Rilzabrutinib, an Oral BTK Inhibitor, in Immune Thrombocytopenia.

BACKGROUND: Rilzabrutinib, an oral, reversible covalent inhibitor of Bruton's tyrosine kinase, may increase platelet counts in patients with immune thrombocytopenia by means of dual mechanisms of action: decreased macrophage (Fcγ receptor)-mediated platelet destruction and reduced production of pathogenic autoantibodies. METHODS: In an international, adaptive, open-label, dose-finding, phase 1-2 clinical trial, we evaluated rilzabrutinib therapy in previously treated patients with immune thrombocytopenia. We used intrapatient dose escalation of oral rilzabrutinib over a period of 24 weeks; the lowest starting dose was 200 mg once daily, with higher starting doses of 400 mg once daily, 300 mg twice daily, and 400 mg twice daily. The primary end points were safety and platelet response (defined as at least two consecutive platelet counts of ≥50×103 per cubic millimeter and an increase from baseline of ≥20×103 per cubic millimeter without the use of rescue medication). RESULTS: Sixty patients were enrolled. At baseline, the median platelet count was 15×103 per cubic millimeter, the median duration of disease was 6.3 years, and patients had received a median of four different immune thrombocytopenia therapies previously. All the treatment-related adverse events were of grade 1 or 2 and transient. There were no treatment-related bleeding or thrombotic events of grade 2 or higher. At a median of 167.5 days (range, 4 to 293) of treatment, 24 of 60 patients (40%) overall and 18 of the 45 patients (40%) who had started rilzabrutinib treatment at the highest dose met the primary end point of platelet response. The median time to the first platelet count of at least 50×103 per cubic millimeter was 11.5 days. Among patients with a primary platelet response, the mean percentage of weeks with a platelet count of at least 50×103 per cubic millimeter was 65%. CONCLUSIONS: Rilzabrutinib was active and associated with only low-level toxic effects at all dose levels. The dose of 400 mg twice daily was identified as the dose for further testing. Overall, rilzabrutinib showed a rapid and durable clinical activity that improved with length of treatment. (Funded by Sanofi; ClinicalTrials.gov number, NCT03395210; EudraCT number, 2017-004012-19.).

A thorough QTc study to evaluate the effects of oral rilzabrutinib administered alone and with ritonavir in healthy subjects.

This study aimed to define the clinically relevant supratherapeutic dose of rilzabrutinib, an oral Bruton tyrosine kinase (BTK) inhibitor, and evaluate potential effects of therapeutic and supratherapeutic exposures on cardiac repolarization in healthy subjects. This was a two-part phase I study (anzctr.org.au ACTRN12618001036202). Part A was a randomized, open-label, three-period, single-dose crossover study (n = 12) with rilzabrutinib 100 mg ± ritonavir 100 mg or rilzabrutinib 1200 mg. Part B was a randomized, double-blind, placebo-controlled, four-way, single-dose crossover study (n = 39) with matched placebo, rilzabrutinib 400 mg ± ritonavir 100 mg, or moxifloxacin (positive control). Primary objectives: part A - pharmacokinetics (PK) of rilzabrutinib ± ritonavir, safety, and optimal dose for Part B; Part B - effect of rilzabrutinib therapeutic and supratherapeutic concentration on electrocardiogram (ECG) parameters. ECGs and PK samples were serially recorded before and post-dose. In part A, rilzabrutinib 100 mg + ritonavir led to 17-fold area under the concentration-time curve (AUC0-∞ ) and 7-fold maximum plasma concentration (Cmax ) increases over rilzabrutinib alone. Rilzabrutinib 1200 mg was discontinued due to mild-to-moderate gastrointestinal intolerance. In Part B, rilzabrutinib 400 mg + ritonavir increased rilzabrutinib mean AUC0-∞ from 454 to 3800 ng h/mL and Cmax from 144 to 712 ng/mL. The concentration-QTc relationship was slightly negative, shallow (-0.01 ms/ng/mL [90% CI -0.016 to -0.001]), and an effect >10 ms on QTcF could be excluded within the observed range of plasma concentrations, up to 2500 ng/mL. Safety was similar to other studies of rilzabrutinib. In conclusion, rilzabrutinib, even at supratherapeutic doses, had no clinically relevant effects on ECG parameters, including the QTc interval.

