Natural killer (NK) cells can enhance engraftment and mediate graft-versus-leukemia following allogeneic hematopoietic stem cell transplantation (HSCT), but the potency of graft-versus-leukemia ...mediated by naturally reconstituting NK cells following HSCT is limited. Preclinical studies demonstrate that activation of NK cells using interleukin-15 (IL-15) plus 4-1BBL upregulates activating receptor expression and augments killing capacity. In an effort to amplify the beneficial effects of NK cells post-HSCT, we conducted a first-in-human trial of adoptive transfer of donor-derived IL-15/4-1BBL–activated NK cells (aNK-DLI) following HLA-matched, T-cell–depleted (1-2 × 104 T cells/kg) nonmyeloablative peripheral blood stem cell transplantation in children and young adults with ultra-high-risk solid tumors. aNK-DLI were CD3+-depleted, CD56+-selected lymphocytes, cultured for 9 to 11 days with recombinant human IL-15 plus 4-1BBL+IL-15Rα+ artificial antigen-presenting cells. aNK-DLI demonstrated potent killing capacity and displayed high levels of activating receptor expression. Five of 9 transplant recipients experienced acute graft-versus-host disease (GVHD) following aNK-DLI, with grade 4 GVHD observed in 3 subjects. GVHD was more common in matched unrelated donor vs matched sibling donor recipients and was associated with higher donor CD3 chimerism. Given that the T-cell dose was below the threshold required for GVHD in this setting, we conclude that aNK-DLI contributed to the acute GVHD observed, likely by augmenting underlying T-cell alloreactivity. This trial was registered at www.clinicaltrials.gov as #NCT01287104.
•Acute GVHD occurred in 5 of 9 patients after major histocompatibility–matched, T-cell–depleted peripheral blood stem cell transplantation plus IL-15/4-1BBL aNK-DLI.•GVHD was more common in matched unrelated donor transplants and associated with higher CD3 chimerism, suggesting that aNK-DLI may augment T-cell alloreactivity.
In the adult mouse testis, germ cells of various developmental cell states co-exist. FACS isolation of cells stained with the DNA dye Hoechst 33342 has been used for many years to sub-divide these ...cells based on their DNA content. This approach provides an efficient way to obtain broad categories of male germ cells: pre-meiotic spermatogonia, meiotic spermatocytes and post-meiotic spermatids. The addition of a red filter for Hoechst staining enables further sub-division of spermatocytes depending on sub-stages of meiotic prophase. However, separation of different stage spermatids using Hoechst staining alone is not possible. We recently reported a methodology, combining Hoechst staining with a second DNA dye (SYTO16) that enables the further separation of these cells into three sub-populations: round, early elongating, and late elongating spermatids (Gill et al., Cytometry A 101:529-536, 2022). This method makes it possible to obtain rapidly and simply pure fractions of male germ cells from multiple developental stages from the same animal.
During spermatogenesis, mammalian male germ cells undergo multiple developmental processes, including meiosis and post‐meiotic differentiation (spermiogenesis). To understand the transitions between ...different cellular states it is essential to isolate pure populations of cells at different stages of development. Previous approaches enabled the isolation of cells from different stages of meiotic prophase I, but techniques to sub‐fractionate unfixed, post‐meiotic spermatids have been lacking. Here we report the development of a protocol enabling simultaneous isolation of cells at different stages of meiotic prophase and post‐meiotic differentiation from testes of adult mice. This approach builds on existing fluorescence activated cell sorting protocols designed to purify cells in different stages of meiotic prophase I. By utilizing the specific spectral properties that two different DNA dyes (Hoechst 33342 and SYTO 16) exhibit when bound to chromatin of different stage male germ cells, we obtain highly pure populations of cells in relatively large numbers. This FACS protocol will enable immunocytological and molecular characterization studies of fractionated meiotic and haploid germ cells from both wild type and genetically mutant animals.
