Non-donor specific anti-human leukocyte antigen (HLA) antibodies are not associated with poor outcome in hematopoietic stem cell transplant recipients.

Testing for anti-human leukocyte antigen (HLA) antibodies has now become standard practice in allogeneic hematopoietic stem cell transplantation (HSCT), and anti-HLA antibodies (both donor specific and non-donor specific) are being identified and have many potential consequences. Most studies suggest that donor-specific HLA antibodies lead to adverse outcomes, though little is reported on non-donor specific anti-HLA antibodies. We present the results of a retrospective cohort analysis of 157 patients who received HSCT at the University of Rochester over a period of four years. We identified 45 patients (28.7%) who had detectable anti-HLA antibodies, while only one patient (0.6%) had donor-specific anti-HLA antibodies. Patients with prior pregnancies and multiple transfusions were at increased risk to develop antibodies. In our cohort, the presence of non-donor specific anti-HLA antibodies did not significantly impact overall survival, progression free survival, graft failure, or transplant-related mortality.

Insights about transport mechanisms and fracture flow channeling from multi-scale observations of tracer dispersion in shallow fractured crystalline rock.

In fractured media, solute transport is controlled by advection in open and connected fractures and by matrix diffusion that may be enhanced by chemical weathering of the fracture walls. These phenomena may lead to non-Fickian dispersion characterized by early tracer arrival time, late-time tailing on the breakthrough curves and potential scale effect on transport processes. Here we investigate the scale dependency of these processes by analyzing a series of convergent and push-pull tracer experiments with distance of investigation ranging from 4m to 41m in shallow fractured granite. The small and intermediate distances convergent experiments display a non-Fickian tailing, characterized by a -2 power law slope. However, the largest distance experiment does not display a clear power law behavior and indicates possibly two main pathways. The push-pull experiments show breakthrough curve tailing decreases as the volume of investigation increases, with a power law slope ranging from -3 to -2.3 from the smallest to the largest volume. The multipath model developed by Becker and Shapiro (2003) is used here to evaluate the hypothesis of the independence of flow pathways. The multipath model is found to explain the convergent data, when increasing local dispersivity and reducing the number of pathways with distance which suggest a transition from non-Fickian to Fickian transport at fracture scale. However, this model predicts an increase of tailing with push-pull distance, while the experiments show the opposite trend. This inconsistency may suggest the activation of cross channel mass transfer at larger volume of investigation, which leads to non-reversible heterogeneous advection with scale. This transition from independent channels to connected channels when the volume of investigation increases suggest that both convergent and push-pull breakthrough curves can inform the existence of characteristic length scales.

Flavaglines target primitive leukemia cells and enhance anti-leukemia drug activity.

Identification of agents that target human leukemia stem cells is an important consideration for the development of new therapies. The present study demonstrates that rocaglamide and silvestrol, closely related natural products from the flavagline class of compounds, are able to preferentially kill functionally defined leukemia stem cells, while sparing normal stem and progenitor cells. In addition to efficacy as single agents, flavaglines sensitize leukemia cells to several anticancer compounds, including front-line chemotherapeutic drugs used to treat leukemia patients. Mechanistic studies indicate that flavaglines strongly inhibit protein synthesis, leading to the reduction of short-lived antiapoptotic proteins. Notably though, treatment with flavaglines, alone or in combination with other drugs, yields a much stronger cytotoxic activity toward leukemia cells than the translational inhibitor temsirolimus. These results indicate that the underlying cell death mechanism of flavaglines is more complex than simply inhibiting general protein translation. Global gene expression profiling and cell biological assays identified Myc inhibition and the disruption of mitochondrial integrity to be features of flavaglines, which we propose contribute to their efficacy in targeting leukemia cells. Taken together, these findings indicate that rocaglamide and silvestrol are distinct from clinically available translational inhibitors and represent promising candidates for the treatment of leukemia.

The role of iconic memory in change-detection tasks.

