Cytomegalovirus Immunoglobulin After Thoracic Transplantation: An Overview

Cytomegalovirus (CMV) is a highly complex pathogen which, despite modern prophylactic regimens, continues to affect a high proportion of thoracic organ transplant recipients. The symptomatic manifestations of CMV infection are compounded by adverse indirect effects induced by the multiple immunomodulatory actions of CMV. These include a higher risk of acute rejection, cardiac allograft vasculopathy after heart transplantation, and potentially bronchiolitis obliterans syndrome in lung transplant recipients, with a greater propensity for opportunistic secondary infections. Prophylaxis for CMV using antiviral agents (typically oral valganciclovir or intravenous ganciclovir) is now almost universal, at least in high-risk transplants (D+/R-). Even with extended prophylactic regimens, however, challenges remain. The CMV events can still occur despite antiviral prophylaxis, including late-onset infection or recurrent disease, and patients with ganciclovir-resistant CMV infection or who are intolerant to antiviral therapy require alternative strategies. The CMV immunoglobulin (CMVIG) and antiviral agents have complementary modes of action. High-titer CMVIG preparations provide passive CMV-specific immunity but also exert complex immunomodulatory properties which augment the antiviral effect of antiviral agents and offer the potential to suppress the indirect effects of CMV infection. This supplement discusses the available data concerning the immunological and clinical effects of CMVIG after heart or lung transplantation

Immune reconstitution inflammatory syndrome in patients starting antiretroviral therapy for HIV infection: a systematic review and meta-analysis

In patients with HIV-1 infection who are starting combination antiretroviral therapy (ART), the incidence of immune reconstitution inflammatory syndrome (IRIS) is not well defined. We did a meta-analysis to establish the incidence and lethality of the syndrome in patients with a range of previously diagnosed opportunistic infections, and examined the relation between occurrence and the degree of immunodeficiency. Systematic review identified 54 cohort studies of 13 103 patients starting ART, of whom 1699 developed IRIS. We calculated pooled cumulative incidences with 95% credibility intervals (CrI) by Bayesian methods and did a random-effects metaregression to analyse the relation between CD4 cell count and incidence of IRIS. In patients with previously diagnosed AIDS-defining illnesses, IRIS developed in 37.7% (95% CrI 26.6-49.4) of those with cytomegalovirus retinitis, 19.5% (6.7-44.8) of those with cryptococcal meningitis, 15.7% (9.7-24.5) of those with tuberculosis, 16.7% (2.3-50.7) of those with progressive multifocal leukoencephalopathy, and 6.4% (1.2-24.7) of those with Kaposi's sarcoma, and 12.2% (6.8-19.6) of those with herpes zoster. 16.1% (11.1-22.9) of unselected patients starting ART developed any type of IRIS. 4.5% (2.1-8.6) of patients with any type of IRIS died, 3.2% (0.7-9.2) of those with tuberculosis-associated IRIS died, and 20.8% (5.0-52.7) of those with cryptococcal meningitis died. Metaregression analyses showed that the risk of IRIS is associated with CD4 cell count at the start of ART, with a high risk in patients with fewer than 50 cells per microL. Occurrence of IRIS might therefore be reduced by initiation of ART before immunodeficiency becomes advanced.

Prolonged amelioration of acute lung allograft rejection by sequential overexpression of human interleukin-10 and hepatocyte growth factor in rats

The effect of prolonged electroporation-mediated human interleukin-10 (hIL-10) overexpression 24 hours before transplantation, combined with sequential human hepatocyte growth factor (HGF) overexpression into skeletal muscle on day 5, on rat lung allograft rejection was evaluated. Left lung allotransplantation was performed from Brown-Norway to Fischer-F344 rats. Gene transfer into skeletal muscle was enhanced by electroporation. Three groups were studied: group I animals (n = 5) received 2.5 μg pCIK-hIL-10 (hIL-10/CMV [cytomegalovirus] early promoter enhancer) on day -1 and 80 μg pCIK-HGF (HGF/CMV early promoter enhancer) on day 5. Group II animals (n = 4) received 2.5 μg pCIK-hIL-10 and pUbC-hIL-10 (hIL-10/pUbC promoter) on day -1. Control group III animals (n = 4) were treated by sham electroporation on days -1 and 5. All animals received daily nontherapeutic intraperitoneal dose of cyclosporin A (2.5 mg/kg) and were sacrificed on day 15. Graft oxygenation and allograft rejection were evaluated. Significant differences were found between study groups in graft oxygenation (Pao(2)) (P = .0028; group I vs. groups II and III, P < .01 each). Pao(2) was low in group II (31 ± 1 mm Hg) and in group III controls (34 ± 10 mm Hg), without statistically significant difference between these 2 groups (P = .54). In contrast, in group I, Pao(2) of recipients sequentially transduced with IL-10 and HGF plasmids was much improved, with 112 ± 39 mm Hg (vs. groups II and III; P < .01 each), paralleled by reduced vascular and bronchial rejection (group I vs. groups II and III, P < .021 each). Sequential overexpression of anti-inflammatory cytokine IL-10, followed by sequential and overlapping HGF overexpression on day 5, preserves lung function and reduces acute lung allograft rejection up to day 15 post transplant as compared to prolonged IL-10 overexpression alone.

