Regression of Peritubular Capillaries Coincides with Angiogenesis and Renal Cyst Growth in Experimental Polycystic Kidney Disease.

BACKGROUND/AIM: The natural history of the renal microvasculature changes in PKD is not known. The aim of this study was to test the hypothesis that angiogenesis is coupled with kidney cyst expansion, and the loss of peritubular capillary networks precedes the onset of interstitial fibrosis. METHODS: The renal microvasculature (RECA-1 and CD34) was evaluated in groups of Lewis polycystic kidney (LPK) rats and juvenile cystic kidney (jck) mice during the early, mid and late stage of disease. In addition, LPK rats and jck mice received sirolimus to determine if the reduction in renal cyst growth is in part mediated by the suppression of angiogenesis. RESULTS: In LPK rats, the loss of peritubular capillaries occurred in early-stage disease and paralleled cyst formation whereas in jck mice it was delayed to the mid stage. In both models, vasa recta were displaced by growing cysts and regressed in LPK rats with disease progression but lengthened in jck mice. Cortical and medullary capillary neoangiogenesis occurred during the early stage in both models and persisted with progression. Treatment with sirolimus reduced cyst enlargement but did not alter the progression of renal microvasculature changes in either model. CONCLUSION: Regression of peritubular capillaries and disruption of vasa recta occur in parallel with angiogenesis and the progressive enlargement of kidney cysts. These data suggest that the regrowth of peritubular capillaries together with inhibition of angiogenesis are potential strategies to be considered in the treatment of PKD.

Role of cyclin-dependent kinase 2 in the progression of mouse juvenile cystic kidney disease.

A hallmark of polycystic kidney diseases (PKDs) is aberrant proliferation, which leads to the formation and growth of renal cysts. Proliferation is mediated by cyclin-dependent kinases (Cdks), and the administration of roscovitine (a pan-Cdk inhibitor) attenuates renal cystic disease in juvenile cystic kidney (jck) mice. Cdk2 is a key regulator of cell proliferation, but its specific role in PKD remains unknown. The aim of this study was to test the hypothesis that Cdk2 deficiency reduces renal cyst growth in PKD. Three studies were undertaken: (i) a time course (days 28, 56, and 84) of cyclin and Cdk activity was examined in jck mice and compared with wild-type mice; (ii) the progression was compared in jck mice with or without Cdk2 ablation from birth; and (iii) the effect of sirolimus (an antiproliferative agent) on Cdk2 activity in jck mice was investigated. Renal disease in jck mice was characterized by diffuse tubular cyst growth, interstitial inflammation and fibrosis, and renal impairment, peaking on day 84. Renal cell proliferation peaked during earlier stages of disease (days 28-56), whereas the expression of Cdk2-cyclin partners (A and E) and Cdk1 and 2 activity, was maximal in the later stages of disease (days 56-84). Cdk2 ablation did not attenuate renal disease progression and was associated with persistent Cdk1 activity. In contrast, the postnatal treatment of jck mice with sirolimus reduced both Cdk2 and Cdk1 activity and reduced renal cyst growth. In conclusion, (i) the kinetics of Cdk2 and Cdk2-cyclin partners did not correlate with proliferation in jck mice; and (ii) the absence of Cdk2 did not alter renal cyst growth, most likely due to compensation by Cdk1. Taken together, these data suggest that Cdk2 is dispensable for the proliferation of cystic epithelial cells and progression of PKD.

Establishment and Operation of a Third-Party Virus-Specific T Cell Bank within an Allogeneic Stem Cell Transplant Program.

Hematopoietic stem cell transplantation (HSCT) donor-generated virus-specific T cells (VSTs) can provide effective treatment for viral infection post-HSCT but are not readily accessible to all patients. Off-the-shelf cryopreserved VSTs suitable for treatment of multiple patients are an attractive alternative. We generated a bank of 17 cytomegalovirus (CMV)-, 14 Epstein-Barr virus (EBV)-, and 15 adenovirus (AdV)-specific T cell products from 30 third-party donors. Donors were selected for expression of 6 core HLA antigens expressed at high frequency in the local transplant population. T cells were generated by co-culturing venous blood or mobilized hematopoietic stem cell (HSC)-derived mononuclear cells with monocyte-derived dendritic cells pulsed with overlapping peptides covering CMV pp65, AdV5 hexon, or EBV BZLF1/LMP2A/EBNA1 proteins. Addition of a CD14+ selection step instead of plate adherence to isolate monocytes before culture initiation significantly improved expansion in cultures from HSC material. Phenotyping showed the CD8+ subset to have significantly higher numbers of terminal effector T cells (CD45RA+62L-) and lower numbers of effector memory T cells (CD45RA-62L-) when compared with the CD4+ subset. Increased expression of the immunoinhibitory markers PD-1 and TIM-3 was noted on CD4+ but not CD8+ cells when compared with the control group. VST showed antiviral activity restricted through a variety of common HLAs, and modelling suggested a suitably HLA-matched product would be available for >90% of HSCT patients. Only a small number of carefully selected third-party donors are required to generate a VST bank of broad coverage, indicating the feasibility of local banking integrated into existing allogeneic HSCT programs.