In three experiments, subjects attempted to detect the change of a single item in a visually presented array of items. Subjects' ability to detect a change was greatly reduced if a blank interstimulus interval (ISI) was inserted between the original array and an array in which one item had changed ('change blindness'). However, change detection improved when the location of the change was cued during the blank ISI. This suggests that people represent more information of a scene than change blindness might suggest. We test two possible hypotheses why, in the absence of a cue, this representation fails to produce good change detection. The first claims that the intervening events employed to create change blindness result in multiple neural transients which co-occur with the to-be-detected change. Poor detection rates occur because a serial search of all the transient locations is required to detect the change, during which time the representation of the original scene fades. The second claims that the occurrence of the second frame overwrites the representation of the first frame, unless that information is insulated against overwriting by attention. The results support the second hypothesis. We conclude that people may have a fairly rich visual representation of a scene while the scene is present, but fail to detect changes because they lack the ability to simultaneously represent two complete visual representations.

Patients' concerns prior to undergoing total hip and total knee arthroplasty.

OBJECTIVE: To document and examine the concerns patients have prior to undergoing primary total hip or total knee arthroplasty in a tertiary care center or an orthopedic private practice group. PATIENTS AND METHODS: In this prospective survey, 136 patients from a tertiary care center and 130 from an orthopedic private practice group completed a questionnaire covering 54 items regarding their concerns prior to undergoing primary total hip or total knee arthroplasty. Patients responded on a visual analog scale, and concern was ranked by mean responses (1, not concerned at all; 2, somewhat concerned; 3, very concerned; or 4, extremely concerned). RESULTS: Responses to only 6 items averaged scores higher than 1.9: pain immediately after the surgery (2.07), length of recovery (2.07), ability to walk as much as you wish (2.03), ability to return to recreational activities (1.97), ability to go up and down stairs (1.94), and risk of getting acquired immunodeficiency syndrome from a transfusion (1.92). Older patients (> or = 65 years) were less concerned than younger patients (< 65 years) in 34 of the 54 questions asked. Women were more concerned than men in 19 of the 54 questions asked. CONCLUSION: These data provide information that will be helpful in preoperative patient discussions and in development of educational materials for patients undergoing total hip or total knee arthroplasty.

Dissociation of vasoconstrictor-stimulated basic fibroblast growth factor expression from hypertrophic growth in cultured vascular smooth muscle cells. Relevant roles of protein kinase C.

Thromboxane A2 (TXA2) and angiotensin II (Ang II) stimulate vascular smooth muscle hypertrophy by upregulating endogenous synthesis of basic fibroblast growth factor (bFGF). Because mitogenic phorbol esters can also stimulate bFGF formation, we investigated the role of protein kinase C (PKC) in vascular smooth muscle cell (VSMC) bFGF formation and hypertrophy. Preliminary characterization of PKC isoform expression in VSMC by use of polymerase chain reaction identified PKC alpha, delta, epsilon, and zeta. Western analysis confirmed the presence of these isoforms in cultured VSMC lines and demonstrated downregulation of PKC alpha, delta, and epsilon by phorbol 12-myristate 13-acetate (PMA) but not TXA2 or Ang II. PKC activation with 100 nmol/L PMA stimulated VSMC mitogenesis measured as incorporation of [3H]leucine and [3H]thymidine and increased cell number. Like TXA2 and Ang II, PMA increased endogenous VSMC bFGF in a time-dependent manner, whereas an inactive phorbol ester had no such effect. Addition of an antisense oligodeoxynucleotide against bFGF prevented PMA-stimulated bFGF expression and inhibited PMA-stimulated growth, suggesting that bFGF synthesis is necessary for VSMC growth stimulated by PMA. To clarify the role of PKC in vasoconstrictor-stimulated VSMC production of bFGF and hypertrophy, PKC was down-regulated by prolonged exposure to PMA or was inhibited with calphostin C or staurosporine before the addition of TXA2 or Ang II. PKC inhibition prevented TXA2-stimulated and Ang II--stimulated VSMC hypertrophy without attenuating the observed increase in bFGF expression. Furthermore, PKC inhibition with calphostin C inhibited VSMC mitogenesis stimulated by exogenous bFGF.(ABSTRACT TRUNCATED AT 250 WORDS)

Thromboxane A2 stimulates vascular smooth muscle hypertrophy by up-regulating the synthesis and release of endogenous basic fibroblast growth factor.

