the ability of different peptides to bind beads. The aim of this study was to develop a peptide coupling method while demonstrating the utility of these peptide epitopes from multiple stage antigens of Plasmodium falciparum for measuring antibodies. Successful coupling of peptide epitopes to beads followed three steps: 1) development of a peptide tag appended to the C-terminus of each peptide epitope consisting of beta-alanine-lysine (x 4)--cysteine, 2) bead modification with a high concentration of adipic acid dihydrazide, and 3) use of the peptide epitope as a blocker in place of the traditional choice, bovine serum albumin (BSA). This new method was used to couple 12 peptide epitopes from multiple stage specific antigens of P. falciparum, 1 Anopheles mosquito salivary gland peptide, and 1 Epstein-Barr virus peptide as an assay control. The new method was applied to testing of IgG in pooled samples from 30 individuals with previously repeated malaria exposure in western Kenya and IgM and IgG in samples from 37 U.S. travelers with recent exposure to malaria. The new peptide-bead coupling method and subsequent multiplex Luminex assay showed reliable detection of IgG to all 14 peptides in Kenyan samples. Among 37 samples from U.S. travelers recently diagnosed with malaria, IgM and IgG to the peptide epitopes were detected with high sensitivity and variation. Overall, the U.S. travelers had a much lower positivity rates of IgM than IgG to different peptide epitopes, ranging from a high of 62.2% positive for one epitope to a low of only 5.4% positive for another epitope. In contrast, the travelers had IgG positive rates from 97.3% to 91.9% to various peptide epitopes. Based on the different distribution in IgM and IgG positivity to overall number of peptide epitopes and to the number of pre-erythrocytic, erythrocytic, gametocytic, and salivary stage epitopes at the individual level, four distinct patterns of IgM and IgG responses among the 37 samples from US travelers were observed. Independent peptide-bead coupling and antibody level readout between two different instruments also showed comparable results. Overall, this new coupling method resolves the peptide-bead coupling challenge, is reproducible, and can be applied to any other immunogenic peptide epitopes. The resulting all peptide bead-based multiplex Luminex assay can be expanded to include other peptide epitopes of P. falciparum, different malaria species, or other diseases for surveillance, either in US travelers or endemic areas.

The temporomandibular joint of California sea lions (Zalophus californianus): part 1 - characterisation in health and disease.

OBJECTIVES: This study aimed to characterise the histologic, biomechanical and biochemical properties of the temporomandibular joint (TMJ) of California sea lions. In addition, we sought to identify structure-function relationships and to characterise TMJ lesions found in this species. DESIGN: Temporomandibular joints from fresh cadaver heads (n=14) of California sea lions acquired from strandings were examined macroscopically and microscopically. The specimens were also evaluated for their mechanical and biochemical properties. Furthermore, if TMJ arthritic changes were present, joint characteristics were described and compared to healthy joints. RESULTS: Five male and 9 female specimens demonstrated macroscopically normal fibrocartilaginous articular surfaces and fibrous discs in the TMJ. Out of the 9 female specimens, 4 specimens had TMJ lesions were seen either in the articular surface or the disc. Histologically, these pathologic specimens demonstrated subchondral bone defects, cartilage irregularities and inflammatory cell infiltrates. The normal TMJ discs did not exhibit significant direction dependence in tensile stiffness or strength in the rostrocaudal direction compared with the mediolateral direction among normal discs or discs from affected joints. The TMJ discs were not found to be anisotropic in tensile properties. This feature was further supported by randomly oriented collagen fibres as seen by electron microscopy. Furthermore, no significant differences were detected in biochemical composition of the discs dependent upon population. CONCLUSION: The TMJ and its disc of the California sea lion exhibit similarities but also differences compared to other mammals with regards to structure-function relationships. A fibrous TMJ disc rich in collagen with minimal glycosaminoglycan content was characterised, and random fibre organisation was associated with isotropic mechanical properties in the central region of the disc.

Characterization of degenerative changes in the temporomandibular joint of the bengal tiger (Panthera tigris tigris) and siberian tiger (Panthera tigris altaica).