Abstract Doxorubicin (DOX) is one of the most effective chemotherapeutic agents, but cardiotoxicity limits DOX therapy. Although the mechanisms are not entirely understood, reactive oxygen species ...(ROS) appear to be involved in DOX cardiotoxicity. Ca/calmodulin dependent protein kinase II (CaMKII) can be activated by ROS through oxidation and is known to contribute to myocardial dysfunction through Ca leakage from the sarcoplasmic reticulum (SR). We hypothesized that CaMKII contributes to DOX-induced defects in intracellular Ca (Cai ) handling. Cardiac myocytes were isolated from wild-type (WT) adult rat hearts and from mouse hearts lacking the predominant myocardial CaMKII isoform (CaMKIIδ−/− , KO) vs. WT. Isolated cardiomyocytes were investigated 30 min after DOX (10 μmol/L) superfusion, using epifluorescence and confocal microscopy. Intracellular ROS-generation (ROSi ) and Cai handling properties were assessed. In a subset of experiments, KN-93 or AIP (each 1 μmol/L) were used to inhibit CaMKII. Melatonin (Mel, 100 μmol/L) served as ROS-scavenger. Western blots were performed to determine the amount of CaMKII phosphorylation and oxidation. DOX increased ROSi and led to significant diastolic Cai overload in rat myocytes. This was associated with reduced Cai transients, a 5.8-fold increased diastolic SR Ca leak and diminished SR Ca content. ROS-scavenging partially rescued Ca handling. Western blots revealed increased CaMKII phosphorylation, but not CaMKII oxidation after DOX. Pharmacological CaMKII inhibition attenuated diastolic Cai overload after DOX superfusion and led to partially restored Cai transients and SR Ca content, presumably due to reduced Ca spark frequency. In line with this concept, isoform-specific CaMKIIδ-KO attenuated diastolic Cai overload and Ca spark frequency. DOX exposure induces CaMKII-dependent SR Ca leakage, which partially contributes to impaired cellular Cai homeostasis. Pharmacological and genetic CaMKII inhibition attenuated but did not completely abolish the effects of DOX on Cai . In light of the clinical relevance of DOX, further investigations seem appropriate to determine if CaMKII inhibition could reduce DOX-induced cardiotoxicity.
Abstract
Phase I CD19 and CD22 targeting Chimeric Antigen Receptor (CAR) T cell trials have shown tremendous results against acute lymphoid leukemia (ALL). Across these trials there were variations ...in the types of costimulatory molecules included in the CAR constructs and this lead to the discovery that CAR T cells with CD28 result in earlier potency and activation with decreased persistence, while CARs with 4-1BB show increased expansion and persistence. There is great interest in translating these results in ALL trials into other diseases such as acute myeloid leukemia (AML). It is unclear which costimulatory domain in a CAR will be most effective in treating AML. To look at how co-stimulatory domains impact CAR functionality in AML, we developed CD33 targeting CD28 and 4-1BB CARs.
In vitro testing of the constructs revealed that CD33 CD28 CAR consistently produced more IL2 and Interferon-gamma than CD33 4-1BB across multiple AML cell lines. To translate these findings in vivo, xenograft models were injected with Molm 14 AML cells and treated with either CD33 CD28 or CD33 4-1BB CAR T cells. By bioluminescence imaging, CD33 CD28 treated mice had no detectable disease while CD33 4-1BB treated mice were ridden with leukemia. Combined, the in vitro and in vivo results suggest that the co-stimulatory domain does play a critical role in CAR T cell functionality and may improve CAR potency. To confirm the presence of AML in mice detected by bioluminescence, flow cytometry was performed on tissues from mock and CD33 4-1BB treated mice. No leukemia was found in the bone marrow of mock T cell treated mice. In contrast, CD33 4-1BB treated animals were clear of any leukemia in the bone marrow, suggesting the presence of extra medullary disease (EMD). The development of EMD in the less potent CD33 4-1BB CAR treated mice suggests that CAR immune pressure may be potent enough to clear primary sites of leukemia such as the bone marrow, but unable to eliminate disease in secondary tissues that AML can seed. This is not surprising since treatment of AML with chemotherapy often leads to the development of extramedullary disease in the form of chloromas.
