The combined effects of water and nitrogen on the relationship between a native hemiparasite and its invasive host.

Stem hemiparasites are dependent on their hosts for water and nitrogen. Most studies, however, have assessed the influence of one factor on parasite : host associations, thus limiting our mechanistic understanding of their performance in nature. We investigated the combined effects of water and nitrogen (N) availability on both a host (Ulex europaeus) and its parasite (Cassytha pubescens). Parasite infection significantly decreased host shoot biomass and shoot : root ratio more severely in high water than low water, irrespective of N supply. Parasite stem [N] was significantly higher in high water than low water treatments, regardless of N supply, but parasite biomass did not vary among treatments. Irrespective of water and N supply, infected plants had significantly lower total, root and nodule biomass, predawn and midday quantum yields, maximum electron transport rates, water potentials and nitrogen concentration [N]. Parasite δ13 C was significantly higher than that of the host. Our results suggested that stem hemiparasites can better extract resources from hosts when water availability is high, resulting in a greater impact on the host under these conditions. When hemiparasitic plants are being investigated as a biocontrol for invasive weeds, they may be more effective in wetter habitats than in drier ones.

Does phosphorus influence performance of a native hemiparasite and its impact on a native legume?

Phosphorus (P) is an essential plant nutrient and can become limiting in terrestrial ecosystems where parasitic plant:host associations occur. Yet little is known on how P availability influences parasite performance and its impact on hosts. We investigated the performance of the Australian native stem hemiparasite Cassytha pubescens and its impact on the native leguminous shrub Acacia paradoxa in high or low P conditions in a glasshouse experiment. Infected plants had significantly lower total, shoot, root and nodule biomass and shoot:root ratio than uninfected plants, regardless of P supply. The significant negative effect of infection on arbuscular mycorrhizal colonisation of host roots was more severe in the high P treatment. Infection significantly decreased predawn quantum yield of A. paradoxa in low P but not high P conditions. This finding may be due to the parasite-induced significant enrichment of aluminium in host foliage in low P but not high P treatments. A. paradoxa had significantly lower foliar phosphorus concentration [P] and nitrogen concentration in low P than high P conditions. Parasite biomass and photosynthetic performance were unaffected by P, whereas C. pubescens had significantly lower stem [P] in the low P than high P treatment. Parasite carbon isotope composition was significantly higher than that of the host, especially in low P conditions. Our results show that: (a) native parasite growth and its negative impact on growth of this native shrub was unaffected by P supply and (b) soil P conditions may have no influence on stem hemiparasite:host associations in nature.

The impact of a native hemiparasite on a major invasive shrub is affected by host size at time of infection.

Many studies have investigated the effect of parasitic plants on their hosts; however, few have examined how parasite impact is affected by host size. In a glasshouse experiment, we investigated the impact of the Australian native hemiparasitic vine, Cassytha pubescens, on a major invasive shrub, Ulex europaeus, of different sizes. Infected plants had significantly lower total, shoot, and root biomass, but the parasite's impact was more severe on small than on large hosts. When infected, small but not large hosts had significantly lower nodule biomass. Irrespective of size, infection significantly decreased the host shoot/root ratio, pre-dawn and midday quantum yields, maximum electron transport rates, and carbon isotope composition, and the host nodule biomass per gram of root biomass significantly increased in response to infection. Infection did not affect host foliar nitrogen concentration or midday shoot water potential. Parasite biomass was significantly lower on small relative to large hosts, but was similar when expressed on a per gram of host total biomass basis. Parasite stem nitrogen, phosphorus, and potassium concentrations were significantly greater when C. pubescens was growing on small than on large hosts. Our results clearly show that C. pubescens strongly decreases performance of this major invasive shrub, especially when hosts are small. This suggests that C. pubescens could be used most effectively as a native biocontrol when deployed on smaller hosts.

Does nitrogen affect the interaction between a native hemiparasite and its native or introduced leguminous hosts?

