Rejection resistant CD30.CAR-modified Epstein-Barr virus-specific T cells as an off-the-shelf platform for CD30+ lymphoma.

Off-the-shelf (OTS) adoptive T cell therapies have many benefits such as immediate availability, improved access and reduced cost, but face the major challenges of graft-vs-host disease (GVHD) and graft rejection, mediated by alloreactive T cells present in the graft and host, respectively. We have developed a platform for OTS T cell therapies by using Epstein-Bar virus (EBV)-specific T cells (EBVSTs) expressing a chimeric antigen receptor (CAR) targeting CD30. Allogeneic EBVSTs have not caused GVHD in several clinical trials, while the CD30.CAR, that is effective for the treatment of lymphoma, can also target alloreactive T cells that upregulate CD30 on activation. Although EBVSTs express high levels of CD30, they were protected from fratricide in cis, by the CD30.CAR. Hence, they could proliferate extensively and maintained function both through their native EBV-specific T cell receptor and the CD30.CAR. The CD30.CAR enabled EBVSTs to persist in co-cultures with naive and primed alloreactive T cells and eliminate activated natural killer cells that can also be alloreactive. In conclusion, we show that CD30.CAR EBVSTs have the potential to be an effective OTS therapy against CD30+ tumors and, if successful, could then be used as a platform to target other tumor antigens.

Overexpression of an Engineered SERPINB9 Enhances Allogeneic T-cell Persistence and Efficacy.

Allogeneic chimeric antigen receptor (CAR)-expressing T cells offer many advantages over autologous therapies, but their benefits are curtailed by graft-versus-host disease and elimination by recipient immune cells. Moreover, just as with autologous therapies, allogeneic CAR T cells are susceptible to activation-induced cell death (AICD) caused by chronic antigen exposure (CAE). Granzyme B- and Fas/Fas ligand-initiated caspase-mediated apoptoses are key mechanisms of T-cell death caused by T/NK cell-mediated allorejection or CAE. We explored a protective strategy of engineering CAR T cells to overexpress variants of the Granzyme B-specific serine protease inhibitor SERPINB9 (SB9) to improve allogeneic T-cell persistence and antitumor efficacy. We showed that the overexpression of an SB9 variant with broadened caspase specificity, SB9(CAS), not only significantly reduced rejection of allogeneic CAR T cells but also increased their resistance to AICD and enabled them to thrive better under CAE, thus improving allogeneic T-cell persistence and antitumor activity in vitro and in vivo. In addition, although SB9(CAS) overexpression improved the efficacy of allogeneic CAR T-cell therapy by conferring protection to cell death, we did not observe any autonomous growth, and the engineered CAR T cells were still susceptible to an inducible suicide switch. Hence, SB9(CAS) overexpression is a promising strategy that can strengthen current development of cell therapies, broadening their applications to address unmet medical needs.

Banking on virus-specific T cells to fulfill the need for off-the-shelf cell therapies.

Adoptively transferred virus-specific T cells (VSTs) have shown remarkable safety and efficacy for the treatment of virus-associated diseases and malignancies in hematopoietic stem cell transplant (HSCT) recipients, for whom VSTs are derived from the HSCT donor. Autologous VSTs have also shown promise for the treatment of virus-driven malignancies outside the HSCT setting. In both cases, VSTs are manufactured as patient-specific products, and the time required for procurement, manufacture, and release testing precludes their use in acutely ill patients. Further, Good Manufacturing Practices-compliant products are expensive, and failures are common in virus-naive HSCT donors and patient-derived VSTs that are rendered anergic by immunosuppressive tumors. Hence, highly characterized, banked VSTs (B-VSTs) that can be used for multiple unrelated recipients are highly desirable. The major challenges facing B-VSTs result from the inevitable mismatches in the highly polymorphic and immunogenic human leukocyte antigens (HLA) that present internally processed antigens to the T-cell receptor, leading to the requirement for partial HLA matching between the B-VST and recipient. HLA mismatches lead to rapid rejection of allogeneic T-cell products and graft-versus-host disease induced by alloreactive T cells in the infusion product. Here, we summarize the clinical outcomes to date of trials of B-VSTs used for the treatment of viral infections and malignancies and their potential as a platform for chimeric antigen receptors targeting nonviral tumors. We will highlight the properties of VSTs that make them attractive off-the-shelf cell therapies, as well as the challenges that must be overcome before they can become mainstream.

