Increased apoptosis of immunoreactive host cells and augmented donor leukocyte chimerism, not sustained inhibition of B7 molecule expression are associated with prolonged cardiac allograft survival in mice preconditioned with immature donor dendritic cells plus anti-CD40L mAb.

BACKGROUND: We previously reported the association among donor leukocyte chimerism, apoptosis of presumedly IL-2-deficient graft-infiltrating host cells, and the spontaneous donor-specific tolerance induced by liver but not heart allografts in mice. Survival of the rejection-prone heart allografts in the same strain combination is modestly prolonged by the pretransplant infusion of immature, costimulatory molecule-(CM) deficient donor dendritic cells (DC), an effect that is markedly potentiated by concomitant CM blockade with anti-CD40L (CD154) monoclonal antibody (mAb). We investigated whether the long survival of the heart allografts in the pretreated mice was associated with donor leukocyte chimerism and apoptosis of graft-infiltrating cells, if these end points were similar to those in the spontaneously tolerant liver transplant model, and whether the pretreatment effect was dependent on sustained inhibition of CM expression of the infused immature donor DC. In addition, apoptosis was assessed in the host spleen and lymph nodes, a critical determination not reported in previous studies of either spontaneous or "treatment-aided" organ tolerance models. METHODS: Seven days before transplantation of hearts from B10 (H-2b) donors, 2x10(6) donor-derived immature DC were infused i.v. into C3H (H-2k) recipient mice with or without a concomitant i.p. injection of anti-CD40L mAb. Donor cells were detected posttransplantation by immunohistochemical staining for major histocompatibility complex class II (I-Ab) in the cells of recipient lymphoid tissue. CM expression was determined by two-color labeling. Host responses to donor alloantigen were quantified by mixed leukocyte reaction, and cytotoxic T lymphocyte (CTL) assays. Apoptotic death in graft-infiltrating cells and in areas of T-dependent lymphoid tissue was visualized by terminal deoxynucleotidyltransferase-catalyzed dUTP-digoxigenin nick-end labeling and quantitative spectrofluorometry. Interleukin-2 production and localization were estimated by immunohistochemistry. RESULTS: Compared with control heart transplantation or heart transplantation after only DC administration, concomitant pretreatment with immature donor DC and anti-CD40L mAb caused sustained elevation of donor (I-Ab+) cells (microchimerism) in the spleen including T cell areas. More than 80% of the I-Ab+ cells in combined treatment animals also were CD86+, reflecting failure of the mAb to inhibit CD40/ CD80/CD86 up-regulation on immature DC in vitro after their interaction with host T cells. Donor-specific CTL activity in graft-infiltrating cells and spleen cell populations of these animals was present on day 8, but decreased strikingly to normal control levels by day 14. The decrease was associated with enhanced apoptosis of graft-infiltrating cells and of cells in the spleen where interleukin-2 production was inhibited. The highest levels of splenic microchimerism were found in mice with long surviving grafts (>100 days). In contrast, CTL activity was persistently elevated in control heart graft recipients with comparatively low levels of apoptotic activity and high levels of interleukin-2. CONCLUSION: The donor-specific acceptance of rejection-prone heart allografts by recipients pretreated with immature donor DC and anti-CD40L mAb is not dependent on sustained inhibition of donor DC CM (CD86) expression. Instead, the pretreatment facilitates a tolerogenic cascade similar to that in spontaneously tolerant liver recipients that involves: (1) chimerism-driven immune activation, succeeded by deletion of host immune responder cells by apoptosis in the spleen and allograft that is linked to interleukin-2 deficiency in both locations and (2) persistence of comparatively large numbers of donor-derived leukocytes. These tolerogenic mechanisms are thought to be generic, explaining the tolerance induced by allografts spontaneously, or with the aid of various kinds of immunosuppression.     

Hip fracture in Hong Kong over the last decade--a comparison with the UK.

BACKGROUND: Hip fracture is a major public health problem in Asia and the UK. The objectives of this study were to describe the trends of hip fracture in Hong Kong over the last decade, and to compare the incidence in Hong Kong with that from the Wessex Health Region of the UK in 1995. METHODS: The number of hip fractures was calculated using hospital discharge records for all public hospitals in Hong Kong in 1991 and 1995. Age-specific incidence rates were then calculated using the mid-year census population for the two years. These rates were presented with previously reported age-specific rates for Hong Kong in 1966 and 1985. These age-specific rates for Hong Kong in 1995 were compared with rates for the Wessex Health Region of the UK. The total number of hip fracture expected in 2010 was calculated by applying the age-specific rates of 1995 to the projected population for 2010. RESULTS: In 1995, a total of 1138 men and 2782 women in Hong Kong fractured their hip. The age-specific rates had remained static from 1985 to 1995, after substantial rise from 1966 to 1985. In 1995, the rates of hip fracture rates were 11/1000 in women and 5/1000 in men who were 70 years and older. These rates were almost identical to those observed in the Wessex Health Region of the UK. CONCLUSION: The age-specific incidence rates of hip fracture had not risen in Hong Kong in the last decade. The incidence of hip fracture in Hong Kong was similar to that in the UK in 1995. The total number of patients with hip fracture in Hong Kong will increase substantially in the future, as a result of the ageing of the population.     

