The clinical significance of flow cytometry crossmatching in heart transplantation.

BACKGROUND: Flow cytometry crossmatching is more sensitive than cytotoxic methods in identifying preformed antibodies to donor alloantigens. However, the significance of a positive flow crossmatch remains unknown for a recipient of a heart transplant who has a negative anti-human globulin crossmatch. METHODS: Flow crossmatching was performed retrospectively for 92 recipients of a primary cardiac allograft who underwent transplantation with a negative AHG crossmatch. RESULTS: Forty-six patients were flow crossmatch-positive for alloantibody: 20 were positive on both T and B lymphocytes, 12 were positive only on B lymphocytes, and 13 were positive only on T lymphocytes. Eleven had autoantibody invalidating the flow crossmatch with donor cells. Thirty-six patients had negative flow crossmatch. A significantly higher incidence of graft dysfunction with vascular rejection by 6 months was found for patients who had a positive flow crossmatch on B lymphocytes. This group also had an increased incidence of mortality within this same period. Patients who were flow crossmatch-positive on T and B lymphocytes were more likely to experience greater than two episodes of treated cellular rejection within the first 6 months. Flow crossmatch-positive patients stayed longer in the hospital in comparison to the other two groups, although the increases were not statistically significant. There were no differences between groups with regard to time to first rejection, absence of rejection episodes, episodes of decreased cardiac index (<2.3 L/m2), depressed left and right ventricular ejection fraction, or development of transplant atherosclerosis. CONCLUSION: A positive flow crossmatch identified a subset of patients who are predisposed to development of vascular rejection or are more likely to have frequent cellular rejection.

Conversion from Sandimmune to Neoral in organ transplant recipients.

The pharmacokinetic profiles of Sandimmune and Neoral vary considerably among transplant recipients. Cyclosporine exposure is far more consistent with Neoral than it is with Sandimmune. Because intrapatient variability of drug exposure has been demonstrated to be a risk factor for chronic rejection, this difference becomes important. Neoral also has a linear dose response and a stronger correlation between trough level and drug exposure. Dose linearity greatly facilitates accurate dose titration. Results of controlled studies in which kidney, liver, and heart transplant recipients were converted from Sandimmune to Neoral have shown that conversion on a 1:1 mg basis results in more predictable bioavailability and often in reductions in cyclosporine dose. Carefully monitored conversion has not been associated with increased side effects, and any side effects that do emerge can usually be managed by taking Neoral with food, changing the dose from every 12 hours to every 8 hours, or through dose reduction.

VCAM-1 and E-selectin expression during cytomegalovirus infection in post-transplant myocardial biopsies.

Adhesion molecule expression may become useful in monitoring cardiac graft rejection, but first the question of whether expression is upregulated by cytomegalovirus (CMV), a common post-transplant infection, must be answered. To study this, all cardiac biopsies (n = 201) on 12 cardiac transplant recipients were examined for rejection grade and VCAM-1, ICAM-1, and E-selectin expression over the first 6-15 months post-transplant. Adhesion molecule expression in biopsies taken during documented CMV infections were compared to those taken in the absence of infection, both overall and sorted as to rejection grade. There were 17 CMV infections in this patient group. VCAM-1 was expressed in 82% of biopsies coincident with CMV infections, compared to 43% of biopsies unrelated to CMV infection, a significant difference (p < 0.01). E-selectin was expressed in 65% of biopsies with CMV infection, compared to 30% of biopsies unrelated to CMV infection, also statistically significant (p = 0.01). Both VCAM-1 and E-selectin were expressed in 80% of biopsies without rejection taken during CMV infections, significantly greater than the 24% incidence of VCAM-1 and 14% incidence of E-selectin expression in biopsies without rejection that were not concomitant with CMV infection. In the absence of CMV infection, both VCAM-1 and E-selectin expression correlated significantly with rejection grade, but this relationship became invalid in the presence of CMV infection. ICAM-1 expression bore no relation to CMV infection. VCAM-1 and E-selectin expression in cardiac biopsies can be upregulated with CMV infection in the absence of graft rejection.

