Hind Leg Paralysis Research Articles

Combined therapy with laser puncture and wheelchair aids in cases of paralysis of the cat's hind legs

A male cat named Kucil was brought by his owner to the Cimanggu Animal Clinic with complaints that the animal was unable to stand on its hind legs. The results of the physical examination showed that the cat Kucil was unable to walk, urinate, and defaecate, and paralysed in the tail and hind legs. Radiographic examination revealed a buildup of faeces in the colon and narrowing of the lumbar intervertebral spaces 5-6 and 6-7. The Cat was diagnosed with Intervertebral Disc Disease (IVDD). Acupuncture therapy using laser puncture at points ST-36, GB-34, BL-35, BL-54, SP-10, and Hua Tuo Jia-Ji, assisted by the use of a wheelchair, is an option for treating neurological disorders in this case. Improvement in the condition resulting from the therapy occurred after the 2nd therapy. To support the use of a wheelchair applied to the cat until the 4th therapy, the cat appears to be able to move its hind legs, and after the 9th therapy the cat appears to be able to walk.

Vector competence evaluation of mosquitoes for Tahyna virus PJ01 strain, a new Orthobunyavirus in China.

Tahyna virus (TAHV), an arbovirus of the genus Orthobunyavirus, is a cause of human diseases and less studied worldwide. In this study, a new strain of TAHV was isolated from Aedes sp. mosquitoes collected in Panjin city, Liaoning province. However, the competent vector of TAHV in China is still unknown. The genome of newly isolated TAHV was sequenced and phylogenetic analysis is performed. Aedes albopictus and Culex pipiens pallens were orally infected with artificial virus blood meals (1:1 of virus suspension and mouse blood), the virus was detected in the midgut, ovary, salivary gland and saliva of the mosquitoes. Then, the transmission and dissemination rates, vertical transmission and horizontal transmission of the virus by the mosquitoes were assessed. Phylogenetic analysis revealed that the virus shared high similarity with TAHV and was named the TAHV PJ01 strain. After oral infection with virus blood meals, Ae. albopictus showed positive for the virus in all tested tissues with an extrinsic incubation period of 2 days and a fluctuating increasement of transmission and dissemination rates. Whereas no virus was detected in the saliva of Cx. pipiens pallens. Suckling mice bitten by infectious Ae. albopictus developed obvious neurological symptoms, including inactivity, hind-leg paralysis and difficulty turning over, when the virus titer reached 1.70×105 PFU/mL in the brain. Moreover, TAHV was detected in the eggs, larvae and adults of F1 offspring of Ae. albopictus. Ae. albopictus is an efficient vector to transmit TAHV but Cx. pipiens pallens is not. Ae. albopictus is also a reservoir host that transmits the virus vertically, which further increases the risk of outbreaks. This study has important epidemiological implications for the surveillance of pathogenic viruses in China and guiding comprehensive vector control strategies to counteract potential outbreaks in future.

Abstract 15: Investigating the spectrum of brain tumors associated with Adgrb3 and Tp53 loss in a mouse model of Li-Fraumeni syndrome

