Promoting Bone Formation and Healing in Segmental Defects Through Ectopic Induced Membrane.

We aimed to determine whether addition of an in vivo ectopic induced membrane (EM) to the Masquelet Technique enhanced angiogenesis and bone formation in a segmental defect. After generating and stabilizing a diaphyseal femur defect, 10 rats received a polymethylmethacrylate (PMMA) spacer within the defect (control); 10 received another PMMA spacer implanted subcutaneously (EM). We removed the spacers and added autograft; the excised EM was added to their autograft (EM group). Post-mortem x-rays assessed bone formation and bridging. Osteogenesis in the proximal defect was significantly more uniform (p < 0.01), and there was greater amount of bone remodeling distally in the EM group (p < 0.05). There was no difference in bone formation (p = 0.19) but greater degrees of bridging in the EM group (2.20 vs. 1.20, p = 0.09). The EM resulted in more homogeneous proximal osteogenesis and increased bone remodeling distally. These findings could lead to more consistent and predictable bone healing. (Journal of Surgical Orthopaedic Advances 31(3):161-165, 2022).

Treatment of pancreatic ductal adenocarcinoma with tumor antigen specific-targeted delivery of paclitaxel loaded PLGA nanoparticles.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) remains the most aggressive cancers with a 5-year survival below 10%. Systemic delivery of chemotherapy drugs has severe side effects in patients with PDA and does not significantly improve overall survival rate. It is highly desirable to advance the therapeutic efficacy of chemotherapeutic drugs by targeting their delivery and increasing accumulation at the tumor site. MUC1 is a membrane-tethered glycoprotein that is aberrantly overexpressed in > 80% of PDA thus making it an attractive antigenic target. METHODS: Poly lactic-co-glycolic acid nanoparticles (PLGA NPs) conjugated to a tumor specific MUC1 antibody, TAB004, was used as a nanocarrier for targeted delivery into human PDA cell lines in vitro and in PDA tumors in vivo. The PLGA NPs were loaded with fluorescent imaging agents, fluorescein diacetate (FDA) and Nile Red (NR) or isocyanine green (ICG) for in vitro and in vivo imaging respectively or with a chemotherapeutic drug, paclitaxel (PTX) for in vitro cytotoxicity assays. Confocal microscopy was used to visualize internalization of the nanocarrier in vitro in PDA cells with high and low MUC1 expression. The in vivo imaging system (IVIS) was used to visualize in vivo tumor targeting of the nanocarrier. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was used to determine in vitro cell survival of cells treated with PTX-loaded nanocarrier. One-sided t-test comparing treatment groups at each concentration and two-way ANOVAs comparing internalization of antibody and PLGA nanoparticles. RESULTS: In vitro, TAB004-conjugated ICG-nanocarriers were significantly better at internalizing in PDA cells than its non-conjugated counterpart. Similarly, TAB004-conjugated PTX-nanocarriers were significantly more cytotoxic in vitro against PDA cells than its non-conjugated counterpart. In vivo, TAB004-conjugated ICG-nanocarriers showed increased accumulation in the PDA tumor compared to the non-conjugated nanocarrier while sparing normal organs. CONCLUSIONS: The study provides promising data for future development of a novel MUC1-targeted nanocarrier for direct delivery of imaging agents or drugs into the tumor microenvironment.

Mucin-1-Targeted Chimeric Antigen Receptor T Cells Are Effective and Safe in Controlling Solid Tumors in Immunocompetent Host.

