Human leukocyte antigen-DR matching improved skin graft survival from transgenic pigs to accommodate SCID mice reconstituted with human peripheral blood mononuclear cells

The shortage of human organs has encouraged scientists to develop genetically modified pigs for xenotransplantation, such as CD55 or CD46, and CD59 transgenesis as well as alpha-galactosyl transferase gene knockouts. In allotransplantation, the match of human leukocyte antigen class II (HLA-II) may improve graft survival although the role of HLA-II in xenotransplantation is unknown. HLA-II transgenic pigs, including DP, DQ, and DR, have been successfully generated and HLA-DR15+ transgenic pig skin pieces grafted onto severe congenital immunodeficiency (SCID) mice reconstituted intraperitoneally with HLA-DR15+ or HLA-DR15(-) human peripheral blood mononuclear cells (hPBMCs). This study sought to develop an animal model to evaluate the effects of HLA-DR matching on xenograft survival. Human CD4+ and CD8+ were detected from days 7 to 29 after hPBMC reconstitution in SCID mice. Both CD4+ and CD8+ cells of HLA-DR15(-) reconstituted SCID mice were significantly higher at day 29 postgrafting compared with HLA-DR15+ reconstituted SCID mice. An HLA-DR15+ transgenic pig dermal graft survived and integrated into SCID mice reconstituted with hPBMCs/HLA-DR15+ as proven by the histopathological finding that the collagen layer remained intact with little lymphocytic response. In contrast, the transgenic pig dermal graft showed more collagen disruption as well as mild to moderate lymphocytic infiltration when reconstituted in an hPBMC/HLA-DR15(-) SCID mouse. The results suggested that HLA-DR matching eased xenograft rejection; however, it was not yet clear that the response was mediated by T cells.     

Long-term survival of HLA-DR15+ pig skin in SCID mice after reconstitution with human peripheral blood mononuclear cells and under short-term immunosuppression

In allotransplantation, donor-recipient human leukocyte antigen (HLA) matches improve graft survival. For studies of the role of donor-recipient HLA II matching on xenotransplantation, we successfully generated HLA-DR15+ transgenic pigs the the skins of which were transplanted to SCID mice, which were thereafter reconstituted with HLA-DR15+ or -DR15(-) hPBMC. Cyclosporine was given intraperitoneally to SCID mice for 12 days. Human T cells were observed in SCID mice after reconstitution. Mixed lymphocytes responses showed greater responses by HLA-DR15(-) human peripheral blood mononuclear cells (hPBMC) against HLA-DR15+ porcine PBMC. HLA-DR15+ porcine skins survived more than 100 days in all SCID mice. HLA-DR15+ porcine skins were rejected in all non-SCID (Balb/c) mice. The histologic pictures of transplanted HLA-DR15+ porcine skins showed surviving porcine epithelium in remodeling murine dermis and little lymphocyte infiltration into the murine dermis. The long-term survival of HLA-DR15+ pig skin in all hPBMC-SCID mice might be due to poor engraftment or function of reconstituted T cells. Further studies are needed to clarify the role of donor-recipient matching of HLA-DR15.     

C4d deposition is correlated with the level of antivimentin antibody in rat kidneys undergoing chronic allograft nephropathy

Aims

Antivimentin antibody is often produced as an autoantibody after transplantation. C4d deposition, a marker of humoral immunity during transplantation, is believed to reflect alloantibodies. This study investigated the relationship between C4d deposition and humoral immunity to vimentin among rat kidneys undergoing chronic allograft nephropathy (CAN).

