Donor-specific indirect pathway analysis reveals a B-cell-independent signature which reflects outcomes in kidney transplant recipients.

To investigate the role of donor-specific indirect pathway T cells in renal transplant tolerance, we analyzed responses in peripheral blood of 45 patients using the trans-vivo delayed-type hypersensitivity assay. Subjects were enrolled into five groups-identical twin, clinically tolerant (TOL), steroid monotherapy (MONO), standard immunosuppression (SI) and chronic rejection (CR)-based on transplant type, posttransplant immunosuppression and graft function. The indirect pathway was active in all groups except twins but distinct intergroup differences were evident, corresponding to clinical status. The antidonor indirect pathway T effector response increased across patient groups (TOL < MONO < SI < CR; p < 0.0001) whereas antidonor indirect pathway T regulatory response decreased (TOL > MONO = SI > CR; p < 0.005). This pattern differed from that seen in circulating naïve B-cell numbers and in a cross-platform biomarker analysis, where patients on monotherapy were not ranked closest to TOL patients, but rather were indistinguishable from chronically rejecting patients. Cross-sectional analysis of the indirect pathway revealed a spectrum in T-regulatory:T-effector balance, ranging from TOL patients having predominantly regulatory responses to CR patients having predominantly effector responses. Therefore, the indirect pathway measurements reflect a distinct aspect of tolerance from the recently reported elevation of circulating naïve B cells, which was apparent only in recipients off immunosuppression.

Neutralizing IL-17 prevents obliterative bronchiolitis in murine orthotopic lung transplantation.

Obliterative bronchiolitis (OB) is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining OB immunopathogenesis. In the current study, lungs from C57BL/10 H-2(b) mice that are MHC compatible, but minor histocompatability antigen incompatible, were transplanted into C57BL/6 mice. Histological features and cytokine profiles of OB were assessed. Moderate rejection (grade A3) developed by day 14, with evidence of OB at that time point. At 21 days, OB was present in 55% of grafts and moderate to severe rejection (grade A3-A4) was present in all mice. At 28 days, OB was present in 44% of mice and severe rejection (grade A4) was present in all. IL-17A, but not IL-17F, splenic mRNA transcripts and serum protein levels were increased only in mice that developed OB, whereas IL-10 transcripts and protein were increased only in non-OB mice. Neutralizing IL-17 prevented OB, down regulated acute rejection, and upregulated systemic IL-10. Collectively, these data show that transplantation of minor histoincompatible lungs from C57BL/10 mice into C57BL/6 mice results in a highly reproducible preclinical model of OB. In addition, these data indicate that neutralizing IL-17A or augmenting IL-10 could be therapeutic interventions to prevent OB.

Genotypic and phenotypic characterization of human immunodeficiency virus type 1 variants isolated from patients treated with the protease inhibitor nelfinavir.

Nelfinavir mesylate (formerly AG1343) is a potent and selective inhibitor of human immunodeficiency virus (HIV) protease approved for the treatment of individuals infected with HIV. Nucleotide sequence analysis of protease genes from plasma HIV type 1 (HIV-1) RNA revealed a unique aspartic acid (D)-to-asparagine (N) substitution at residue 30 (D30N) in 25 of 55 patients treated with nelfinavir for a median of 13 weeks. Although the appearance of D30N was occasionally associated with concurrent or sequential emergence of other changes (e.g., at residues 35, 36, 46, 71, 77, and 88), genotypic changes associated with phenotypic resistance to other protease inhibitors were not observed (e.g., at residues 48, 50, 82, and 84) or were only rarely observed (e.g., at residue 90). In phenotypic assays, viral isolates with high-level resistance to nelfinavir remained susceptible to indinavir, saquinavir, ritonavir, and amprenavir (formerly VX-478/141W94). Similar results were observed in phenotypic assays utilizing HIV-1 NL4-3, which contained the D30N substitution alone or in combination with substitutions at other residues (e.g., residues 46, 71, and 88). These data indicate that the initial pathway of resistance to nelfinavir is unique and suggest that individuals failing short courses of nelfinavir-containing regimens may respond to regimens containing other protease inhibitors.

Effect of supplemental fish meal on the lactation and reproductive performance of dairy cows.