The chimeric antigen receptor (CAR) T-cell therapy in solid epithelial tumors has been explored, however, with limited success. As much of the preclinical work has relied on xenograft models in immunocompromised animals, the immune-related efficacies and toxicities may have been missed. In this study, we engineered syngeneic murine CAR T cells targeting the tumor form of human mucin-1 (tMUC1) and tested the MUC1 CAR T cells' efficacy and toxicity in the immunocompetent human MUC1-expressing mouse models. The MUC1 CAR T cells significantly eliminated murine pancreatic and breast cancer cell lines in vitro. In vivo, MUC1 CAR T cells significantly slowed the mammary gland tumor progression in the spontaneous PyVMT×MUC1.Tg (MMT) mice, prevented lung metastasis, and prolonged survival. Most importantly, there was minimal short or long-term toxicity with acceptable levels of transient liver toxicity but no kidney toxicity. In addition, the mice did not show any signs of weight loss or other behavioral changes with the treatment. We also report that a single dose of MUC1 CAR T-cell treatment modestly reduced the pancreatic tumor burden in a syngeneic orthotopic model of pancreatic ductal adenocarcinoma given at late stage of an established tumor. Taken together, these findings suggested the further development of tMUC1-targeted CAR T cells as an effective and relatively safe treatment modality for various tMUC1-expressing solid tumors.

Upper airways sensory irritation responses of mice exposed to mainstream smoke from four cigarette types.

Relative sensory irritation responses for Swiss-Webster mice exposed nose-only to mainstream tobacco smoke were evaluated for several cigarette types using a smoking regimen consisting of a 35-ml puff, 2 s in duration, taken once per minute. The degree of sensory irritation for each cigarette type was evaluated as the smoke concentration inducing a 50% reduction in breathing frequency. The smoke concentration inducing 50% respiratory depression is called the RD(50) value. Study findings suggest that mainstream tobacco smoke from the Eclipse cigarette, which primarily heats rather than burns tobacco, yielded an RD(50) that was significantly higher (approximately twofold) than a tobacco-burning leading ultralight or the 2R4F or 1R5F reference cigarettes. This is indicative of reduced upper airways irritation by Eclipse that may be due to its distinct design. Study findings suggest that the irritating nature of mainstream tobacco smoke from different cigarette types can be evaluated effectively in terms of smoke concentration using the relative sensory irritation assessment. These findings constitute the first report about use of the RD(50) sensory irritation response during comparative evaluations of mainstream tobacco smoke.

Overcoming Immunological Resistance Enhances the Efficacy of A Novel Anti-tMUC1-CAR T Cell Treatment against Pancreatic Ductal Adenocarcinoma.

Chimeric antigen receptor (CAR) T cells have shown remarkable success in treating hematologic cancers. However, this efficacy has yet to translate to treatment in solid tumors. Pancreatic ductal adenocarcinoma (PDA) is a fatal malignancy with poor prognosis and limited treatment options. We have developed a second generation CAR T cell using the variable fragments of a novel monoclonal antibody, TAB004, which specifically binds the tumor-associated-MUC1 (tMUC1). tMUC1 is overexpressed on ~85% of all human PDA. We present data showing that TAB004-derived CAR T cells specifically bind to tMUC1 on PDA cells and show robust killing activity; however, they do not bind or kill normal epithelial cells. We further demonstrated that the tMUC1-CAR T cells control the growth of orthotopic pancreatic tumors in vivo. We witnessed that some PDA cells (HPAFII and CFPAC) were refractory to CAR T cell treatment. qPCR analysis of several genes revealed overexpression of indoleamine 2, 3-dioxygenases-1 (IDO1), cyclooxygenase 1 and 2 (COX1/2), and galectin-9 (Gal-9) in resistant PDA cells. We showed that combination of CAR T cells and biological inhibitors of IDO1, COX1/2, and Gal-9 resulted in significant enhancement of CAR T cell cytotoxicity against PDA cells. Overcoming PDA resistance is a significant advancement in the field.

Early detection of pancreatic cancer in mouse models using a novel antibody, TAB004.