To further investigate the effects of these two factors, we moved onto another AML model, THP1, that regularly presents with EMD even in the absence of CAR pressure. With CD33 CD28 against THP1, there was clearance in compartments bone marrow, however CD33 CD28 CAR was not able to prevent the development of EMD. These experiments suggest that although the CD28 costimulatory domain is more potent than 4-1BB in Molm14, the potency of CD28 is still not able to overcome EMD in all models. Using further studies of different AML models, we will continue to tease apart the contribution the impact of immune pressure from CARs, the natural progression of AML models, and effect of CAR potency on leukemia distribution.
Citation Format: Lila Yang, Samiksha Tarun, Christopher D. Chien, Mark E. Kohler, Haiying Qin, Terry J. Fry. Analysis of CAR 41-BB versus CD28 co-stimulatory domains exposes emergence of extramedullary disease in acute myeloid leukemia abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1534.
Trace elements sustain biological productivity, yet the significance of trace element mobilization and export in subglacial runoff from ice sheets is poorly constrained at present. Here, we present ...size-fractionated (0.02, 0.22, and 0.45 μm) concentrations of trace elements in subglacial waters from the Greenland Ice Sheet (GrIS) and the Antarctic Ice Sheet (AIS). Concentrations of immobile trace elements (e.g., Al, Fe, Ti) far exceed global riverine and open ocean mean values and highlight the importance of subglacial aluminosilicate mineral weathering and lack of retention of these species in sediments. Concentrations are higher from the AIS than the GrIS, highlighting the geochemical consequences of prolonged water residence times and hydrological isolation that characterize the former. The enrichment of trace elements (e.g., Co, Fe, Mn, and Zn) in subglacial meltwaters compared with seawater and typical riverine systems, together with the likely sensitivity to future ice sheet melting, suggests that their export in glacial runoff is likely to be important for biological productivity. For example, our dissolved Fe concentration (20,900 nM) and associated flux values (1.4 Gmol y−1) from AIS to the Fe-deplete Southern Ocean exceed most previous estimates by an order of magnitude. The ultimate fate of these micronutrients will depend on the reactivity of the dominant colloidal size fraction (likely controlled by nanoparticulate Al and Fe oxyhydroxide minerals) and estuarine processing. We contend that ice sheets create highly geochemically reactive particulates in subglacial environments, which play a key role in trace elemental cycles, with potentially important consequences for global carbon cycling.
An extraordinary new technique using hyperpolarized (13)C-labeled pyruvate and taking advantage of increased glycolysis in cancer has the potential to improve the way magnetic resonance imaging is ...used for detection and characterization of prostate cancer. The aim of this study was to quantify, for the first time, differences in hyperpolarized 1-(13)C pyruvate and its metabolic products between the various histologic grades of prostate cancer using the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Fast spectroscopic imaging techniques were used to image lactate, alanine, and total hyperpolarized carbon (THC = lactate + pyruvate + alanine) from the entire abdomen of normal mice and TRAMP mice with low- and high-grade prostate tumors in 14 s. Within 1 week, the mice were dissected and the tumors were histologically analyzed. Hyperpolarized lactate SNR levels significantly increased (P < 0.05) with cancer development and progression (41 +/- 11, 74 +/- 17, and 154 +/- 24 in normal prostates, low-grade primary tumors, and high-grade primary tumors, respectively) and had a correlation coefficient of 0.95 with the histologic grade. In addition, there was minimal overlap in the lactate levels between the three groups with only one of the seven normal prostates overlapping with the low-grade primary tumors. The amount of THC, a possible measure of substrate uptake, and hyperpolarized alanine also increased with tumor grade but showed more overlap between the groups. In summary, elevated hyperpolarized lactate and potentially THC and alanine are noninvasive biomarkers of prostate cancer presence and histologic grade that could be used in future three-dimensional (13)C spectroscopic imaging studies of prostate cancer patients.