Trends in Opioid Use Among Cancer Patients in the United States: 2013-2018.

Background: In response to the US opioid epidemic, the Centers for Disease Control and Prevention updated their guideline on prescription opioids for chronic pain management in March 2016. The aim of this study was to provide detailed analysis of trends in opioid claims among cancer patients in the United States during 2013-2018. Methods: We analyzed pharmaceutical dispensing data from Symphony Health's Integrated Dataverse database, which covers approximately 80% of the US population. We examined annual trends in dispensed opioids in cancer patients during 2013-2018. We examined quarterly trends of the prevalence, mean number of days, and dose (stated as morphine milligram equivalents) of opioid dispensing in cancer patients. Results: Dispensing records of an average of over 3.7 million cancer patients contributed to the study annually in 2013-2018. The annual prevalence of opioid dispensing claims declined from 40.2% in 2013 to 34.5% in 2018. Annual declines occurred across cancer sites, and particularly among patients with metastatic cancer (decline of 19.8%), breast cancer (18.2%), and lung cancer (13.8%). By quarter, the prevalence of opioid claims declined statistically significantly from 26.6% in Q1 2013 to 21.2% in Q4 2018; this decline was more pronounced after Q3 2016 (2-sided P = .004). Both quarterly trends in mean days and morphine milligram equivalents of opioids supplied showed a gradual decline from 2013 to 2018, with a slightly larger decline after 2016. Conclusions: We observed a decline in opioid use among cancer patients, particularly after 2016, coinciding with the publication of the Centers for Disease Control and Prevention's guideline on prescription opioids for chronic pain management.

Strategic Design of Catalytic Lysine-Targeting Reversible Covalent BCR-ABL Inhibitors*.

Targeted covalent inhibitors have re-emerged as validated drugs to overcome acquired resistance in cancer treatment. Herein, by using a carbonyl boronic acid (CBA) warhead, we report the structure-based design of BCR-ABL inhibitors via reversible covalent targeting of the catalytic lysine with improved potency against both wild-type and mutant ABL kinases, especially ABLT315I bearing the gatekeeper residue mutation. We show the evolutionarily conserved lysine can be targeted selectively, and the selectivity depends largely on molecular recognition of the non-covalent pharmacophore in this class of inhibitors, probably due to the moderate reactivity of the warhead. We report the first co-crystal structures of covalent inhibitor-ABL kinase domain complexes, providing insights into the interaction of this warhead with the catalytic lysine. We also employed label-free mass spectrometry to evaluate off-targets of our compounds at proteome-wide level in different mammalian cells.

Engineered off-the-shelf therapeutic T cells resist host immune rejection.

Engineered T cells are effective therapies against a range of malignancies, but current approaches rely on autologous T cells, which are difficult and expensive to manufacture. Efforts to develop potent allogeneic T cells that are not rejected by the recipient's immune system require abrogating both T- and natural killer (NK)-cell responses, which eliminate foreign cells through various mechanisms. In the present study, we engineered a receptor that mediates deletion of activated host T and NK cells, preventing rejection of allogeneic T cells. Our alloimmune defense receptor (ADR) selectively recognizes 4-1BB, a cell surface receptor temporarily upregulated by activated lymphocytes. ADR-expressing T cells resist cellular rejection by targeting alloreactive lymphocytes in vitro and in vivo, while sparing resting lymphocytes. Cells co-expressing chimeric antigen receptors and ADRs persisted in mice and produced sustained tumor eradication in two mouse models of allogeneic T-cell therapy of hematopoietic and solid cancers. This approach enables generation of rejection-resistant, 'off-the-shelf', allogeneic T-cell products to produce long-term therapeutic benefit in immunocompetent recipients.

Amelioration of Both Central and Peripheral Neuropathy in Mouse Models of Type 1 and Type 2 Diabetes by the Neurogenic Molecule NSI-189.

