Vascularized bone marrow-based immunosuppression inhibits rejection of vascularized composite allografts in nonhuman primates.

Vascularized composite allograft (VCA) transplantation (also referred to as composite tissue allotransplantation) has demonstrated clinical success in cases of hand, arm and face transplantation despite prior belief that skin provides an insurmountable barrier to allograft rejection. These overall good outcomes are facilitated by substantial immunosuppressive requirements in otherwise healthy patients, yet still demonstrate frequent rejection episodes. We developed a nonhuman primate model of facial segment allotransplantation to elucidate the unique pathophysiology and immunosuppressive requirements of VCA with addition of concomitant vascularized bone marrow (VBM). Heterotopically transplanted facial segment VCA with VBM treated only with tacrolimus and mycophenolate mofetil (MMF) demonstrated prolonged rejection-free survival, compared to VCA without VBM that demonstrated early rejection episodes and graft loss. While VCA with VBM demonstrated sporadic macrochimerism, acute and chronic rejection and graft loss occurred after discontinuation of immunosuppression. These data support an immunomodulatory role of VBM in VCA that reduces immunosuppressive requirements while providing improved outcomes.

The impact of c4d pattern and donor-specific antibody on graft survival in recipients requiring indication renal allograft biopsy.

We examined the pattern of PTC C4d by immunohistochemistry and DSA in 297 kidney recipients with indication biopsies, and evaluated their predictive value for graft survival. Median biopsy time was 5.1 months posttransplant. Patients were followed for 17.9 +/- 9.4 months postbiopsy. An 18.5% had focal and 15.2% had diffuse C4d, with comparable graft survival (adjusted graft failure HR: 2.3, p = 0.001; HR:1.9, p < 0.02, respectively). 31.3% were DSA+, 19.5% class I and 22.9% class II DSA. Only those with class II DSA had worse outcome (adjusted HR:2.5, p = 0.001 for class II only; HR:2.7, p < 0.001 for class I/II DSA). Among patients with <10%C4d, 23.9% had DSA, compared to 68.9% with diffuse staining. For patients biopsied in first-year posttransplant presence of DSA, regardless of C4d positivity in biopsy, was a poor prognostic factor (adjusted graft failure HR: 4.2, p < 0.02 for C4d-/DSA+; HR:4.9, p = 0.001 for C4d+/DSA+), unlike those biopsied later. We have shown that focal C4d had similar impact on graft survival as diffuse pattern. During the first-year posttransplant either class I or II DSA, and afterward only class II DSA were associated with worse graft survival. DSA was predictive of worse outcome regardless of C4d for patients biopsied in first year and only with C4d positivity afterward, supporting the importance of assessment of both DSA and C4d pattern in biopsy.

Positive cross-match living donor kidney transplantation: longer-term outcomes.

The long-term graft outcomes after positive cross-match (PXM) living donor kidney transplantation (LDKT) are unknown and the descriptive published data present short-medium term results. We conducted a retrospective cohort study of LDKT with PXM by flow cytometry performed at our center during February 1999 to October 2006, compared to a control group, matched 1:1 for age, sex, race, retransplantation and transplant year. The PXM group was treated with a course of plasmapheresis/low-dose intravenous immunoglobulin (IVIg) preoperatively, and OKT3 or thymoglobulin induction. Both groups (n = 41 each) were comparable except for duration of end-stage renal disease (ESRD), induction, HLA mismatch and panel-reactive antibody (PRA). During the period of up to 9 years, 14 PXM and 7 controls lost their grafts (p < 0.04). Graft survival rates at 1 and 5 years were 89.9% and 69.4% for PXM group and 97.6% and 80.6% for the controls, respectively. PXM was associated with higher risk of graft loss (HR 2.6, p = 0.04; 95%CI 1.03-6.4) (t(1/2)= 6.8 years), but not with patient survival (HR 1.96, p = 0.29; 95%CI 0.6-7.0) or 1-year serum creatinine (beta= 0.06, p = 0.59 for ln (SCr); 95% CI -0.16 to 0.28). These results suggest that despite the favorable short-term results of PXM LDKT after PP/IVIg conditioning, medium-long-term outcomes are notably worse than expected, perhaps comparable to non-ECD deceased donor kidney transplantation (KT).

Characterization of multidrug resistance by fluorescent dyes.