Associations between plants and nitrogen (N)-fixing rhizobia intensify with decreasing N supply and come at a carbon cost to the host. However, what additional impact parasitic plants have on their leguminous hosts' carbon budget in terms of effects on host physiology and growth is unknown. Under glasshouse conditions, Ulex europaeus and Acacia paradoxa either uninfected or infected with the hemiparasite Cassytha pubescens were supplied (high nitrogen (HN)) or not (low nitrogen (LN)) with extra N. The photosynthetic performance and growth of the association were measured. Cassytha pubescens significantly reduced the maximum electron transport rates and total biomass of U. europaeus but not those of A. paradoxa, regardless of N. Infection significantly decreased the root biomass of A. paradoxa only at LN, while the significant negative effect of infection on roots of U. europaeus was less severe at LN. Infection had a significant negative impact on host nodule biomass. Ulex europaeus supported significantly greater parasite biomass (also per unit host biomass) than A. paradoxa, regardless of N. We concluded that rhizobia do not influence the effect of a native parasite on overall growth of leguminous hosts. Our results suggest that C. pubescens will have a strong impact on U. europaeus but not A. paradoxa, regardless of N in the field.

High water availability increases the negative impact of a native hemiparasite on its non-native host.

Environmental factors alter the impacts of parasitic plants on their hosts. However, there have been no controlled studies on how water availability modulates stem hemiparasites' effects on hosts. A glasshouse experiment was conducted to investigate the association between the Australian native stem hemiparasite Cassytha pubescens and the introduced host Ulex europaeus under high (HW) and low (LW) water supply. Cassytha pubescens had a significant, negative effect on the total biomass of U. europaeus, which was more severe in HW than LW. Regardless of watering treatment, infection significantly decreased shoot and root biomass, nodule biomass, nodule biomass per unit root biomass, F v/F m, and nitrogen concentration of U. europaeus. Host spine sodium concentration significantly increased in response to infection in LW but not HW conditions. Host water potential was significantly higher in HW than in LW, which may have allowed the parasite to maintain higher stomatal conductances in HW. In support of this, the δ(13)C of the parasite was significantly lower in HW than in LW (and significantly higher than the host). C. pubescens also had significantly higher F v/F m and 66% higher biomass per unit host in the HW compared with the LW treatment. The data suggest that the enhanced performance of C. pubescens in HW resulted in higher parasite growth rates and thus a larger demand for resources from the host, leading to poorer host performance in HW compared with LW. C. pubescens should more negatively affect U. europaeus growth under wet conditions rather than under dry conditions in the field.

Does light influence the relationship between a native stem hemiparasite and a native or introduced host?

BACKGROUND AND AIMS: There have been very few studies investigating the influence of light on the effects of hemiparasitic plants on their hosts, despite the fact that hemiparasites are capable of photosynthesis but also access carbon (C) from their host. In this study we manipulated light availability to limit photosynthesis in an established hemiparasite and its hosts, and determined whether this affected the parasite's impact on growth and performance of two different hosts. We expected that limiting light and reducing autotrophic C gain in the parasite (and possibly increasing its heterotrophic C gain) would lead to an increased impact on host growth and/or host photosynthesis in plants grown in low (LL) relative to high light (HL). METHODS: The Australian native host Leptospermum myrsinoides and the introduced host Ulex europaeus were either infected or not infected with the native stem hemiparasite Cassytha pubescens and grown in either HL or LL. Photosynthetic performance, nitrogen status and growth of hosts and parasite were quantified. Host water potentials were also measured. KEY RESULTS: In situ midday electron transport rates (ETRs) of C. pubescens on both hosts were significantly lower in LL compared with HL, enabling us to investigate the impact of the reduced level of parasite autotrophy on growth of hosts. Despite the lower levels of photosynthesis in the parasite, the relative impact of infection on host biomass was the same in both LL and HL. In fact, biomass of L. myrsinoides was unaffected by infection in either HL or LL, while biomass of U. europaeus was negatively affected by infection in both treatments. This suggests that although photosynthesis of the parasite was lower in LL, there was no additional impact on host biomass in LL. In addition, light did not affect the amount of parasite biomass supported per unit host biomass in either host, although this parameter was slightly lower in LL than HL for U. europaeus (P = 0·073). We also found no significant enhancement of host photosynthesis in response to infection in either host, regardless of light treatment. CONCLUSIONS: Despite lower photosynthetic rates in LL, C. pubescens did not increase its dependency on host C to the point where it affected host growth or photosynthesis. The impact of C. pubescens on host growth would be similar in areas of high and low light availability in the field, but the introduced host is more negatively affected by infection.