While peripheral neuropathy is the most common complication of long-term diabetes, cognitive deficits associated with encephalopathy and myelopathy also occur. Diabetes is a risk factor for Alzheimer disease (AD) and increases the risk of progression from mild cognitive impairment to AD. The only current recommendation for preventing or slowing the progression of peripheral neuropathy is to maintain close glycemic control, while there is no recommendation for central nervous system disorders. NSI-189 is a new chemical entity that when orally administered promotes neurogenesis in the adult hippocampus, increases hippocampal volume, enhances synaptic plasticity, and reduces cognitive dysfunction. To establish the potential for impact on peripheral neuropathy, we first showed that NSI-189 enhances neurite outgrowth and mitochondrial functions in cultured adult rat primary sensory neurons. Oral delivery of NSI-189 to murine models of type 1 (female) and type 2 (male) diabetes prevented multiple functional and structural indices of small and large fiber peripheral neuropathy, increased hippocampal neurogenesis, synaptic markers and volume, and protected long-term memory. NSI-189 also halted progression of established peripheral and central neuropathy. NSI-189, which is currently in clinical trials for treatment of major depressive disorder, offers the opportunity for the development of a single therapeutic agent against multiple indices of central and peripheral neuropathy.

A strategy to protect off-the-shelf cell therapy products using virus-specific T-cells engineered to eliminate alloreactive T-cells.

BACKGROUND: The use of "off-the-shelf" cellular therapy products derived from healthy donors addresses many of the challenges associated with customized cell products. However, the potential of allogeneic cell products to produce graft-versus-host disease (GVHD), and their likely rejection by host alloreactive T-cells are major barriers to their clinical safety and efficacy. We have developed a molecule that when expressed in T-cells, can eliminate alloreactive T-cells and hence can be used to protect cell therapy products from allospecific rejection. Further, expression of this molecule in virus-specific T-cells (VSTs) should virtually eliminate the potential for recipients to develop GVHD. METHODS: To generate a molecule that can mediate killing of cognate alloreactive T-cells, we fused beta-2 microglobulin (B2M), a universal component of all human leukocyte antigen (HLA) class I molecules, to the cytolytic endodomain of the T cell receptor ζ chain, to create a chimeric HLA accessory receptor (CHAR). To determine if CHAR-modified human VSTs could eliminate alloreactive T-cells, we co-cultured them with allogeneic peripheral blood mononuclear cells (PBMC), and assessed proliferation of PBMC-derived alloreactive T-cells and the survival of CHAR-modified VSTs by flow cytometry. RESULTS: The CHAR was able to transport HLA molecules to the cell surface of Daudi cells, that lack HLA class I expression due to defective B2M expression, illustrating its ability to complex with human HLA class I molecules. Furthermore, VSTs expressing CHAR were protected from allospecific elimination in co-cultures with allogeneic PBMCs compared to unmodified VSTs, and mediated killing of alloreactive T-cells. Unexpectedly, CHAR-modified VSTs eliminated not only alloreactive HLA class I restricted CD8 T-cells, but also alloreactive CD4 T-cells. This beneficial effect resulted from non-specific elimination of activated T-cells. Of note, we confirmed that CHAR-modified VSTs did not affect pathogen-specific T-cells which are essential for protective immunity. CONCLUSIONS: Human T-cells can be genetically modified to eliminate alloreactive T-cells, providing a unique strategy to protect off-the-shelf cell therapy products. Allogeneic cell therapies have already proved effective in treating viral infections in the stem cell transplant setting, and have potential in other fields such as regenerative medicine. A strategy to prevent allograft rejection would greatly increase their efficacy and commercial viability.

Remediation of Radiation-Induced Cognitive Dysfunction through Oral Administration of the Neuroprotective Compound NSI-189.