Fluorimetric techniques were used to examine accumulation of fluorescent probes by the P388 murine leukemia and an anthracycline-resistant subline, P388/Adriamycin(ADR), which expresses the multidrug-resistant phenotype. P388 could be differentiated from P388/ADR on the basis of fluorescence intensity measurements using 3 classes of cationic dyes that are sensitive to membrane potential differences: rhodamine esters, cyanines, and styrylpyridinium dyes. But fluorescence intensity differences were also observed with potential-insensitive dyes: zwitterionic rhodamines and an acridine orange derivative. In all cases, fluorescence intensity differences were caused by impaired dye accumulation, and could be eliminated by treatment of P388/ADR cells with verapamil. Moreover, fluorescence signals from 2 anionic potential-sensitive dyes, merocyanine 540 and a bis-oxonol, were identical in P388 and P388/ADR. None of these dyes could be used to delineate CCRF-CEM, a lymphoblastic leukemia of human origin from the CEM/VM-1 subline that exhibits a markedly atypical drug resistance pattern not based on an enhanced outward transport. But accumulation of both neutral and cationic dyes was impaired in CEM/VLB100, a subline of CCRF-CEM expressing mdr. These studies show that many cationic and neutral fluorescent probes are substrates for the enhanced outward drug transport system associated with P388/ADR cells, and cannot be used to probe membrane-potential differences in cells expressing the mdr phenotype. With several dyes, differences in fluorescence intensity were sufficient so that flow cytometry could be used to delineate P388 from P388/ADR and CCRF-CEM from CEM-VLB100. The latter technique may be useful for identifying malignant cell populations expressing multidrug resistance in patients with neoplastic disease.

Mixed hematopoietic chimerism at day 90 following allogenic myeloablative stem cell transplantation is a predictor of relapse and survival.

We retrospectively analyzed the prognostic significance of mixed chimerism and associated clinical parameters in 80 patients following unmanipulated allogenic stem cell transplantation. Chimerism studies were performed on marrow aspirates using fluorescent in situ hybridization and variable number tandem repeats techniques at day +30, day +90 and +12 months. The median overall survival (OS) was 24 months (range, 1-56 months). Mixed chimerism was found in 23, 28 and 14% of patients at day +30 (1 month), +90 (3 months), and +12 months, respectively. Day +30 chimerism studies failed to provide any prognostic information. Day +90 mixed chimeras (MC) had significantly higher relapse rates compared to day +90 complete chimeras (CC) at 6 months (P=0.03) and 18 months when compared to MC (P=0.03) following transplant. The median OS in day +90 MC and day+90 CC were, respectively (95% CI, 2-35 months), compared to 47 months (95% CI, 20-74 months) (P=0.02). In conclusion, chimerism studies on day +30 could be reserved for patients who fail to demonstrate engraftment. Day +90 MC had higher relapse rates and lower OS, and therefore may be considered for novel therapies and future studies.

A flow cytometric method for measuring lymphocyte proliferation directly from tissue culture plates using Ki-67 and propidium iodide.

A new method for measuring lymphocyte proliferation in response to mitogens and allogeneic cells without using radiolabelling is described. It utilizes flow cytometry and the monoclonal antibody, Ki-67, which detects a nuclear proliferation antigen. The entire test is performed in standard, 96-well tissue culture plates. Stable, clean nuclear suspensions rather than whole cells were used to avoid non-specific staining. The nuclei were stained by the indirect fluorescent method. Simultaneous measurements of DNA content were possible by dual staining with propidium iodide (PI). The percentage of Ki-67-positive nuclei correlated well with [3H]thymidine uptake and morphologic quantitation of blasts. This method avoids use of radioactive material and is less time consuming.

A special report: histocompatibility testing guidelines for hematopoietic stem cell transplantation using volunteer donors. Quality Assurance and Donor Registries Working Groups of the World Marrow Donor Association.