Sensitive and rapid detection of ganciclovir resistance by PCR based MALDI-TOF analysis

Cytomegalovirus (CMV) infection is associated with significant morbidity and mortality in transplant recipients. Resistance against ganciclovir is increasingly observed. According to current guidelines, direct drug resistance testing is not always performed due to high costs and work effort, even when resistance is suspected.

Clinical Characteristics of Children with Viral Single- and Co-Infections and a Petechial Rash

BACKGROUND:: Children with petechial rash are more likely to undergo invasive diagnostics, to be treated with antibiotics for potential bacterial infection and to be hospitalized. However, viruses have also been associated with petechial rash. Nonetheless, a systematic analysis of viral infections with modern available techniques as quantitative real time polymerase chain reaction (q-PCR) in the context of petechial rash is lacking. The purpose of this pediatric study was to prospectively uncover viral pathogens that may promote the emergence of petechiae and to analyze the correlation with the clinical characteristics and course. METHODS:: We conducted a prospective study in children (0 to 18 years) presenting with petechiae and signs or symptoms of infection at the emergency department between November 2009 and March 2012. In nasopharyngeal aspirates the following viruses were analyzed by q-PCR: Cytomegalovirus, Epstein-Barr virus, parvovirus B19, Influenza A and B, parainfluenza viruses, human respiratory syncytial virus A and B, human metapneumovirus, rhinovirus, enterovirus, adenovirus, human coronavirus OC43, 229E, NL63 and human bocavirus. RESULTS:: A viral pathogen was identified in 67% of the analyzed 58 cases with petechial rash. Virus positive patients showed a significantly higher incidence of lower respiratory tract infections. Forty-one percent were viral co-infections, which were significantly younger than virus negative patients, had a higher leukocyte count and were hospitalized for a longer time. CONCLUSIONS:: A petechial rash is frequently associated viral single- and co-infections and can rapidly be identified via q-PCR.

Gene transfer of hepatocyte growth factor by electroporation reduces bleomycin-induced lung fibrosis

Abnormal alveolar wound repair contributes to the development of pulmonary fibrosis after lung injury. Hepatocyte growth factor (HGF) is a potent mitogenic factor for alveolar epithelial cells and may therefore improve alveolar epithelial repair in vitro and in vivo. We hypothesized that HGF could increase alveolar epithelial repair in vitro and improve pulmonary fibrosis in vivo. Alveolar wound repair in vitro was determined using an epithelial wound repair model with HGF-transfected A549 alveolar epithelial cells. Electroporation-mediated, nonviral gene transfer of HGF in vivo was performed 7 days after bleomycin-induced lung injury in the rat. Alveolar epithelial repair in vitro was increased after transfection of wounded epithelial monolayers with a plasmid encoding human HGF, pCikhHGF [human HGF (hHGF) gene expressed from the cytomegalovirus (CMV) immediate-early promoter and enhancer] compared with medium control. Electroporation-mediated in vivo HGF gene transfer using pCikhHGF 7 days after intratracheal bleomycin reduced pulmonary fibrosis as assessed by histology and hydroxyproline determination 14 days after bleomycin compared with controls treated with the same vector not containing the HGF sequence (pCik). Lung epithelial cell proliferation was increased and apoptosis reduced in hHGF-treated lungs compared with controls, suggesting increased alveolar epithelial repair in vivo. In addition, profibrotic transforming growth factor-beta1 (TGF-beta1) was decreased in hHGF-treated lungs, indicating an involvement of TGF-beta1 in hHGF-induced reduction of lung fibrosis. In conclusion, electroporation-mediated gene transfer of hHGF decreases bleomycin-induced pulmonary fibrosis, possibly by increasing alveolar epithelial cell proliferation and reducing apoptosis, resulting in improved alveolar wound repair.