Differentiation of regulatory myeloid and T-cells from adult human hematopoietic stem cells after allogeneic stimulation.

Introduction: Donor hematopoietic stem cell (DHSC) infusions are increasingly being studied in transplant patients for tolerance induction. Methods: To analyze the fate of infused DHSCs in patients, we developed an in vitro culture system utilizing CD34+DHSCs stimulated with irradiated allogeneic cells in cytokine supplemented medium long-term. Results: Flow cytometric analyses revealed loss of the CD34 marker and an increase in CD33+ myeloid and CD3+ T-cell proportion by 10.4% and 72.7%, respectively, after 21 days in culture. T-cells primarily expressed TcR-αß and were of both CD4+ and CD8+ subsets. Approximately 80% of CD3+ T cells lacked expression of the co-stimulatory receptor CD28. The CD4+ compartment was predominated by CD4+CD25+CD127-FOXP3+ Tregs (>50% CD4+CD127- compartment) with <1% of all leukocytes exhibiting a CD4+CD127+ phenotype. Molecular analyses for T-cell receptor excision circles showed recent and increased numbers of TcR rearrangements in generated T cells over time suggesting de novo differentiation from DHSCs. CD33+ myeloid cells mostly expressed HLA-DR, but lacked expression of co-stimulatory receptors CD80 and CD83. When studied as modulators in primary mixed lymphocyte reactions where the cells used to stimulate the DHSC were used as responders, the DHSC-lines and their purified CD8+, CD4+, CD33+ and linage negative subsets inhibited the responses in a dose-dependent and non-specific fashion. The CD8+ cell-mediated inhibition was due to direct lysis of responder cells. Discussion: Extrapolation of these results into the clinical situation would suggest that DHSC infusions into transplant recipients may generate multiple subsets of donor "chimeric" cells and promote recipient Treg development that could regulate the anti-donor immune response in the periphery. These studies have also indicated that T cell maturation can occur in vitro in response to allogeneic stimulation without the pre-requisite of a thymic-like environment or NOTCH signaling stimulatory cell line.

A native parasitic plant affects the performance of an introduced host regardless of environmental variation across field sites.

Increasing evidence from glasshouse studies shows that native hemiparasitic plants can significantly impact the performance and growth of introduced host plants. We investigated the effect of the native Australian hemiparasite Cassytha pubescens R.Br. on the introduced shrub Ulex europaeus L. at three field sites in South Australia. Parasite infection significantly decreased midday PSII efficiency (ΦPSII) and the maximum electron transport rates (ETRmax) of U. europaeus across sites. The impact of C. pubescens on the photosynthetic performance of U. europaeus may have been caused by infected plants having significantly lower N and K, but higher Fe and Al than uninfected plants at all sites. Significant Al and Fe enrichment in infected plants may be possibly due to the parasite indirectly inducing rhizosphere acidification. At two sites, C. pubescens significantly affected host Fv/Fm, indicating chronic photoinhibition in response to infection. The impact of infection on Fv/Fm was greatest at the wettest site, in line with an experiment where C. pubescens had more impact under high water availability. At this site, infected plants also had the highest foliar Fe and Al. The C isotope (δ13C) of infected plants was significantly lower than that of uninfected plants at only one site. Unusually, the δ13C of the parasite was the same as or significantly higher than that of the hosts. There were no site effects on parasite Fv/Fm or ΦPSII; however, ETRmax and δ13C varied across sites. The results suggest that this native parasite has negative effects on U. europaeus in the field, as was found for glasshouse studies. The abundance of this introduced weed in Australia could be negatively affected by C. pubescens infection.