A phase I pharmacokinetics trial comparing PF-05280586 (a potential biosimilar) and rituximab in patients with active rheumatoid arthritis.

AIMS: Pharmacokinetic (PK) similarity was assessed among PF-05280586 (a proposed biosimilar) vs. rituximab sourced from the European Union (rituximab-EU) and the United States (rituximab-US). Pharmacodynamics (PD), overall safety and immunogenicity were also evaluated. METHODS: Patients with active rheumatoid arthritis on a background of methotrexate and inadequate response to one or more tumour necrosis factor antagonist therapies were randomized to intravenous PF-05280586, rituximab-EU or rituximab-US 1000 mg doses on study days 1 and 15. RESULTS: A total of 220 patients were randomized to receive study treatment as assigned. Of these, 198 met per-protocol population criteria for inclusion in the PK data analysis. PF-05280586, rituximab-EU and rituximab-US exhibited similar PK profiles following administration of assigned study drug on days 1 and 15. The 90% confidence intervals of test-to-reference ratios for Cmax , AUCT , AUC0-∞ and AUC2-week were within the bioequivalence margin of 80.00-125.00% for comparisons of PF-05280586 with rituximab-EU, PF-05280586 with rituximab-US, and rituximab-EU with rituximab-US. All treatments resulted in a rapid and profound reduction in CD19+ B cells and sustained profound B cell suppression up to week 25. The incidence of antidrug antibody (ADA) response (n = 7, 10 and 9 for PF-05280586, rituximab-EU and rituximab-US, respectively), time to ADA emergence and ADA titres were similar across treatments. None of the ADA-positive samples was positive for neutralizing activity. No clinically meaningful differences in adverse events were identified. CONCLUSIONS: The study demonstrated PK similarity among PF-05280586, rituximab-EU and rituximab-US. In addition, all treatments showed comparable CD19+ B cell depletion PD responses, as well as safety and immunogenicity profiles.

Development of biosimilars in an era of oncologic drug shortages.

Acute and chronic shortages of various pharmaceuticals and particularly of sterile injectable products are being reported on a global scale, prompting evaluation of more effective strategies to manage current shortages and development of new, high-quality pharmaceutical products to mitigate the risk of potential future shortages. Oncology drugs such as liposomal doxorubicin and 5-fluorouracil represent examples of first-choice drugs critically affected by shortages. Survey results indicate that the majority of hospitals and practicing oncologists have experienced drug shortages, which may have compromised patient safety and clinical outcomes, and increased health care costs, due to delays or changes in treatment regimens. Clinical trials evaluating novel agents in combination with standard-of-care drugs are also being affected by drug shortages. Clinical and ethical considerations on treatment objectives, drug indication, and availability of alternative options may help in prioritizing cancer patients involved in active drug shortages. The United States Food and Drug Administration and the European Medicines Agency have identified manufacturing problems, delays in supply, and lack of available active ingredients as the most frequent causes of recent or ongoing drug shortages, and have released specific guidance to monitor, manage, and reduce the risk of shortages. The upcoming loss of exclusivity for a number of anticancer biologics, together with the introduction of an abbreviated approval pathway for biosimilars, raises the question of whether these products will be vulnerable to shortages. Future supply by reliable manufacturers of well characterized biosimilar monoclonal antibodies, developed in compliance with regulatory and manufacturing guidelines and with substantial investments, may contribute to prevent future biologics shortages and ensure access to effective and safe treatment options for patients with cancer. Preclinical and clinical characterization is ongoing for potential biosimilars of trastuzumab, rituximab, and bevacizumab, with promising results.

Clinical considerations for the development of biosimilars in oncology.