Prolonged amelioration of acute lung allograft rejection by overexpression of human interleukin-10 under control of a long acting ubiquitin C promoter in rats

BACKGROUND: The prolonged effect of electroporation-mediated human interleukin-10 (hIL-10) overexpression in skeletal muscle under the control of the constitutional polyubiquitin C promoter (pUb hIL-10) on rat lung allograft rejection was evaluated. METHODS: Left lung allotransplantation was performed from Brown-Norway to Fischer-F344 rats. Either 2.5 mug pCIK hIL-10 (hIL-10/cytomegalovirus early promoter enhancer) alone (Group I/sacrifice Day 5 and II/sacrifice Day 10) or in combination with 2.5 mug pUb hIL-10 (hIL-10/UbC promoter; Group III/sacrifice Day 10) were injected into the tibialis anterior muscle of the recipient, followed by electroporation 24 hours before transplantation. Animals in Control Groups IV and V without gene transfer were euthanized on Day 5 and 10, respectively. All animals received a daily non-therapeutic dose of cyclosporine A (2.5 mg/kg). RESULTS: In Control Group IV, complete rejection (median A3B3) was noted on Day 5 with a Pao(2) of 43 +/- 9 mm Hg. In recipients of Control Group V, measurement of gas exchange on Day 10 and rejection grading was impossible because of complete destruction of the allograft. Group I animals on Day 5 (233 +/- 123 mm Hg; p = 0.02 vs Group IV) and Group II animals on Day 10 (150 +/- 139 mm Hg; p = 0.15 vs Group IV) demonstrated improved graft function. Graft function in Group III was further improved on Day 10 (299 +/- 123 mm Hg; p = 0.002 vs Group IV; p = 0.05 vs Group II; p = 0.36 vs Group I). Rejection was significantly reduced in Group III (median, A2B2) compared with Group II (median, A4B3; p < 0.05). CONCLUSIONS: Interleukin-10 overexpression under control of the constitutive ubiquitin C promoter ameliorates acute rejection and preserves lung graft function for a prolonged time.

In vivo electroporation and ubiquitin promoter--a protocol for sustained gene expression in the lung

BACKGROUND: Gene therapy applications require safe and efficient methods for gene transfer. Present methods are restricted by low efficiency and short duration of transgene expression. In vivo electroporation, a physical method of gene transfer, has evolved as an efficient method in recent years. We present a protocol involving electroporation combined with a long-acting promoter system for gene transfer to the lung. METHODS: The study was designed to evaluate electroporation-mediated gene transfer to the lung and to analyze a promoter system that allows prolonged transgene expression. A volume of 250 microl of purified plasmid DNA suspended in water was instilled into the left lung of anesthetized rats, followed by left thoracotomy and electroporation of the exposed left lung. Plasmids pCiKlux and pUblux expressing luciferase under the control of the cytomegalovirus immediate-early promoter/enhancer (CMV-IEPE) or human polyubiquitin c (Ubc) promoter were used. Electroporation conditions were optimized with four pulses (200 V/cm, 20 ms at 1 Hz) using flat plate electrodes. The animals were sacrificed at different time points up to day 40, after gene transfer. Gene expression was detected and quantified by bioluminescent reporter imaging (BLI) and relative light units per milligram of protein (RLU/mg) was measured by luminometer for p.Pyralis luciferase and immunohistochemistry, using an anti-luciferase antibody. RESULTS: Gene expression with the CMV-IEPE promoter was highest 24 h after gene transfer (2932+/-249.4 relative light units (RLU)/mg of total lung protein) and returned to baseline by day 3 (382+/-318 RLU/mg of total lung protein); at day 5 no expression was detected, whereas gene expression under the Ubc promoter was detected up to day 40 (1989+/-710 RLU/mg of total lung protein) with a peak at day 20 (2821+/-2092 RLU/mg of total lung protein). Arterial blood gas (PaO2), histological assessment and cytokine measurements showed no significant toxicity neither at day 1 nor at day 40. CONCLUSIONS: These results provide evidence that in vivo electroporation is a safe and effective tool for non-viral gene delivery to the lungs. If this method is used in combination with a long-acting promoter system, sustained transgene expression can be achieved.