FoxP3 mRNA transcripts and regulatory cells in renal transplant recipients 10 years after donor marrow infusion.

BACKGROUND: We update more favorable 10-year deceased donor kidney transplant survival in 63 recipients infused perioperatively with donor vertebral body bone marrow (DBMC-i) vs. 219 noninfused controls having equivalent immunosuppression and demographics. We questioned if this was associated with putatively regulatory FoxP3 mRNA and cell phenotypes (CD4+CD25+high percentages and high DC2:DC1 ratios) in DBMC-i vs. noninfused controls. METHODS: Baseline studies were performed on peripheral blood lymphocytes (PBLs) vs. marrow in normal laboratory volunteers of CD4+CD25+high percentages and DC2:DC1 by flow cytometry, and FoxP3 mRNA in CD3+ cells by real-time polymerase chain reaction. Similar studies were performed on PBL of the majority of the 10-year patients remaining with graft function: 21 (of the remaining 37) DBMC-i vs. 55 (of the remaining 105) controls. RESULTS: In normal subjects, all parameters were significantly higher in marrow than in PBL, supporting our previous reports of ex vivo DBMC immunoregulation. At 9.8+/-.02 years posttransplant in DBMC-i vs. controls, death-censored percent graft failure was 17.5% vs. 32.9% (P=0.02) with 247.6+/-24 vs. 79.9+/-3.1 (mean+/-SE) FoxP3 copies/5,000 CD3+ cells (P=0.0001). PBL CD4+CD25+high percentages were lower, but DC2:DC1 values higher in both recipient groups than in end-stage renal disease patients who had lower FoxP3 levels (40.8+/-5.9, P<0.0001), consistent with non-CD4+CD25+high T regulatory cells generated long-term posttransplant. Individual higher FoxP3 values correlated with higher iliac crest chimerism in DBMC-i, but not in controls (with 50-fold lower chimerism). In chronically rejecting controls, FoxP3 was further decreased. CONCLUSIONS: Peritransplant DBMC-i has higher 10-year renal transplant acceptance, chimerism, and FoxP3 mRNA in thus-far unclarified regulatory cell phenotypes.

A novel approach to detect donor/recipent immune responses between HLA-identical pairs.

Renal transplant rejection and graft versus host reactions between HLA genetically-identical sibling (HLAgi) donor/recipient (D/R) pairs are thought to result from minor histocompatibility antigen (mHAg) disparities. We have compared two methods of measuring HLAgi D/R T lymphocyte responses to "matured" dendritic cells: 1.) a modified Cylex assay of CD4(+) ATP levels (MLDC-ATP) versus 2.) (3)H-thymidine uptake (MLDC-(3)H). The MLDC-ATP kinetics peaked at 48 hours versus the MLDC-(3)H at 7 days, and appeared more sensitive. We tested HLAgi (normal) volunteer siblings (NLs), and D/R sibling pairs before and after renal transplantation (pre-Tx and post-Tx). The overall frequencies of positive responses in the MLDC-ATP for HLAgi NLs, pre-Tx, and post-Tx D/R pairs were 63%, 50%, and 42%, respectively. The percentage with reciprocal responses was 37.5%, 20%, and 22.22%, respectively. In one set of three HLAgi (NLs) siblings (two males and one female), there was a nongender-associated differential response. There was no MLDC correlation with class I MHC-associated mHAg (SSP) incompatibility, nor could some MLDC positive reactive pairs theoretically process the necessary HLA-class I restriction molecules for presentation of known (nanomeric) mHAg peptides. Speculatively, the MLDC reflects class II MHC-restricted mHAg reactions (not yet definable), with possible effects of other polymorphic (nonhistocompatibility) immune response genes, and thereby may be a useful measurement of CD4(+) T-cell HLAgi transplantation immunity.