Abstract Adhesion G protein-coupled receptor 3 (ADGRB3), also known as brain specific angiogenesis inhibitor (BAI3) is a member of the ADGRB1-3 subfamily of adhesion transmembrane proteins, which are highly expressed in the brain specifically in cerebellum and hippocampal neurons. ADGRB3 has been shown to play diverse roles under physiological and pathological conditions which include dendritic morphogenesis, synaptic plasticity, synaptogenesis, and myogenesis. Loss of ADGRB3 expression and point mutations in the gene have been observed in sporadic tumors, including brain tumors, but the significance of this observation has not been investigated. Moreover, no mouse models to understand the role of ADGRB3 in brain tumor susceptibility and pathobiology have been developed thus far. Li-Fraumeni syndrome (LFS) is a rare inherited autosomal dominant disorder caused by a germline mutation in one TP53 allele, predisposing patients to the development of a variety of tumors from a pediatric age, including gliomas and medulloblastoma. The secondary molecular alterations that predispose LFS patients to brain tumors are currently unknown. Interestingly, a LFS patient was reported to carry a translocation in ADGRB3 and HGMLL genes. To investigate the molecular mechanisms underlying brain tumor formation in LFS patients and the role of p53 and ADGRB3 in the process, we have generated a LFS mouse model. The mice harbor a germline p53 deleted allele and a second floxed allele. Loss of the second allele is induced in the brain by crossing with mice harboring a Nestin promoter driven Cre recombinase, since Nestin is expressed along the craniospinal axis and in glial precursor cells. We observed that the majority of nestin-Cre p53m/f mice all died between 8 and 10 months of age while nestin-Cre p53m/+ mice did not. About 20% of the mice developed hind leg paralysis and harbored large gliomas, which likely caused their demise. The remainder mice lacked brain tumors, but had other malignancies (sarcomas, etc.) as observed in patients. Remarkably, the addition of Adgrb3 deletion to the genotype led to a dramatic increase (from 20% to 60%??) in the number of brain tumors, including medulloblastoma formation, which are also reported in LFS patients. The Adgrb3-/- p53+/- Nestin-Cre mouse model constitutes a useful tool to understand the tumorigenic landscape caused by the loss of Adgrb3. We are now performing genomic analyses on the excised tumors and derived neurosphere cultures to further study the transformation process and the molecular changes induced by Adgrb3 loss. Citation Format: Alex B. Torrelli-Diljohn, Svetlana Komarova, Vipul Sheth, Benjamin Lin, Paran Goel, Ryan Miller, Robert Welner, Erwin G. Van Meir. Investigating the spectrum of brain tumors associated with Adgrb3 and Tp53 loss in a mouse model of Li-Fraumeni syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 15.

Comparison of toxicities between Vibrio fluvialis and Vibrio furnissii strains isolated from environmental samples

Gastrointestinal episodes associated with Vibrio species have been rising worldwide in the last few years. In this study, toxicities of two very well characterized V. fluvialis and V. furnissii strains, isolated from environmental samples, were used to investigate the comparative pathogenicity of these strains. The results showed that there is a positive relationship between toxin production as well as a high interspecies correlation. In the analysis of toxicity, both species showed positive results with close similarity in cell death on BHK-21 cell line and destruction of RBC, which were indicative of cytotoxic and hemolytic activity, respectively. Besides, molecular analysis showed the absence of ctx, tdh, trh, stx1 and stx2 genes. On the other hand, reduced fluid accumulation ratio in rabbit ileal loop assay and paralysis of hind legs of Swiss Albino mice were observed for V. furnissi strain, which indicates the differences in pathogenicity between the two species.
 Bangladesh J Microbiol, Volume 38, Number 2, December 2021, pp 73-77

Preclinical Development of Anti-FLT3 CAR-T Therapy for the Treatment of Acute Myeloid Leukemia