We have shown previously that thromboxane A2 stimulates hypertrophy of cultured rat aortic smooth muscle cells defined as protooncogene expression and protein synthesis without DNA synthesis or cellular proliferation (Dorn, G.W., II, Becker, M.W., Davis, M.G. (1992) J. Biol. Chem. 267, 24897-24905). Since endogenous growth modulators could possibly regulate vascular smooth muscle growth to this vasoconstrictor, we tested the hypothesis that thromboxane-stimulated vascular smooth muscle hypertrophy was due to increased expression of endogenously produced basic fibroblast growth factor (bFGF). The thromboxane mimetic (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619) (1 microM) increased cultured rat aorta derived smooth muscle cell immunoreactive bFGF content by 331 +/- 40% over untreated controls after 24 h. Co-incubation of vascular smooth muscle cells with a specific antisense oligodeoxynucleotide (AS) against codon 60 of bFGF coding sequence reduced thromboxane-stimulated bFGF expression by 72 +/- 5% and prevented thromboxane-stimulated hypertrophy (nonsense oligonucleotide had no effects). Addition of exogenous bFGF (20 ng/ml) restored growth to AS-treated/thromboxane-stimulated vascular smooth muscle cells. Furthermore, addition to the culture medium of neutralizing antibody against bFGF inhibited U46619-stimulated vascular smooth muscle hypertrophy by 69 +/- 17%, whereas nonimmune IgG had no effect. Since protein tyrosine phosphorylation is a cell signal associated with growth, thromboxane-stimulated tyrosine phosphorylation was also examined. Exposure to 1 microM U46619 for 10 min increased vascular smooth muscle immunoreactive phosphotyrosine content of 130-144-, 86-, 80-, 75-, and 58-kDa proteins. The tyrosine kinase inhibitor herbimycin A (5 microM) prevented thromboxane-stimulated tyrosine phosphorylation, but not thromboxane-stimulated hypertrophy, suggesting that tyrosine phosphorylation was not required for thromboxane-stimulated vascular smooth muscle growth. These results indicate that increased expression and release of endogenous bFGF, but not direct tyrosine phosphorylation, mediates the hypertrophic vascular smooth muscle response to thromboxane.

Thromboxane A2 stimulated signal transduction in vascular smooth muscle.

Thromboxane A2 (TXA2) is a potent, labile vasoconstrictor which stimulates vessel contraction through vascular smooth muscle TXA2 receptors differing from those in platelets. We studied TXA2-stimulated events in cultured adult rat aortic smooth muscle cells. The stable TXA2 mimetic (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z, 13E-dienoic acid (U46619) competed for TXA2 agonist binding to vascular smooth muscle cells with an IC50 of 10 +/- 1 nM. In fura-2-loaded cells, U46619 increased free cytosolic Ca++ concentration with an EC50 of 49 +/- 14 nM. The increase in free cytosolic Ca++ was rapid, transient and independent of extracellular Ca++ or Ca++ antagonists and thus was due to release from intracellular stores. U46619-mediated Ca++ release was temporally associated with phosphorylation of myosin light chains, increased accumulation of 1,4,5-inositol trisphosphate (EC50 = 32 +/- 4 nM) and cytoplasmic acidification from pH 7.06 +/- 0.01 to 7.00 +/- 0.02 (P = .02). Ca++ release was 53% attenuated by the phospholipase C inhibitor, 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H- pyrrole-2,5-dione. In rat aortic rings U46619 caused TXA2 receptor-mediated contractions (EC50 of 28 +/- 2 nM) which were not attenuated by removal of extracellular Ca++ from the superfusion buffer. Together, these results suggest that agonist occupation of TXA2 receptors produces vascular smooth muscle contraction through initial activation of phospholipase C with production of 1,4,5-inositol phosphate, release of intracellular calcium stores and phosphorylation of myosin light chains associated with cellular acidification, presumably via activation of Ca++ ATPase.

Unicondylar knee arthroplasty. An evaluation of selection criteria.