The articulation of the temporomandibular joint (TMJ) is composed of the temporal bone dorsally, the mandibular condyle ventrally and a fibrous articular disc. The TMJ disc plays an essential role in distributing load between the two articular surfaces. Degeneration of the disc in the presence of joint pathology has been shown in man; however, TMJ pathology has not been documented previously in tigers (Panthera tigris). The mandibular condyle and TMJ disc of a Bengal tiger (P. tigris tigris) and a Siberian tiger (P. tigris altaica) were evaluated grossly and the TMJ disc was characterized biochemically and mechanically. Characterization of the TMJ disc verified region- and direction-dependent biochemical and mechanical properties, reflective of the functional demands on the joint. Degenerative joint disease was observed in both cases and this was more severe in the Siberian tiger. Simultaneous evaluation of joint pathology, biochemical composition and mechanical properties of the TMJ disc revealed a loss in functional properties (tensile anisotropy) of the disc as joint pathology advanced from moderate to severe. TMJ degeneration may compromise the ability of the animal to eat and thrive and may be a factor contributing to the endangered status of these species.

Tensile characterization of porcine temporomandibular joint disc attachments.

The frequency and impact of temporomandibular joint (TMJ) disorders necessitate research in characterizing the joint's function. The 6 discal attachments have not yet been systematically characterized under tension. Understanding their role in joint function may guide our study of TMJ pathologies, including disc displacement. In the present study, a porcine model was used to characterize the attachments in tension anteroposteriorly and mediolaterally, based on previously identified similarities in the porcine and human masticatory behaviors and discal properties. Tensile stiffness, strength, toughness, and maximum strain were quantified. Collagen alignment was characterized via polarized light and scanning electron microscopy. Anisotropy was demonstrated in all attachments, with the exception of the anterior inferior attachment. Anteroposteriorly, the lateral attachment was stiffest (8.3 MPa) and the anterior superior was least stiff (1.4 MPa). Mediolaterally, the posterior superior attachment was stiffest (16.3 MPa) and the medial was least stiff (1.4 MPa). The greatest strain was observed in the lateral attachment in the mediolateral direction and the posterior superior attachment in the anteroposterior direction. With greatest strains in the most commonly observed directions of disc displacement, it is suggested that compromise in the posterior and lateral attachments contributes to partial lateral and anterior disc displacement.

Criteria for polycystic ovarian morphology in polycystic ovary syndrome as a function of age.

CONCEPT: Ovaries meeting criteria for polycystic ovary morphology during peak reproductive years may no longer meet the criteria with age. OBJECTIVE: Ovarian volume and follicle number decrease with age in women with polycystic ovary syndrome (PCOS), permitting age-dependent criteria for PCOM. DESIGN AND SETTING: We conducted longitudinal (7-15 year interval) and cross-sectional studies to examine polycystic ovarian morphology over time at an outpatient clinic and pathology laboratory in a tertiary care hospital. PATIENTS: Subjects included those with PCOS defined by the National Institutes of Health criteria (n = 11 and 483 for longitudinal and cross-sectional, respectively) and control women with regular menstrual cycles and no hyperandrogenism (n = 15 and 367), age 18-64 yr. INTERVENTIONS: Subjects underwent an ovarian ultrasound by a single observer. MAIN OUTCOME MEASURES: Ovarian volume and follicle number were measured and ultrasound findings confirmed by a pathologist in a subset (n = 9). RESULTS: Ovarian volume (15.2 +/- 7.4 vs. 7.1 +/- 3.7 ml; P < 0.01) and follicle number (12.8 +/- 3.2 vs. 8.1 +/- 3.9; P < 0.05) decreased longitudinally in PCOS and control women (volume 11.6 +/- 4.4 vs. 5.4 +/- 2.2 ml and follicle number 8.3 +/- 1.9 vs. 6.3 +/- 1.8; both P < 0.005). Using cross-sectional data, log ovarian volume and follicle number decreased in both groups, but the decrease in log ovarian volume was less pronounced in women with PCOS than in controls (P < 0.01). A combination of age, log ovarian volume, follicle number, and testosterone distinguished PCOS subjects from controls with a receiver operator characteristic curve area of 0.90. CONCLUSIONS: Ovarian volume and follicle number decrease with age in women with PCOS and controls necessitating age-based criteria to define polycystic ovarian morphology. It is possible to use these criteria to distinguish PCOS in women over age 40 yr.