E-selectin expression in human cardiac grafts with cellular rejection.

BACKGROUND: E-selectin expression has recently been documented to occur with lymphocytic infiltration in the skin and synovium. The question of whether E-selectin is expressed in the context of cardiac graft rejection was addressed in this study. METHODS AND RESULTS: One hundred ninety-five human posttransplant cardiac biopsy specimens were immunoreacted with antibodies to E-selectin and VCAM-1, and endothelial expression of both adhesion molecules was recorded as present or absent. Cardiac graft rejection was graded in blinded fashion. The frequency of E-selectin expression was 11% in biopsies without rejection, 36% in mild rejection, and 58% in moderate rejection, a significant correlation (P < .001). VCAM-1 expression was present in 11% of biopsies with no rejection, 37% with mild rejection, and 85% with moderate rejection, corroborating the previously reported strong correlation between VCAM-1 expression and graft rejection (P < .0001). In 71% of specimens, E-selectin expression coincided with VCAM-1 expression. In the remaining 29% of specimens in which E-selectin and VCAM-1 expression were not both present, isolated E-selectin expression was found more frequently in biopsies with early, increasing rejection, whereas isolated VCAM-1 expression was found more frequently in specimens with established moderate rejection and later, resolving rejection. CONCLUSIONS: E-selectin is expressed in cardiac allograft rejection and may play a role in recruitment of lymphocytes into the graft. Rejection trend analysis suggests that E-selectin expression may be prominent early in the course of rejection.

Vascular cell adhesion molecule-1 is induced on endothelium during acute rejection in human cardiac allografts.

An infiltration of mononuclear leukocytes into the myocardium of a cardiac allograft is diagnostic of transplant rejection. The presence of these leukocytes implies their adhesion to, and subsequent migration through, the vascular endothelium. Intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 are endothelial proteins that have been shown to be involved in the binding of mononuclear leukocytes to the endothelium in vitro. We investigated the induction of these proteins in a random series from 99 endomyocardial biopsy specimens obtained from 1 week to 4 years after cardiac allograft transplantation. Intercellular adhesion molecule-1 was found to be expressed constitutively by the myocardial microvasculature in the recipient's original heart and in the posttransplantation biopsy specimens. No correlation was found between the presence or absence of intercellular adhesion molecule-1 expression and cellular rejection. In contrast, no endothelial expression of vascular cell adhesion molecule-1 was observed in the recipient heart or in endomyocardial biopsy specimens lacking cellular rejection. The presence of vascular cell adhesion molecule-1 significantly correlated with the presence of mild or moderate rejection. The de novo induction of vascular cell adhesion molecule-1 on the myocardial vasculature during periods of rejection, in addition to the recruitment of mononuclear leukocytes that are known to bind to this protein, suggests that the expression of this endothelial adhesion protein could be of use in diagnosing rejection.

Restrictive hemodynamic patterns after cardiac transplantation: relationship to histologic signs of rejection.