Clinical management of primary and secondary central nervous system (CNS) malignancies frequently includes radiotherapy to forestall tumor growth and recurrence after surgical resection. While cranial radiotherapy remains beneficial, adult and pediatric brain tumor survivors suffer from a wide range of debilitating and progressive cognitive deficits. Although this has been recognized as a significant problem for decades, there remains no clinical recourse for the unintended neurocognitive sequelae associated with these types of cancer treatments. In previous work, multiple mechanisms have been identified that contribute to radiation-induced cognitive dysfunction, including the inhibition of neurogenesis caused by the depletion of radiosensitive populations of stem and progenitor cells in the hippocampus. To explore the potential neuroprotective properties of a pro-neurogenic compound NSI-189, Long-Evans rats were subjected to a clinically relevant fractionated irradiation protocol followed by four weeks of NSI-189 administered daily by oral gavage. Animals were then subjected to five different behavioral tasks followed by an analysis of neurogenesis, hippocampal volume and neuroinflammation. Irradiated cohorts manifested significant behavioral decrements on all four spontaneous exploration tasks. Importantly, NSI-189 treatment resulted in significantly improved performance in four of these tasks: novel place recognition, novel object recognition, object in place and temporal order. In addition, there was a trend of improved performance in the contextual phase of the fear conditioning task. Importantly, enhanced cognition in the NSI-189-treated cohort was found to persist one month after the cessation of drug treatment. These neurocognitive benefits of NSI-189 coincided with a significant increase in neurogenesis and a significant decrease in the numbers of activated microglia compared to the irradiated cohort that was given vehicle alone. The foregoing changes were not accompanied by major changes in hippocampal volume. These data demonstrate that oral administration of a pro-neurogenic compound exhibiting anti-inflammatory indications could impart long-term neurocognitive benefits in the irradiated brain.

NSI-189, a small molecule with neurogenic properties, exerts behavioral, and neurostructural benefits in stroke rats.

Enhancing neurogenesis may be a powerful stroke therapy. Here, we tested in a rat model of ischemic stroke the beneficial effects of NSI-189, an orally active, new molecular entity (mol. wt. 366) with enhanced neurogenic activity, and indicated as an anti-depressant drug in a clinical trial (Fava et al., , Molecular Psychiatry, DOI: 10.1038/mp.2015.178) and being tested in a Phase 2 efficacy trial (ClinicalTrials.gov, , ClinicalTrials.gov Identifier: NCT02695472) for treatment of major depression. Oral administration of NSI-189 in adult Sprague-Dawley rats starting at 6 hr after middle cerebral artery occlusion, and daily thereafter over the next 12 weeks resulted in significant amelioration of stroke-induced motor and neurological deficits, which was maintained up to 24 weeks post-stroke. Histopathological assessment of stroke brains from NSI-189-treated animals revealed significant increments in neurite outgrowth as evidenced by MAP2 immunoreactivity that was prominently detected in the hippocampus and partially in the cortex. These results suggest NSI-189 actively stimulated remodeling of the stroke brain. Parallel in vitro studies further probed this remodeling process and demonstrated that oxygen glucose deprivation and reperfusion (OGD/R) initiated typical cell death processes, which were reversed by NSI-189 treatment characterized by significant attenuation of OGD/R-mediated hippocampal cell death and increased Ki67 and MAP2 expression, coupled with upregulation of neurogenic factors such as BDNF and SCF. These findings support the use of oral NSI-189 as a therapeutic agent well beyond the initial 6-hr time window to accelerate and enhance the overall functional improvement in the initial 6 months post stroke.

Prevalence of hypercalcemia among cancer patients in the United States.

Hypercalcemia of malignancy (HCM) is a serious metabolic complication whose population-based prevalence has not been quantified. Rates of HCM differ by tumor type, with highest rates reported in multiple myeloma and lowest among colorectal and prostate cancer patients. This analysis estimates HCM prevalence in the US. This retrospective study used the Oncology Services Comprehensive Electronic Records (OSCER) warehouse of electronic health records (EHR) including laboratory values from 569000 patients treated at 565 oncology outpatient sites. OSCER data were projected to the national level by linking EHR to claims data. Cancer patients included were ≥18 years, and had serum calcium (Ca) and albumin (for corrected serum Ca [CSC]) records. Period prevalence was estimated by HCM CTCAE grade, tumor type, and year (2009-2013). Estimates were adjusted to capture patients diagnosed with HCM outside oncology practices based on a subset of patients linkable to office and hospital data. The analysis included 68023 (2009) to 121482 (2013) cancer patients. In 2013, patients with HCM had a median of six Ca tests, 69.7% had chemotherapy, and 34% received bone modifying agents. HCM rates were highest for multiple myeloma patients (7.5% [2012]-10.2% [2010]), lowest for prostate cancer (1.4% [2012]-2.1% [2011]).The estimated adjusted annual prevalence of HCM from 2009 to 2013 was 95441, 96281, 89797, 70158, and 71744, respectively. HCM affected 2.0-2.8% of all cancer patients. EHR data from oncology clinics were critical for this study because these data contain results from laboratory studies (i.e., serum calcium values) that are routinely ordered in that setting. We estimated that the prevalence of HCM in the US in 2013 is 71744, affecting approximately 2% of cancer patients overall. This percentage differs by tumor type and appears to have decreased over the five-year study period.