Despite availability of biologic therapies, limited patient access to many of the most-effective cancer treatments affects overall health outcomes. To address this issue, many governments have enacted legislation for the approval of biosimilars. The term "biosimilar" refers to a biologic product that is developed to be highly similar, as opposed to identical, to a licensed biologic product (the reference or innovator product), such that, per US Food and Drug administration draft guidelines, "no clinically meaningful differences [exist] between the biological product and the reference product in terms of safety, purity, and potency." This article presents some considerations about the development of biosimilars in cancer treatment through an overview of biosimilars from a clinical perspective. Topics covered include the development requirements and unique regulatory requirements for biosimilars, labeling considerations, potential limitations to the uptake of biosimilars, and review of some biosimilars in development for oncology indications.

Improvement in renal function in kidney transplant recipients switched from cyclosporine or tacrolimus to belatacept: 2-year results from the long-term extension of a phase II study.

Kidney transplant recipients who switched from a calcineurin inhibitor (CNI) to belatacept demonstrated higher calculated glomerular filtration rates (cGFRs) at 1 year in a Phase II study. This report addresses whether improvement was sustained at 2 years in the long-term extension (LTE). Patients receiving cyclosporine or tacrolimus were randomized to switch to belatacept or continue CNI. Of 173 randomized patients, 162 completed the 12-month main study and entered the LTE. Two patients (n = 1 each group) had graft loss between Years 1-2. At Year 2, mean cGFR was 62.0 ml/min (belatacept) vs. 55.4 ml/min (CNI). The mean change in cGFR from baseline was +8.8 ml/min (belatacept) and +0.3 ml/min (CNI). Higher cGFR was observed in patients switched from either cyclosporine (+7.8 ml/min) or tacrolimus (+8.9 ml/min). The frequency of acute rejection in the LTE cohort was comparable between the belatacept and CNI groups by Year 2. All acute rejection episodes occurred during Year 1 in the belatacept patients and during Year 2 in the CNI group. There were more non-serious mucocutaneous fungal infections in the belatacept group. Switching to a belatacept-based regimen from a CNI-based regimen resulted in a continued trend toward improved renal function at 2 years after switching.

Summary of roundtable discussion meeting: non-human primates to assess risk for EBV-related lymphomas in humans.

Epstein-Barr virus (EBV)-associated lymphomas are a known risk for immunosuppressed individuals. Non-clinical methods to determine the potential of new immunomodulatory compounds to produce EBV-associated lymphomas (hazard identification) have not been developed. Since lymphocryptovirus (LCV) in non-human primates (NHP) has similar characteristics to EBV in humans, a Roundtable meeting was held in October 2010 to explore how the potential for EBV-related lymphomas in humans can be assessed by using surrogate biomarkers for lymphoma risk in NHP toxicity studies. Stakeholders from regulatory agencies, academia, and industry came together to determine the research gaps and potential benefits and considerations of such an approach given the current state-of-the-science. Key conclusions from the discussion included considerations raised about the potential usefulness of LCV-related biomarkers from NHP studies since there is significant controversy over the reliability of using EBV viral load or EBV-specific T-lymphocytes to predict for lymphoproliferative disorders in transplant patients. In addition, there are technical challenges that need to be further addressed in order to develop methods to measure LCV viral load and LCV-specific T-lymphocytes from cynomolgus monkeys.

Belatacept-versus cyclosporine-based immunosuppression in renal transplant recipients with pre-existing diabetes.