Adjuvant Peptide Pulsed Dendritic Cell Vaccination in Addition to T Cell Adoptive Immunotherapy for Cytomegalovirus Infection in Allogeneic Hematopoietic Stem Cell Transplantation Recipients.

Adoptive cellular immunotherapy has been shown to be effective in the management of cytomegalovirus (CMV) reactivation and disease. Whether adjuvant dendritic cell (DC) vaccination will provide additional benefit in prophylaxis or treatment of CMV in hematoietic cell transplantation (HSCT) recipients is unknown. In this study, we administered prophylactic CMV-peptide specific T cell infusions, followed by 2 doses of intradermal CMV peptide-pulsed DC vaccine, to 4 HSCT recipients. There were no immediate adverse events associated with T cell infusion or DC vaccinations. One of the 4 patients developed grade III acute gut graft-versus-host disease. Immune reconstitution against CMV was detected in all 4 patients. Patients receiving CMV peptide-specific T cells and DC vaccination had peak immune reconstitution at least 10 days after the second DC vaccination. In summary, combining DC vaccine with T cell infusion appears feasible, although further study is required to ascertain its safety and efficacy in augmenting the effects of infusing donor-derived CMV-specific T cells.

Long-term control of recurrent or refractory viral infections after allogeneic HSCT with third-party virus-specific T cells.

Donor-derived adoptive T-cell therapy is a safe and effective treatment of viral infection posttransplant, but it is limited by donor serostatus and availability and by its personalized nature. Off-the-shelf, third-party virus-specific T cells (VSTs) appear promising, but the long-term safety and durability of responses have yet to be established. We conducted a prospective study of 30 allogeneic hemopoietic stem cell transplant (HSCT) patients with persistent or recurrent cytomegalovirus (CMV) (n = 28), Epstein-Barr virus (n = 1), or adenovirus (n = 1) after standard therapy. Patients were treated with infusions of partially HLA-matched, third-party, ex vivo-expanded VSTs (total = 50 infusions) at a median of 75 days post-HSCT (range, 37 to 349 days). Safety, viral dynamics, and immune recovery were monitored for 12 months. Infusions were safe and well tolerated. Acute graft versus host disease occurred in 2 patients, despite a median HLA match between VSTs and the recipient of 2 of 6 antigens. At 12 months, the cumulative incidence of overall response was 93%. Virological control was durable in the majority of patients; the reintroduction of antiviral therapy after the final infusion occurred in 5 patients. CMV-specific T-cell immunity rose significantly and coincided with a rise in CD8+ terminal effector cells. PD-1 expression was elevated on CD8+ lymphocytes before the administration of third-party T cells and remained elevated at the time of viral control. Third-party VSTs show prolonged benefit, with virological control achieved in association with the recovery of CD8+ effector T cells possibly facilitated by VST infusion. This trial was registered at www.clinicaltrials.gov as #NCT02779439 and www.anzctr.org.au as #ACTRN12613000603718.

Addition of varicella zoster virus-specific T cells to cytomegalovirus, Epstein-Barr virus and adenovirus tri-specific T cells as adoptive immunotherapy in patients undergoing allogeneic hematopoietic stem cell transplantation.