Estimating high-risk castration resistant prostate cancer (CRPC) using electronic health records.

INTRODUCTION: Canadian guidelines define castration-resistant prostate cancer (CRPC) at high risk of developing metastases using PSA doubling time (PSADT) < 8 months, whereby men may be offered more frequent bone scans/imaging. We evaluated PSA data from nonmetastatic (M0) prostate cancer patients treated at urology and oncology clinics across the United States (US) to describe the proportion and characteristics of patients who met CRPC and high-risk criteria. MATERIALS AND METHODS: We identified M0 prostate cancer patients aged = 18 years receiving androgen deprivation therapy (ADT) in 2011 from electronic health records (EHR), covering 129 urology and 64 oncology practices across the US. We estimated the proportion of prostate cancer patients with evidence of CRPC (consecutive rising PSAs) and subsets that may be at high risk (using several PSA and PSADT cut-points). RESULTS: Among 3121 M0 prostate cancer patients actively treated with ADT, 1188 (38%) had evidence of CRPC. Of these, 712 (60%) qualified as high risk in 2011 based on PSADT < 8 months (equivalent to = 8 months in these data). Men = 65 years were more likely to have evidence of CRPC than younger men, although younger men were more likely to have evidence of high-risk disease. CRPC was more common among men receiving ADT in the oncology setting than the urology setting (48% versus 37%). CONCLUSIONS: In this large EHR study with patient-level PSA data, 38% of men with M0 prostate cancer treated with ADT had CRPC. Approximately 60% of M0 CRPC patients may experience a PSADT of < 8 months. These findings require validation in a Canadian patient population.

Prevalence of renal impairment and use of nephrotoxic agents among patients with bone metastases from solid tumors in the United States.

The renal status of patients with bone metastases secondary to solid tumors and their treatment with nephrotoxic agents is not well characterized. This retrospective study analyzed electronic medical records data from US-based oncology clinics to identify adult (age ≥18) solid tumor patients with first bone metastasis diagnosis and ≥1 serum creatinine recorded between January 1, 2009 and December 31, 2013. Patients with multiple myeloma, multiple primary tumor types, acute renal failure, and/or end-stage renal disease were excluded. Using the Chronic Kidney Disease Epidemiology Collaboration formula, we determined the prevalence of renal impairment (RI: single estimated glomerular filtration rate [eGFR] value <60 mL/min per 1.73 m(2) ) and chronic kidney disease (CKD: ≥2 eGFR values <60, at least 90 days apart). We also examined the use of intravenous bisphosphonates (IV BP) and other nephrotoxic agents. Approximately half of the 11,809 patients were female. Breast (34%) and lung (28%) tumors were the most common. At bone metastasis diagnosis, mean age was 67 years and 24% of patients exhibited RI. The 5-year prevalence was 43% for RI and 71% for CKD among RI patients. Nearly half (46%) of CKD patients received IV BP in the 12 months following their confirming eGFR and 13% of these patients received at least one other nephrotoxic agent during that period. This is the first US-based study to examine the prevalence of RI among patients with bone metastases from solid tumors. RI is common at bone metastases diagnosis, and a substantial proportion of patients develop RI or CKD as their disease progresses. Whenever possible, treatments that are potentially less damaging for the kidney should be considered for patients with or predisposed to RI.

Prevalence of women with early-stage breast cancer receiving active management using electronic health records from oncology clinics in the United States.

The purpose of this study was to estimate the prevalence of women receiving treatment or active surveillance for stage I-III breast cancer in the United States from 2009 to 2012, stratified by patient age and tumor characteristics. In each study year, electronic medical records were used to identify women aged ≥18 years with stage I-III breast cancer and treated or under active surveillance (≥4 visits) at an oncology clinic that contributes data to the Oncology Services Comprehensive Electronic Records database. Prevalence was projected to the national level overall and within strata (by tumor characteristics, year of breast cancer diagnosis, and age). We identified 5,219 female breast cancer patients (18 %

Patients with bone metastases from solid tumors initiating treatment with a bone-targeted agent in 2011: a descriptive analysis using oncology clinic data in the US.