Percutaneous nitroglycerin absorption in rats.

Percutaneous nitroglycerin absorption was studied in shaved rats by monitoring unchanged plasma drug concentrations for up to 4 hr. Drug absorption from the neat liquid state or from an alcoholic solution was considerably poorer than that from a commercial ointment. This observation was unanticipated since the driving force for percutaneous drug absorption was assumed to be drug thermodynamics. Potential artifacts such as drug volatilization from the skin, reduction of surface area through droplet formation, and vehicle occlusion were investigated, but they did not appear to be responsible for the observed results. Two experimental aqueous nitroglycerin gels were prepared with polyethylene glycol 400. One gel contained just sufficient polyethylene glycol to solubilize the nitroglycerin; the other had excess polyethylene glycol to solubilize nitroglycerin far below saturation. Both gels gave extremely low plasma nitroglycerin levels. The composite data suggested that percutaneous nitroglycerin absorption is highly vehicle dependent and that this dependency cannot be explained by simple consideration of drug thermodynamic activity.     

Effects of Nicotine on Bone and Calciotropic Hormones in Growing Female Rats

Limited research in young adults and immature animals suggests a detrimental effect of tobacco on bone during growth. This study investigated the effects of nicotine, the major alkaloid component of tobacco, on calciotropic hormone concentrations and bone status in growing female rats. One-month-old animals received either saline (n = 10), nicotine at 3.0 mg/kg/day (n = 10), or nicotine at 4.5 mg/kg/day (n = 10) administered subcutaneously via osmotic minipumps for either 2 or 3 months. Sera, femora, tibiae, and lumbar vertebrae (3–5) were collected at necropsy. The concentrations of serum calcium, phosphorus, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, parathyroid hormone, calcitonin, and insulin-like growth factor-I were determined. Bone variables evaluated included mineral content and density (vertebrae and femora), cancellous and cortical histomorphometry (tibiae), and bone strength (vertebrae and femora). Statistically significant differences in serum mineral and hormone concentrations were not associated with nicotine dose or exposure time. No significant nicotine treatment effects were detected for bone mineral content and density, bone histomorphometry, or bone strength. We conclude that nicotine treatment for 2 or 3 months at serum concentrations in the upper range of those found in smokers has no detrimental effect on bone mass, volume, or strength in the growing rat. Received: 20 May 1999 / Accepted: 21 January 2000     

Immunomodulation for intestinal transplantation by allograft irradiation, adjunct donor bone marrow infusion, or both

BACKGROUND: The passenger leukocytes in the intestine have a lineage profile that predisposes to graft-versus-host disease (GVHD) in some animal models and have inferior tolerogenic qualities compared with the leukocytes in the liver, other solid organs, and bone marrow. Elimination by ex vivo irradiation of mature lymphoid elements from the bowel allografts is known to eliminate the GVHD risk. We hypothesized that infusion of donor bone marrow cells (BMC) in recipients of irradiated intestine would improve tolerogenesis without increasing the risk of GVHD. METHODS: Orthotopic small intestine transplantation was performed with the GVHD-prone Lewis (LEW)-to-Brown Norway (BN) combination and the reverse GVHD-resistant BN-to-LEW model under a short course of tacrolimus treatment (1 mg/kg/day, days 0-13, 20, 27). Grafts were irradiated ex vivo, using a 137Cs source. In selected experimental groups, donor BMC (2.5 x 10(8)) were infused on the day of small intestine transplantation. RESULTS: The unmodified LEW intestine remained intact, whether transplanted alone or with adjunct donor BMC infusion, but all of the BN recipients died of GVHD after approximately 2 months. Intestinal graft irradiation (10 Gy) effectively prevented the GVHD and prolonged survival to 92.5 days, but all of the BN recipients died with chronic rejection of the LEW grafts, which was prevented by infusion of adjunct donor BMC without causing GVHD. In the GVHD-resistant reverse strain direction (BN-->LEW), all intestinal recipients treated for 27 days with tacrolimus survived > or =150 days without regard for graft irradiation or adjunct BMC, but chronic rejection was severe in the irradiated intestine, moderate in the unaltered graft, and least in the irradiated intestine transplanted with adjunct BMC. Mild arteritis in the 150 day allografts of both strain combinations (i.e., LEW--> BN and BN-->LEW) may have been irradiation associated, but this was prevented when weekly doses of tacrolimus were continued for the duration of the experiment rather than being stopped at 27 days. CONCLUSIONS: Recipients are protected from GVHD by irradiating intestinal allografts, but the resulting leukocyte depletion leads to chronic rejection of the transplanted bowel. The chronic rejection is prevented with adjunct donor BMC without causing GVHD. Although application of the strategy may be limited by the possibility of radiation injury, the results are consistent with the paradigm that we have proposed to explain organ-induced graft acceptance, tolerance, and chronic rejection.     