The World Marrow Donor Association has formulated guidelines for establishing the extent and quality of histocompatibility testing for unrelated donor registries, umbilical cord blood banks, and transplant centers involved in international exchange of hematopoietic stem cells for allogeneic transplantation. Registry and cord blood bank guidelines suggest that, at a minimum, initial HLA typing should be performed for three HLA loci, HLA-A, -B, and -DR, at low resolution/split antigen level. DNA-based testing methods should be utilized for HLA-DR typing. DNA-based testing for HLA-A and -B should replace serologic testing of new volunteer donors and cord blood units as robust protocols and reagents become available to the laboratories. Transplant center guidelines for typing of patient, family and to confirm the HLA types of potential unrelated donors should include, at the minimum, typing HLA-A, B, and -DR loci using primarily DNA-based testing methods at allele level resolution for DRB1 and low resolution/split antigen level for HLA-A and -B. It is strongly recommended that the typing of a patient and the selected donor be performed using the same set of reagents, methodology, and interpretation criteria with fresh tissue samples to ensure HLA identity. Guidelines for laboratory accreditation, approaches to quality assurance and quality control for HLA testing, and suggestions for the format of the HLA database of donor types are also outlined.

Mutation analysis of the cystic fibrosis and cationic trypsinogen genes in patients with alcohol-related pancreatitis.

Cationic trypsinogen and cystic fibrosis mutations have been identified in pancreatitis patients, although no study has looked for mutations in both genes in the same patient. Pancreatitis can be induced by alcohol, although not all alcoholics develop pancreatitis. We hypothesize that this phenomenon is due to a genetic predisposition in persons with alcohol-related pancreatitis. We performed sequence analysis of the cationic trypsinogen-coding region in 46 alcohol-related pancreatitis patients and 16 patients with pancreatitis due to causes other than alcohol. We also screened for 40 cystic fibrosis mutations including the 5T allele. No cationic trypsinogen mutations were identified. Cystic fibrosis mutation screening identified the DeltaF508 mutation in two Caucasian alcoholic patients (P<0.025). The cystic fibrosis mutation carrier frequency in African-American alcoholic patients was 3%, which was not significantly increased compared with the normal carrier frequency. The frequency of the 5T allele was not significantly increased compared with the normal population carrier frequency in either racial group. These results may suggest a role for the cystic fibrosis gene in alcohol-related pancreatitis but indicate that cationic trypsinogen mutations are not a common predisposing risk factor for alcohol-related pancreatitis. A multicenter study is necessary to attain sufficient numbers to come to a conclusion.

Histocompatibility testing guidelines for hematopoietic stem cell transplantation using volunteer donors: report from The World Marrow Donor Association. Quality Assurance and Donor Registries Working Groups of the World Marrow Donor Association.

The World Marrow Donor Association has formulated guidelines for establishing the extent and quality of histocompatibility testing for unrelated donor registries, umbilical cord blood banks, and transplant centers involved in international exchange of hematopoietic stem cells for allogeneic transplantation. The ability to identify unrelated stem cell donors in one country for patients in another country requires cooperation and standardization in many areas. The adoption of guidelines for histocompatibility testing, such as those summarized in this report, will facilitate these opportunities and rapidly provide accurately typed donors for patients in need.

Hematopoietic stem cell donor registry strategies for assigning search determinants and matching relationships.

Registries and cord blood banks around the world collect and store the HLA types of volunteers in order to identify matched unrelated donors for patients requiring hematopoietic stem cell transplantation. This task is complicated by the many formats in which HLA types are provided by the testing laboratories (types obtained by serology vs by DNA-based methods; high vs intermediate vs low resolution) and by the need to identify which of these diverse types are most likely to match the HLA assignments of a searching patient as closely as possible. Conversion of the assignments to 'search determinants' may be included within the algorithm used to select and prioritize a list of potentially suitable donors, either as an aid to matching or as a tool to optimize the performance of comparisons within large data files. The strategies used by registries to create search determinants are described. A set of search determinants, utilized by the National Marrow Donor Program, is provided as an example and is intended to initiate further discussion aimed at understanding the process used by each registry with the possibility of developing a standard process among registries worldwide.

A method for measuring lymphocyte proliferation in mixed lymphocyte cultures using a nuclear proliferation antigen, Ki-67, and flow cytometry.

The mixed lymphocyte culture procedure using tritiated thymidine (3H-TdR) incorporation is time consuming and labor intensive, therefore costly. With the use of a fluorescent antibody to a human nuclear proliferation antigen, Ki-67, and flow cytometry, mixed lymphocyte cultures on 20 families of renal and bone marrow transplant patients and normal controls were performed. In this method for measuring lymphocytic proliferation, previously developed by the authors, the entire culture and staining procedures are performed in microculture plates. Finally, the cell suspensions are aspirated with a microsampler to be analyzed by a flow cytometer. Excellent correlation of the percentage of Ki-67-positive cells and the counts per minute (CPM) of 3H-TdR incorporated into the DNA was obtained. This method eliminates the use of radioactive labels, is less time consuming, and yields results two to three days earlier than the radioactive method. In addition, the authors dual-labeled the lymphocyte nuclei with Ki-67 and propidium iodide (Ki-67/PI). This permitted the comparison of the appearance of nuclear antigen with the various phases of the cell cycle.