PURPOSE: Three bone-targeted agents (BTAs) are approved in the USA for prevention of bone complications among solid tumor patients with bone metastases: two intravenous bisphosphonates (IV BP) (pamidronate and zoledronic acid), and one subcutaneous receptor activator of nuclear factor-kappaB (RANK) ligand inhibitor (denosumab). Using electronic medical record data from outpatient community and hospital-affiliated oncology clinics, we examined the characteristics of patients who initiated treatment with a BTA in 2011 and followed them for a maximum of 12 months. METHODS: Adult patients with bone metastasis secondary to solid tumors newly treated with a BTA during 2011 were identified from the Oncology Services Comprehensive Electronic Records (OSCER) database. We examined patient characteristics at BTA initiation, treatment patterns, and compliance during a 12-month period. Sensitivity analyses were performed in a subgroup of patients who had confirmed 12 months of follow-up data. RESULTS: Denosumab patients (N = 1,594) were older (65 % ≥65 years vs. 60 % ≥65 years), further along in their disease progression (time since bone metastasis diagnosis: 16 % ≥2 years vs. 10 % ≥2 years), less likely to switch BTA (overall: 6 vs. 14 %; subgroup: 8 vs. 19 %), and more compliant with treatment (overall: median doses of 7 vs. 4; subgroup: 11 vs. 8) compared to IV BP patients (N = 1,975). Findings were consistent across gender, age, tumor type, naïve, and transition strata. CONCLUSIONS: Patients receiving denosumab and IV BPs may differ. Despite higher age and more advanced disease, patients treated with denosumab are more likely to stay on treatment and have better compliance.

Behavioral and histopathological assessment of adult ischemic rat brains after intracerebral transplantation of NSI-566RSC cell lines.

Stroke is a major cause of death and disability, with very limited treatment option. Cell-based therapies have emerged as potential treatments for stroke. Indeed, studies have shown that transplantation of neural stem cells (NSCs) exerts functional benefits in stroke models. However, graft survival and integration with the host remain pressing concerns with cell-based treatments. The current study set out to investigate those very issues using a human NSC line, NSI-566RSC, in a rat model of ischemic stroke induced by transient occlusion of the middle cerebral artery. Seven days after stroke surgery, those animals that showed significant motor and neurological impairments were randomly assigned to receive NSI-566RSC intracerebral transplants at two sites within the striatum at three different doses: group A (0 cells/µl), group B (5,000 cells/µl), group C (10,000 cells/µl), and group D (20,000 cells/µl). Weekly behavioral tests, starting at seven days and continued up to 8 weeks after transplantation, revealed dose-dependent recovery from both motor and neurological deficits in transplanted stroke animals. Eight weeks after cell transplantation, immunohistochemical investigations via hematoxylin and eosin staining revealed infarct size was similar across all groups. To identify the cell graft, and estimate volume, immunohistochemistry was performed using two human-specific antibodies: one to detect all human nuclei (HuNu), and another to detect human neuron-specific enolase (hNSE). Surviving cell grafts were confirmed in 10/10 animals of group B, 9/10 group C, and 9/10 in group D. hNSE and HuNu staining revealed similar graft volume estimates in transplanted stroke animals. hNSE-immunoreactive fibers were also present within the corpus callosum, coursing in parallel with host tracts, suggesting a propensity to follow established neuroanatomical features. Despite absence of reduction in infarct volume, NSI-566RSC transplantation produced behavioral improvements possibly via robust engraftment and neuronal differentiation, supporting the use of this NSC line for stroke therapy.

Orientation distribution of highly oriented type I collagen deposited on flat samples with different geometries.

