Loss of tolerance to a maternal kidney transplant is selective for HLA class II: evidence from trans-vivo DTH and alloantibody analysis

We studied late graft rejection in a patient who had received a kidney transplant 9–10 years earlier from his mother and who had been off all immunosuppressive drugs for 7 years at the time of graft rejection onset. The mother differed for one HLA-A (A3) and one HLA-B (B62) antigen but had only a subtype mismatch at the HLA-DRβ1 locus (donor: DRβ1*1104; recipient: DRβ1*1102). A gradual rise in serum creatinine from 1.8 to 2.0 mg/dl at year 9 prompted a biopsy, which was negative for rejection (focal infiltrates but no tubulitis). Ten months later the patient’s creatinine had risen to > 3.4 mg/dl, and a second biopsy revealed extensive tubulitis, cellular rejection, and glomerular sclerosis. Sonicates of donor leukocytes triggered no delayed-type hypersensitivity (DTH) response above background (PBMC only) in the patient’s peripheral blood leukocytes obtained prior to year 9. A gradual recovery of antidonor DTH response between year 9 and 10 closely paralleled the change from tolerant to rejection status. Antidonor antibody was also undetectable in serum prior to year 9, but a donor-reactive antibody did develop at year 10.2 shortly after the peak of DTH response. The serum level of soluble donor HLA class I B62 antigen rose > 10-fold over prerejection level at the time of the biopsy-proven rejection, suggesting a possible trigger for both the cellular and humoral immune response. Nonetheless, we found no evidence for the de-velopment of humoral or cellular immunity to maternal HLA class I. Instead, DTH analysis of memory T cells of the patient obtained after rejection showed that a single maternal HLA DRβ1*1104 allopeptide, differing by two amino acids in sequence from the peptide of the recipient (DRβ1*1102), stimulated a strong memory DTH response. Similarly, we found an anti-HLA class II donor-specific antibody in serum that appeared to be crossreactive with DRβ1*1104 and DRβ1*1101 but not with the recipient DRβ1*1102 antigen. The data support the idea of a profound unresponsive state at both the cellular (DTH) and humoral level toward maternal HLA class I antigens that was not reversed even during late cellular rejection, despite the release of high levels of soluble HLA class I. Furthermore, the data suggest that DTH recovery was a close correlate of the onset of rejection and this “indirect” alloresponse, like the anti-donor alloantibody response that followed, was directed not to noninherited maternal HLA-A,B antigens but to the maternal HLA DRβ1*1104 subtype.     

Microdissection genotyping of archival fixative treated tissue for Gaucher disease

The genetic diagnosis of Gaucher disease by molecular methods is complicated by the existence of a highly homologous transcribed pseudogene (96% identity) that is found in close proximity to the true gene on chromosome 1q21. In addition, the pseudogene sequence can mimic disease-causing mutations in the true gene. Selective polymerase chain reaction (PCR) amplification of the true gene can be accomplished in extracted DNA from fresh-frozen samples by designing oligonucleotide primers to hybridize to defined regions that are not present in the pseudogene. This standard molecular approach, which entails amplification of relatively long segments of intact DNA, is not feasible in archival, paraffin-embedded, solid-tissue specimens in which the negative effects of chemical fixation result in DNA strand scission and breakdown of nucleic acid. A novel approach, specifically created for use with archival, fixative-treated tissue specimens, was developed for detection and characterization of common mutations of Gaucher disease. Three separate robust PCR reactions were formulated, 2 for selective amplification of portions of only the true gene exons 2 and 9, with a third reaction targeting exon 10, wherein both the true and pseudogene were coamplified. In the latter, DNA sequencing was used to determine the presence of true and pseudogene allele content in addition to identification of base sequence alterations. This method, requiring a single, 4-microm-thick histologic section, was successfully applied to archival paraffin block tissue specimens that had been in storage for up to 75 years. It was capable of accurately genotyping common Gaucher disease mutations as well as discovering a novel mutation and genetic polymorphism. We recommend our approach when only fixative-treated tis sue is available for molecular genotyping.     

Upregulation of VEGF and FGF2 in normal rat brain after experimental intraoperative radiation therapy

The expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)2 in the irradiated brain was examined to test how a single high dose radiation, similar to that used for intraoperative radiation therapy given to the normal cerebrum, can affect the vascular endothelium. After a burr hole trephination in the rat skull, the cerebral hemisphere was exposed to a single 10 Gy dose of gamma rays, and the radiation effect was assessed at 1, 2, 4, 6, and 8 weeks after irradiation. Histological changes, such as reactive gliosis, inflammation, vascular proliferation and necrosis, were correlated with the duration after irradiation. Significant VEGF and FGF2 expression in the 2- and 8-week were detected by enzyme-linked immunosorbent assay quantification in the radiation group. Immunohistochemical study for VEGF was done and the number of positive cells gradually increased over time, compared with the sham operation group. In conclusion, the radiation injuries consisted of radiation necrosis associated with the expression of VEGF and FGF2. These findings indicate that VEGF and FGF2 may play a role in the radiation injuries after intraoperative single high-dose irradiation.     

Expression of cell cycle regulators during smooth muscle cell proliferation after balloon catheter injury of rat artery

Intimal hyperplasia is defined as the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) with deposition of extracellular matrix. However, the cell cycle regulatory mechanisms of injury-induced VSMC proliferation are largely unknown. To examine the expression kinetics of cell cycle regulatory factors which is known to be worked positively or negatively, we used rat balloon injury model. Marked induction of proliferating cell nuclear antigen (PCNA), G1/S cyclin-dependent kinase (cdk2), and its regulatory subunit (cyclin E) occurred between 1 and 3 days after balloon arterial injury, and this was sustained for up to 7 days and then declined. However, the induction of the negative regulators, p21 and p27, occurred between 3 and 5 days of injury, peaked after 7 and 14 days and was then sustained. VSMC proliferation after balloon catheter injury of the rat iliac artery is associated with coordinated expression of positive (cdk2, cyclin E and PCNA) and negative (p21, p27) regulators. Cell cycle regulators such as cdk2, cyclin E, p21, p27 may be suitable targets for the control of intimal hyperplasia.     