FMS-like tyrosine kinase 3 (FLT3) is a class III transmembrane receptor tyrosine kinase involved in survival, proliferation, and differentiation of hematopoietic stem/progenitor cells. It is preferentially expressed on the leukemic cells of myeloid lineage including acute myeloid leukemia (AML) and is mutated in approximately one-third of patients with AML, resulting in constitutive signaling associated with poor disease prognosis. Although small molecule inhibitors targeting FLT3 have shown some success in clinical trials, they only work transiently while resistance develops in virtually all patients. The only proven curative treatment for the relapsed or refractory (R/R) AML is allogenic hematopoietic stem cell transplantation (HSCT) which requires highly toxic conditioning regimens often associated with fatal side effects. Thus, there still remains an urgent need for the development of safe yet effective new therapies for the treatment of AML. We developed a novel chimeric antigen receptor modified T (CAR-T) cell therapy targeting FLT3 to eliminate FLT3+ R/R AML leukemia via cytotoxic T lymphocytes (CTL)-mediated cytolysis. Since FLT3 is also expressed on hematopoietic stem cells (HSCs) as well as on early hematopoietic progenitors (HPs), we evaluated the conditioning efficacy of our anti-FLT3 CAR-T in addition to its anti-leukemic activity. We first discovered a novel mouse monoclonal antibody that binds to the extracellular domain of human FLT3 with high affinity (0.8 nM EC50 in FLT3+ leukemic cell line REH) while not competing with FLT3 ligand in order to achieve unobstructed and efficient binding to FLT3. We next generated humanized single-chain variable fragment (scFv) antibodies and characterized their binding affinities. The scFv clone that exhibited highest binding to FLT3 (3.42 nM EC50 in REH cells) was used to design a third generation CAR construct with CD28 and 4-1BB costimulatory and CD3ζ activation domains. T cells isolated from peripheral blood (PB) were transduced with a lentiviral vector encoding the FLT3-CAR. Transduced cells exhibited stable expression of CAR protein and expanded over 120-fold after 18 days in culture. We demonstrated high cytotoxicity of FLT3-CAR-T cells towards AML-derived cell lines in co-culture experiments, even at effector-to-target cells ratios as low as 1:10. In vivo functionality of FLT3-CART was determined by flow cytometry analysis of leukemia burden in the peripheral blood of mice engrafted with GFP+ MOLM-13 (FLT3+ AML cell line) and treated with two doses of 4x106 control or FLT3-CAR-T cells. Compared to control, the appearance of MOLM-13 cells in peripheral blood was significantly delayed in FLT3-CAR-T treated mice. AML progression in mice was also assessed by detection of physical symptoms such as cachexia and hind-leg paralysis in terminal stages. FLT3-CAR-T treatment extended the median survival to 47 days compared to 24 days in control. Moreover, to test if our CAR-T therapy can also efficiently eliminate FLT3+ HSCs and HPs, humanized mice generated by engrafting human cord blood CD34+ cells were injected with autologous control or FLT3-CAR-T cells. Analysis of bone marrow 18 days post treatment, showed that mice that received FLT3-CAR-T cells exhibited dramatically lower frequencies (by 57% in CD38+ and 86% in CD38-) of human CD34+ hematopoietic stem and progenitor cells than control mice, suggesting the potential of CAR-T therapy for HSCT conditioning. In conclusion, our CAR-T therapy shows robust cytolytic activity against FLT3+ cells, demonstrates high efficacy in eradicating FLT3+ R/R AML leukemia in vivo and enables bone marrow conditioning for potentially curative HSCTs by specifically targeting FLT3+ HSCs and early HPs. To prevent the potentially harmful side effects associated with CAR-T therapies, such as cytokine release syndrome and cytotoxicity towards newly transplanted HSCs post conditioning, we are currently testing FLT3-CAR-T cells equipped with inducible caspase9 or EGFRT expression based safety switch to specifically eliminate CAR-Ts by administering FDA-approved small molecules or biologics. Disclosures Shrestha: Hemogenyx Pharmaceuticals LLC: Current Employment. Liang:Hemogenyx Pharmaceuticals LLC: Current Employment. Sirochinsky:Hemogenyx Pharmaceuticals LLC: Current Employment. Ben Jehuda:Hemogenyx Pharmaceuticals LLC: Current Employment. Sandler:Hemogenyx Pharmaceuticals LLC: Current Employment, Current equity holder in publicly-traded company.

FLT3-CD3 Bispecific Antibody Specifically Eliminates Normal Hematopoietic Progenitors and AML in Humanized Mouse Models