Methods

Fisher 344 rat renal grafts were orthotopically transplanted into Lewis rats following the procedure of Kamada with our modification. All recipients were administered cyclosporine (CsA) (10 mg/kg⁻¹ · d⁻¹ × 10 d) before being divided into 3 groups of oral treatments: (1) vehicle, (2) CsA (6 mg/kg⁻¹ · d⁻¹), and (3) mycophenolate mofetil (MMF; 20 mg/kg⁻¹ · d⁻¹). At 4, 8 and 12 weeks after transplantation, the rats were killed, the renal allografts harvested, and the sera collected. Serum creatinine (SCr) was measured and pathologic changes assessed according to the Banff 97 criteria. The antivimentin antibody was quantified by enzyme-linked immunosorbent assay. The deposition of C4d detected by immunofluorescence was analyzed by integrated optical density (IOD).

Results

Antivimentin antibody was observed in sera of all transplanted rats. The level of antivimentin antibody (IgGΔOD) increased gradually during the development of CAN from 4 weeks. Simultaneously, C4d deposition in peritubular capillaries also progressively strengthened. There was a strong positive correlation between the content of antivimentin antibody and C4d deposition (r = 0.892; P = .000). MMF simultaneously decreased antivimentin antibody formation and C4d deposition. In contrast, CsA had no significant effect.

Conclusions

We demonstrated the production of antivimentin antibodies and the deposition of C4d during the development of CAN. There was a positive correlation between them. Whether humoral immunity to vimentin contributes to C4d deposition is not clear and further studies are needed to elucidate this issue.     

The upregulation of osteoblast marker genes in mesenchymal stem cells prove the osteoinductivity of hydroxyapatite/tricalcium phosphate biomaterial

Background

Calcium phosphate (Ca-P), mainly concerning hydroxyapatite (HA), is the main inorganic component of the body's hard tissue. It is acknowledged that Ca-P biomaterial not only has osteoconduction but also can form bone bonding to host bone, making an ideal tissue-engineering scaffold. However, whether Ca-P biomaterial possesses osteoinductivity is still debated. The present study was performed to explore the expression level of osteoblast maker genes in human mesenchymal stem cells (hMSCs) grown on a Ca-P biomaterial.

Materials and methods

hMSCs were cultured on the HA/tricalcium phosphate-(TCP) double-phase ceramic. After coculture for 5, 10, 15, or 20 days, the cells were digested for isolation of total RNA. Fluorescence quantitative polymerase chain reaction was used to detect the relative mRNA levels of Runx2, collagen type I, osteopontin, and osteocalcin, all of which are the marker genes for osteoblasts. The mg63 cell was recruited as the reference and un-cocultured hMSCs as the negative controls. Alkaline phosphatase (ALP) activity in the cells was also examined after culture for 10 or 20 days.

Results

Our results showed that the expression levels of all four genes continued to rise during the first 10 days. Then, both collagen type I and Runx2 decreased. In contrast, osteocalcin mRNA reached its maximum at day 15 and osteopontin mRNA kept increasing throughout the whole experimental period. Additionally, ALP activity increased in a time-dependent manner.

Conclusion

The up-regulation of all four osteoblast marker genes in hMSCs grown on Ca-P biomaterial suggested that HA/TCP biomaterials possess osteoinductivity on hMSCs, cells a mechanism that requires further investigation.     

Diffusion magnetic resonance imaging as an evaluation of the response of brain metastases treated by stereotactic radiosurgery

BACKGROUND: Loss of tumor cell results in a relative increase in extracellular space that may lead to alteration of ADC. Our purpose was to see if the ADC could be used, rather than methods depending on changes in tumor size, to predict treatment success after treatment of brain metastases with SRS. METHODS: In a prospective study, the ADCs of 21 patients with 32 solid or solid-dominated brain metastases were taken before and 1 week, 1 month, and at 3-month intervals after SRS. Mean ADC values at the various time intervals were compared with each other to see whether or not the ADC might be used as an early indicator of treatment success or failure. RESULTS: The mean pretreatment value of the ADC in the metastatic tumors was 1.05 +/- 0.12 x 10(-3) mm2/s (mean +/- SD). This value for the tumors rose significantly (P = .009) 7 days after SRS and continued to rise with time. Magnetic resonance imaging showed that 91% of these tumors had been controlled by the SRS. The ADC values in cystic/necrotic tumor tissue (2.13 +/- 0.18 x 10(-3) mm2/s) were significantly (P < .001) higher than those in noncentral necrotic tumor tissue (1.61 +/- 0.14 x 10(-3) mm2/s). CONCLUSIONS: The ADC values might eventually be useful to evaluate treatment success-and in some patients, detected even at early time points-and to distinguish radiation-induced central necrosis from tumor regrowth in cases where other imagery is not definitive.     