The influence of high protein diets (21% CP, DM basis), containing varied percentages of RUP, on lactation performance and fertility was evaluated. Sixty-two Holstein cows (65% multiparous) were blocked by age and randomly assigned to a 2 x 2 x 2 factorial design from d 12 to 125 postpartum. Factor 1 was 0 or 3.5% fish meal diet, factor 2 was location (Calan door versus free stall), and factor 3 was parity (first versus second or later). The soybean meal diet consisted of alfalfa hay, corn silage, barley, and soybean meal. The fish meal diet contained 3.5% fish meal (ruminant grade menhaden) that replaced a portion of the soybean meal. Cows fed the fish meal diet (40% RUP) had DMI, BW, and body condition similar to those of cows offered the soybean meal diet (34% RUP). Cows receiving the fish meal supplement tended to have higher milk protein production throughout the trial, higher milk production during the first 6 wk, and significantly lower ruminal ammonia concentrations than cows receiving the soybean meal diet. Differences in reproductive performance were not significant except for a diet by housing location interaction for conception rates from first AI.

Stereoselective actions of thiadiazinones on canine cardiac myocytes and myofilaments.

Thiadiazinones are cardiotonic agents that have potent, direct, and stereoselective actions on the myofilament response to Ca2+ in intact myocardium. Their mechanism of action is unknown. We studied the effects of racemic thiadiazinone, EMD 53998 (5-[1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydro-6-quinolyl]-6-meth yl-3,6- dihydro-2H-1,3,4-thiadiazin-2-one), and its enantiomers on Ca2+ signaling in myocytes, myofilaments, and myofilament proteins. Intact canine ventricular myocytes responded to the positive enantiomer, EMD 57033, with an increase in the extent of shortening during twitch contractions without increasing the peak amplitude of the Ca2+ transient. The negative enantiomer, EMD 57439, also increased the extent of shortening, but in this case there was a concentration-dependent increase in the peak amplitude of the Ca2+ transient. This is predicted from in vitro data showing that this enantiomer is a relatively potent inhibitor of phosphodiesterase activity. There was no effect of EMD 57439 on the relation between pCa and actomyosin Mg-ATPase activity of canine heart myofibrils. In contrast, EMD 57033 shifted the pCa-Mg-ATPase activity relation to the left. There was no effect of either enantiomer on Ca2+ binding to myofilament troponin C. Moreover EMD 57033, but not EMD 57439, stimulated actomyosin ATPase activity of myofilament preparations in which either troponin or troponin-tropomyosin had been extracted. EMD 57033 had no effect on Mg-ATPase activity of pure ventricular myosin. EMD 57033 also stimulated the velocity of actin filament sliding on myosin heads adhered to nitrocellulose-coated glass coverslips. We propose that the action of EMD 57033 is at the actin-myosin interface on a "receptor" that may be on actin or the crossbridge. Drug binding to this domain appears to reverse the inhibition of actin-myosin interactions by troponin-tropomyosin and also to promote transition of crossbridges from weak to strong force-generating states.

Explanation of cerebral white--gray contrast in computed tomography.

The difference in Hounsfield number between white and gray brain tissue is calculated from published chemical compositions and density values. The result of 5.5 Hounsfield units at typical CT energies is in excellent agreement with experiments. The difference arises because gray matter contains 8% more oxygen and 8% less carbon, due to its higher water and lower lipid content, and this increases the photoelectric absorption. Efforts to obtain greater contrast by lowering the X-ray energy will be offset by the noise increase at low energies.

Optimum energy for performing CT iodinated constrast studies.

A study has been made of CT contrast for iodine as a function of X-ray energy from 80 to 140 kV constant potential. The EMI CT 1010 scanner was used for phantom measurements of contrast signal, noise, artefact and dose. The results show that optimum detectability occurs at 80-100 kV, with the close efficiency being somewhat better at 80kV.

Evaluation of edge-induced streaking artifacts in CT scanners.

A phantom is presented which permits the evaluation of streaking artifacts produced in CT reconstructions by abrupt edges. Its application is demonstrated by results obtained from nine CT scanners. It is observed that even in regions where streaking is not readily apparent, edge-induced artifacts can significantly increase the variance in the reconstruction.

Computed tomography profiles of periventricular hypodensity in hydrocephalus and leukoencephalopathy.

Profiles of hypodense periventricular white matter, drawn from the interior of the frontal horns through the surrounding white matter to the peripheral gray matter, were analyzed in 65 patients. Abnormal curves were grouped into four patterns: linear, double-slope, plateau, and bimodal. The first two, found primarily in hydrocephalic patients, appear to be due to transependymal passage of cerebrospinal fluid (CSF). The linear pattern was found more frequently in acute cases and indicates breakdown of the ventricular wall as a barrier. The double-slope pattern probably indicates a persistent or restored barrier effect at the wall, with residual CSF infiltration of the parenchyma. Most of the other patients' curves showed either a plateau pattern or (especially in primary leukoencephalopathy) a bimodal ("bumpy") pattern which is believed to correspond to the nonuniform nature of the pathological process.