The American Cancer Society (ACS), the Centers for Disease Control and Prevention (CDC), the National Cancer Institute (NCI), and the North American Association of Central Cancer Registries (NAACCR) ...collaborate annually to provide updates on cancer incidence and death rates and trends in these outcomes for the United States. This year's report includes incidence trends for human papillomavirus (HPV)-associated cancers and HPV vaccination (recommended for adolescents aged 11-12 years).
Data on cancer incidence were obtained from the CDC, NCI, and NAACCR, and data on mortality were obtained from the CDC. Long- (1975/1992-2009) and short-term (2000-2009) trends in age-standardized incidence and death rates for all cancers combined and for the leading cancers among men and among women were examined by joinpoint analysis. Prevalence of HPV vaccination coverage during 2008 and 2010 and of Papanicolaou (Pap) testing during 2010 were obtained from national surveys.
Death rates continued to decline for all cancers combined for men and women of all major racial and ethnic groups and for most major cancer sites; rates for both sexes combined decreased by 1.5% per year from 2000 to 2009. Overall incidence rates decreased in men but stabilized in women. Incidence rates increased for two HPV-associated cancers (oropharynx, anus) and some cancers not associated with HPV (eg, liver, kidney, thyroid). Nationally, 32.0% (95% confidence interval CI = 30.3% to 33.6%) of girls aged 13 to 17 years in 2010 had received three doses of the HPV vaccine, and coverage was statistically significantly lower among the uninsured (14.1%, 95% CI = 9.4% to 20.6%) and in some Southern states (eg, 20.0% in Alabama 95% CI = 13.9% to 27.9% and Mississippi 95% CI = 13.8% to 28.2%), where cervical cancer rates were highest and recent Pap testing prevalence was the lowest.
The overall trends in declining cancer death rates continue. However, increases in incidence rates for some HPV-associated cancers and low vaccination coverage among adolescents underscore the need for additional prevention efforts for HPV-associated cancers, including efforts to increase vaccination coverage.
Abstract
Context
Patients with adrenal insufficiency require increased hydrocortisone cover during major stress to avoid a life-threatening adrenal crisis. However, current treatment recommendations ...are not evidence-based.
Objective
To identify the most appropriate mode of hydrocortisone delivery in patients with adrenal insufficiency who are exposed to major stress.
Design and Participants
Cross-sectional study: 122 unstressed healthy subjects and 288 subjects exposed to different stressors (major trauma N = 83, sepsis N = 100, and combat stress N = 105). Longitudinal study: 22 patients with preserved adrenal function undergoing elective surgery. Pharmacokinetic study: 10 patients with primary adrenal insufficiency undergoing administration of 200 mg hydrocortisone over 24 hours in 4 different delivery modes (continuous intravenous infusion; 6-hourly oral, intramuscular or intravenous bolus administration).
Main Outcome Measure
We measured total serum cortisol and cortisone, free serum cortisol, and urinary glucocorticoid metabolite excretion by mass spectrometry. Linear pharmacokinetic modeling was used to determine the most appropriate mode and dose of hydrocortisone administration in patients with adrenal insufficiency exposed to major stress.
Results
Serum cortisol was increased in all stress conditions, with the highest values observed in surgery and sepsis. Continuous intravenous hydrocortisone was the only administration mode persistently achieving median cortisol concentrations in the range observed during major stress. Linear pharmacokinetic modeling identified continuous intravenous infusion of 200 mg hydrocortisone over 24 hours, preceded by an initial bolus of 50–100 mg hydrocortisone, as best suited for maintaining cortisol concentrations in the required range.
Conclusions
Continuous intravenous hydrocortisone infusion should be favored over intermittent bolus administration in the prevention and treatment of adrenal crisis during major stress.
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