Electroporation enhances reporter gene expression following delivery of naked plasmid DNA to the lung

BACKGROUND: Existing methods of non-viral airway gene transfer suffer from low levels of efficiency. Electroporation has been used to enhance gene transfer in a range of tissues. Here we assess the usefulness of electroporation for enhancing gene transfer in the lungs of mice and sheep. METHODS: Naked plasmid DNA (pDNA) expressing either luciferase or green fluorescent protein (GFP) was delivered to mouse lungs by instillation. Following surgical visualisation, the lungs were directly electroporated and the level and duration of luciferase activity was assessed and cell types that were positive for GFP were identified in lung cryosections. Naked pDNA was nebulised to the sheep lung and electrodes attached to the tip of a bronchoscope were used to electroporate airway segment bifurcations, Luciferase activity was assessed in electroporated and control non-electroporated regions, after 24 h. RESULTS: Following delivery of naked pDNA to the mouse lung, electroporation resulted in up to 400-fold higher luciferase activity than naked pDNA alone when luciferase was under the control of a cytomegalovirus (CMV) promoter. Following delivery of a plasmid containing the human polyubiquitin C (UbC) promoter, electroporation resulted in elevated luciferase activity for at least 28 days. Visualisation of GFP indicated that electroporation resulted in increased GFP detection compared with non-electroporated controls. In the sheep lung electroporation of defined sites in the airways resulted in luciferase activity 100-fold greater than naked pDNA alone. CONCLUSIONS: These results indicate that electroporation can be used to enhance gene transfer in the lungs of mice and sheep without compromising the duration of expression.

Infection with human herpesvirus 8 and transplant-associated gammopathy

BACKGROUND: The role of human herpesvirus (HHV)-8 in the pathogenesis of multiple myeloma and its pre-malignant state of monoclonal gammopathy is unclear. HHV-8 is transmitted by organ transplantation, representing a unique model with which to investigate primary HHV-8 infection. METHODS: The authors studied the incidence of clonal gammopathy in renal transplant recipients and correlated it with previous and recent HHV-8 infection. RESULTS: Clonal gammopathy was observed in 31 of 162 (19%) HHV-8-seronegative patients, in 5 of 17 (29%) HHV-8-seropositive patients, and in 9 of 24 (38%) HHV-8 seroconverters within 5 years after transplantation. Gammopathy was often transient, and no progression to myeloma was observed. Two patients with persistent gammopathy developed B-cell lymphoma. In a logistic regression model, HHV-8 serostatus of the graft recipient was significantly associated with subsequent development of gammopathy, with a relative risk (RR) of 1.9 and a 95% confidence interval (CI) of 0.5 to 6.4 for an HHV-8-seropositive recipient and an RR of 2.9 and a 95% CI of 1.01 to 8.0 for seroconverters as compared with baseline (HHV-8 seronegative). Other significant variables were cytomegalovirus (CMV) serostatus and the intensity of immunosuppression (RR of 10.4 and 95% CI of 2.6-41.7 for a CMV-negative recipient with a CMV-positive donor vs. a CMV-negative recipient with a CMV-negative donor and RR of 17.6 and 95% CI of 2.0-150.8 if OKT3 was used vs. no use of antilymphocytic substances). CONCLUSIONS: Transplant recipients with HHV-8 infection are more likely to develop clonal gammopathy. However, this risk is much lower than the risk conferred by CMV infection and antilymphocytic therapy, arguing against a major role of HHV-8 infection in the pathogenesis of clonal plasma cell proliferation.

Infection prevention strategies in a stem cell transplant unit: impact of change of care in isolation practice and routine use of high dose intravenous immunoglobulins on infectious complications and transplant related mortality

OBJECTIVE: Nursing in 'live islands' and routine high dose intravenous immunoglobulins after allogeneic hematopoietic stem cell transplantation were abandoned by many teams in view of limited evidence and high costs. METHODS: This retrospective single-center study examines the impact of change from nursing in 'live islands' to care in single rooms (SR) and from high dose to targeted intravenous immunoglobulins (IVIG) on mortality and infection rate of adult patients receiving an allogeneic stem cell or bone marrow transplantation in two steps and three time cohorts (1993-1997, 1997-2000, 2000-2003). RESULTS: Two hundred forty-eight allogeneic hematopoetic stem cell transplantations were performed in 227 patients. Patient characteristics were comparable in the three cohorts for gender, median age, underlying disease, and disease stage, prophylaxis for graft versus host disease (GvHD) and cytomegalovirus constellation. The incidence of infections (78.4%) and infection rates remained stable (rates/1000 days of neutropenia for sepsis 17.61, for pneumonia 6.76). Cumulative incidence of GvHD and transplant-related mortality did not change over time. CONCLUSIONS: Change from nursing in 'live islands' to SR and reduction of high dose to targeted IVIG did not result in increased infection rates or mortality despite an increase in patient age. These results support the current practice.