Acute myeloid leukemia (AML) is a blood cancer arising from clonal proliferation of myeloid progenitors harboring oncogenic mutations. AML patients have poor clinical prognosis and limited therapeutic options, with myeloablation followed by hematopoietic stem cell transplantation (HSCT) as the only curative treatment. The conditioning regimens used indiscriminately kill all highly proliferative cell types, leading to life threatening side effects, and are also potentially ineffective against quiescent AML subpopulations. To address the challenges of efficacy and resistance in targeting AML, we developed a humanized bispecific antibody (CDX) that recruits T cells through CD3 to target and kill Fms-like Tyrosine Kinase 3 (FLT3) expressing cells. Normal FLT3 expression is mostly restricted to hematopoietic stem and progenitor cells (HSPCs) and its activation, through binding of FLT3 ligand (FLT3L), promotes normal differentiation of downstream blood lineages. FLT3 is also expressed on AML blasts in a majority of patients and is thought to promote survival and proliferation of AML. Although multiple promising tyrosine kinase inhibitors (TKIs) have been and are being developed to specifically target FLT3, secondary mutations leading to resistance against FLT3-TKIs remain a major obstacle. Surface expression of FLT3 in leukemic cells, as well as in HSCs, makes it an excellent target for T cell mediated conditioning that specifically eliminates both AML blasts and HSPCs, allowing for subsequent HSCT. In addition, HDX mass spectrometry revealed that our CDX binds an extracellular, membrane proximal FLT3 domain, which does not compete with FLT3L and is outside the regions commonly mutated in AML, therefore, unlike other therapies, targets all FLT3-expressing cells, regardless of mutations in the receptor. We found that in vitro treatment with CDX redirected primary human T cells to kill multiple AML-derived FLT3+ cell lines, some harboring common FLT3 mutations, at EC50 ranging from 0.3 pM to 15 pM. Additionally, the presence of FLT3L (10 nM) had no effect on the EC50 of CDX binding to FLT3+ target cells. To test CDX in vivo, we generated xenograft mice engrafted with primary human peripheral blood mononuclear cells (PBMCs) and MV4;11 cells (FLT3+ AML cell line), which were engineered to stably express EGFP. Mice were treated intravenously with 3 doses of CDX at a concentration of 0.1 mg/kg or left untreated over 1 week (n=5). Progression of AML was tracked by monitoring the frequency of EGFP-MV4;11 cells in peripheral blood (PB) of xenografted mice, which were euthanized if they presented with cachexia, hind-leg paralysis, and severe weight loss (symptoms of AML progression). Treatment with CDX significantly delayed the proliferation of MV4;11 cells, based on lower PB frequencies of EGFP-MV4;11, and increased median survival by 28 days relative to untreated mice. To more stringently model CDX efficacy in a clinical setting, we injected EGFP-MV4;11 cells into humanized mice that were generated by transplantation of human cord blood derived, CD34+ HSPCs and displayed stable multi-lineage engraftment (~20% human CD45+) with significant populations of T cells at 35 weeks. We observed that T cells in CDX-treated mice over-expressed the immune checkpoint receptor PD1, relative to untreated mice, and hypothesized that PD1 inhibition could enhance MV4;11 clearance via CDX treatment by preventing T cell exhaustion. Treatment regimens consisted of 3 doses of CDX (0.1 mg/kg), anti-PD1 antibody (100 μg), or a combination of CDX and anti-PD1 (n=5), followed by transplant with autologous mouse bone marrow cells. Treatment with CDX alone led to decreased PB frequencies of EGFP-MV4;11 cells, but only modestly improved median survival relative to anti-PD1 alone (9 days). In contrast, co-treatment with CDX and anti-PD1 antibody significantly increased median survival (16 days) and was associated with decreased PD1 expression on T cells. In addition, administration of CDX alone or in combination with PD1 in humanized mice resulted in efficient elimination of the human hematopoietic compartment from the mouse bone marrow. Based on our findings, CDX shows promise for treatment of AML with concurrent conditioning for HSCT with improved specificity compared to standard of care and even displays synergy with checkpoint inhibition of PD1. Disclosures Sirochinsky: Hemogenyx Pharmaceuticals LLC: Current Employment. Liang:Hemogenyx Pharmaceuticals LLC: Current Employment. Shrestha:Hemogenyx Pharmaceuticals LLC: Current Employment. Ben Jehuda:Hemogenyx Pharmaceuticals LLC: Current Employment. Sandler:Hemogenyx Pharmaceuticals LLC: Current Employment, Current equity holder in publicly-traded company.

The Spinal Cord Damage in a Rat Asphyxial Cardiac Arrest/Resuscitation Model.

After cardiac arrest/resuscitation (CA/R), animals often had massive functional restrictions including spastic paralysis of hind legs, disturbed balance and reflex abnormalities. Patients who have survived CA also develop movement restrictions/disorders. A successful therapy requires detailed knowledge of the intrinsic damage pattern and the respective mechanisms. Beside neurodegenerations in the cerebellum and cortex, neuronal loss in the spinal cord could be a further origin of such movement artifacts. Thus, we aimed to evaluate the CA/R-induced degeneration pattern of the lumbar medulla spinalis by immunocytochemical expression of SMI 311 (marker of neuronal perikarya and dendrites), IBA1 (microglia marker), GFAP (marker of astroglia), calbindin D28k (marker of the cellular neuroprotective calcium-buffering system), MnSOD (neuroprotective antioxidant), the transcription factor PPARγ and the mitochondrial marker protein PDH after survival times of 7 and 21days. The CA/R specimens were compared with those from sham-operated and completely naïve rats. RESULTS& CONCLUSION: The main ACA/R-mediated results were: (1) degeneration of lumbar spinal cord motor neurons, characterized by neuronal pyknotization and peri-neuronal tissue artifacts; (2) attendant activation of microglia in the short-term group; (3) attendant activation of astroglia in the long-term group; (4) degenerative pattern in the intermediate gray matter; (5) activation of the endogenous anti-oxidative defense systems calbindin D28k and MnSOD; (6) activation of the transcription factor PPARγ, especially in glial cells of the gray matter penumbra; and (7) activation of mitochondria. Moreover, marginal signs of anesthesia-induced cell stress were already evident in sham animals when compared with completely naïve spinal cords. A correlation between the NDS and the motor neuronal loss could not be verified. Thus, the NDS appears to be unsuitable as prognostic tool.