In a prospective evaluation of 165 consecutive patients (228 knees), intraoperative evaluation of the knees was performed at the time of total knee arthroplasty. Each of the three knee compartments was independently graded for arthritic changes depending on the extent of articular degeneration visualized. Patients were believed to be suitable candidates for unicondylar knee arthroplasty (UKA) if they fulfilled the Kozinn and Scott criteria. Specific attention was given to patient age, weight, preoperative range of motion, angular deformity, as well as the extent of intraoperative cartilage erosions seen. Thirty-five knees (15%) were candidates for UKA based solely on inspection of the articular surfaces at the time of surgery. Further analysis revealed that of these 35 knees, 22 failed to meet the other selection criteria. Thus, of the original 228 knees, only 13 knees (6%) fulfilled all of the stringent selection requirements and were considered suitable candidates for UKA. With proper patient selection, the number of UKAs performed could become relatively small.

Dissociation of the contractile and hypertrophic effects of vasoconstrictor prostanoids in vascular smooth muscle.

To more clearly define the physiologic roles of thromboxane (TX)A2 and primary prostaglandins (PG) in vascular tissue we examined vascular contractility, cell signaling, and growth responses. The growth-promoting effects of (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619; TXA2 agonist), PGF2 alpha, and PGE2 consisted of protein synthesis and proto-oncogene expression, but not DNA synthesis or cell proliferation. U46619 contracted rat aortas and increased cultured rat aortic vascular smooth muscle cell intracellular free calcium concentration [Ca2+]i, [3H]inositol monophosphate (IP) accumulation, myosin light chain phosphorylation, and protein synthesis ([3H]leucine incorporation) with EC50 values ranging from 10 to 50 nM. Each of these responses was inhibitable with the TXA2 receptor antagonist [1S]1 alpha,2 beta(5Z),3 beta,4 alpha-7-(3-[2- [(phenylamino)carbonyl]hydrazino]methyl)-7-oxabicyclo[2.2.1]hept-2- yl-5-heptenoic acid (SQ29548). In contrast, PGF2 alpha increased [Ca2+]i, [3H]IP, and protein synthesis with EC50 values of 30-230 nM but contracted rat aortas with an EC50 of 4800 nM. PGE2 increased [Ca2+]i, [3H]IP accumulation, protein synthesis, and contracted rat aortas with EC50 values of 2.5-3.5 microM. TXA2 receptor blockade prevented PGF2 alpha- and PGE2-induced aortic contraction and cell myosin light chain phosphorylation, but not cell signaling or protein synthesis. Binding studies to vascular smooth muscle TXA2 receptors using 1S-[1 alpha,2 beta(5Z),3 alpha(1E,3S),4 alpha]-7-(3-[3-hydroxy-4-(p- [125I]iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-hepte noic acid ([125I]BOP) showed U46619, SQ29548, PGF2 alpha, and PGE2 competition for TXA2 receptor binding at concentrations similar to their EC50 values for aortic contraction, while binding competition with [3H]PGF2 alpha and [3H]PGE2 demonstrated the specificity of [125I]BOP and SQ29548 for TXA2 receptors. The results suggest that 1) PGF2 alpha- and E2-stimulated vessel contraction is due to cross-agonism at vascular TXA2 receptors; 2) PGF2 alpha stimulates TXA2 receptor-independent vascular smooth muscle protein synthesis at nanomolar concentrations, consistent with an interaction at its primary receptor; and 3) TXA2 is a potent stimulus for vascular smooth muscle contraction and protein synthesis. We suggest that the main physiologic effect of PGF2 alpha may be as a stimulus for vascular smooth muscle cell hypertrophy, not as a contractile agonist.

The significance of calf thrombi after total knee arthroplasty.

We reviewed the records of 1257 patients having 1625 total knee arthroplasties; all had pre-operative and postoperative perfusion lung scans and postoperative venograms which were classified as showing no thrombi, calf thrombi or proximal thrombi. Patients with calf thrombi were found to have a significantly greater risk for both symptomatic and asymptomatic pulmonary embolism compared with patients with no venographic thrombi. There were positive lung scans in 6.9% of patients with calf thrombi compared with 2.0% of patients with negative venograms (p < 0.001). Symptomatic pulmonary embolism occurred in 1.6% of patients with calf thrombi compared with 0.2% of patients with negative venograms (p = 0.034). The risk of pulmonary embolism was not significantly different between patients with treated proximal thrombi, and those with calf thrombi. Patients who develop deep-vein thrombosis despite prophylaxis are at increased risk for pulmonary embolism; these patients should receive treatment, or undergo follow-up studies to detect proximal propagation.