Inhibition of NF-kappa B during human dendritic cell differentiation generates anergy and regulatory T-cell activity for one but not two human leukocyte antigen DR mismatches.

We examined the in vitro inhibition of human monocyte-derived dendritic cells (DC) maturation via NF-kappaB blockade on T-cell allostimulation, cytokine production, and regulatory T-cell generation. DC were generated from CD14+ monocytes isolated from peripheral blood using GM-CSF and IL-4 for differentiation and TNF-alpha, IL-1beta, and PGE2 as maturational stimuli with or without the NF-kappaB inhibitors, BAY 11-7082 (BAY-DC) or Aspirin (ASA-DC). Stimulator and responder cells were one versus two HLA-DR mismatched in direct versus indirect presentation assays. Both BAY-DC and ASA-DC expressed high levels of HLA-DR and CD86 but always expressed less CD40 compared with controls. Some experiments showed slightly lower levels of CD80. Both BAY- and ASA-allogeneic DC and autologous alloantigen pulsed DC were weaker stimulators of T cells (by MLR) compared with controls, and there was reduced IL-2 and IFN-gamma production by T cells stimulated with BAY-DC or ASA-DC (by ELISPOT) (more marked results were always observed with ASA-treated DC). In addition, NF-kappaB blockade of DC maturation caused the generation of T cells with regulatory function (T regs) but only when T cells were stimulated by either allogeneic (direct presentation) or alloantigen pulsed autologous DC (indirect presentation) with one HLA-DR mismatch and not with two HLA-DR mismatches (either direct or indirect presentation). However, the T regs generated from these ASA-DC showed similar FoxP3 mRNA expression to those from nontreated DC. Extension of this study to human organ transplantation suggests potential therapies using one DR-matched NF-kappaB blocked DC to help generate clinical tolerance.

A randomized trial of three renal transplant induction antibodies: early comparison of tacrolimus, mycophenolate mofetil, and steroid dosing, and newer immune-monitoring.

BACKGROUND: New trends in immunosuppression in clinical transplantation include the use of antibody induction agents in protocols that emphasize reduction or avoidance of steroids and calcineurin inhibitors. METHODS: In a randomized trial using three different antibody induction agents in 90 first renal transplant recipients from cadaver donors, group A received Thymoglobulin, group B received Alemtuzumab, and group C received Daclizumab. Maintenance immunosuppression included tacrolimus and mycophenolate in all three arms, and methylprednisolone in groups A and C only (standard clinical institutional practice). The targeted trough level of tacrolimus was between 8 and 10 ng/mL for groups A and C, respectively, with a targeted mycophenolate dose of 1 g twice daily. However, in group B, the target tacrolimus trough level was 4 to 7 ng/mL to reduce long-term nephrotoxicity, with 500 mg twice-daily doses of mycophenolate, without steroid maintenance. RESULTS: In this 15-month median postoperative interval report, there were no notable differences in demographics and patient and graft survivals. Acute rejection rates at 1 year were equivalent, that is, 5 of 30 in all three groups (16.6%). In group B, there was slightly lower renal function at 1 month, but no difference at 1 year. There was also significantly more leukopenia, but a greater percentage of T-regulatory cells and number of Fox-P3 mRNA copies by flow cytometry and semiquantitative polymerase chain reaction analysis, respectively, in group B. CONCLUSIONS: This preliminary analysis indicates that 80% of the patients in group B remained steroid-free 1 year postoperatively, with lower tacrolimus trough levels and no difference in other adverse events.

Native hemiparasite and light effects on photoprotection and photodamage in a native host.

