Co-infection of polyomavirus-BK and cytomegalovirus in renal transplant recipients.

BACKGROUND: Polyomavirus-BK (BK) is a significant cause of allograft dysfunction in renal transplant recipients. Cytomegalovirus (CMV) and BK infection are thought to be possible risk factors for one another, but no supporting data are yet available. METHODS: The authors monitored BK and CMV infection by quantitative polymerase chain reaction (PCR) in 69 renal transplant recipients with serum creatinine elevation to determine the prevalence of co-infection. In addition, 150 adult renal transplant recipients were also retrospectively analyzed for both infections. RESULTS: Of 69 recipients, 12 were plasma BK-PCR-positive. Eight of the 12 showed high BK levels (>10 copies) and BK nephropathy. Six of the 12 were also CMV-PCR-positive compared with only 3 of 57 plasma BK-negative patients (50% vs. 5.3%, P=0.001). Comparatively, the incidence of Epstein-Barr virus infection was similar in both groups (1 of 12 [8.3%] vs. 2 of 57 [3.5%], P =not significant). In addition, retrospective analysis of CMV-PCR-positivity in 150 adult renal transplant recipients showed similar results (5 of 6 in BK-PCR-positive [83%] vs. 8 of 144 in BK-PCR-negative [5.6%], P=0.00001). More plasma BK-PCR-positive patients had concomitant CMV infection than CMV-PCR-positive patients with BK infection (5 of 6 [83%] vs. 4 of 13 [31%], P=0.05). CONCLUSIONS: In conclusion, high plasma BK-positivity (>10) is significantly associated with BK nephropathy. Plasma BK-positivity is highly associated with co-infection of CMV, suggesting possible risk factors for one another. Therefore, detection of either infection strongly suggests the need to monitor for the other. This strategy may lead to the prevention of virus-induced complications by preemptive antiviral therapy in renal allografts.

Human growth hormone-transferrin fusion protein for oral delivery in hypophysectomized rats.

Transferrin (Tf)-based recombinant fusion protein approach was investigated to achieve oral delivery for human growth hormone (hGH). Plasmid constructs expressing the fusion proteins were established by fusing coding sequences of both hGH and Tf in frame. Fusion proteins were produced in serum free media by transient transfection of human embryonic kidney HEK293 cells. The SDS-PAGE analysis of conditioned media showed that fusion proteins expressed at high purity with a 100 kDa molecular weight; the Western blot analysis with anti-hGH and anti-Tf antibodies verified the identity of fusion proteins. The Nb2 cell proliferation and Caco-2 cell Tf receptor (TfR) binding assays demonstrated that fusion proteins retained bioactivity of both hGH and Tf, respectively. A helical linker was inserted as spacer between hGH- and Tf-domain to enhance the bioactivity and the yield of the fusion protein. Two fusion proteins, hGH-Tf (GT) and hGH-(H4)(2)-Tf (GHT) were obtained and assessed in hGH-deficient hypophysectomized rats for in vivo biological activity. Results from seven-day subcutaneous dosing (1.25mg/kg/day) demonstrated that both GT and GHT fusion proteins were bioactive in vivo, comparable to native hGH. However, only the GHT, but not GT, fusion protein promoted a modest but statistically significant weight gain after oral dosing with 12.5mg/kg/day.

In vitro effects of everolimus and intravenous immunoglobulin on cell proliferation and apoptosis induction in the mixed lymphocyte reaction.

Targeting multiple pathways in the activation of alloimmune responses by multi-drug immunosuppressive regimens with complementary mechanisms of action enhances allograft survival and improves quality of life, owing to the reduction of adverse drug effects. In this report we investigated the effect of the combination of everolimus and intraveneous immunoglobulin (IVIG) on cell proliferation and apoptosis induction in human two-way mixed lymphocyte reaction (MLR). Everolimus alone (0.1-50 ng/ml) and IVIG (1-10 mg/ml) alone inhibited cell proliferation in a dose-dependent manner (16.4-67.2% and 12.1-66.3% inhibition, respectively). The inhibition by everolimus was not enhanced in the presence of 1 mg/ml IVIG. Addition of 10 and 50 ng/ml everolimus increased the inhibitory effect of 5 and 10 mg/ml IVIG, but only by 10-27%. Addition of 0.1 and 1 ng/ml everolimus did not increase IVIG's inhibitory effects. Apoptosis was significantly higher in IVIG (5 mg/ml)-treated CD19+ cells and less so in CD3+ cells as assessed by Annexin V and TUNEL assays. However, everolimus (0.1-50 ng/ml) did not induced apoptosis or alter apoptosis induced by IVIG. These results suggest that everolimus is a potent inhibitor of immune cell proliferation but does not act additively or synergistically with IVIG when analyzed in this in vitro system.

Mycophenolic acid and intravenous immunoglobulin exert an additive effect on cell proliferation and apoptosis in the mixed lymphocyte reaction.

