The First Cold Atmospheric Plasma Phase I Clinical Trial for the Treatment of Advanced Solid Tumors: A Novel Treatment Arm for Cancer.

Local regional recurrence (LRR) remains the primary cause of treatment failure in solid tumors despite advancements in cancer therapies. Canady Helios Cold Plasma (CHCP) is a novel Cold Atmospheric Plasma device that generates an Electromagnetic Field and Reactive Oxygen and Nitrogen Species to induce cancer cell death. In the first FDA-approved Phase I trial (March 2020-April 2021), 20 patients with stage IV or recurrent solid tumors underwent surgical resection combined with intra-operative CHCP treatment. Safety was the primary endpoint; secondary endpoints were non-LRR, survival, cancer cell death, and the preservation of surrounding healthy tissue. CHCP did not impact intraoperative physiological data (p > 0.05) or cause any related adverse events. Overall response rates at 26 months for R0 and R0 with microscopic positive margin (R0-MPM) patients were 69% (95% CI, 19-40%) and 100% (95% CI, 100-100.0%), respectively. Survival rates for R0 (n = 7), R0-MPM (n = 5), R1 (n = 6), and R2 (n = 2) patients at 28 months were 86%, 40%, 67%, and 0%, respectively. The cumulative overall survival rate was 24% at 31 months (n = 20, 95% CI, 5.3-100.0). CHCP treatment combined with surgery is safe, selective towards cancer, and demonstrates exceptional LRR control in R0 and R0-MPM patients. (Clinical Trials identifier: NCT04267575).

Canady Cold Helios Plasma Reduces Soft Tissue Sarcoma Viability by Inhibiting Proliferation, Disrupting Cell Cycle, and Inducing Apoptosis: A Preliminary Report.

Soft tissue sarcomas (STS) are a rare and highly heterogeneous group of solid tumors, originating from various types of connective tissue. Complete removal of STS by surgery is challenging due to the anatomical location of the tumor, which results in tumor recurrence. Additionally, current polychemotherapeutic regimens are highly toxic with no rational survival benefit. Cold atmospheric plasma (CAP) is a novel technology that has demonstrated immense cancer therapeutic potential. Canady Cold Helios Plasma (CHCP) is a device that sprays CAP along the surgical margins to eradicate residual cancer cells after tumor resection. This preliminary study was conducted in vitro prior to in vivo testing in a humanitarian compassionate use case study and an FDA-approved phase 1 clinical trial (IDE G190165). In this study, the authors evaluate the efficacy of CHCP across multiple STS cell lines. CHCP treatment reduced the viability of four different STS cell lines (i.e., fibrosarcoma, synovial sarcoma, rhabdomyosarcoma, and liposarcoma) in a dose-dependent manner by inhibiting proliferation, disrupting cell cycle, and inducing apoptosis-like cell death.

BCL2A1 regulates Canady Helios Cold Plasma-induced cell death in triple-negative breast cancer.

Breast cancer is the leading cause of cancer death among women. Triple-negative breast cancer (TNBC) has a poor prognosis and frequently relapses early compared with other subtypes. The Cold Atmospheric Plasma (CAP) is a promising therapy for prognostically poor breast cancer such as TNBC. The Canady Helios Cold Plasma (CHCP) induces cell death in the TNBC cell line without thermal damage, however, the mechanism of cell death by CAP treatment is ambiguous and the mechanism of resistance to cell death in some subset of cells has not been addressed. We investigate the expression profile of 48 apoptotic and 35 oxidative gene markers after CHCP treatment in six different types of breast cancer cell lines including luminal A (ER+ PR+/-HER2-), luminal B (ER+PR+/-HER2+), (ER-PR-HER2+), basal-like: ER-PR-HER2- cells were tested with CHCP at different power settings and at 4 different incubation time. The expression levels of the gene markers were determined at 4 different intervals after the treatment. The protein expression of BCL2A1 was only induced after CHCP treatment in TNBC cell lines (p < 0.01), whereas the HER2-positive and ER, PR positive cell lines showed little or no expression of BCL2A1. The BCL2A1 and TNF-alpha expression levels showed a significant correlation within TNBC cell lines (p < 0.01). Silencing BCL2A1 mRNA by siRNA increased the potency of the CHCP treatment. A Combination of CHCP and CPI203, a BET bromodomain inhibitor, and a BCL2A1 antagonist increased the CHCP-induced cell death (p < 0.05). Our results revealed that BCL2A1 is a key gene for resistance during CHCP induced cell death. This resistance in TNBCs could be reversed with a combination of siRNA or BCL2A1 antagonist-CHCP therapy.