Polycystic ovarian morphology in normal women does not predict the development of polycystic ovary syndrome.

CONTEXT: Polycystic ovarian morphology (PCOM) is present in 25% of normal women in the absence of polycystic ovary syndrome (PCOS); however, the natural history of PCOM is unknown. OBJECTIVE: We hypothesized that the presence of PCOM predisposes the development of PCOS. DESIGN: The study was a longitudinal follow-up study over 8.2 +/- 5.2 yr (mean +/- sd; range 1.7-17.5 yr). SETTING: The study took place in an outpatient setting. SUBJECTS: Women who took part in a previous study as a normal control and had an ultrasound examination (n = 40) participated. INTERVENTION: Subjects underwent an interval menstrual history, physical exam, blood sampling, and repeat ultrasound in the follicular phase. MAIN OUTCOME MEASURE: Development of PCOS was diagnosed by irregular menses and hyperandrogenism, in the absence of other disorders. Changes in ovarian morphology over time were evaluated. RESULTS: At the baseline visit, 23 women (57.5%) had PCOM and 17 (42.5%) had normal ovarian morphology. One subject with PCOM developed irregular menses and presumptive PCOS. Eleven subjects with PCOM no longer met the criteria for PCOM at follow-up. There was no factor that predicted the change to normal ovarian morphology at the follow-up visit. CONCLUSIONS: These data suggest that PCOM in women with regular ovulatory cycles does not commonly predispose the development of PCOS. Although it is unusual to develop PCOM if the ovaries are normal on first assessment, ovaries in women with PCOM no longer meet the criteria for PCOM in approximately half of cases over time.

Characteristics and performance of the Sunna high dose dosemeter using green photoluminescence and UV absorption readout methods.

Growth in the use of ionising radiation for medical sterilisation and the potential for wide-scale international food irradiation have created the need for robust, mass-producible, inexpensive, and highly accurate radiation dosemeters. The Sunna dosemeter, lithium fluoride injection-moulded in a polyethylene matrix, can be read out using either green photoluminescence or ultraviolet (UV) absorption. The Sunna dosemeter can be mass-produced inexpensively with high precision. Both the photoluminescent and the UV absorption reader are simple and inexpensive. Both methods of analysis display negligible humidity effects, minimal dose rate dependence, acceptable post-irradiation effects, and permit measurements with a precision of nearly 1% 1sigma. The UV method shows negligible irradiation temperature effects from -30 degrees C to +60 degrees C. The photoluminescence method shows negligible irradiation temperature effects above room temperature for sterilisation dose levels and above. The dosimetry characteristics of these two readout methods are presented along with performance data in commercial sterilisation facilities.

Arachidonic acid metabolism in intestinal hypothermic preservation injury.

The effect of hypothermic intestinal ischemia and short-term reperfusion on mucosal arachidonic acid metabolism was studied in a dog model of intestinal preservation injury. Canine intestinal segments were flushed with cold Collins solution, cold stored (4 degrees C) for either 24 or 48 h, and subsequently reperfused in the donor for 1 h. Samples of intestinal mucosa obtained before ischemia, after the ischemia period, and after the reperfusion period were placed into tissue culture, and arachidonic acid metabolites were measured in the tissue incubation media. Prostaglandin E2 (PGE2) and prostacyclin (PGI2) production significantly increased after 24 h of cold ischemia and after 1 h of reperfusion, respectively. Intestines cold stored for 48 h and after 1 h of reperfusion produced significantly elevated quantities of thromboxane B2, PGI2, PGE2, and leukotriene B4, relative to the production rates from nonischemic control tissue or tissue subjected to 48 h of hypothermic ischemia without reperfusion. Mucosal production of thiol ether leukotrienes (LTC4, LTD4, LTE4) was not altered by ischemia or reperfusion at any time of cold ischemia. The synthesis of the lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE) was not altered by hypothermic ischemia or reperfusion, but this arachidonate metabolite was produced by small intestinal mucosa in the greatest quantities. Specifically, nanogram quantities of 12-HETE were produced by intestinal mucosa compared to picogram quantities of the other metabolites measured. Significant synthesis of the delta lactone derivative of 5-hydroxyeicosatetraenoic acid was detected by HPLC in many tissue samples undergoing 48 h of ischemia and reperfusion, relative to nonischemic tissue samples. In conclusion, significant increases in arachidonate cyclooxygenase and lipoxygenase metabolites have been identified in intestinal mucosa subjected to long-term hypothermic ischemia and short-term reperfusion. Synthesis of these products increases with the duration of cold ischemia and may play a role in intestinal preservation injury.