The structural arrangement of type I collagen in vivo is critical for the normal functioning of tissues, such as bone, cornea, tendons, and blood vessels. At present, there are no established low-cost techniques for fabricating aligned collagen structures for applications in regenerative medicine. Here, we report on a straightforward approach to fabricate collagen films, with defined orientation distributions of collagen fibrillar aggregates within a matrix of oriented collagen molecules on flat sample surfaces. Langmuir-Blodgett (LB) technology was used to deposit thin films of oriented type I collagen onto flat substrates exhibiting various shapes. By varying the shapes of the substrates (e.g., rectangles, squares, circles, parallelograms, and various shaped triangles) as well as their sizes, a systematic study on collagen alignment patterns was conducted. It was found that the orientation and the orientation distribution of collagen along these various shaped substrates are directly depending on the geometry of the substrate and the dipping direction of that sample with respect to the collagen/water subphase. An important factor in tissue engineering is the stability, durability, and endurance of the constructed artificial tissue and thus its functioning in regenerative medicine applications. By testing these criteria, we found that the coated films and their alignments were stable for at least three months under different conditions and, moreover, that these films can withstand temperatures of up to 60 °C for a short time. Therefore, these constructs may have widespread applicability in the engineering of collagen-rich tissues.

A sympathetic neuron autonomous role for Egr3-mediated gene regulation in dendrite morphogenesis and target tissue innervation.

Egr3 is a nerve growth factor (NGF)-induced transcriptional regulator that is essential for normal sympathetic nervous system development. Mice lacking Egr3 in the germline have sympathetic target tissue innervation abnormalities and physiologic sympathetic dysfunction similar to humans with dysautonomia. However, since Egr3 is widely expressed and has pleiotropic function, it has not been clear whether it has a role within sympathetic neurons and if so, what target genes it regulates to facilitate target tissue innervation. Here, we show that Egr3 expression within sympathetic neurons is required for their normal innervation since isolated sympathetic neurons lacking Egr3 have neurite outgrowth abnormalities when treated with NGF and mice with sympathetic neuron-restricted Egr3 ablation have target tissue innervation abnormalities similar to mice lacking Egr3 in all tissues. Microarray analysis performed on sympathetic neurons identified many target genes deregulated in the absence of Egr3, with some of the most significantly deregulated genes having roles in axonogenesis, dendritogenesis, and axon guidance. Using a novel genetic technique to visualize axons and dendrites in a subpopulation of randomly labeled sympathetic neurons, we found that Egr3 has an essential role in regulating sympathetic neuron dendrite morphology and terminal axon branching, but not in regulating sympathetic axon guidance to their targets. Together, these results indicate that Egr3 has a sympathetic neuron autonomous role in sympathetic nervous system development that involves modulating downstream target genes affecting the outgrowth and branching of sympathetic neuron dendrites and axons.

Egr3 dependent sympathetic target tissue innervation in the absence of neuron death.

Nerve Growth Factor (NGF) is a target tissue derived neurotrophin required for normal sympathetic neuron survival and target tissue innervation. NGF signaling regulates gene expression in sympathetic neurons, which in turn mediates critical aspects of neuron survival, axon extension and terminal axon branching during sympathetic nervous system (SNS) development. Egr3 is a transcription factor regulated by NGF signaling in sympathetic neurons that is essential for normal SNS development. Germline Egr3-deficient mice have physiologic dysautonomia characterized by apoptotic sympathetic neuron death and abnormal innervation to many target tissues. The extent to which sympathetic innervation abnormalities in the absence of Egr3 is caused by altered innervation or by neuron death during development is unknown. Using Bax-deficient mice to abrogate apoptotic sympathetic neuron death in vivo, we show that Egr3 has an essential role in target tissue innervation in the absence of neuron death. Sympathetic target tissue innervation is abnormal in many target tissues in the absence of neuron death, and like NGF, Egr3 also appears to effect target tissue innervation heterogeneously. In some tissues, such as heart, spleen, bowel, kidney, pineal gland and the eye, Egr3 is essential for normal innervation, whereas in other tissues such as lung, stomach, pancreas and liver, Egr3 appears to have little role in innervation. Moreover, in salivary glands and heart, two tissues where Egr3 has an essential role in sympathetic innervation, NGF and NT-3 are expressed normally in the absence of Egr3 indicating that abnormal target tissue innervation is not due to deregulation of these neurotrophins in target tissues. Taken together, these results clearly demonstrate a role for Egr3 in mediating sympathetic target tissue innervation that is independent of neuron survival or neurotrophin deregulation.