Canady Helios Cold Plasma Induces Breast Cancer Cell Death by Oxidation of Histone mRNA.

Breast cancer is the most common cancer among women worldwide. Its molecular receptor marker status and mutational subtypes complicate clinical therapies. Cold atmospheric plasma is a promising adjuvant therapy to selectively combat many cancers, including breast cancer, but not normal tissue; however, the underlying mechanisms remain unexplored. Here, four breast cancer cell lines with different marker status were treated with Canady Helios Cold Plasma™ (CHCP) at various dosages and their differential progress of apoptosis was monitored. Inhibition of cell proliferation, induction of apoptosis, and disruption of the cell cycle were observed. At least 16 histone mRNA types were oxidized and degraded immediately after CHCP treatment by 8-oxoguanine (8-oxoG) modification. The expression of DNA damage response genes was up-regulated 12 h post-treatment, indicating that 8-oxoG modification and degradation of histone mRNA during the early S phase of the cell cycle, rather than DNA damage, is the primary cause of cancer cell death induced by CHCP. Our report demonstrates for the first time that CHCP effectively induces cell death in breast cancer regardless of subtyping, through histone mRNA oxidation and degradation during the early S phase of the cell cycle.

The synergistic effect of Canady Helios cold atmospheric plasma and a FOLFIRINOX regimen for the treatment of cholangiocarcinoma in vitro.

Cholangiocarcinoma (CCA) is a rare biliary tract cancer with a low five-year survival rate and high recurrence rate after surgical resection. Currently treatment approaches include systemic chemotherapeutics such as FOLFIRINOX, a chemotherapy regimen is a possible treatment for severe CCA cases. A limitation of this chemotherapy regimen is its toxicity to patients and adverse events. There exists a need for therapies to alleviate the toxicity of a FOLFIRINOX regimen while enhancing or not altering its anticancer properties. Cold atmospheric plasma (CAP) is a technology with a promising future as a selective cancer treatment. It is critical to know the potential interactions between CAP and adjuvant chemotherapeutics. In this study the aim is to characterize the efficacy of FOLFIRINOX and CAP in combination to understand potential synergetic effect on CCA cells. FOLFIRINOX treatment alone at the highest dose tested (53.8 µM fluorouracil, 13.7 µM Leucovorin, 5.1 µM Irinotecan, and 3.7 µM Oxaliplatin) reduced CCA cell viability to below 20% while CAP treatment alone for 7 min reduced viability to 3% (p < 0.05). An analysis of cell viability, proliferation, and cell cycle demonstrated that CAP in combination with FOLFIRINOX is more effective than either treatment alone at a lower FOLFIRINOX dose of 6.7 µM fluorouracil, 1.7 µM leucovorin, 0.6 µM irinotecan, and 0.5 µM oxaliplatin and a shorter CAP treatment of 1, 3, or 5 min. In conclusion, CAP has the potential to reduce the toxicity burden of FOLFIRINOX and warrants further investigation as an adjuvant therapy.

Randomized Controlled Trial Evidence of Cost-Effectiveness of a Multifaceted AKI Intervention Approach.