The role of dose rate in the induction of micronuclei in deep-lung fibroblasts in vivo after exposure to cobalt-60 gamma rays.

To evaluate the influence of low-dose-rate exposures on biological damage, it is necessary to have cells that can be maintained in the same stage of the cell cycle for long periods. Normal rat lung fibroblasts represent a stable cell type with a slow turnover rate in vivo. These cells can be stimulated to divide by placing them in tissue culture. Therefore, a constant cell population can be exposed over a protracted time and stimulated to divide, and the cytogenetic damage can be evaluated at the first cell division after exposure. By placing rats at different distances from a 60Co source, they were exposed to graded doses of gamma rays--0.0, 3.9, 7.4 and 11.3 Gy--protracted over either 4 or 67 h. Fibroblasts were isolated from the lung and cultured for 24 h; after cytochalasin B was added, the cells were cultured for an additional 69 to 72 h. The percentage binucleated cells in fibroblasts of animals exposed for 4 or 67 h was 47.1 +/- 4.3 and 62.1 +/- 3.9. There was no influence of dose on the percentage binucleated cells, but the fraction of cells that divided at 67 h was significantly higher (P < 0.05) than observed at 4 h. Cells were scored for micronuclei on coded slides. The dose-response data from animals exposed for 4 and 67 h were fitted to the following linear dose-response relationships, where D = dose; micronuclei/binucleated cell = 0.02 +/- 0.03 + 2.38 +/- 0.44 x 10(-2) D, and micronuclei/binucleated cell = 0.01 +/- 0.06 + 1.01 +/- 0.10 x 10(-2) D, respectively. The r2 values for the two curves were 0.67 and 0.91, indicating the goodness of fit for the data for the 4- and 67-h treatments. The slopes were different from zero and each other at the P < 0.05 level of significance. The effectiveness of the 60Co exposure decreased as the dose rate decreased. At dose rates below 0.17 Gy/h, the effectiveness remained constant over the range of doses and dose rates used. Comparing the slope of the dose response for the lowest exposure rate to that from information published previously, the dose-rate effectiveness factor was 6.14 +/- 0.65 for the induction of micronuclei in deep-lung fibroblasts.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of the arachidonate 5-lipoxygenase synthesis inhibitor A-64077 in intestinal ischemia-reperfusion injury.

This study was designed to characterize the role of arachidonate 5-lipoxygenase metabolism during experimental intestinal ischemia-reperfusion (I/R) injury. Canines were subjected to 3 hr of intestinal ischemia followed by 1 hr of normobaric reperfusion. Intestinal ischemia followed by 1 hr of normobaric reperfusion. Intestinal mucosal leukotriene B4 and leukotriene C4 synthesis tripled after ischemia and ischemia-reperfusion, relative to non-ischemic intestinal mucosa. The flux of fluid and protein from the capillary to the lumen also increased 3-fold after I/R. The selective 5-lipoxygenase synthesis inhibitor A-64077 (Ziluten, 5 mg/kg, p.o.) abolished I/R-induced leukotriene synthesis and reduced transluminal protein flux (50%) but did not influence the lumenal accumulation of fluid after I/R. In animals treated with the leukotriene synthesis inhibitor, intestinal vascular resistance significantly declined during the imposed ischemia period and after 60 min of reperfusion. Mucosal myeloperoxidase activity, a biochemical marker for tissue neutrophils, rose significantly after I/R, and these increases were prevented with the 5-lipoxygenase synthesis inhibitor. In other experiments, the lipoxygenase inhibitor nondihydroguaretic acid produced similar results to those of A64077. In an attempt to determine the source of mucosal leukotrienes during intestinal I/R, we imposed in vitro ischemia and reperfusion on normal mucosal tissue in a blood-free environment. Mucosal tissue was incubated in Krebs buffer under oxygen for 3 hr to simulate the control condition, under nitrogen for 3 hr to simulate ischemia and under nitrogen for 2 hr followed by oxygen for 1 hr to simulate I/R.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of platelet-activating factor receptors on canine T lymphocytes.