BACKGROUND AND OBJECTIVES: Renal transplant recipients with pre-existing diabetes (PD) have reduced graft survival and increased risk of mortality and ischemic heart disease compared with nondiabetic transplant recipients. To assess the effect of belatacept in this high-risk group, we evaluated outcomes of the subpopulation with PD from previously published BENEFIT and BENEFIT-EXT trials. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A post hoc analysis evaluated pooled data from BENEFIT (living donors or standard criteria donors) and BENEFIT-EXT (extended criteria donors). Patients were randomized to receive cyclosporine or a more intensive (MI) or less intensive (LI) belatacept regimen. RESULTS: Of 1209 intent-to-treat patients, 336 had PD. At 12 months, the belatacept LI arm demonstrated a numerically higher rate of patients surviving with a functioning graft (90.4% MI [103 of 114], 92.8% LI [90 of 97], and 80.8% cyclosporine [101 of 125]), and fewer serious adverse events than cyclosporine or MI patients. Three cases of posttransplant lymphoproliferative disorder were reported in LI patients, one involving the central nervous system. Higher rates (% [95% confidence interval]: 22.8% MI [15.1 to 30.5]; 20.6% LI [12.6 to 28.7]; 14.4% cyclosporine (8.2 to 20.6]) and grades of acute rejection were observed with belatacept. Measured GFR (ml/min per 1.73 m(2), 59.8 MI; 62.5 LI; 45.4 cyclosporine), and cardiovascular risk profile were better for belatacept versus cyclosporine. CONCLUSIONS: In post hoc analysis of patients with PD, patient/graft survival and renal function at 12 months were numerically higher with belatacept versus cyclosporine, but not statistically significant. Further study is necessary to confirm the benefits belatacept may provide in these patients.

Lymphoproliferative disorders after adult kidney transplant: epidemiology and comparison of registry report with claims-based diagnoses.

BACKGROUND: Posttransplant lymphoproliferative disorder (PTLD) is a major complication of kidney transplant. STUDY DESIGN: Retrospective cohort study comparing PTLD incidence rates using US Medicare claims and Organ Procurement and Transplantation Network (OPTN) data, examining risk factors for PTLD in OPTN data, and studying recipient and graft survival after PTLD diagnosis. SETTING & PARTICIPANTS: All adult first-transplant patients who underwent deceased or living donor kidney-only transplants in 2000-2006 (n = 89,485) followed up through 3 years posttransplant. PREDICTORS: Recipient and donor characteristics, HLA mismatches, viral serologic test results, and initial immunosuppression. OUTCOMES: OPTN-reported or Medicare claims-based PTLD diagnosis, recipient and graft survival after OPTN-reported PTLD diagnosis. MEASUREMENTS: Adjusted HRs for PTLD diagnosis estimated using a Cox proportional hazards model; probability of survival free of all-cause graft failure estimated using the Kaplan-Meier method. RESULTS: The incidence rate of PTLD during the first posttransplant year was 2-fold higher in Medicare claims (0.46/100 patient-years; 95% CI, 0.39-0.53) than in OPTN data (0.22/100 patient-years; 95% CI, 0.17-0.27). Factors associated with increased rates of PTLD included older age, white race (vs African American), induction with T-cell-depleting antibodies, Epstein-Barr virus seronegativity at the time of transplant, and cytomegalovirus seronegativity at the time of transplant. The adjusted risk of death with graft function was 17.5 (95% CI, 14.3-21.4) times higher after a report of PTLD, and the risk of death-censored graft failure was 5.5 (95% CI, 3.9-7.7) times higher. LIMITATIONS: Shortcomings inherent in large databases, including inconsistencies in patient follow-up, reporting, and coding practices by transplant centers; insufficient registry data to analyze acute rejection episodes and antirejection treatment; no available data for potential effects of different types of PTLD treatment on patient outcomes. CONCLUSIONS: Despite the limitations of data collected by registries, PTLD clearly is an important complication; both mortality and death-censored graft failure increase after PTLD.

Switching from calcineurin inhibitor-based regimens to a belatacept-based regimen in renal transplant recipients: a randomized phase II study.