INTRODUCTION: Acute kidney injury (AKI) is associated with increased health care utilization and higher costs. The Tackling AKI study was a multicenter, pragmatic, stepped-wedge cluster randomized trial that demonstrated a reduced hospital length of stay after implementation of a multifaceted AKI intervention (e-alerts, care bundle, and an education program). We tested whether this would result in cost savings. METHODS: A decision-analytic tree model from the payer perspective (National Health Service in the United Kingdom) was generated on which cost-effectiveness analyses were performed using a probabilistic sensitivity analysis, accounting only for direct medical costs. Clinical data from the Tackling AKI study were used as inputs and economic and utility data derived from relevant published literature. RESULTS: A total of 24,059 AKI episodes occurred during the study period, and in 18,887 admissions the patient was discharged alive. When all AKI stages were considered together, the cost per AKI admission was £5065 in the control arm and £4333 in the intervention arm, representing an incremental cost saving of £732 per admission with the intervention. Similar results were obtained when AKI stages were included as separate variables. Costs per quality-adjusted life year were £61,194 in the control group and £51,161 in the intervention group. At a willingness to pay threshold of £20,000 per quality-adjusted life year, the probability of the intervention being cost-effective compared with standard care was 90%. CONCLUSION: An organizational level approach to improve standards of AKI care reduces the cost of hospital admissions and is cost effective within the National Health Service in the United Kingdom.

A Translational Model for Venous Thromboembolism: MicroRNA Expression in Hibernating Black Bears.

BACKGROUND: Hibernating American black bears have significantly different clotting parameters than their summer active counterparts, affording them protection against venous thromboembolism during prolonged periods of immobility. We sought to evaluate if significant differences exist between the expression of microRNAs in the plasma of hibernating black bears compared with their summer active counterparts, potentially contributing to differences in hemostasis during hibernation. MATERIALS AND METHODS: MicroRNA sequencing was assessed in plasma from 21 American black bears in summer active (n = 11) and hibernating states (n = 10), and microRNA signatures during hibernating and active state were established using both bear and human genome. MicroRNA targets were predicted using messenger RNA (mRNA) transcripts from black bear kidney cells. In vitro studies were performed to confirm the relationship between identified microRNAs and mRNA expression, using artificial microRNA and human liver cells. RESULTS: Using the bear genome, we identified 15 microRNAs differentially expressed in the plasma of hibernating black bears. Of these microRNAs, three were significantly downregulated (miR-141-3p, miR-200a-3p, and miR-200c-3p), were predicted to target SERPINC1, the gene for antithrombin, and demonstrated regulatory control of the gene mRNA expression in cell studies. CONCLUSIONS: Our findings suggest that the hibernating black bears' ability to maintain hemostasis and achieve protection from venous thromboembolism during prolonged periods of immobility may be due to changes in microRNA signatures and possible upregulation of antithrombin expression.

Dehydrated Amnion/Chorion Improves Achilles Tendon Repair in a Diabetic Animal Model.

INTRODUCTION: Healing of tendon injuries is often plagued by significant scar formation and compromised biomechanical function. For those with diabetes, these injuries are further complicated by alterations to the extracellular matrix of the tendon, poor circulation, and delayed wound healing; consequently, complications and re-rupture rates for patients with diabetes are reported higher than the typical patient population. Placental derived membranes, specifically dehydrated human amnion/chorion membranes (dACMs), have been utilized clinically as an adhesion barrier, and these membranes have been shown to reduce scarring and aid in tissue repair. OBJECTIVE: The purpose of this study was to evaluate the effect of dACMs on tendon repair in a diabetic model with impaired healing. MATERIALS AND METHODS: Using a type II diabetic model (BBZDR/WOR rats), a full-thickness injury was made through the Achilles tendon and repaired using a modified Kessler method. Repaired tendons were wrapped with dACM or left unwrapped as a control (n = 15/group; n = 30 total). Tendons were retrieved at 14 (n = 5/group; n = 10 total) or 28 days (n = 10/group; n = 20 total) and evaluated using histology, immunofluorescence, and biomechanical testing. RESULTS: Treatment of tendons with dACM resulted in reduced failure rates, increased cell migration, and improved mechanical properties (compared with unwrapped controls). The dACM-treated tendons also showed changes in the production of several important biomarkers to tendon healing at both 14 and 28 days; most notably, Scleraxis was found to be upregulated in dACM-treated tendons. CONCLUSIONS: This study highlights a promising treatment option for this challenging clinical population.

The rationale for the new deceased donor pancreas allocation schema.