BACKGROUND AIMS: Virus-specific T-cell immunotherapy is emerging as a promising management strategy for virus infections in patients after hematopoietic stem cell transplant (HSCT). Here we present outcomes of 10 adult patients who received multi-virus-specific T cells prophylactically after HSCT. METHODS: Donor-derived cytomegalovirus (CMV)-, Epstein-Barr virus (EBV)-, adenoviral- and varicella zoster virus (VZV)-specific T cells were generated in a single culture and administered to HSCT patients at a dose of 2 × 10(7)/m(2) virus-specific T cells at a median of 63 days post-transplant. Patients were monitored for 12 months for evidence of viral reactivation and graft-versus-host disease. RESULTS: There was no acute infusion-related toxicity. Six patients developed CMV reactivation after T-cell infusion with a median peak CMV DNA titer of 600 copies per milliliter, and 1 received CMV-specific pharmacotherapy post-infusion. No EBV, adenoviral or VZV reactivation or disease was reported. Using interferon-γ Elispot analysis on post-infusion samples, we identified anti-viral immunity against all viruses including VZV. Three patients (30%) developed grade II-IV acute graft-versus-host disease. CONCLUSIONS: This is the first description of the use of a multi-virus-specific T-cell product containing cells specific for VZV after allogeneic HSCT. The T-cell product appears safe in the setting of HSCT and confirms our previous findings regarding CMV control and treatment. A larger study with longer follow-up is required to determine the efficacy of VZV-specific T cells given prophylactically in controlling episodes of herpes zoster and disseminated varicella infection after cessation of prophylactic anti-viral treatment.

Central implementation strategies outperform local ones in improving HIV testing in Veterans Healthcare Administration facilities.

BACKGROUND: Pilot data suggest that a multifaceted approach may increase HIV testing rates, but the scalability of this approach and the level of support needed for successful implementation remain unknown. OBJECTIVE: To evaluate the effectiveness of a scaled-up multi-component intervention in increasing the rate of risk-based and routine HIV diagnostic testing in primary care clinics and the impact of differing levels of program support. DESIGN: Three arm, quasi-experimental implementation research study. SETTING: Veterans Health Administration (VHA) facilities. PATIENTS: Persons receiving primary care between June 2009 and September 2011 INTERVENTION: A multimodal program, including a real-time electronic clinical reminder to facilitate HIV testing, provider feedback reports and provider education, was implemented in Central and Local Arm Sites; sites in the Central Arm also received ongoing programmatic support. Control Arm sites had no intervention MAIN MEASURES: Frequency of performing HIV testing during the 6 months before and after implementation of a risk-based clinical reminder (phase I) or routine clinical reminder (phase II). KEY RESULTS: The adjusted rate of risk-based testing increased by 0.4 %, 5.6 % and 10.1 % in the Control, Local and Central Arms, respectively (all comparisons, p < 0.01). During phase II, the adjusted rate of routine testing increased by 1.1 %, 6.3 % and 9.2 % in the Control, Local and Central Arms, respectively (all comparisons, p < 0.01). At study end, 70-80 % of patients had been offered an HIV test. CONCLUSIONS: Use of clinical reminders, provider feedback, education and social marketing significantly increased the frequency at which HIV testing is offered and performed in VHA facilities. These findings support a multimodal approach toward achieving the goal of having every American know their HIV status as a matter of routine clinical practice.

Cytomegalovirus-specific cytotoxic T lymphocytes can be efficiently expanded from granulocyte colony-stimulating factor-mobilized hemopoietic progenitor cell products ex vivo and safely transferred to stem cell transplantation recipients to facilitate immune reconstitution.

Uncontrolled cytomegalovirus (CMV) reactivation after allogeneic hematopoietic stem cell transplantation causes significant morbidity and mortality. Adoptive transfer of CMV-specific cytotoxic T lymphocytes (CTLs) is a promising therapy to treat reactivation and prevent viral disease. In this article, we describe the generation of clinical-grade CMV-specific CTLs directly from granulocyte colony-stimulating factor-mobilized hemopoietic progenitor cell (G-HPC) products collected for transplantation. This method requires less than 2.5% of a typical G-HPC product to reproducibly expand CMV-specific CTLs ex vivo. Comparison of 11 CMV CTL lines generated from G-HPC products with 52 CMV CTL lines generated from nonmobilized peripheral blood revealed similar expansion kinetics and phenotype. G-HPC-derived CTLs produced IFN-γ after reexposure to CMVpp65 antigen and exhibited CMV-directed cytotoxicity but no alloreactivity against transplantation recipient-derived cells. Seven patients received CMV-specific CTL lines expanded from G-HPC products in a prophylactic adoptive immunotherapy phase I/II clinical trial. Use of G-HPC products will facilitate integration of CTL generation into established quality systems of transplantation centers and more rapid inclusion of T cell therapies into routine clinical care.

Donor-derived CMV-specific T cells reduce the requirement for CMV-directed pharmacotherapy after allogeneic stem cell transplantation.