Systematic evaluation of skeletal fractures caused by induction of electroconvulsive seizures in rat state a need for attention and refinement of the procedure

Electroconvulsive therapy (ECT) is one of the most efficient treatments for major depression. Electroconvulsive seizures (ECS), the animal model of ECT, is widely used to study both mechanisms of action and adverse effects of ECT. As the treatment itself serves as an instant anaesthetic and anaesthetic agents may affect memory functions and behaviour, ECS is traditionally administered without muscle relaxation and anaesthesia. A major problem of unmodified ECS, which has only been addressed peripherally in the literature, is that some animals sustain spinal fractures and subsequent hind leg paralysis (paraplegia). This phenomenon leads to a higher degree of suffering and these animals need to be excluded from the studies. To reach sufficient statistical power, the group sizes are therefore often increased and this may lead to a pre-selected study group in risk of skewing the results. Moreover, the study design of the experiments do not comply with the 3R principles, which advocate for both refinement and reduction of animal experiments. The objective of this study is to systematically evaluate injuries caused by ECS. We summarise the incidence of spinal fractures from 24 studies conducted during 2009-2015 in six different rat strains and report preliminary findings on scapular fractures following auricular ECS. In total, 12.8% of all tested animals suffered from spinal fractures and we find an increase in spinal fracture incidence over time. Furthermore, X-ray analyses revealed that some animals displayed scapular fractures. We discuss consequences of and possible explanations for ECS-induced fractures. Modifications of the method are highly warranted and we furthermore suggest that all animals are thoroughly examined for discrete fractures.

IL-21-Expressing Mesenchymal Stem Cells Prevent Lethal B-Cell Lymphoma Through Efficient Delivery of IL-21, Which Redirects the Immune System to Target the Tumor

Interleukin (IL)-21, a proinflammatory cytokine, has been developed as an immunotherapeutic approach due to its effects on various lymphocytes, including natural killer (NK) cells and T cells; however, the clinical success in cancer patients has been limited. Recently, mesenchymal stem cells (MSCs) have emerged as vehicles for cancer gene therapy due to their inherent migratory abilities toward tumors. In the present study, we hypothesized that MSCs, genetically modified to express high levels of IL-21 (IL-21/MSCs), can enhance antitumor responses through localized delivery of IL-21. For tumor induction, BALB/c mice were injected intravenously with syngeneic A20 B-cell lymphoma cells to develop a disseminated B-cell lymphoma model. Then, 6 days following tumor induction, the tumor-bearing mice were treated with IL-21/MSCs weekly, four times. Systemic infusion of A20 cells led to hind-leg paralysis as well as severe liver metastasis in the control group. The IL-21/MSC-treated group showed delayed tumor incidence as well as improved survival, whereas the MSC- and recombinant adenovirus-expressing IL-21 (rAD/IL-21)-treated groups did not show significant differences from the untreated mice. These therapeutic effects were associated with high levels of IL-21 delivered to the liver, which prevented the formation of tumor nodules. Furthermore, the infusion of IL-21/MSCs led to induction of effector T and NK cells, while potently inhibiting immune suppressor cells. Our findings demonstrate that IL-21-expressing MSCs have the therapeutic potential to induce potent antitumor effects against disseminated B-cell lymphoma through localized IL-21 delivery and induction of systemic antitumor immunity.