BACKGROUND AND OBJECTIVES: Prolonged use of calcineurin inhibitors (CNIs) in kidney transplant recipients is associated with renal and nonrenal toxicity and an increase in cardiovascular risk factors. Belatacept-based regimens may provide a treatment option for patients who switch from CNI-based maintenance immunosuppression. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This is a randomized, open-label Phase II trial in renal transplant patients with stable graft function and receiving a CNI-based regimen. Patients who were ≥6 months but ≤36 months after transplantation were randomized to either switch to belatacept or continue CNI treatment. All patients received background maintenance immunosuppression. The primary end point was the change in calculated GFR (cGFR) from baseline to month 12. RESULTS: Patients were randomized either to switch to belatacept (n=84) or to remain on a CNI-based regimen (n=89). At month 12, the mean (SD) change from baseline in cGFR was higher in the belatacept group versus the CNI group. Six patients in the belatacept group had acute rejection episodes, all within the first 6 months; all resolved with no allograft loss. By month 12, one patient in the CNI group died with a functioning graft, whereas no patients in the belatacept group had graft loss. The overall safety profile was similar between groups. CONCLUSIONS: The study identifies a potentially safe and feasible method for switching stable renal transplant patients from a cyclosporine- or tacrolimus-based regimen to a belatacept-based regimen, which may allow improved renal function in patients currently treated with CNIs.

An integrated safety profile analysis of belatacept in kidney transplant recipients.

BACKGROUND: Belatacept is associated with better renal function and an improved cardiovascular/metabolic risk profile versus cyclosporine in kidney transplant recipients. The current analysis examined pooled safety data for belatacept versus cyclosporine used in combination with basiliximab, mycophenolate mofetil, and steroids. METHODS: Patients enrolled in three core studies in de novo kidney transplantation were randomized to a more intensive (MI) or less intensive (LI) regimen of belatacept or cyclosporine. The pooled analysis included 1425 patients (MI: 477; LI: 472; cyclosporine: 476). Median follow-up was approximately 2.4 years. RESULTS: Belatacept was generally well tolerated. The frequency of deaths (MI: 7%; LI: 5%; cyclosporine: 7%) and serious infections (MI: 37%; LI: 32%; cyclosporine: 36%) were lower in the LI group versus cyclosporine. The frequency of malignancies was 10%, 6%, and 7% in the MI, LI, and cyclosporine groups, respectively. Sixteen cases of posttransplant lymphoproliferative disorder (PTLD) occurred (n=8 MI; n=6 LI; n=2 cyclosporine), including nine cases involving the central nervous system (CNS) (n=6 MI; n=3 LI). The risk of CNS PTLD was highest in Epstein-Barr virus(-) recipients; more CNS PTLD cases occurred in the MI group. One case of progressive multifocal leukoencephalopathy was reported in the MI group. CONCLUSIONS: Treatment with belatacept-based regimens was generally safe for a period of at least 2 years. There was a greater risk of PTLD--specifically CNS PTLD--in the belatacept groups versus cyclosporine, especially in Epstein-Barr virus(-) patients and with the MI dose. The number of deaths and serious infections was lower in the LI regimen versus MI and cyclosporine. The overall safety profile favored the LI over the MI regimen.

Tonsilar NK cells restrict B cell transformation by the Epstein-Barr virus via IFN-gamma.

Cells of the innate immune system act in synergy to provide a first line of defense against pathogens. Here we describe that dendritic cells (DCs), matured with viral products or mimics thereof, including Epstein-Barr virus (EBV), activated natural killer (NK) cells more efficiently than other mature DC preparations. CD56(bright)CD16(-) NK cells, which are enriched in human secondary lymphoid tissues, responded primarily to this DC activation. DCs elicited 50-fold stronger interferon-gamma (IFN-gamma) secretion from tonsilar NK cells than from peripheral blood NK cells, reaching levels that inhibited B cell transformation by EBV. In fact, 100- to 1,000-fold less tonsilar than peripheral blood NK cells were required to achieve the same protection in vitro, indicating that innate immune control of EBV by NK cells is most efficient at this primary site of EBV infection. The high IFN-gamma concentrations, produced by tonsilar NK cells, delayed latent EBV antigen expression, resulting in decreased B cell proliferation during the first week after EBV infection in vitro. These results suggest that NK cell activation by DCs can limit primary EBV infection in tonsils until adaptive immunity establishes immune control of this persistent and oncogenic human pathogen.