Magnesium lithospermate B possesses inhibitory activity on Na^＋,K^＋-ATPase and neuroprotective effects against ischemic stroke

AIM: To examine if magnesium lithospermate B (MLB) extracted from Danshen, the dried roots of Salvia miltiorrhiza, may act as an active component responsible for the cardiac therapeutic effect of this traditional Chinese herb via the same molecular mechanism triggered by cardiac glycosides, such as ouabain and digoxin. Moreover, we wanted to test if MLB may provide neuroprotection against ischemic stroke as observed for cardiac glycosides. METHODS: Similarity in the chemical structure and molecular configuration between MLB and ouabain was analyzed. The inhibition potency of MLB and ouabain on Na( +),K( +) -ATPase activity of a commercial product, as well as in purified membrane fractions from rat brain and heart tissues, was examined and compared. Neuroprotective effect of MLB against ischemic stroke was also evaluated using a cortical brain slice-based assay model. RESULTS: Dose-dependent inhibition on the commercial Na( +),K( +)-ATPase equivalent to that for ouabain was observed for MLB of approximately half dosage by weight. This relative potency of ouabain and MLB was also observed for their inhibition on Na( +),K( +)-ATPase activity of plasma membrane purified from rat tissues, although these 2 inhibitors exhibited somewhat lower competence in these crude extracts. In ischemic gerbil brains, post-treatment with MLB significantly reduced the infarct size, visualized by 2,3,5-triphenyltetrazolium chloride staining, by approximately 55% when compared with the control group. CONCLUSION: These results evidently suggest that the cardiac therapeutic effect of Danshen should be at least partly attributed to the effective inhibition of Na( +),K( +)-ATPase by MLB, and that MLB provides anti-ischemic neuroprotection in gerbils subjected to focal ischemia and reperfusion.     

Individualized estimates of second cancer risks after contemporary radiation therapy for Hodgkin lymphoma

BACKGROUND: Estimates of radiation-related second cancer risk among Hodgkin lymphoma survivors are largely based on radiation therapy (RT) fields and doses no longer in use, and these estimates do not account for differences in normal tissue dose among individual patients. This study gives individualized estimates for the risks of lung and female breast cancer expected with contemporary involved-field RT and low-dose (20 Gy) RT for mediastinal Hodgkin lymphoma. METHODS: Three RT plans were constructed for 37 consecutive patients with mediastinal Hodgkin lymphoma: 35 Gy mantle RT, 35 Gy involved-field RT (IFRT), and 20 Gy IFRT. For each of the 111 RT plans, individual-level dosimetry data were incorporated into a cell initiation/inactivation/proliferation model to estimate the excess relative risk (ERR) and cumulative incidence of radiation-induced second cancer. RESULTS: ERR estimates were compatible with results of epidemiological studies. Compared with 35 Gy mantle radiation therapy, 35 Gy IFRT was predicted to reduce the 20-year ERRs of breast and lung cancer by 63% and 21%, respectively, primarily because of lower normal tissue doses with the omission of axillary RT. Low-dose (20 Gy) IFRT was associated with a 77% and 57% decrease in these ERRs. Patient-specific differences in normal tissue dose with IFRT led to 11-fold and 3.6-fold variations among individual's estimates of breast and lung cancer ERR, respectively. CONCLUSIONS: Contemporary IFRT is predicted to substantially reduce risk of secondary breast and lung cancer compared with mantle RT, with considerable variation in risk among individuals. Individualized prospective risk estimates could facilitate patient-specific counseling and the development of more effective RT techniques.     