The ability of purified canine T lymphocytes to selectively bind platelet activating factor (PAF) was characterized. Authentic radiolabeled PAF rapidly and selectively bound to T lymphocytes and reached saturation within 1 min. This binding was reversible and highly selective for (R) PAF because (S) PAF, lyso-PAF, and diacyl PAF did not displace the bound (R) PAF probe. Only increasing quantities of chemically pure (R) PAF displaced the radiolabeled (R) PAF probe. The binding maximum of PAF was determined to be 35 pM per 2 x 10(6) lymphocytes. Competitive radioligand binding studies and Scatchard analysis indicated a single class of high affinity receptors with a dissociation constant of 0.077 nM and a receptor density of 6419 receptors per cell. The ability of purified canine T lymphocytes to hydrolyze PAF to the biologically inactive metabolite lyso-PAF was also studied. Over a 30-min incubation period, about 5% of PAF was metabolized to lyso PAF. This rate of PAF hydrolysis was the same as the rate observed with the media without cells, suggesting a small degree of nonenzymatic hydrolysis. The effects of varying concentrations of authentic PAF on intracellular free Ca2+ release in purified T lymphocytes was evaluated using the fluorescent probe Fura-2 and excitation-emission spectrofluorometry. PAF below the concentration of 1.0 nM did not significantly increase intracellular Ca2+ in T lymphocytes. More than 1 nM PAF, intracellular-free Ca2+ modestly, but significantly, increased in T lymphocytes. In other experiments, canine PBMC proliferated in response to Con A and in the one way MLR. These proliferative responses were abolished when the selective PAF receptor antagonist SC-47014A was added to the culture medium. In the MLR, this inhibitory effect was dependent on the length of time that the antagonist was in the culture. Specifically, inhibition of proliferation was incrementally reversed when the PAF antagonist was introduced progressively later into the 7-day MLR stimulation period, suggesting that PAF receptor blockade prevents an MLR response from occurring, but is unable to suppress an existing MLR response. Although the Con A-induced mononuclear cell proliferation was abolished with PAF receptor antagonists, the addition of authentic biologically active PAF or PAF analogs did not alter the proliferative response to Con A. In conclusion, canine T lymphocytes possess high affinity receptors for PAF. These binding sites are highly selective and reversible. PAF binding slightly increases intracellular free Ca2+ in T lymphocytes and appears to be involved in lymphocyte proliferation in response to soluble plant mitogen and alloantigen.

Long-chain acyl-coenzyme A thioesters and renal hypothermic ischemic injury: effects of glycine flush.

The effects of hypothermic ischemia utilizing Euro-Collins flush on renal tissue long-chain activated fatty acid content was studied in dogs. Also, the ability of the simple amino acid glycine to complex these acyl thioesters was also investigated. Renal inner cortex was found to contain (in increasing amounts) myristoyl-, palmitoleoyl-, palmitoyl-, arachidonyl-, and oleoyl-coenzyme A throughout the 3 days of cold ischemia. Although the amounts of individual long-chain acyl-CoA compounds varied considerably, the concentrations were not found to differ significantly with increasing ischemia times. The presence of 5 mM of glycine in the flush also did not influence the amount or species of long-chain acyl-CoA esters in renal tissue during cold ischemia. Ischemic renal tissue content of most long-chain acyl-CoA compounds was reduced by about 50% when the tissue underwent in vitro reperfusion with 37 degrees C O2-saturated media. Glycine included in the flush storage solution did not alter acyl-CoA levels in tissue undergoing hypothermic ischemia and short-term in vitro reperfusion with O2-saturated buffer. In conclusion, long-chain acyl-CoA thioesters are present during hypothermic renal ischemia and the levels of most of these species are reduced during in vitro reperfusion after ischemia. The quality and production mass of these metabolites appears to be unaltered by progressive hypothermic ischemia times. Finally, the protective effects of glycine in this model of renal organ preservation injury are not associated with reductions of renal tissue long-chain activated fatty acids.