DEC-205/CD205+ dendritic cells are abundant in the white pulp of the human spleen, including the border region between the red and white pulp.

The distribution of dendritic cells (DCs) and macrophages in the human spleen has received less attention than that of lymphocytes. Here we have addressed this problem with the human DEC-205/CD205 marker ('DEC'), which is an endocytic receptor on DCs that mediates efficient presentation of antigens. DEC was abundant on dendritic profiles in the white pulp but absent from the red pulp, the latter defined with antibodies to two antigens, mannose receptor/CD206 on sinusoidal lining cells, and macrosialin/CD68 on macrophages. Double staining with anti-DEC and anti-CD3 showed the expected concentration of DEC+ cells in the relatively small T-cell areas of the human spleen. DEC+ cells were also found in other regions of the white pulp. In all regions, the DEC+ cells were positive for major histocompatibility complex (MHC) class II and the CD11c integrin but largely immature, with low expression of B7-2/CD86 costimulator and DC-lysosome-associated membrane protein (LAMP)/CD208. When we concentrated on the perifollicular region between the red pulp and the marginal zone, we found macrophages that stained with antibodies to sialoadhesin/CD169 and DC-specific ICAM-3 grabbing non-integrin (SIGN)/CD209, and just inside these cells were DEC+ profiles. The DEC+ DCs were intertwined with cells that stained for the vascular addressin mucosal addressin cell adhesion molecule (MAdCAM). Therefore, anti-DEC-205/CD205 antibodies are useful for identifying DCs in human splenic white pulp and its border region with the red pulp.

Hyperexpression of Foxp3 and IDO during acute rejection of islet allografts.

BACKGROUND: We investigated the hypothesis that Foxp3+ cells are an integral component of antiallograft immunity but are dominated by pathogenic effectors. METHODS: Wild-type H-2b C57BL/6 (B6) mice or B6 mice with a targeted disruption of c-Rel gene (c-Rel-/-) were used as recipients of islet grafts from allogeneic DBA/2 (H-2d) mice or syngeneic B6 mice. We developed kinetic quantitative polymerase chain reaction assays and measured intragraft expression of mRNA for Foxp3, IDO, cytolytic molecules, proinflammatory cytokines, and chemokines/receptors. RESULTS: Intraislet levels of mRNA for Foxp3, IDO, CD3, CD25, tumor necrosis factor-alpha, RANTES, IP-10, and CXCR3 were highest in DBA/2 islet allografts from WT B6 recipients compared to DBA/2 islet allografts from c-Rel-/- B6 recipients or syngeneic B6 islet grafts from WT B6 mice. The ratio of granzyme B or IFN-gamma to Foxp3 was higher with the DBA/2 islet allografts from the WT B6 recipients compared to DBA/2 islet allografts from c-Rel-/- B6 recipients or B6 islet grafts from WT B6 recipients. CONCLUSIONS: Foxp3+ cells are an integral component of acute rejection of allografts but may be dominated by pathogenic effectors.

Mitochondrial targeting with antioxidant peptide SS-31 prevents mitochondrial depolarization, reduces islet cell apoptosis, increases islet cell yield, and improves posttransplantation function.

Apoptotic cell death is a defined pathway for islet cell demise, and mitochondrial dysfunction contributes to islet cell apoptosis. The hypothesis that the novel peptide D-Arg-2', 6'-dimethyltyrosine-Lys-Phe-NH2 (SS-31), previously shown to target inner mitochondrial membrane and prevent oxidative damage of neuronal cells and other cell types, optimizes pancreatic islet isolation and improves posttransplantation function in recipients with diabetes was investigated. Herein is demonstrated that SS-31 readily penetrates intact mouse islets, preserves mitochondrial polarization, reduces islet cell apoptosis, and increases islet cell yield. Optimization of islet isolation is demonstrable after SS-31 pretreatment of islet (pancreas) donor mice and with the addition of SS-31 to reagents that are used in the isolation of mouse islets. The supplementation of in vitro culture medium with SS-31 reduced islet cell apoptosis and increased the viability of human islets, as ascertained by dual-parameter flow cytometry analysis. In a stringent marginal islet cell mass transplantation model (200 mouse islets transplanted under the renal capsule of syngeneic diabetic mice) and using islets that were derived from old mice (>24 wk), SS-31 treatment was associated with prompt and sustained normoglycemia, whereas the untreated islet graft recipients remained diabetic. Our data suggest a novel strategy to optimize islet isolation and reduce the need for multiple pancreata to achieve insulin independence in the recipient with type 1 diabetes. Because SS-31 was effective with "extended" islet donors, it is hypothesized that the antioxidant SS-31 may serve to increase the pool of eligible organ donors.