We investigated the use of adoptively transferred donor-derived cytomegalovirus (CMV) specific cytotoxic T lymphocytes (CTL) as immune reconstitution postallogeneic transplant in a phase 2 study. Fifty patients were infused with a single dose of 2 × 10(7)cells/m(2) after day 28 post-transplant. Twenty-six patients reactivated CMV posttransplant (only 5 post-CTL infusion) and 9 required therapy with ganciclovir or foscarnet (only 1 post-CTL infusion). There was 1 case of fatal CMV disease, attributable to high levels of antithymocyte globulin at the time of T cell infusion. We compared the patients in the phase 2 study with a group of contemporaneous controls also treated at the trial centers. There was no increase in acute or chronic graft-versus-host disease attributable to CTL infusion; overall and progression-free survival were similar in both groups. There was a reduction in the percentage of patients who required CMV directed antiviral therapy (17% vs 36%, P = .01) and in the total number of treatment days in the cohort receiving CTL (3.4 days vs 8.9 days, P = .03) without a reduction in CMV reactivation rates. We postulate that adoptively transferred cells are able to expand in response to viral antigen, limit viral replication, and prevent progression to tissue infection. This study was registered on the Australian Clinical Trial Registry as #ACTRN12605000213640 and #ACTRN12607000224426.

Early cyst growth is associated with the increased nuclear expression of cyclin D1/Rb protein in an autosomal-recessive polycystic kidney disease rat model.

BACKGROUND: In this study we hypothesised that proliferation, and the increased expression of G(1)-phase cyclins (D1, E) and phosphorylated retinoblastoma protein (p-Rb) is restricted to the early period of synchronized cyst growth in autosomal-recessive polycystic kidney disease (ARPKD). METHODS: Lewis polycystic kidney disease (lpk) rats (model of ARPKD; postnatal weeks 1, 3, 6, 12 and 24; n = 6 each) as well as human juvenile cystic renal disease tissue (n = 2) were examined. RESULTS: Between weeks 1 and 3, the percentage cyst area increased 6-fold in lpk rats, followed by a more progressive rise (1.5-fold increase) until week 24. The number of Ki-67-, cyclin D1- and p-Rb-positive cells increased in lpk rats and peaked at week 3, declining thereafter. By serial sections, cysts co-expressed Ki-67, cyclin D1 and p-Rb. The expression of cyclin E was variable, and peaked at week 24. In human tissue, small cysts had a higher expression of p-Rb. CONCLUSION: Proliferation and the increased nuclear expression of cyclin D1 and p-Rb coincide with the early phase of cyst growth in rats and humans, suggesting that there might be a therapeutic window in which cyclin-dependent kinase inhibitors are most effective in preventing kidney enlargement in ARPKD.

A provider participatory implementation model for HIV testing in an ED.

BACKGROUND: The Centers for Disease Control and Prevention recommends routine HIV screening for adults. OBJECTIVES: Community-based participatory research incorporates subjects in the design and conduct of research. We included nurses and physicians in the implementation of HIV rapid test use in the emergency department (ED). We explored the process, facilitators, and barriers. METHODS: We identified clinical champions and trained staff. Physicians obtained consent and ordered HIV testing; nurses performed rapid testing. Testing rates were tracked by electronic medical record. We conducted regular meetings between staff and researchers. Semistructured qualitative interviews with providers were conducted at 3 months. RESULTS: By week 15, we administered 121 tests. After the eligibility protocol evolved to incorporate ED nursing concerns regarding staffing limitations from a random sampling model to one focused on testing during nonpeak hours, the weekly number of tests increased. Eighteen percent of providers favored nontargeted HIV screening, 27% favored the current model of testing at nonpeak hours, 32% supported diagnostic testing, and 18% favored no testing or "other." Barriers include written consent, electronic documentation, time constraints, and belief that screening is not a core ED duty. Facilitators include ease of test administration, belief that ED patients are at higher risk, and flexibility to tailor screening efforts according to patient volume. CONCLUSIONS: The ED-based HIV testing is feasible within a Veterans Hospital Administration setting. Involvement of nursing in a community-based participatory research implementation model may facilitate staff acceptance of nontargeted HIV screening and be a mechanism to initiate administration of clinical preventive services to ED patients with limited primary care contact.

Therapeutic role of sirolimus in non-transplant kidney disease.