Pancreatic ductal adenocarcinoma (PDA) is the fourth-leading cause of cancer death in the United States with a 5-year overall survival rate of 8% for all stages combined. But this decreases to 3% for the majority of patients that present with stage IV PDA at time of diagnosis. The lack of distinct early symptoms for PDA is one of the primary reasons for the late diagnosis. Common symptoms like weight loss, abdominal and back pains, and jaundice are often mistaken for symptoms of other issues and do not appear until the cancer has progressed to a late stage. Thus the development of novel imaging platforms for PDA is crucial for the early detection of the disease. MUC1 is a tumor-associated antigen (tMUC1) expressed on 80% of PDA. The goal of this study was to determine the targeting and detection capabilities of a tMUC1 specific antibody, TAB004. TAB004 antibody conjugated to a near infrared fluorescent probe was injected intraperitoneally into immune competent orthotopic and spontaneous models of PDA. Results show that fluorophore conjugated TAB004 specifically targets a) 1 week old small tumor in the pancreas in an orthotopic PDA model and b) very early pre-neoplastic lesions (PanIN lesions) that develop in the spontaneous PDA model before progression to adenocarcinoma. Thus, TAB004 is a promising antibody to deliver imaging agents directly to the pancreatic tumor microenvironment, significantly affecting early detection of PDA.

Microbiological evaluation of a newly constructed animal facility.

Newly constructed animal facilities require microbiological evaluation prior to occupation to ensure that all facets of the design not only function accordingly and that the building is ready for introduction of new, healthy animal species and personnel. Our objective was to perform microbiological monitoring to ensure the integrity of systems implemented within our facility, both new and those transferred from existing facilities. This monitoring included environmental sampling of the autoclaves, cage washers, tunnel washers, and water system to ensure operant conditions. A sentinel surveillance program also was conducted using Crl:CD1(ICR) mice, C57BL/6J mice, and Crl:CD(SD) rats to make certain the facility had not been contaminated by any infectious agents that could affect animal health prior to their introduction into the facility. On the basis of these results, we introduced new animals, transferred existing animals, relocated personnel, and implemented new measures for sanitation that previously had not been used in any of our other facilities.

Yeast metallothionein in transgenic tobacco promotes copper uptake from contaminated soils.

Metallothioneins (MTs) are metal-binding proteins that confer heavy metal tolerance and accumulation in yeast. To augment higher plant metal sequestration, the yeast metallothionein (CUP 1) was introduced into tobacco plants. The CUP 1 gene expression and copper and cadmium phytoextraction were determined. To confirm transformation, selfed and kanamycin-resistant third generation plants were subjected to DNA blot and polymerase chain reaction (PCR) analysis. A 4 mM CuSO(4) stress for 7 days resulted in a decline in CUP 1 transcripts versus nonstress conditions. Despite low mRNA levels, CUP 1 transformants accumulated up to seven times more copper in older versus younger leaves during copper stress. Pooled leaves of transgenic plants grown in soils from copper stamp-sands contained two to three times the copper content as that of the control plants. Unlike some previous reports featuring MT overexpression in plants, CUP 1 seedlings did not significantly sequester or demonstrate tolerance to CdCl(2). Using this transgenic approach, yeast CUP 1 expression under nonstressed conditions contributed to copper metal phytoextraction during a subsequent copper challenge. This strategy could be incorporated into plants designed for enhanced phytoremediation of metal contaminants.

Toxicological evaluation of smokeless tobacco: 90-day rodent feeding studies.

This manuscript presents data from 90-day toxicology studies designed to characterize the subchronic effects of a smokeless tobacco blend and an aqueous extract of that blend when administered to rodents in NTP-2000 feed. Positive control (nicotine tartrate) and treatment groups were matched for a range of nicotine levels. The doses evaluated were 0.3, 3, and 6 mg nicotine/kg body weight/day in Wistar Hannover rats and 6, 60, and 120 mg nicotine/kg/day in CD-1 mice. Variables evaluated included plasma nicotine and cotinine, body weights, feed consumption, clinical observations, clinical and anatomic pathology (including organ weights), and histopathology. Plasma nicotine and cotinine levels were dose-responsive. Key effects such as body weight reductions and organ weight changes occurred in rats and mice predominantly at the highest doses of test articles and positive control in the absence of treatment-related gross or histopathological changes. Organ weight changes were attributed mainly to the lower body weights of treated vs. control groups. The blend- and extract-induced effects generally paralleled each other and the nicotine-induced effects. Based on these studies, the doses evaluated spanned the no observable adverse effect level, the lowest observable adverse effect level and the maximum tolerated dose.