Plants infected with hemiparasites often have lowered rates of photosynthesis, which could make them more susceptible to photodamage. However, it is also possible that infected plants increase their photoprotective capacity by changing their pigment content and/or engagement of the xanthophyll cycle. There are no published studies investigating infection effects on host pigment dynamics and how this relates to host susceptibility to photodamage whether in high (HL) or low light (LL). A glasshouse experiment was conducted where Leptospermum myrsinoides Schltdl. either uninfected or infected with Cassytha pubescens R.Br. was grown in HL or LL and pigment content of both host and parasite were assessed. Infection with C. pubescens significantly decreased all foliar pigment concentrations (except chlorophyll b) in L. myrsinoides in both HL and LL. Xanthophyll cycle (violaxanthin, antheraxanthin, zeaxanthin; VAZ) and chlorophyll (Chl) pigments decreased in parallel in response to infection, hence, VAZ/Chl of the host was unaffected by C. pubescens in either HL or LL. Pre-dawn and midday de-epoxidation state [(A+Z)/(V+A+Z)] of L. myrsinoides was also unaffected by infection in both HL and LL. Thus, L. myrsinoides infected with C. pubescens maintained similar photoprotective capacity per unit chlorophyll and engagement of the xanthophyll cycle as uninfected plants. Even though midday quantum yield (ΦPSII) of HL plants was affected by infection, pre-dawn maximum quantum yields (Fv/Fm) of hosts were the same as uninfected plants whether in HL or LL. This ability of L. myrsinoides to maintain photoprotective capacity/engagement when infected by C. pubescens thereby preventing photodamage could explain this host's tolerance to hemiparasite infection.

Use of liver grafts from donors positive for antihepatitis B-core antibody (anti-HBc) in the era of prophylaxis with hepatitis-B immunoglobulin and lamivudine.

BACKGROUND: The increasing demand for transplantation has resulted in a trend toward using virologically compromised donors. We reviewed our experience with liver grafts from hepatitis-B surface antigen (HBsAg)(-), antibody to core antigen (anti-HBc)(+) donors. METHODS: Sixty-two liver transplants using HBsAg(-), anti-HBc(+) donors were studied. The decision to use prophylaxis was based on the presence or absence of donor and recipient risk factors for posttransplant hepatitis-B virus (HBV) transmission or reinfection. If the donor or recipient showed positive HBVDNA, hepatitis-B immunoglobulin (HBIg) and lamivudine were used. If both donor and recipient HBVDNA were negative, a choice between lamivudine and no prophylaxis was made on the basis of presence or absence of HBsAg and antibody to the surface antigen (anti-HBs) in the recipient. RESULTS: No death or graft loss could be ascribed to HBV. Mild HBV infection occurred in two patients who were not taking the recommended prophylaxis. Among the other 60 patients, 1 showed positive e antigen (HBeAg) early after transplantation, and 2 (1 with recurrent cancer, 1 with HIV infection) showed HBsAg(+). None of the three patients had any other evidence of HBV infection. Forty-seven patients underwent liver biopsies. Changes consistent with hepatitis were observed in 26, and 24 had HCV infection; immunostains for HBV antigens were negative in all cases, and 7 showed positive HBVDNA. CONCLUSIONS: A selective protocol based on donor and recipient risk factors for post-liver transplant HBV infection can prevent hepatitis-B infection and avoid unnecessary administration of antiviral prophylaxis in recipients of HBsAg(-), anti-HBc(+) liver allografts.

Successful treatment of posttransplant lymphoproliferative disease with prolonged rituximab treatment in intestinal transplant recipients.