Ex vivo generation of effective Epstein-Barr virus (EBV)-specific CD8+ cytotoxic T lymphocytes from the peripheral blood of immunocompetent Epstein Barr virus-seronegative individuals

BACKGROUND: Although readily accomplished from immunocompetent Epstein-Barr virus- (EBV) seropositive individuals, the effective ex vivo generation of EBV-specific cytotoxic T lymphocytes (CTL) from the peripheral blood mononuclear cells (PBMC) of EBV-seronegative subjects has proven to be a challenge. The focus of our study was to ascertain optimized culture conditions required for the ex vivo generation of EBV-reactive autologous CTL from the PBMC of EBV-seronegative volunteers. METHOD: Freshly isolated PBMC obtained from immunocompetent EBV-seronegative and -seropositive individuals were used to generate EBV-specific autologous CTL lines using both conventional and a novel, modified ex vivo culture technique. RESULTS: In contrast to responses observed in EBV-seropositives after two to three rounds of ex vivo stimulation, gamma-irradiated autologous lymphoblastoid cell lines (LCL) were incapable of eliciting an effective anti-EBV cytotoxic response when freshly-isolated PBMC from EBV-seronegative individuals were used as responders. Under these culture conditions, CD4+ T cells with preferential expression of the Th2-type cytokine IL-4 were predominantly expanded in the PBMC obtained from EBV-seronegative individuals. However, the addition of recombinant human (rh) IL-12 during the primary phase of ex vivo stimulation resulted in augmentation of EBV-specific cytolysis of autologous LCL by CD8+ T cells. Furthermore, there was down-regulation in the secretion of IL-4 and up-regulation in that of the Th1-type cytokine IFN-gamma by responder CD4+ and CD8+ T cells. CONCLUSIONS: Taken together these data suggest that the addition of rhIL-12 during the primary phase of ex vivo stimulation of freshly isolated PBMC from EBV-seronegative individuals results in skewing of the immune response predominantly towards a CD4+ Th1-type (IFN-gamma) with the generation of an efficacious CTL-mediated anti-EBV reactivity. This novel ex vivo approach for generating effective autologous EBV-specific CTL could be adopted to treat refractory post-transplant lymphoproliferative disorders, which may be encountered in EBV-seropositive-->EBV-seronegative organ transplant recipients. Additionally, these ex vivo generated anti-EBV T cells could also be infused perioperatively to enhance prophylactically immunity against EBV infection in high-risk EBV-seronegative organ allograft recipients.     

Epstein-Barr virus-negative post-transplant lymphoproliferative disorders: a distinct entity?

Post-transplant lymphoproliferative disorders (PTLDs) are usually but not invariably associated with Epstein-Barr virus (EBV). The reported incidence, however, of EBV-negative PTLDs varies widely, and it is uncertain whether they should be considered analogous to EBV-positive PTLDs and whether they have any distinctive features. Therefore, the EBV status of 133 PTLDs from 80 patients was determined using EBV-encoded small ribonucleic acid (EBER) in situ hybridization stains with or without Southern blot EBV terminal repeat analysis. The morphologic, immunophenotypic, genotypic, and clinical features of the EBV-negative PTLDs were reviewed, and selected features were compared with EBV-positive cases. Twenty-one percent of patients had at least one EBV-negative PTLD (14% of biopsies). The initial EBV-negative PTLDs occurred a median of 50 months post-transplantation compared with 10 months for EBV-positive cases. Although only 2% of PTLDs from before 1991 were EBV negative, 23% of subsequent PTLDs were EBV negative (p <0.001). Of the EBV-negative PTLDs, 67% were of monomorphic type (M-PTLD) compared with 42% of EBV-positive cases (p <0.05). The other EBV-negative PTLDs were of infectious mononucleosis-like, plasma cell-rich (n = 2), small B-cell lymphoid neoplasm, large granular lymphocyte disorder (n = 4) and polymorphic (P) types. B-cell clonality was established in 14 specimens and T-cell clonality was established in three (two patients). None of the remaining specimens were studied with Southern blot analysis and some had no ancillary studies. Rearrangement of c-MYC was identified in two M-PTLDs with small noncleaved-like features, and rearrangement of BCL-2 was found in one large noncleaved-like M-PTLD. Ten patients were alive at 3 to 63 months (only three patients received chemotherapy). Seven patients, all with M-PTLDs, are dead at 0.3 to 6 months. Therefore, EBV-negative PTLDs have distinct features, but some do respond to decreased immunosuppression, similar to EBV-positive cases, suggesting that EBV positivity should not be an absolute criterion for the diagnosis of a PTLD.