APC I1307K and the E1317Q variants are not present in Chinese colorectal cancer patients.

PURPOSE: The APC I1307K and E1317Q variants predispose to colorectal adenomas and carcinomas in Caucasians, but data are lacking in Asians. METHODS AND RESULTS: We sequenced the APC gene from codons 1261 to 1409 and found none of 147 Chinese, 20 Malay, and 11 Indian colorectal cancer patients in Singapore to carry the APC I1307K or E1317Q variants. CONCLUSION: These variants are rare in these Asian populations, and play little role in colorectal cancer causation in Chinese.     

Accurate Monte Carlo simulations for nozzle design, commissioning and quality assurance for a proton radiation therapy facility

Monte Carlo dosimetry calculations are essential methods in radiation therapy. To take full advantage of this tool, the beam delivery system has to be simulated in detail and the initial beam parameters have to be known accurately. The modeling of the beam delivery system itself opens various areas where Monte Carlo calculations prove extremely helpful, such as for design and commissioning of a therapy facility as well as for quality assurance verification. The gantry treatment nozzles at the Northeast Proton Therapy Center (NPTC) at Massachusetts General Hospital (MGH) were modeled in detail using the GEANT4.5.2 Monte Carlo code. For this purpose, various novel solutions for simulating irregular shaped objects in the beam path, like contoured scatterers, patient apertures or patient compensators, were found. The four-dimensional, in time and space, simulation of moving parts, such as the modulator wheel, was implemented. Further, the appropriate physics models and cross sections for proton therapy applications were defined. We present comparisons between measured data and simulations. These show that by modeling the treatment nozzle with millimeter accuracy, it is possible to reproduce measured dose distributions with an accuracy in range and modulation width, in the case of a spread-out Bragg peak (SOBP), of better than 1 mm. The excellent agreement demonstrates that the simulations can even be used to generate beam data for commissioning treatment planning systems. The Monte Carlo nozzle model was used to study mechanical optimization in terms of scattered radiation and secondary radiation in the design of the nozzles. We present simulations on the neutron background. Further, the Monte Carlo calculations supported commissioning efforts in understanding the sensitivity of beam characteristics and how these influence the dose delivered. We present the sensitivity of dose distributions in water with respect to various beam parameters and geometrical misalignments. This allows the definition of tolerances for quality assurance and the design of quality assurance procedures.     

Modifications in the organization and expression of collagen genes associated with skeletal disorders.

Fibril-forming collagens represent an evolutionary related group of structurally similar molecules within the larger family of collagen proteins. Characterization of naturally occurring mutations has provided a model whereby clinically distinct phenotypes are predicted on the basis of how specific mutations alter normal fibrillogenesis. This model, originally derived from studies of type I collagen defects in osteogenesis imperfecta and Ehlers Danlos syndrome type VII, has been modified and extended by recent correlations of type II collagen defects with several chondrodysplasias and of type III collagen defects with Ehlers Danlos syndrome type IV. From analysis of the skeletal dysplasias, the pathogenic role of fibrillar collagen defects in more common clinical entities has been suggested and awaits rigorous proof. Although informative, these collective studies have revealed important exceptions to predictions of the original pathobiochemical paradigm, and, thus, they have initiated a more rigorous reconsideration of the deductive model. As an alternative, investigations are currently underway to generate transgenic mouse models of human collagenopathies. This task will not only clarify the complexity of collagen pathophysiology, but it will also permit the development of therapeutic strategies.     

Blood and Marrow Transplant Clinical Trials Network State of the Science Symposium 2007.

Outcomes of hematopoietic cell transplantation are steadily improving. New techniques have reduced transplant toxicities, and there are new sources of hematopoietic stem cells from unrelated donors. In June 2007 the Blood and Marrow Transplant Clinical Trials Network convened a State of the Science Symposium of more than 200 participants in Ann Arbor to identify the most compelling clinical research opportunities in the field. This report summarizes the symposium's discussions and identifies eleven high priority clinical trials that the network plans to pursue over the course of the next several years.     

Co-culture of Trypanosoma musculi with spleen-derived adherent fibroblasts: possible transfer of small molecules via connexons

Trypanosoma musculi, a protozoan parasite specific to mouse, was cultured in vitro in the presence of spleen-derived adherent cells. T. musculi co-cultured with adherent cells survived and proliferated indefinitely as long as cellular contact was retained. Scanning and transmission electron microscopy confirmed intimate membrane-to-membrane contact between the adherent cells and parasites. Cellular contact, therefore, seemed to be essential for trypanosomal survival and growth. Immunocytochemical studies demonstrated intense fibroblast growth factor (FGF) activity in adherent cells, and FGFR-2 in associated trypanosomes. BioPorter Lucifer yellow protein delivery reagent studies demonstrated that Lucifer yellow transfected into fibroblast was incorporated into associated trypanosomes. The results suggest the existence of viable channels reminiscent of gap junctions between associated cells. Such transfer of low molecular weight molecules might represent antiapoptotic metabolic factors that support survival of adherent trypanosomes in vitro. Immunocytochemical studies also detected connexin-32 and connexin-43 in the cytoplasm of fibroblasts and associated trypanosomes, however, restriction of connexons to trypanosome/fibroblast adherent sites was not observed. Western blots confirmed the presence of connexin protein molecules in trypanosomes.