Hemodynamic and echocardiographic data from 33 consecutive patients undergoing cardiac transplantation were correlated with endomyocardial biopsy results to determine whether reversible restrictive hemodynamics accompany histologic evidence of transplant rejection. During the study period 251 biopsy specimens were obtained during periods of no histologic evidence of transplant rejection and 52 episodes of mild, 20 episodes of moderate, and one episode of severe rejection. Right atrial mean pressure increased significantly during episodes of moderate transplant rejection (9.9 +/- 6.2 mm Hg, p less than 0.001) compared with pressures obtained during periods when there was no evidence of rejection (4.6 +/- 3.2 mm Hg), mild rejection (5.8 +/- 3.9 mm Hg), or resolving rejection (4.3 +/- 3.4 mm Hg). Y descent was elevated during moderate rejection (9.6 +/- 4.2 mm Hg, p less than 0.001) compared with pressures during episodes of no rejection (5.6 +/- 2.5 mm Hg), mild rejection (6.6 +/- 2.7 mm Hg), and resolving rejection (5.8 +/- 3.1 mm Hg) and showed a wave morphology consistent with a restrictive hemodynamic pattern. Pulmonary capillary wedge pressure was increased during moderate rejection (14.4 +/- 6.4 mm Hg) when compared with pressures obtained during episodes of no rejection (10.2 +/- 5.8 mm Hg) or resolving rejection (10.2 +/- 5.4 mm Hg) (p less than 0.02). Sensitivity for a right atrial mean pressure of 11 mm Hg indicating moderate rejection was 41% with a specificity of 96%. Sensitivity for Y descent (greater than or equal to 10 mm Hg) was 52% and specificity was 94%.(ABSTRACT TRUNCATED AT 250 WORDS)

A new method for phase identification for electron diffractionists.

An accurate analytical procedure for phase identification for electron diffractionists has been developed. The method opens new frontiers in the identification of solid-state materials, as crystalline samples in the size range 10 microns to 10 A can be accurately characterized. Research with NIST CRYSTAL DATA (a large database with chemical, physical, and crystallographic data on solid-state materials) has proved that a material can be uniquely characterized on the basis of its lattice and chemical composition. To characterize a material, it is sufficient to determine any primitive cell of the lattice and the element types present. Using a modern analytical electron microscope (AEM), the experimentalist can collect the required data on an unknown sample. The lattice information is obtained by rotation of the sample to obtain two or more planes of data. From these planes, a unit cell defining the lattice can be deduced. The chemical data are determined by energy-dispersive spectroscopy (EDS). Once the experimental data are measured, the unknown is identified against the database of knows using lattice/element-type matching techniques. The basic strategy consists of three conceptual steps. First, the unknown lattice is searched against the database to find all lattices that are the same or related; the results are kept in set 1. Second, the unknown is searched against the database to find all materials with the same or similar element types; the results are kept in set 2. Finally, the results in sets 1 and 2 are combined to obtain the answer set. Experience has proved that the procedure is highly selective and reliable.

NIST/Sandia/ICDD Electron Diffraction Database: A Database for Phase Identification by Electron Diffraction.

A new database containing crystallographic and chemical information designed especially for application to electron diffraction search/match and related problems has been developed. The new database was derived from two well-established x-ray diffraction databases, the JCPDS Powder Diffraction File and NBS CRYSTAL DATA, and incorporates 2 years of experience with an earlier version. It contains 71,142 entries, with space group and unit cell data for 59,612 of those. Unit cell and space group information were used, where available, to calculate patterns consisting of all allowed reflections with d-spacings greater than 0.8 A for ~ 59,000 of the entries. Calculated patterns are used in the database in preference to experimental x-ray data when both are available, since experimental x-ray data sometimes omits high d-spacing data which falls at low diffraction angles. Intensity data are not given when calculated spacings are used. A search scheme using chemistry and r-spacing (reciprocal d-spacing) has been developed. Other potentially searchable data in this new database include space group, Pearson symbol, unit cell edge lengths, reduced cell edge length, and reduced cell volume. Compound and/or mineral names, formulas, and journal references are included in the output, as well as pointers to corresponding entries in NBS CRYSTAL DATA and the Powder Diffraction File where more complete information may be obtained. Atom positions are not given. Rudimentary search software has been written to implement a chemistry and r-spacing bit map search. With typical data, a full search through ~ 71,000 compounds takes 10~20 seconds on a PDP 11/23-RL02 system.

Attachment and growth of mammalian cells on microcarriers with different ion exchange capacities.