Sirolimus is a member of a novel class of immunosuppressant drug that potently suppresses T cell proliferation and expansion by inhibition of the Target of Rapamycin Complex 1 (TORC1) protein kinase. Sirolimus also has anti-proliferative effects on intrinsic cells of the kidney, and increasing evidence suggests that it may have a therapeutic role in non-transplant renal diseases. In the normal kidney, sirolimus is considered to be non-nephrotoxic. In the diseased kidney, sirolimus may be beneficial or detrimental, depending on the type of renal injury. In polycystic kidney disease, TORC1 activation mediates renal tubular epithelial cell (TEC) proliferation and cyst growth in animals, and Phase III clinical trials are underway to determine the effect of sirolimus in attenuating disease progression in humans. In contrast, in acute kidney injury, sirolimus transiently impairs proximal TEC regeneration and delays renal recovery. In animal models of lupus nephritis and diabetic kidney disease, sirolimus prevents disease progression. However, the efficacy of sirolimus in human glomerulonephritis as well as in diabetic chronic kidney disease remains unclear, as it paradoxically exacerbates renal dysfunction when the baseline glomerular filtration rate is low (< 40 ml/min/1.73 m(2)) and there is heavy proteinuria (> 300 mg/day). This may, in part, be due to inhibition of compensatory glomerular capillary repair through the suppression of endothelial cell proliferation and angiogenic growth factor production by podocytes. Therefore, at present, polycystic kidney disease is the most promising therapeutic application for sirolimus in non-transplant renal diseases, and further studies are needed to clarify its role in other situations.

Requirement of MyD88 for macrophage-mediated islet xenograft rejection after adoptive transfer.

BACKGROUND: Porcine antigen primed and CD4+ T-cell activated macrophages are able to migrate to and destroy porcine xenografts. However, the specific signaling mechanisms involved remain to be identified. METHODS: In this study macrophages which lack the universal toll-like receptor (TLR) adaptor MyD88 were used to investigate the role of TLR in the recognition and activation of macrophages in islet xenograft rejection. Macrophages were isolated from rejecting MyD88(-/-) and wild-type C57BL/6 mice that were recipients of neonatal porcine pancreatic cell cluster (NPCC) xenografts, and were transferred to NPCC recipient NOD-SCID mice. RESULTS: Both wild-type C57BL/6 and MyD88(-/-) mice rejected NPCC xenografts 8 and 10 days, respectively after transplantation, and the grafts were heavily infiltrated with CD4+ T cells and macrophages. However, graft infiltrating macrophages from rejecting MyD88(-/-) recipients demonstrated impaired up-regulation of TLR expression and impaired activation phenotype, when compared to those from rejecting C57BL/6 recipients. Transfer of NOD-SCID recipients with macrophages from rejecting C57BL/6 mice resulted in NPCC xenograft rejection along with massively infiltrated macrophages 8 days after transfer, whereas NPCC xenografts in NOD-SCID mice transferred with macrophages from rejecting MyD88(-/-) mice remained intact until the end of this study, 90 days after transfer, with insulin-positive islets and no infiltration by macrophages. CONCLUSION: This study demonstrates that deletion of MyD88 causes impaired macrophage activation after pig islet xenotransplantation. However, graft survival is not prolonged and xenografts are rejected rapidly by alternate mechanisms.

A training programme for prevention of occupational exposure to bloodborne pathogens: impact on knowledge, behaviour and incidence of needle stick injuries among student nurses in Changsha, People's Republic of China.

BACKGROUND: Although the human immunodeficiency virus (HIV) epidemic in China is expanding rapidly, the principles of universal precautions (UP) are not widely disseminated in Chinese hospitals. Lack of training about occupational prevention of bloodborne pathogens (BBP) may place student nurses at risk when they are in clinical practice. AIM: To examine the impact of structured training on prevention of occupational exposure to BBP on knowledge, behaviour, and incidence of medical sharp injuries among student nurses in Changsha, China. METHODS: A quasi-experimental study evaluated changes in knowledge, self-reported UP behaviours, observed adherence to UP, and needle stick/sharp injuries during a 4-month follow-up period. The study population consisted of 106 student nurses in two classes. One class served as the experimental group, while the other served as a control group. Students in the experimental group participated in a structured training intervention consisting of lectures and demonstrations. RESULTS: At 4 months, the group that received the BBP training scored significantly higher than the standard education group on both knowledge (P < 0.001) and behaviour (P = 0.002). Although students in the experimental group were not observed to practise UP significantly more frequently than those in the control group, they were less likely to experience needle stick/sharp injuries (OR = 0.29; 95% CI 0.11, 0.74; P = 0.004). CONCLUSION: Structured training in prevention of occupational exposure to BBP improved knowledge and behaviour and reduced the number of needle stick/sharp injuries among Chinese student nurses, compared with students who did not receive the training. Training in the techniques of UP could play a role in reducing the risk for occupational exposure to BBP among Chinese future nurses. In view of the accelerating HIV epidemic, implementation and evaluation of such training programmes are urgently needed.