Improved and simplified tissue extraction method for quantitating long-chain acyl-coenzyme A thioesters with picomolar detection using high-performance liquid chromatography.

A method has been developed that permits rapid and easy tissue extraction of long-chain acyl-coenzyme A (acyl-CoA) thioesters with sensitive quantitation by reversed-phase high-performance liquid chromatography (RP-HPLC). Tissue homogenants are extracted using a reserve Bligh-Dyer technique, and long-chain acyl-CoA esters are harvested in the methanolic aqueous phase. Complex lipids and phospholipids are removed in the chloroform-rich organic Bligh-Dyer second phase, and long-chain acyl-CoA compounds are further purified from the methanolic aqueous Bligh-Dyer first phase on C18 extraction columns after removal of the methanol. The eluted and purified acyl-CoA esters are then quantitated by RP-HPLC using heptadecanoyl-CoA as an internal standard resulting in a detector sensitivity of about 12 pmol. Ten long-chain acyl-CoA esters from C12:0 to C20:4 were identified and separated from canine renal cortex and murine liver samples. The predominant acyl-CoA peaks from both kidney and liver were 14:0, 16:1, 16:0, 18:1, 18:2 and 20:4. Murine liver also produced 18:0 and all peaks disappeared after alkaline hydrolysis of the samples. This extraction and quantitation technique can successfully be used for tissue samples as small as 20 mg, and many samples can be processed in a short period of time. The simplicity of the extraction procedure and the sensitivity of the assay make this an attractive alternative approach to quantitating long-chain acyl-CoA thioesters from complex biological samples such as tissues.

Protective effects of glycine during hypothermic renal ischemia-reperfusion injury.

The objective of this investigation was to test the effects of glycine, a cytoprotectant in normothermic in vitro models of renal ischemia, in a model of hypothermic renal preservation injury. This study also probes possible physiological mechanisms of glycine protection during renal hypothermic ischemia-reperfusion injury. Canine kidneys were subjected to 48 h of hypothermic ischemia (4 degrees C) after intravascular flush with cold conventional Collins solution (G. H. Collins, M. B. Bravo-Shugarman, and P. I. Terasaki, Lancet 2: 1219-1223, 1969) and were subsequently revascularized for 1 h. After 1 h of reperfusion, glomerular filtration rate, urine production, and electrolyte excretion were dramatically higher when the Collins flush contained 5 mM glycine, compared with the 0 mM glycine controls. Renal tissue adenine nucleotides and glutathione levels progressively declined with graded cold ischemia times, and glycine had no effect on these levels. However, renal tissue ATP levels (but not glutathione) were significantly higher when kidneys were flushed with glycine, stored for 48 h, and reoxygenated in vitro for 1 h at 37 degrees C, compared with kidneys flushed without glycine. Analysis of CoA esters from ischemic renal tissue indicated altered production of only butyryl CoA after 48 and 72 h of cold ischemia, but no differences were detected in glycine or control kidneys. In conclusion, this study reports dramatic functional preservation with glycine in kidneys subjected to hypothermic ischemia and in vivo reperfusion. The mechanisms of these effects appear not to be attributable to the maintenance of cellular adenine nucleotide or glutathione levels nor to the scavenging of accumulated amphipathic acyl CoA esters.

Renal allograft platelet activating factor synthesis during acute cellular rejection.