BACKGROUND: Intravenous immunoglobulin (IVIG) has known immunomodulatory effects in autoimmune diseases and transplantation and is commonly used in desensitization protocols and for treatment of antibody-mediated rejection (AMR). IVIG inhibits the MLR and induces apoptosis in immune cells. Mycophenolate mofetil inhibits immune cell proliferation and is an effective immunsuppressive agent. Here, we examined the possible synergistic effects of combined MMF and IVIG on cell proliferation and apoptosis induction in the MLR. METHODS: Two-way MLRs were performed with mycophenolic acid (MPA), IVIG and both in combination. Cell proliferation and apoptosis were detected by 3H-thymidine incorporation and Annexin flow cytometry, respectively. RESULTS: IVIG (1-10mg/ml) or MPA (0.01-0.25 microg/ml) alone inhibited cell proliferation in the MLR in a dose-dependent manner. MPA at 0.01-0.03 microg/ml showed minimal inhibition, but the addition of 5 and 10mg/ml IVIG increased inhibition significantly (p<0.05) to 43% and 64%, respectively. Annexin V positive cell number was significantly higher in IVIG (5mg/ml) treated CD19+ cells (68+/-13% vs. 43+/-12%, p=0.001) compared to untreated cells and to a lesser degree in CD3+ cells (29+/-7% vs. 25+/-10 %, p=0.02). MPA (0.25-10 microg/ml) alone neither induced nor inhibited apoptosis. Addition of MPA had no effect on apoptosis induced by IVIG. CONCLUSION: 1) Combining low concentrations of IVIG (5-10 mg/ml) and MPA (0.01-0.03 microg/ml)has an additive effect on inhibition of cell proliferation in the MLR. 2) MPA alone neither induces nor inhibits apoptosis in T or B cells in the MLR, and has no effect on apoptosis induced by IVIG. These in vitro observations may have implications for modification of therapeutic approaches to protocols utilizing IVIG for desensitization and immune modulation.

Insertion of the designed helical linker led to increased expression of tf-based fusion proteins.

PURPOSE: To demonstrate a high-level expression of transferrin (Tf)-based fusion proteins by inserting a helical linker between two protein domains. METHODS: Tf-based fusion proteins were designed to contain oligonucleotides encoding a helical linker inserted between the protein domains. Plasmid constructs were transfected into HEK293 cells and the secreted fusion proteins were purified from conditioned serum free media. Expression was assessed using both SDS-PAGE and Western Blot using anti-hGH, G-CSF, or Tf antibodies; protein bands were analyzed using Quantity One software. The function of fusion proteins consisting of human growth hormone (hGH) and Tf was evaluated in Nb2 cell proliferation assays. RESULTS: The fusion proteins containing a helical linker, hGH-(H4)(2)-Tf and Tf-(H4)(2)-hGH, were expressed 1.7-and 2.4-fold higher, respectively, with a twofold lower ED(50) than the hGH-Tf fusion protein without a helical linker. The Tf-(H4)(2)-G-CSF fusion protein exhibited a greater expression with an 11.2-fold increase compared with Tf-G-CSF fusion protein. CONCLUSIONS: The helical linker introduced in Tf-fusion proteins resulted in a high-level of expression with improved in vitro bioactivity. This approach provides a simple method to increase poor expression of other fusion proteins.

Isolated heart and liver transplant recipients are at low risk for polyomavirus BKV nephropathy.

BACKGROUND: BKV infection and nephropathy is a significant cause of allograft dysfunction in kidney transplantation. BKV viremia, rather than viruria, corresponds to BKV nephropathy. The prevalence of BKV viremia in non-renal solid organ transplants has not been systematically evaluated. METHODS: We determined prevalence of BKV viremia in kidney, combined kidney-heart, kidney-liver, kidney-pancreas, kidney-heart-liver, and heart and liver transplant recipients using BKV-PCR. RESULTS: Seven out of 173 (4%) kidney transplant recipients had BKV viremia, with BKV>2 x 10(5) copies/mL in 6/7 and 1.9 x 10(3) in the remaining one patient. BKV viremia was not found in 24 heart transplant recipients, whereas 1/37 (2.7%) liver transplants showed low copy numbers (< or =10(3)). BKV-PCR< or =10(3) copies/mL were also found in one of each combined kidney-heart and kidney-liver transplant recipients. BKV nephropathy was proven by biopsy in 4/6 patients with high BKV viral loads. All six patients showed renal dysfunction, requiring reduction in immunosuppression and antiviral therapy. All four patients with low BKV viral loads (1.9 x 10(3) or < or =10(3)) showed stable renal function after reduction of immunosuppression or no treatment, respectively. CONCLUSION: Higher BKV levels in plasma are associated with renal dysfunction. Kidney transplant recipients are at high risk compared with recipients of isolated heart or liver allografts, for development of BKV nephropathy.

Cellular immune responses to cytomegalovirus in renal transplant recipients.

Control of CMV replication depends primarily on anti-CMV T lymphocyte activity. However, the functional T-cell responses to CMV in immunosuppressed solid organ transplant recipients are not well understood. In this study we employed cytokine flowcytometry (CFC) using pooled CMV peptides and viral lysates to detect CMV-specific T-cell responses in 17 healthy controls, 33 stable renal transplant recipients (Tx recipients) and 6 transplant recipients with active CMV infection (CMV(+)). We found that pooled peptides and lysates provide optimal detection of IFN gamma production in anti-CMV CD8(+) and CD4(+) T cells, respectively. In both healthy controls and Tx recipients, CMV-specific T-cell levels strongly correlated with serostatus. Seropositive Tx recipients have significantly higher levels of CMV-specific CD8(+) T-cell responses compared to healthy controls, which may signify an effort to control enhanced viral replication in immunosuppressed Tx recipients. In some individuals, absence of anti-CMV T-cell response may correlate with lack of viral clearance by ganciclovir therapy, even when CMV isolates are not ganciclovir resistant. Thus, monitoring cellular immunity with CFC along with viral load by PCR merits further exploration for identification of patients at the risk of developing CMV disease, tailoring prophylactic and therapeutic decisions and preventing complications.