Systemic transforming growth factor-beta1 gene therapy induces Foxp3+ regulatory cells, restores self-tolerance, and facilitates regeneration of beta cell function in overtly diabetic nonobese diabetic mice.

BACKGROUND: Type 1 diabetes results from auto-aggressive T-cell-mediated destruction of beta cells of the pancreas. Recent data suggest that restoration of self-tolerance may facilitate islet-cell regeneration/recovery. In view of the immunoregulatory activity of transforming growth factor (TGF)-beta1, we investigated whether systemic TGF-beta1 gene therapy blocks islet destructive autoimmunity and facilitates regeneration of beta-cell function in overtly diabetic nonobese diabetic (NOD) mice. METHODS: We used site-directed mutagenesis to create cysteine to serine mutation at sites 224 and 226 and constructed a replication deficient adenovirus (Ad) vector encoding active form of human TGF-beta1 (Ad-hTGF-beta1). Overtly diabetic NOD mice received intravenous injection of Ad-hTGF-beta1. Seven to 14 days after the injection, the mice received transplants with 500 syngeneic islets under the kidney capsule. Islet-graft survival and regeneration of endogenous beta-cell function were examined. RESULTS: Syngeneic islet grafts failed by day 17 in all untreated mice, whereas Ad-hTGF-beta1 therapy prolonged survival of islet grafts. Islet grafts from treated mice showed well-preserved islets with a peri-islet infiltrate primarily of CD4+ T cells and expression of CD25 and Foxp3. Systemic TGF-beta1 gene therapy was associated with islet regeneration in the native pancreas. Native pancreas of treated mice revealed islets staining strongly for insulin. Similar to what was found in the syngeneic islet graft, there were well-demarcated peri-islet infiltrates that were positive for CD4, TGF-beta1, and Foxp3. CONCLUSIONS: Our data demonstrate that systemic TGF-beta1 gene therapy blocks islet destructive autoimmunity, facilitates islet regeneration, and cures diabetes in diabetic NOD mice.

Measurement of apoptosis of intact human islets by confocal optical sectioning and stereologic analysis of YO-PRO-1-stained islets.

Apoptosis is an established pathway for islet cell demise. Current protocols for assessment of islet cell apoptosis are time-consuming (as with terminal deoxynucleotide transferase-mediated dUTP nick-end labeling reaction) and involve disruption of the islet architecture (as with flow cytometry) or destruction of cell integrity (as with enzyme-linked immunosorbent assay). The membranes of apoptotic cells, but not those of live cells, are permeant to the DNA-intercalant dye YO-PRO-1. We report a novel methodology for the rapid quantification of apoptosis of human islets: confocal laser optical sectioning and stereologic analysis of intact human islets stained with YO-PRO-1 and Hoechst 33342. The advantages include (1) rapid quantification of apoptosis without disrupting islet architecture and (2) identification of significant heterogeneity in the extent of apoptosis among islets from the same isolate. Confocal laser scanning microscopy microscopic imaging of YO-PRO-1-stained islets may advance investigation of islet cell apoptosis and help develop islet parameters predictive of posttransplant function.

Distinct roles of IL-12 and IL-15 in human natural killer cell activation by dendritic cells from secondary lymphoid organs.