JC virus-specific immune responses in human immunodeficiency virus type 1 patients with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a frequently fatal disease caused by uncontrolled polyomavirus JC (JCV) in severely immunodeficient patients. We investigated the JCV-specific cellular and humoral immunity in the Swiss HIV Cohort Study. We identified PML cases (n = 29), as well as three matched controls per case (n = 87), with prospectively cryopreserved peripheral blood mononuclear cells and plasma at diagnosis. Nested controls were matched according to age, gender, CD4(+) T-cell count, and decline. Survivors (n = 18) were defined as being alive for >1 year after diagnosis. Using gamma interferon enzyme-linked immunospot assays, we found that JCV-specific T-cell responses were lower in nonsurvivors than in their matched controls (P = 0.08), which was highly significant for laboratory- and histologically confirmed PML cases (P = 0.004). No difference was found between PML survivors and controls or for cytomegalovirus-specific T-cell responses. PML survivors showed significant increases in JCV-specific T cells (P = 0.04) and immunoglobulin G (IgG) responses (P = 0.005). IgG responses in survivors were positively correlated with CD4(+) T-cell counts (P = 0.049) and negatively with human immunodeficiency virus RNA loads (P = 0.03). We conclude that PML nonsurvivors had selectively impaired JCV-specific T-cell responses compared to CD4(+) T-cell-matched controls and failed to mount JCV-specific antibody responses. JCV-specific T-cell and IgG responses may serve as prognostic markers for patients at risk.

Everolimus immunosuppression in de novo heart transplant recipients: What does the evidence tell us now?

The efficacy of everolimus with reduced cyclosporine in de novo heart transplant patients has been demonstrated convincingly in randomized studies. Moreover, everolimus-based immunosuppression in de novo heart transplant recipients has been shown in two randomized trials to reduce the increase in maximal intimal thickness based on intravascular ultrasound, indicating attenuation of cardiac allograft vasculopathy (CAV). Randomized trials of everolimus in de novo heart transplantation have also consistently shown reduced cytomegalovirus infection versus antimetabolite therapy. In maintenance heart transplantation, conversion from calcineurin inhibitors to everolimus has demonstrated a sustained improvement in renal function. In de novo patients, a renal benefit may only be achieved if there is an adequate reduction in exposure to calcineurin inhibitor therapy. Delayed introduction of everolimus may be appropriate in patients at high risk of wound healing complications, e.g. diabetic patients or patients with ventricular assist device. The current evidence base suggests that the most convincing reasons for use of everolimus from the time of heart transplantation are to slow the progression of CAV and to lower the risk of cytomegalovirus infection. A regimen of everolimus with reduced-exposure calcineurin inhibitor and steroids in de novo heart transplant patients represents a welcome addition to the therapeutic armamentarium.

The sequence of addition of IL-3 and IgE-dependent/IgE-independent stimuli regulates RANKL expression in human basophils

Background: Receptor Activator of Nuclear Factor kappaB Ligand (RANKL), a member of the TNF superfamily, contributes to the imbalance of bone resorption and immunoregulation in rheumatoid arthritis. In mice, collagen induced arthritis was exacerbated by IL-3 and anti-IgER antibodies, two mediators activating basophils that are known as effector cells of allergy. Interestingly, our unpublished microarray data revealed that IL-3 induces RANKL mRNA in human basophils. Here we further investigate under which conditions human basophils express surface and/or soluble RANKL. Methods: One part of purified human basophils was co-stimulated with IL-3 and either IgE-dependent or IgE-independent stimuli. The other part of purified basophils was first primed with IL-3 and subsequently triggered with IgE-dependent or IgE-independent stimuli. Expression of surface and soluble RANKL were detected by flow cytometry, ELISA and real-time PCR. Results: By flow cytometry we show that IL-3 induces de novo expression of surface RANKL on human basophils in a time and dose dependent manner. Co-stimulation of basophils with IL-3 and an IgE-dependent stimulus reduces IL-3-induced expression of surface RANKL in a dose dependent manner while IgE-independent stimuli have no effect. In contrast, both IgE-dependent and IgE-independent stimuli enhance expression of surface and soluble RANKL in basophils that were first primed with IL-3 and then triggered. Real-time PCR analysis shows that surface hRANKL1 and soluble hRANKL3 are induced by IL-3 and reduced by co-stimulation with IL-3 and an IgE-dependent stimulus and thus confirms our flow cytometry data. Conclusion: RANKL expression in human basophils is not only dependent on IL-3 and IgE-dependent/IgE-independent stimuli but also on the sequence of their addition to cell culture. Based on our data, we suggest that basophils might have previously unidentified functions in bone resorption or immunoregulation via RANKL.