BACKGROUND: To ensure the continued success of whole organ pancreas and islet transplantation, deceased donor pancreas allocation policy must continue to evolve. METHODS: To assess the existing system, the Organ Procurement and Transplantation Network (OPTN)/United Network for Organ Sharing Kidney and Pancreas Transplant Committee retrospectively analyzed the disposition and outcomes of deceased donor pancreata in the United States between January 1, 2000 and December 31, 2003. RESULTS: During the time period studied, consent was obtained but the pancreas was not recovered in 48% (11,820) of organ donors. The most common reasons given for nonrecovery were poor quality of the pancreas and difficulty in placement. Of whole organ pancreata that were transplanted, 90% were from donors with a body mass index (BMI) 50 years (P=0.04), and there were trends toward lower graft survival with donor BMI >30 (P=0.06) and increasing cold-ischemia time. CONCLUSIONS: Based on these data, the OPTN adopted a new allocation algorithm in which pancreata from donors >30 kg/m or >50 years of age are, unless accepted for a local whole organ pancreas transplant candidate, preferentially allocated for islet transplantation. These data also suggest that many good quality pancreata are not procured, emphasizing the need for improved communication and cooperation between organ procurement organizations and pancreas and islet transplant programs.

Clinical results of an organ procurement organization effort to increase utilization of donors after cardiac death.

BACKGROUND: To stimulate organ donation, an organ procurement organization (OPO)-wide effort was undertaken to increase donors after cardiac death (DCD) over a 5-year period. This included commonality of protocols, pulsatile perfusion of kidneys, centralization of data and a regional allocation variance designed to minimize cold ischemia times and encourage adoption of DCD protocols at transplant centers. RESULTS: In one OPO, eight centers initiated DCD programs in 11 hospitals. A total of 52 DCD donors were procured, increasing from four in 1999 to 21 in 2003. Eleven donors had care withdrawn in the operating room, whereas 41 had care withdrawn in the ICU. In all, 91 patients received renal transplants from these 52 donors (12 kidneys discarded, one double transplant), whereas 5 patients received liver transplants. One-, two-, and three-year kidney graft survival rates were 90%, 90%, and 82%, respectively. Fifty-five percent of patients needed at least one session of hemodialysis postoperatively. Mean recipient hospital length of stay was 11.1+/-6 days. Mean creatinine levels at 3, 6, 12, and 24 months were 1.65, 1.40, 1.41, and 1.40, respectively. CONCLUSIONS: DCD donors can be an important source of donor organs and provide excellent overall outcomes. Regional cooperation and a prospectively considered allocation and distribution system are important considerations in stimulating DCD programs.

Inhibition of apoptosis by survivin improves transplantation of pancreatic islets for treatment of diabetes in mice.

Survivin is a cancer gene implicated in inhibition of apoptosis and regulation of mitosis, but its function in normal cells has remained elusive. Here, we show that transgenic mice expressing survivin in pancreatic islet beta-cells show no changes in cell proliferation, as determined by islet size or islet number. Transplantation of survivin transgenic islets in diabetic recipient mice affords long-term engraftment and stable correction of hyperglycaemia. This involves intrinsic inhibition of beta-cell apoptosis, in vivo, and global transcriptional changes in pancreatic islets with upregulation of stress response genes, antagonists of cytokine signalling and promoters of angiogenesis. These broad cytoprotective functions of survivin in vivo might be beneficial for gene therapy of diabetes.

Antigen exposure during enhanced CTLA-4 expression promotes allograft tolerance in vivo.