The aim of this study was to characterize the synthesis and metabolism of platelet activating factor (PAF, 1-0-alkyl-2-0 acetyl-sn3-phosphorylcholine) by renal tissue undergoing acute cellular allograft rejection in the canine model. Kidneys were transplanted into outbred mongrel dogs and allowed to reject without immunosuppressive therapy. Five days after transplantation, all kidneys were non-functional and the tissue was assayed for the capacity to produce various molecular species of PAF and lyso-PAF using physical-chemical (GC/MS), immunologic (RIA) and biologic (platelet aggregation) assays. Renal cortical tissue obtained from rejecting allografts produced more PAF than control tissue by the following factors (GC/MS): 18-fold for C16:0 PAF; 3-fold for Lyso-C16:0 PAF; 2-fold for C18:1 PAF; and 6-fold for C18:0 PAF. The control tissue to which comparisons were made was renal cortex obtained from the native contralateral kidney. Increases in the production of various molecular species of PAF were also observed with renal medullary tissue undergoing acute rejection, although the magnitude of change was less dramatic than with renal cortex. The predominant PAF metabolite produced both by normal and allograft tissue was C16:0 Lyso-PAF. The increased PAF production by renal allograft tissue undergoing rejection was mainly attributable to C16:0 PAF and C16:0 Lyso-PAF, but increased production of both C18:0-PAF and of C18: 1-PAF was also detected. Increased renal allograft PAF production was also confirmed with a competitive binding immunoassay specific for PAF. In addition, when PAF-like material was isolated and purified from renal allograft incubation media and added to washed canine platelets, an intense aggregation response was observed that was abolished with prior alkaline methanolysis of the isolated material. Aggregation responses of similar magnitude were not obtained with PAF-like material isolated from native (non-rejection) renal tissue. In other experiment, incubation media obtained from rejecting renal allografts was found to contain factor which catalyzed hydrolysis of exogenous PAF to Lyso- PAF at twice the rate induced by media obtained from normal renal tissue. In conclusion, this study has identified dramatic increases in production of the biologically active molecular species of PAF by renal allograft tissue undergoing untreated cellular rejection. High levels of biologically inactive Lyso-PAF were also detected, and renal allograft tissue elaborates a factor which catalyzes rapid hydrolysis of PAF.

Physical characterization of a 137Cs field used for proficiency-test irradiations.

This paper describes a series of measurements and calculations that were undertaken at the Pacific Northwest Laboratory to determine the physical and dosimetric characteristics of a 137Cs source, which is housed within a commercially manufactured irradiator. These measurements and calculations helped to demonstrate that: (1) the exposure rate for this source was consistent with (traceable to) radiation standards maintained by the National Institute of Standards and Technology; (2) the radiation field at the surface of the irradiation phantom, in a 10 cm X 10 cm central area, was uniform to within +/- 2%; (3) the contribution of scattered photons was minimal; and (4) the ratio of the shallow to the deep dose equivalent was nearly unity. Because there are a number of similar irradiators in use, it is hoped that the methodology and results described in this paper will be of use to others.

Prostanoids and hypothermic renal preservation injury.

The effect of 48 hours of hypothermic renal ischemia utilizing Euro-Collins flush and short term reperfusion on renal prostaglandin synthesis was studied in dogs. Hypothermic ischemia followed by 60 minutes of reperfusion in-vivo resulted in significant elevations in renal Thromboxane B2 (TXB2) production in the outer cortex, inner cortex, and medulla, relative to non-ischemic kidneys. Prostaglandin E2 (PGE2) and 6-keto Prostaglandin F1 alpha (6-K PGF1 alpha) production were not significantly affected by ischemia and reperfusion. Enhanced TXB2 production was not seen with ischemia alone (without reperfusion) or with reperfusion with O2 saturated buffer, indicating a blood born source or stimuli. Early postreperfusion renal blood flow after hypothermic ischemia followed a biphasic pattern; blood flow increased for the first 10 minutes of reperfusion to achieve normal values, and then steadily declined over the next 20 minutes. This pattern was not altered by the cyclooxygenase inhibitors Idomethacin (5 mg/kg, P.O.) or Mefenamic acid (10 mg/kg, I.V.). Administration of the TXA2 synthesis inhibitor CGS-12970 (3 mg/kg, I.V.) or the TXA2/endoperoxide receptor antagonist SQ-29548 (80 micrograms/min, I.A.) significantly increased renal blood flow during reperfusion but neither agent altered the basic time dependent pattern observed in the control group. These data indicate that 48 hours of hypothermic renal ischemia results in dramatic changes in intrarenal TXA2 synthesis at the time of reperfusion. Enhanced TXA2 production is not dependent on reoxygenation per se, but rather requires reperfusion with blood suggesting a circulatory source.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of three radiofrequency coils for NMR studies of conductive samples.

Three rf coil designs of equal volume (approximately 15 ml) were compared using conductive samples. Magnetic loss into the sample was the dominant noise source. At physiological conductivity the sensitivity of the horizontally aligned solenoid and loop-gap resonator was only 1.3 +/- 0.2 times that of the vertically aligned slotted tube resonator.