In the design of microcarriers for animal cell growth, the exchange capacity has been considered a critical factor. However, charge densities of microcarriers under culture conditions are not the same as the exchange capacities. Furthermore, the charge density requirement for optimum attachment is not necessarily the same as that required for optimum growth. We demonstrate that charge is not the sole factor affecting the attachment and growth of animal cells on microcarriers. We also show that supplemental serum in the growth medium has a negative effect on cell attachment to microcarriers.

Effect of glucose on the cultivation of mammalian cells.

Most of the glucose consumed by mammalian cells cultivated in vitro is converted to lactate. The glucose consumption rate appears to be affected by glucose concentration. In a batch cultivation of cells the glucose concentration can be manipulated at a low level by programmed feeding of glucose. In such a culture the specific consumption rate of glucose and the fraction of glucose converted to lactate can be reduced. This reduced conversion rate of glucose to lactate appears to coincide with an increased oxygen uptake rate. A possible consequence of such programmed feeding of glucose is the increased oxidation of glutamine and the concurrent increased production of ammonium. For the cultivation of hybridoma cells high concentrations of ammonium and lactate can be growth inhibitory. It is suggested that the identification of the optimum cultivation conditions is necessary if such a programmed feeding is to be used to increase cell concentration and medium utilization efficiency.

O-imino esters of N,N-bis(2-chloroethyl)phosphorodiamidic acid. Synthesis, X-ray structure determination, and anticancer evaluation.

Nine representatives of the title series of compounds [(ClCH2CH2)2NP(O)(NH2)ON = CRR'] were synthesized as potential anticancer prodrugs, based on the possibility of enzymatic reduction of the N-O bond to release the known cytotoxic agent phosphoramide mustard [1, (ClCH2CH2)2NP(O)(NH2)OH]. The dimethyl derivative (2, R = R' = CH3) exhibited a statistically significant, albeit low, level of anti-L1210 activity in mice. Derivative 2, which was shown by 31P NMR measurements to be very stable toward hydrolysis at 37 degrees C over a pH range of 5.7-7.4 (T1/2 congruent to 7-8 weeks), gave colorimetrically detectable amounts of alkylating material upon incubation with mouse liver slices: approximately 3-5% conversion after 20 min at 37 degrees C. A single-crystal X-ray study of 2 revealed an unusual hydrogen-bonded "ladder" and a very similar steric relationship for the NCH2CH2Cl and ON = CCH3 moieties.

Synthesis and antitumor activity of cyclophosphamide analogues. 3. Preparation, molecular structure determination and anticancer screening of racemic cis- and trans-4-phenylcyclophosphamide.

Cyclization of racemic 3-amino-3-phenyl-1-propranol with bis(2-chloroethyl)phosphoramidic dichloride gave a diastereomeric mixture of 4-phenylcyclophosphamide (3), which was chromatographically separated into the faster and slower eluting components. A combination of 1H/31PNMR and IR spectral data indicated that the faster and slower racemates correspond to cis-3 (mp 129-130 degrees C) and trans-3 (mp 112-114.5 degrees C), respectively. The molecular structure of the former compound was determined by X-ray crystallography and thereby unambiguously established the cis relationship between equatorially disposed phenyl and P = O substituents in a chair conformation. These results confirm the stereochemical assignments for cis- and trans-3 which have been independently deduced by Y. E. Shih, J. S. Wang, and C. T. Chen [Heterocycles, 9, 1277 (1978)]. Anticancer screening tests against L1210 lymphoid leukemia in mice have revealed that, while both diastereomers of 3 afford toxic metabolites, trans-3 led to therapeutic activity and cis-3 did not. The relevance of these findings to results reported for 4-methylcyclophosphamide and cyclophosphamide is briefly discussed.

Inhibition of tubulin assembly by ethylacetylacrylate, a sulfhydryl reagent and potential analog of cytochalasin A.

Ethyl acetylacrylate reacts rapidly with 2-mercaptoethanol and the sulfhydryl groups of tubulin. The structure of ethyl acetylacrylate resembles that of the reactive portion of cytochalasin A and potentially can serve as an analog of this antibiotic.