BACKGROUND: Posttransplant Epstein-Barr virus-associated B-cell lymphoproliferative disease (PTLD) has a higher incidence after intestinal transplantation than after transplantation of other solid organs and is associated with a high mortality. A new anti-CD20 monoclonal antibody, rituximab, has shown efficiency in the treatment of B-cell lymphoma, including PTLD, but its use has not yet been reported in intestinal transplant recipients. METHODS: We retrospectively reviewed five patients who were diagnosed with PTLD from March 1999 to August 2001, after intestinal transplantation. These patients were primarily managed with rituximab, associated with reduction or interruption of immunosuppression and antiviral therapy with ganciclovir and cytomegalovirus immune globulin. Rituximab was administered at weekly doses of 375 mg/m until full remission was ascertained, and the interval between doses was then increased. No patient received chemotherapy. RESULTS: One patient had nonmalignant lymphoproliferation, and four had malignant PTLD, as assessed by histopathology and monoclonality of the tumor. Two pediatric patients had severe generalized disease. All patients had received OKT3 as treatment of rejection before developing PTLD. All tumors showed proliferation of CD20 cells and were positive for Epstein-Barr virus by in situ hybridization. All patients responded to rituximab therapy and have achieved full remission with a follow-up of 3 to 30 (median, 8) months. CONCLUSION: Prolonged rituximab treatment, in association with reduction of immunosuppression and antiviral therapy, is highly efficient as part of the first-line treatment of CD20 B-cell PTLD after intestinal transplantation.

Immune responses and their regulation by donor bone marrow cells in clinical organ transplantation.

Infusions of donor bone marrow derived cells (DBMC) continue to be tested in clinical protocols intended to induce specific immunologic tolerance of solid organ transplants based on the observations that donor-specific tolerance is induced this way in animal models. We studied the immunological effects of human DBMC infusions in renal transplantation using modifications in lymphoproliferation (MLR) and cytotoxicity (CML) assays. The salient observations and tentative conclusions are summarized in this review. Among many types of organs transplanted using DBMC at this center, it was found that the cadaver renal recipients (CAD) had significantly decreased chronic rejection and higher graft survival when compared to equivalent non-infused controls. DBMC infusion was also associated with a marginal and non-specific immune depression. It was also observed that the number of chimeric donor cells gradually increased in the iliac crest bone marrow compartment with a concomitant decrease in the peripheral blood and that the increase was more rapid in living-related donor (LRD)-kidney/DBMC recipients in spite of a lower number of DBMC infused (<25%) than in the CAD-kidney/DBMC group. In the LRD recipients with residual anti-donor responses, purified chimeric cells of either donor or recipient inhibited recipient immune responses to the donor significantly more strongly than the freshly obtained bone marrow from the specific donor or volunteer suggesting an active regulatory role for chimeric cells. A number of (non-chimeric) subpopulations of bone marrow cells including CD34(+) stem cells and the CD34(-) early progeny like CD38(+), CD2(+), CD5(+) and CD1(+) lymphoid cells as well as CD33(+) (but CD15(-)) myeloid cells down-regulated the MLR and CML responses of allogeneic PBMC stimulated with (autologous) donor spleen cells. These regulatory effects appeared to be refractory to the action of commonly used immunosuppressive drugs and occurred during the early phase of the immune response through cell-cell interactions. Most of these DBMC sub-populations had stimulatory capabilities, albeit markedly lower than donor spleen cells, but only through the indirect antigen presentation pathway. When co-cultured with allogeneic stimulators, purified CD34(+) cells were found to give rise both to CD3(-) TCRalphabeta(+), as well as CD3(+) TCRalphabeta(+) cells and, thereby, responded in MLR to allogeneic stimulation (but did not generate cytotoxic effector cells). Also, a number of DBMC subpopulations inhibited the CML and to a lesser extent the MLR, of autologous post-thymic responding T cells stimulated with allogeneic irradiated cells, mediated through soluble factors. Finally, non-chimeric DBMC also inhibited the proliferative and cytotoxic responses of autologous T cells to EBV antigens, inducing T suppressor cells, which in turn could inhibit autologous anti-EBV CTL generation and B cell anti-CMV antibody production. These studies all suggested a strong inhibitory property of a number of DBMC sub-populations in vitro and in vivo with the notion that they promote unresponsiveness.