Dendritic cells (DCs) are known to induce the growth and function of natural killer (NK) cells. Here, we address the capacity of DCs to interact with NK cells in human lymphoid organs and identify the role of specific DC-derived cytokines. We demonstrate that DCs colocalize with NK cells in the T cell areas of lymph nodes. In culture, DCs from either blood or spleen primarily stimulate the CD56(bright)CD16- NK cell subset, which is enriched in secondary lymphoid tissues. Blocking of IL-12 abolished DC-induced IFN-gamma secretion by NK cells, whereas membrane-bound IL-15 on DCs was essential for NK cell proliferation and survival. Maturation by CD40 ligation promoted the highest IL-15 surface presentation on DCs and led to the strongest NK cell proliferation induced by DCs. These results identify secondary lymphoid organs as a potential DC/NK cell interaction site and identify the distinct roles for DC-derived IL-12 and IL-15 in NK cell activation.

Rapamycin inhibits the growth and metastatic progression of non-small cell lung cancer.

PURPOSE: Lung cancer has a dismal prognosis and comprises 5.5% of post-transplant malignancies. We explored whether rapamycin inhibits the growth and metastatic progression of non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Murine KLN-205 NSCLC was used as the model tumor in syngeneic DBA/2 mice to explore the effect of rapamycin on tumor growth and metastastic progression. We also examined the effect of rapamycin on cell cycle progression, apoptosis, and proliferation using murine KLN-205 NSCLC cells and human A-549 NSCLC cells as targets. The in vivo and in vitro effects of cyclosporine and those of rapamycin plus cyclosporine were also investigated. RESULTS: Rapamycin but not cyclosporine inhibited tumor growth; s.c. tumor volume was 1290 +/- 173 mm(3) in untreated DBA/2 mice, 246 +/- 80 mm(3) in mice treated with rapamycin, and 1203 +/- 227 mm(3) in mice treated with cyclosporine (P < 0.001). Rapamycin but not cyclosporine prevented the formation of distant metastases; eight of eight untreated mice and four of six mice treated with cyclosporine developed pulmonary metastases whereas only one of six mice treated with rapamycin developed pulmonary metastases (P = 0.003). In vitro, rapamycin induced cell cycle arrest at the G(1) checkpoint and blocked proliferation of both KLN-205 and A-549 cells but did not induce apoptosis. Cyclosporine did not prevent cell cycle progression and had a minimal antiproliferative effect on KLN-205 and A-549 cells. CONCLUSIONS: The immunosuppressive macrolide rapamycin but not cyclosporine prevents the growth and metastatic progression of NSCLC. A rapamycin-based immunosuppressive regimen may be of value in recipients of allografts.

The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic.

Natural killer cells are important cytolytic cells in innate immunity. We have characterized human NK cells of spleen, lymph nodes, and tonsils. More than 95% of peripheral blood and 85% of spleen NK cells are CD56(dim)CD16(+) and express perforin, the natural cytotoxicity receptors (NCRs) NKp30 and NKp46, as well as in part killer cell Ig-like receptors (KIRs). In contrast, NK cells in lymph nodes have mainly a CD56(bright)CD16(-) phenotype and lack perforin. In addition, they lack KIRs and all NCR expression, except low levels of NKp46. The NK cells of tonsils also lack perforin, KIRs, NKp30, and CD16, but partially express NKp44 and NKp46. Upon IL-2 stimulation, however, lymph node and tonsilar NK cells up-regulate NCRs, express perforin, and acquire cytolytic activity for NK-sensitive target cells. In addition, they express CD16 and KIRs upon IL-2 activation, and therefore display a phenotype similar to peripheral blood NK cells. We hypothesize that IL-2 can mobilize the NK cells of secondary lymphoid tissues to mediate natural killing during immune responses. Because lymph nodes harbor 40% and peripheral blood only 2% of all lymphocytes in humans, this newly characterized perforin(-) NK cell compartment in lymph nodes and related tissues probably outnumbers perforin(+) NK cells. These results also suggest secondary lymphoid organs as a possible site of NK cell differentiation and self-tolerance acquisition.