Characterization of a novel DRB1*14 allele (DRB1*1449) in the Han-Chinese population.

We report here a novel human leukocyte antigen (HLA) allele, DRB1*1449, in the Han-Chinese population. The nature of the new allele was confirmed by the sequencing-based typing (SBT) method. Genomic DNA and six subclones containing DNA fragment of DRB1 exon 2 were sequenced in both forward and reverse directions. The exon 2 nucleotide sequence of DRB1*1449 is closely related to DRB1*1432 allele based on sequence homology. It has four nucleotide (nt) substitutions at positions 71, 196, 244 and 245 in exon 2, which lead to changes of amino acid sequences. The serological assignment of DRB1*1449 is DR14 based on the serological HLA typing result. This novel allele might be a result of recombination between DRB1*1402 and DRB1*140101 like alleles, which are very common among Chinese population.

Identification of an HLA-B*07 allele variant (B*0740) in the Chinese Han population.

A novel HLA-B*07 allele, B*0740, has been identified by sequence-based typing (SBT) in the Chinese Han population. This new allele is identical to B*0705 and B*0706 for exons 2, 3, and 4, except for a single nucleotide at position 605 of codon 202 in exon 3 (AAG-->ATG) leading to an amino acid change from lysine to methionine. SBT was performed following allele separation using the Haploprep method. The serological equivalence of B*0740 to the B7 antigen did not change.

A novel HLA allele, DRB1*1609, identified in the Chinese Han population*.

A novel human leucocyte antigen-DRB1*16 (HLA-DRB1*16) allele (DRB1*1609) has been identified by sequencing-based typing (SBT) in Chinese Han population. This new allele has identical nucleotide sequence to DRB1*160101 in exon 2, except for a single-nucleotide substitution from A to T at position 127. This change leads to an amino acid change from tyrosine to phenylalanin at residue 47 (Y47F). SBT was performed for cloned DRB1*16-specific polymerase chain reaction fragment. The serological phenotype of DRB1*1609 is equivalent to DR16 antigen.

Purification, ultrastructure, and chemical analysis of Alzheimer disease amyloid plaque core protein.

Isolation of Alzheimer disease amyloid plaque core protein (APCP) was carried out by repetitive NaDodSO4/EDTA/sucrose extractions and by Ficoll-400 density-gradient centrifugations. The enriched APCP-Ficoll interface was labeled with the fluorochrome thioflavin T and separated from the contaminating lipofuscin by fluorescence-activated cell sorting. Electron microscopy demonstrated that APCP is made of two different kinds of filaments measuring 5.5-6 nm and 10-12 nm, respectively, and of variable length. Purified APCP and lipofuscin were chemically modified by performic acid oxidation. The amino acid composition of APCP revealed a high content of glycine and valine (30%) and 1% cysteine. By contrast, the protein moiety of the copurified lipofuscin contained 16% cysteine. The amino acid composition of APCP did not resemble that of any known protein.

Flow cytometric purification of Alzheimer's disease amyloid plaque core protein using thioflavin T.

Amyloid plaque core protein (APCP) of Alzheimer's disease obtained from brain tissue homogenate is difficult to recover in pure form, primarily because of contaminating lipofuscin (LF) granules. Thioflavin T, a fluorescent dye previously used to stain amyloid, was found to bind to APCP but not to lipofuscin. The latter, however, is autofluorescent. Fluorometric studies showed that at 370 nm excitation APCP has a maximal emission at 418 nm, whereas the autofluorescent LP has a maximal emission at 450 nm. This difference in emission permitted the use of a flow cytometer-sorter (FACS 440) for purification of APCP. APCP particles fluoresced distinctly from LF granules on the log blue fluorescence parameter. The two entities were sorted using forward light scatter versus fluorescence. A collection apparatus was designed and prepared to facilitate the collection of large volumes of sheath fluid and particles and to minimize fragmentation of particles during the collection process. The sorted APCP fraction was 98% pure. This work demonstrates how old dyes can be used to perform new tricks and provide a useful method for separating complex protein.