The role of CTLA-4 in tolerance is primarily inferred from knockout and blocking studies. Anti-CD45RB mediates allograft tolerance in mice by inducing CTLA-4 expression on CD4 cells, providing a novel opportunity to determine how therapeutic enhancement of CTLA-4 promotes tolerance. We now show that induced CTLA-4 expression normally resolves by day 17. Although thymectomy prolongs enhanced CTLA-4 expression, long-term engraftment is unaffected. To address the temporal relationship between increased CTLA-4 expression and engraftment, transplantation was delayed for various times after anti-CD45RB treatment. Delaying transplantation for 7 days (when CTLA-4 expression had peaked but treatment mAb was no longer detectable), resulted in long-term engraftment comparable to transplantation with no delay (day 0). Delaying transplantation from 10 to 18 days led to a progressively poorer outcome as CTLA-4 expression returned to baseline. This suggested that Ag exposure while CTLA-4 expression is enhanced is sufficient to induce long-term engraftment. To substantiate this, on day 0, anti-CD45RB-treated mice received BALB/c vs unrelated alloantigen, followed by transplantation of BALB/c islets 10 days later. Whereas recipients exposed to unrelated Ag experienced acute rejection, recipients exposed to donor Ag achieved long-term engraftment. Anti-CD45RB-treated mice exposed to alloantigen exhibited anergic CD4(+)CD25(-) effector cells and regulatory CD4(+)CD25(+) cells. Moreover, CD25 depletion in the peritransplant period prevented anti-CD45RB-mediated engraftment. Thus, exposure of CD4 cells expressing CTLA-4 to donor Ag is necessary and sufficient to induce long-term engraftment which appears to be mediated by both regulation and anergy.

Gene silencing in the endocrine pancreas mediated by short-interfering RNA.

OBJECTIVES: RNA interference as mediated by short-interfering RNA (siRNA) offers a nonviral means to silence genes in tissue; however, few data exist about gene therapy using siRNA in pancreas tissue. To determine if siRNA treatment could silence an endogenous gene in pancreatic islets, we developed a murine model using the endocrine pancreas. METHODS: The insulin 2 (Ins2) gene was targeted with siRNA, and quantitative RT-PCR, fluorescent microscopy, and FACS were used to measure transcript levels and siRNA cellular uptake and transfection efficiency. Isolated pancreatic islets were transfected with siRNA in vitro using a liposomal delivery method in a dose titration (50-400 nM) or pooled from BALB/c mice having received siRNA (100 microg) via hydrodynamic tail vein injection. RESULTS: The Ins2 transcript level was significantly reduced by 55% in vitro with FACS data showing a transfection efficiency over 45% with the 400 nM concentration. In vivo delivery of siRNA to pancreatic islets revealed a 33% reduction in Ins2 mRNA levels, although siRNA was able to be detected in 19% of isolated islet cells. CONCLUSION: We have successfully used RNA interference to silence an endogenous tissue-specific gene (Ins2) in pancreatic islets when transfected in vitro or administered in vivo.

Requirements for induction and maintenance of peripheral tolerance in stringent allograft models.

Peripheral tolerance can be achieved in many but not all murine allograft models. The requirements for controlling more aggressive immune responsiveness and generating peripheral tolerance in stringent allograft models are unknown. Understanding these requirements will provide insight toward ultimately achieving tolerance in humans, which are also resistant. We now demonstrate that the combination of donor-specific transfusion, anti-CD45RB, and anti-CD154 uniformly achieves >90-d survival of BALB/c skin allografts on C57BL/6 recipients. Recipients exhibit marked hyporesponsiveness to alloantigen in vitro. In distinct contrast to less rigorous models, engraftment remains absolutely dependent on cytotoxic T lymphocyte antigen 4 signaling, even after grafts are healed, suggesting that prolonged engraftment cannot simply be attributed to more effective depletion of alloreactive T cells but is actively maintained by regulation. Concordantly, we show that both CD4 and CD8 regulatory cells are required and can transfer donor-specific tolerance to naïve recipients. Nonetheless, most recipients ultimately develop gradual graft loss (median survival time = 140 d), suggesting that alloreactive cells emerging from the thymus eventually overwhelm regulatory capacity. In agreement, adding thymectomy to the regimen results in permanent engraftment (>250 d) and donor-specific tolerance not observed previously in this model. These results highlight the potency of both CD4 and CD8 regulatory cells but also suggest that in stringent settings, regulatory T cell longevity and capacity for infectious tolerance compete with prolonged graft immunogenicity and thymic output. These results provide insight into the mechanisms of tolerance in stringent models and provide a rational basis for innovative tolerogenic strategies in humans.

Donor kidney exchanges.