Fluorine-18-labeled benzamide analogues for imaging the sigma2 receptor status of solid tumors with positron emission tomography

A series of fluorine-containing benzamide analogs was synthesized and evaluated as candidate ligands for positron emission tomography (PET) imaging of the sigma-2 (sigma2) receptor status of solid tumors. Four compounds having a moderate to high affinity for sigma2 receptors and a moderate to low affinity for sigma-1 (sigma1) receptors were radiolabeled with fluorine-18 via displacement of the corresponding mesylate precursor with [18F]fluoride. Biodistribution studies in female Balb/c mice bearing EMT-6 tumor allografts demonstrated that all four F-18-labeled compounds had a high tumor uptake (2.5-3.7% ID/g) and acceptable tumor/normal tissue ratios at 1 and 2 h post-i.v. injection. An analysis of the chemistry and biodistribution data suggested that N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-[18F]-fluoroethoxy)-5-methylbenzamide ([18F]3c) and N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-[18F]-fluoroethoxy)-5-iodo-3-methoxybenzamide ([18F]3f) are acceptable compounds for imaging the sigma2 receptor status of solid tumors.     

Upregulation of connective tissue growth factor in a rat model of chronic allograft nephropathy

AIM: To study the expression of connective tissue growth factor (CTGF) in transplanted rat kidney and its relationship with chronic allograft nephropathy (CAN). METHODS: Kidney transplantation was performed from Lewis to Fisher 344 allogeneic rat strain, and kidney grafts were harvested at the eighth, 12th and 16th week. The morphological changes were studied, and collagen deposition was determined by Masson trichrome stain. Serum creatinine was examined. The fibrotic process and the CAN grades were evaluated according to Banff 97 schema. The expressions of transforming growth factor beta, CTGF and alpha-smooth muscle actin were detected to assess the development of grafted kidney fibrosis and to discuss their relationships. Spearman correlation was used for correlation study between CTGF expression and development of CAN. RESULTS: Serum creatinine was promoted in a time-dependent manner. Morphological changes suggested that the grafted kidneys were under abnormalities. At the end stage, focal segmental glomerulosclerosis was seen; tubular epithelial cells lost their phenotype and interstitial fibrosis was notable. Masson trichrome stain showed significant collagen accumulation in a time-dependent manner. Immunohistochemistry and western blotting results showed that the transforming growth factor beta, CTGF and alpha-smooth muscle actin expression were markedly promoted compared with the control group. CTGF was mainly expressed in the plasm of proximal tubular epithelial cells based on the severity of CAN. CONCLUSION: Connective tissue growth factor might play an important role in the pathological changes of CAN after kidney transplantation. The expression of CTGF in epithelial cells could act as a molecular marker of interstitial fibrosis and CAN.     

Validation of the simplified Chinese version of the FACT-B for measuring quality of life for patients with breast cancer

The simplified Chinese version of the FACT-B was evaluated by QOL data measured from 376 patients with breast cancer. The results showed that test–retest reliability for five domains: physical well-being, social/family well-being, emotional well-being, functional well-being, additional concerns, and the overall scales and trial outcome index were 0.82, 0.85, 0.91, 0.86, 0.90, 0.84 and 0.91, 0.89 respectively. The internal consistency α for the five domains mentioned above were 0.85, 0.82, 0.84, 0.84 and 0.59 respectively. Correlation analysis, factor analysis and structural equation model showed good construct validity. Criterion-related validity was also confirmed using QLICP-BR as a criterion. The instrument can find the change of QOL for cancer patients after treatment. It’s concluded that the simplified Chinese version of FACT-B can be used to measure QOL for Chinese patients with breast cancer with good validity, reliability and responsiveness. This research is sponsored by the Natural Sciences Funds of Yunnan Province (99C0016G).