Association between HLA-DQB1 alleles and risk for cervical cancer in African-American women.

Squamous-cell carcinoma of the cervix and its precursor lesions are associated with human papillomavirus (HPV) infection. Epidemiological studies indicate that HPV infection in itself is not sufficient for cervical-cancer induction, suggesting that other factors contribute to carcinogenesis. We have investigated the potential role of host genetic background as one such factor. We screened a series of squamous-cell carcinomas of the cervix for HLA-class-II DQB1* alleles by the polymerase chain reaction and site-specific oligonucleotide probe hybridization and for HPV type from African-American women using a local, ethnically matched control panel. Statistically significant associations for increase in relative risk for cervical cancer were seen for DQB1*0303 and DQB1*0604. DQB1*0201 and the heterozygote DQB1*0301/*0501 showed a decrease in relative risk for cervical cancer. HPV typing revealed no association between virus type and DQB1 alleles. Our results confirm other studies showing an increase in relative risk for cervical cancer associated with HLA-DQ3 alleles in Caucasians.

B lymphocytic lymphoma (large cell) of possible splenic marginal zone origin presenting with prominent splenomegaly and unusual cordal red pulp distribution.

Two cases of large cell lymphoma, B-cell type, primarily involving the red pulp of the spleen rather than the white pulp are described. A number of unusual features suggest that this may be a lymphoma originating from a distinct splenic B-cell lymphocyte whose origin may be the marginal zone of the spleen or the splenic cords. The patients presented with splenomegaly, cytopenias, and no peripheral lymphadenopathy. The gross appearance of the spleens was beefy red without tumor nodules. The tumor cells were primarily in the splenic cords and surrounding residual normal white pulp. There was a minimal hemic phase. The tumor cells had abundant cytoplasm, surface IgM, IgD, kappa, and FC receptors, tartrate-resistant acid phosphatase, but no alkaline phosphatase or interleukin-2 receptors. They had a similar DNA aneuploidy. The most unusual feature was that tumor cells in both cases had phagocytic properties. These lymphomas may be clinically more indolent than their follicular center counterparts.

Large-scale oligonucleotide typing for HLA-DRB1/3/4 and HLA-DQB1 is highly accurate, specific, and reliable.

DNA typing of HLA class II alleles of the DRB1/3/4 and DQB1 loci using sequence-specific oligonucleotide probes and polymerase chain reaction-amplified DNA was used for the large-scale typing of donors for the National Marrow Donor Program unrelated donor registry. The results of quality control analysis for the first 7 months of the project show the typing to be highly accurate, specific, and reliable. The percent of correctly classified HLA oligotypes based on 1652 DRB1 and 1652 DQB1 assignments was greater than 99% for DRB1/DRB3/DRB4 and greater than 98% for DQB1. This level of accuracy is particularly remarkable because the quality control samples could not be distinguished from 9011 donor samples tested at the same time by the laboratories.

A special report: histocompatibility testing guidelines for hematopoietic stem cell transplantation using volunteer donors.

The World Marrow Donor Association has formulated guidelines for establishing the extent and quality of histocompatibility testing for unrelated donor registries, umbilical cord blood banks, and transplant centers involved in international exchange of hematopoietic stem cells for allogeneic transplantation. Registry and cord blood bank guidelines suggest that, at a minimum, initial HLA typing should be performed for three HLA loci, HLA-A, -B, and -DR, at low resolution/split antigen level. DNA-based testing methods should be utilized for HLA-DR typing. DNA-based testing for HLA-A and -B should replace serologic testing of new volunteer donors and cord blood units as robust protocols and reagents become available to the laboratories. Transplant center guidelines for typing of patient, family and to confirm the HLA types of potential unrelated donors should include, at the minimum, typing HLA-A, B, and -DR loci using primarily DNA-based testing methods at allele level resolution for DRB1 and low resolution/ split antigen level for HLA-A and -B. It is strongly recommended that the typing of a patient and the selected donor be performed using the same set of reagents, methodology, and interpretation criteria with fresh tissue samples to ensure HLA identity. Guidelines for laboratory accreditation, approaches to quality assurance and quality control for HLA testing, and suggestions for the format of the HLA database of donor types are also outlined.