Kidney transplantation from live donors achieves an excellent outcome regardless of human leukocyte antigen (HLA) mismatch. This development has expanded the opportunity of kidney transplantation from unrelated live donors. Nevertheless, the hazard of hyperacute rejection has usually precluded the transplantation of a kidney from a live donor to a potential recipient who is incompatible by ABO blood type or HLA antibody crossmatch reactivity. Region 1 of the United Network for Organ Sharing (UNOS) has devised an alternative system of kidney transplantation that would enable either a simultaneous exchange between live donors (a paired exchange), or a live donor/deceased donor exchange to incompatible recipients who are waiting on the list (a live donor/list exchange). This Regional system of exchange has derived the benefit of live donation, avoided the risk of ABO or crossmatch incompatibility, and yielded an additional donor source for patients awaiting a deceased donor kidney. Despite the initial disadvantage to the list of patients awaiting an O blood type kidney, as every paired exchange transplant removes a patient from the waiting list, it also avoids the incompatible recipient from eventually having to go on the list. Thus, this approach also increases access to deceased donor kidneys for the remaining candidates on the list.

Transplantation in the diabetic patient with advanced chronic kidney disease: a task force report.

On May 7 to 8, 2003, more than 60 physicians, surgeons, nurses, and allied health professionals met in Chicago, IL, to discuss state-of-the-art approaches to transplantation in the clinical management of patients with diabetes and advanced chronic kidney disease. Four work groups addressed pretransplantation evaluation and management, therapeutic alternatives for treatment of patients with chronic kidney disease, posttransplantation diabetes mellitus, and improving long-term kidney transplant outcomes in patients with diabetes. This report summarizes the deliberations and recommendations of the task force.

Renal transplant recipients over aged 60 have diminished immune activity and a low risk of rejection.

Strict consideration of the renal transplant candidate's chronologic age is generally supplanted by more subjective reflection on his (her) physiologic state. In the US, patients over 64 years old represented 9.0% of renal transplant recipients in the year 2000, yet little prior experience is available with which to guide the management of geriatric patients. Two hundred and forty six consecutive recipients of primary kidney transplants at the Yale-New Haven Organ Transplant Center between 1990 and 1995 were included in an outcome analysis. Age at transplantation ranged from 2 to 68 years; the study group consisted of the 16 (6.5%) over age 60. The immunosuppressive protocol was uniform for all patients. There was a disproportionately high use of cadaveric organs by older patients; only 1/16 (6.3%) received a living donor kidney. The overall rate of rejection within the first 90 days was 6.7% of cadaveric recipients over 60 versus 37.6% of younger recipients, P=0.001. Actual patient survival rates at 6 years were 100% of patients younger than 11 years versus 69% (11/16) of those older than 60 years. Death censored 5 year graft survival was 100% in older patients versus 85% among the younger patients. The older and younger patients received quantitatively equivalent immunosuppression, but acute rejection was uncommon in the former (6%) versus the younger cohort (34%). It seems logical to consider whether older renal transplant recipients may benefit from a less aggressive immunosuppression strategy.

Cutting edge: transplantation tolerance through enhanced CTLA-4 expression.

Knockout and blocking studies have shown a critical role for CTLA-4 in peripheral tolerance, however, it is unknown whether augmenting CTLA-4 expression actually promotes tolerance. Here we demonstrate a specific and requisite role for CTLA-4 and its up-regulation in tolerance through anti-CD45RB. First, long-term murine islet allograft survival induced by anti-CD45RB is prevented by CTLA4-Ig, which interferes with B7:CTLA-4 interactions. Second, anti-CD45RB is ineffective in recipients lacking CTLA-4, B7-1, and B7-2. In contrast, CTLA4-Ig, which targets B7 on allogeneic cells, promotes long-term engraftment in these mice. Moreover, anti-CD45RB was effective in B7-deficient controls expressing CTLA-4. Finally, in wild-type mice, CTLA-4 expression returned to baseline 17 days after receiving anti-CD45RB, and was refractory to further increase. Transplantation and anti-CD45RB therapy at this time could neither augment CTLA-4 nor prolong engraftment. These data demonstrate a specific role for CTLA-4 in anti-CD45RB-mediated tolerance and indicate that CTLA-4 up-regulation can directly promote allograft survival.