Adenovirus infection in pediatric liver and intestinal transplant recipients: utility of DNA detection by PCR.

To evaluate the incidence of adenovirus (AdV) infection in pediatric liver and intestinal transplant recipients, the records of patients with possible AdV infection were reviewed for demographic data, symptomatology, methods of diagnosis, treatment and outcome. To evaluate the impact of polymerase chain reaction (PCR) amplification and identification of AdV DNA as a diagnostic test, the incidence and outcome of AdV before and after the introduction of PCR were compared. Adenovirus infection was identified in 4.1% of liver recipients and 20.8% of intestinal transplant recipients. The overall incidence of AdV did not increase over time, even following the introduction of PCR for virus detection. The higher incidence of AdV in the pediatric intestinal transplant recipients may be attributed to the frequent application of PCR methodology to intestinal biopsy material. Detection of AdV by PCR was associated with reduced mortality compared with detection by culture, either because of earlier detection of invasive disease or because PCR detects the presence of latent as well as active AdV.

Cytomegalovirus prophylaxis with valganciclovir in kidney, pancreas-kidney, and pancreas transplantation.

Cytomegalovirus, seen in more than 50% of solid organ transplant recipients, is responsible for numerous direct and indirect consequences, including infection with opportunistic pathogens and allograft rejection. Prophylaxis with intravenous ganciclovir has been the gold standard for prevention; however, intravenous treatment is expensive and carries risks of its own. Oral ganciclovir, to be effective, must be given in large, divided doses. Valganciclovir, the valine ester of ganciclovir, at 900 mg/day, provides oral bioavailability equivalent to that of intravenous ganciclovir and superior to that of oral ganciclovir. This retrospective study assessed the efficacy of 3 months prophylaxis with valganciclovir in kidney, simultaneous pancreas-kidney, and pancreas transplantation. Of 161 patients, all of whom received potent immunosuppressive regimens and were followed for a mean of 440 days, only one developed cytomegalovirus infection, at 120 days post-transplantation. Two patients died, and two others lost their grafts. There were seven incidents of acute rejection. Only seven patients discontinued treatment early, five because of leukopenia. These results suggest that prophylaxis with 900 mg/day of valganciclovir for at least 3 months safely and effectively prevents cytomegalovirus infection in kidney, simultaneous pancreas-kidney and pancreas transplant recipients on highly potent immunosuppression.

Prediction of sustained virological response in liver transplant recipients with recurrent hepatitis C virus following combination pegylated interferon alfa-2b and ribavirin therapy using tissue hepatitis C virus reverse transcriptase polymerase chain reaction testing.

The optimal duration of therapy for pegylated interferon combined with ribavirin in recurrent Hepatitis C virus (HCV) following liver transplantation is not known. We wanted to determine if testing for HCV in liver tissue by reverse transcriptase polymerase chain reaction (RT-PCR) was superior in predicting sustained virological response (SVR) in comparison to standard HCV ribonucleic acid (RNA) detection in the serum. All recipients received combination pegylated alpha-2b interferon (1.5 mcg/kg) and ribavirin (200-600 mg/d) therapy for at least 48 weeks of therapy and were found to have nondetectable HCV RNA by PCR serum testing at the end of therapy. Sustained virological response (SVR) was defined as nondetectable serum HCV RNA at 6 months post treatment withdrawal. Ten liver transplant recipients were included in the study; mean time from transplantation was 29.2 months. All had nondetectable serum HCV RNA by RT-PCR. In hepatic tissue 7/10 patients HCV RNA was found to be positive by RT-PCR while 3/10 had nondetectable HCV RNA in their liver by RT-PCR. SVR was attained in all 3/10 that were hepatic tissue HCV PCR negative after 12 months of combination therapy. In conclusion, direct detection of HCV RNA by RT-PCR of liver tissue appears to more effectively predict SVR following pegylated interferon and ribavirin therapy than the conventional use of serum.