VA Lessons From Partnering in COVID-19 Clinical Trials.

Background: The US Department of Veterans Affairs (VA) Office of Research and Development (ORD) supports an extensive clinical trials enterprise. Until recently, external partnerships were limited. The VA's potential value as a partner became more apparent during the COVID-19 pandemic because of its large health care system, diverse patient population, and expertise in conducting clinical trials. Observations: By leveraging its infrastructure, the VA was able to participate in 7 large-scale COVID-19 therapeutic and vaccine trials. A key aspect of this enterprise approach is the ability to provide centralized direction and coordination. The VA's partnerships with external groups offered insights into the challenges associated with conducting important trials, especially when rapidity and coordination were essential. The ORD also developed solutions for reducing study startup time that could be established as best practices. We offer lessons for the challenges VA faced: site infrastructure needs and capabilities; study management roles and responsibilities; educational resources; local review; study design demands; contracting and budgeting; central-level systems; and communication. Conclusions: VA participation in major COVID-19 therapeutic and vaccine trials represented a significant part of its research response to the pandemic. These contributions extended beyond the participants, scientists, and data that helped inform subsequent regulatory approvals. The VA also had an opportunity to directly develop partnerships with non-VA groups. These groups became more familiar with the VA while enabling us to gain more experience in the diverse practices used to conduct multisite clinical studies. Ultimately, these efforts empower the VA to further serve the broader scientific and clinical communities.

Ceramide-dependent regulation of human epidermal keratinocyte CD1d expression during terminal differentiation.

Human keratinocytes (KC), when cultured under conditions to remain undifferentiated or to terminally differentiate, changed their cellular distribution of CD1d. As studied by confocal microscopy, undifferentiated KC had a pool of cytoplasmic CD1d, whereas after terminal differentiation, this molecule localized in the cell membrane, which recapitulates CD1d expression in vivo. A comparison of undifferentiated and differentiated cultured KC did not reveal any differences in the association with beta(2)-microglobulin, invariant chain of class II MHC, or patterns of glycosylation, suggesting that these biochemical properties are not regulating the cellular distribution of CD1d. Time-course studies of CD1d gene expression indicated that KC slowly increased gene expression with CaCl(2)-induced terminal differentiation. Increased CD1d gene expression was dependent on ceramide synthesis, because fumonisin B1, a ceramide synthetase inhibitor, blocked the increase in CD1d gene expression during terminal differentiation. Similarly, exogenous ceramide or the ceramidase inhibitor, B13, induced CD1d gene expression by undifferentiated, but not terminally differentiated, KC. A protein kinase C-zeta (PKC-zeta) inhibitor (a pseudosubstrate oligopeptide), but not a PKC-alphabeta inhibitor, significantly decreased CD1d gene expression by undifferentiated or ceramide-stimulated cultured, undifferentiated KC. As expected, downstream signaling events of PKC-zeta (JNK phosphorylation and NF-kappaBeta accumulation in the nucleus) were also attenuated. The calcineurin phosphatase inhibitor cyclosporine A, which blocks KC terminal differentiation, also blocked CD1d gene expression by cultured KC. In conclusion, this novel function of cellular ceramides extends the importance of this class of biologically active lipids beyond that of terminal differentiation and barrier function in normal human skin.

Cyclooxygenase inhibitors block cell growth, increase ceramide and inhibit cell cycle.

We have shown previously in a model of metastatic breast cancer that murine mammary tumor cells express both cyclooxygenase-1 (Cox-1) and Cox-2 isoforms. Growth and metastasis of these tumors in syngeneic hosts are inhibited by either selective Cox-1 (SC560) or selective Cox-2 (celecoxib) inhibitors. To gain insight into the relevant mechanisms involved in the therapeutic response, we determined the effect of Cox inhibitors on tumor cell behavior in vitro. We now report that either selective Cox-1 or Cox-2 drugs inhibited cell replication, but only at concentrations that are no longer selective for either isoform. Growth delay by either nonselective or selective inhibitors was associated with changes in cell morphology including cell rounding; these changes were reversed upon removal of drug. Unlike many other cell types examined, treatment of these mammary tumor cells with Cox inhibitors was not associated with detectable apoptosis. Growth inhibition, induced by either selective or nonselective Cox inhibitors, was accompanied by increased intracellular levels of the sphingolipid ceramide by 1.7-2.6-fold in comparison to vehicle-treated cells. Ceramide changes are associated with cell cycle arrest and we observed that all the Cox inhibitors examined increased significantly the number of cells in G0/G1 and reduced the S phase fraction. Likewise, addition of a cell-permeable form of ceramide (C6-ceramide) could mimic the effect of Cox inhibitors on both cell cycle and cell growth inhibition. Thus, mammary tumor cells are growth restricted by Cox inhibitors. These effects are associated with changes in ceramide levels and a block in cell cycle progression.

The ceramide analog, B13, induces apoptosis in prostate cancer cell lines and inhibits tumor growth in prostate cancer xenografts.

BACKGROUND: Apoptosis is a therapeutic target for the elimination of cancer cells. As elevations in ceramide levels induce apoptosis, there is much excitement about the use of agents that elevate ceramide levels as novel chemotherapeutic agents. Ceramidases are enzymes involved in degradation of ceramide and inhibition of ceramidase has been proposed as a mechanism to increase ceramide levels. This study provides the first insight into the effect of B13, an inhibitor of acid ceramidase, on human prostate cancer cell lines and xenografts. METHODS: Cell death was evaluated by the trypan blue assay; apoptosis by the Apo2.7 apoptosis assay; and glutathione levels by HPLC. Tumors were irradiated with a dose of 5 Gy of X-rays (250 kVp, 15 mA, 2 Gy/min) and tumor volume was measured during the course of the experiment. At the conclusion of the experiment, tumor weight was determined and the tumors were evaluated histologically. RESULTS: B13 is an inducer of cell death, by apoptosis, in cultured prostate cancer cells. LNCaP and PC3 cells have different responsiveness to the enantiomers of B13. In LNCaP cells, the R enantiomer of B13 (10 microM) was significantly more effective than the S enantiomer at inducing cell death as determined by the trypan blue assay, culminating in approximately 90% cell death at 48 hr. In contrast, the same concentration of B13S induced <20% cell death at 48 hr. In PC3 cells, the S enantiomer was a more effective inducer of cell death, culminating in approximately 30% cell death, relative to 14% for B13R in this model. Evaluation of induction of apoptosis by the Apo2.7 mitochondrial assay confirmed that this induction of cell death was by apoptosis. Concurrent with induction of apoptosis, glutathione levels drop in response to B13. Specifically, B13R caused a significant drop in glutathione levels in LNCaP cells, culminating in a reduction to 40% control values at 48 hr. In PC3 cells, in contrast, the drop in glutathione levels was more dramatic, culminating in a drop to 12% control values in response to B13S at 48 hr. The effects of B13R, however, were not significantly different from control values. In in vivo studies using a model of xenografted androgen-insensitive prostate cancer, B13 sensitized the tumors to the effects of radiation, resulting in a significant reduction in tumor volume and weight after treatment with the combination of B13 and radiation. Microscopic evaluation of the tumors indicated that apoptosis was the primary mechanism of this effect. CONCLUSIONS: Targeting ceramide pathways may be a novel treatment strategy for hormone refractory prostate cancer.