Growth factors downregulate vascular smooth muscle thromboxane receptors independent of cell growth.

Growth factors, in addition to being mitogenic, may modulate vascular smooth muscle differentiation. We tested whether serum or defined growth factors could regulate thromboxane A2 (TxA2) receptors in cultured rabbit aorta smooth muscle cells. Fetal bovine serum (10%) stimulated cell proliferation and DNA synthesis in subconfluent cell cultures. Binding of the thromboxane A2 agonist [1S-(1 alpha 2 beta(5Z),3 alpha(1E,3S),4 alpha)]-7-[3-(3-hydroxy-4-p- iodophenoxy-1-butenyl)-7-oxabicyclo[2.2.1]heptan-2-yl]-5-hep tenoic acid showed a 41% decrease in TxA2 receptors in cells treated with 10% serum compared with serum-deprived (0.1%) controls. Receptor downregulation by serum was gradually reversible upon serum withdrawal. Compared with serum-deprived cells, those exposed to 10% serum also had diminished TxA2-stimulated phosphatidylinositol hydrolysis. Regulatory actions of serum on TxA2 receptors were distinguished from mitogenic effects with heparin, which prevented cell growth but did not inhibit serum-induced downregulation of TxA2 receptors. Furthermore, low concentrations of platelet-derived growth factor and basic fibroblast growth factor decreased TxA2 receptors without stimulating cell proliferation or DNA synthesis. These observations describe a previously unrecognized regulatory action of growth factors on a vascular smooth muscle vasoconstrictor receptor, an action which is independent of effects on cell proliferation or DNA synthesis.

Bilateral total knee arthroplasty. One cruciate retaining and one cruciate substituting.

Thirty bilateral-paired cruciate-retaining and cruciate-substituting total knee arthroplasties (TKAs) were evaluated two to five years after surgery. In general, the more deformed knees received the cruciate-substituting prosthesis. Preoperative Hospital for Special Surgery knee scores averaged 56.4 (range, 44-68) in the cruciate-retaining knees and 53.3 (range, 29-69) in the cruciate-substituting knees. Postoperatively, 25 patients were observed climbing stairs. All patients were scored and asked their preference of knees. Fifteen patients handled stairs normally. Five patients climbed stairs favoring the cruciate-substituting knee and four the cruciate-retaining knee. One patient favored one knee ascending and the other descending. In the cruciate-substituting knees, there were 27 excellent and three good knee scores. There were 28 excellent and two good knee scores in the cruciate-retaining knees. Ten patients preferred the cruciate-retaining knee, eight preferred the cruciate-substituting knee, and 12 had no preference. In this group of patients no clinical advantage of one type of TKA over the other was observed.

Radionuclide imaging of asymptomatic versus symptomatic total knee arthroplasties.

Ninety-eight total knee prostheses were evaluated by roentgenograms and bone scans. Fifty-three were asymptomatic, and 45 were symptomatic. Thirteen prostheses required revision surgery. At a mean of 54 months, asymptomatic knee replacements generally showed only mild uptake in one or more zones. Only one knee had uptake equal to surrounding bone. However, symptomatic knee replacements showed significantly greater uptake in the patella, femur, and medial and lateral tibial plateau regions (Mann-Whitney two-sample rank test). Bone scans in the symptomatic group were obtained at a mean of 44 months. Excluding those patients who had revision surgery, the differences remained significant. Furthermore, symptomatic knee replacements with normal roentgenograms also had significantly greater uptake. Radiolucent lines were noted in 30% of asymptomatic patients, whereas 29% of symptomatic knees had radiolucencies. Radiolucencies were not generally associated with significantly greater uptake. Lateral release had no effect on the patellar score.