Abstract 2462: In Vivo efficacy of a novel anti-CD33 targeted thorium conjugate (TTC) in mouse models of acute myeloid leukemia (AML)

Abstract Introduction: The sialoadhesin receptor CD33 is a well-established target in the treatment of acute myeloid leukemia (AML). CD33 is expressed on leukemic blasts in AML and possibly also on leukemic stem cells in some patients (1). Thorium-227, an alpha-particle emitting radionuclide with a half-life of 18.7 days, is being studied for use as a possible cytotoxic agent. We have developed a novel Targeted Thorium Conjugate (TTC), comprising an anti-CD33 monoclonal antibody conjugated to a chelator for the efficient delivery of high-energy alpha-particles. We present data from in vitro cell cytotoxicity assays as well as biodistribution and efficacy data from preclinical animal models of AML. Experimental procedures: Antibody conjugates were prepared and labeled with 227Th at room temperature giving high radiochemical yields and purity. In vitro cell cytotoxicity experiments were carried out on HL-60 cells and the multi-drug resistant cell line KG-1 by measuring cell viability and monitoring apoptosis. In vivo efficacy and biodistribution were performed in a sub-cutaneous model of AML in athymic nude (nu/nu) mice using HL-60 cells. The 227Th-anti-CD33 conjugate was further tested in a disseminated model of AML using HL-60 cells, injected intravenously 5 days prior to treatment in CB.17 SCID mice with hind-leg paralysis as endpoint. Results: The 227Th-anti-CD33 conjugate was a potent and specific inducer of in vitro cell cytotoxicity independent of MDR-status. Mechanistic studies demonstrated cell arrest in G2 phase with cytotoxicity associated with apoptotic pathways. In vivo biodistribution in a sub-cutaneous model demonstrated a high tumor to blood ratio (TBR ∼ 6) at day 7 post injection based on measured 227Th-activity. Significant efficacy was achieved in two models of AML using human HL-60 cells. In athymic nude mice, a single injection of 227Th-anti-CD33 conjugate at 700 kBq/kg resulted in complete regression of established sub-cutaneous tumors. Importantly, no significant loss in body weight was observed during the course of the study, demonstrating that the dose was well tolerated. In parallel, statistically significant dose dependency of 227Th-anti-CD33 conjugate was demonstrated in a disseminated model of AML with median survival times (MST) of 90 days and 116 days for animals receiving 50 and 150 kBq/kg. Around 80% of animals dosed with either 2 × 150 or 1 × 300 kBq/kg where still alive at the end of the study with no gross pathological findings observed. Conclusions: The data presented are supportive of the further investigation of the 227Th-anti-CD33 conjugate for the treatment of AML.

Biochemical and pharmacological characterization of a toxic fraction and its cytotoxin-like component isolated from Russell's viper (Daboia russelii russelii) venom

The pathophysiological significance of a toxic fraction (GF-VI DEAE-II) isolated from Russell's viper venom (RVV) is characterized. GF-VI DEAE-II represents 1.6% of the total RVV protein and it comprises of a 27.6kDa minor component (RP-I) (0.04%, w/w) and a major 6.6kDa non-enzymatic peptide (1.11%, w/w), named Rusvitoxin. The LC-MS/MS analysis of RP-I showed its identity to snake venom serine proteases, whereas Rusvitoxin demonstrated its close identity with snake venom three finger toxins, cytotoxins and cardiotoxins particularly from Naja sp. GF-VI DEAE-II was found to be non-cytotoxic to the tested mammalian cancer cells and non-hemolytic; nevertheless, it demonstrated α-fibrin(ogen)ase activity and in vivo toxicity in BALB/c mice with an LD50 (i.p.) of 2.3mg/kg. GF-VI DEAE-II induced lethargy and hind-leg paralysis in mice within 10min of i.p. injection. GF-VI DEAE-II induced hyperfibrinogenomia, and significantly altered (p<0.05) the plasma levels of factor X, pro- and anti-inflammatory cytokines viz. TNF-α, IL-6 and IL-10 in treated mice. Histological observations of tissues and biochemical properties of serum from GF-VI DEAE-II-treated mice suggested multiple organ dysfunctions. Conversely, Rusvitoxin at a dose of 5mg/kg did not induce toxicity in BALB/c mice. At 1:15 (antigen: antivenom, w/w) ratio, commercially polyvalent and monovalent antivenoms neutralized more than 80% of the fibrinolytic and anticoagulant activities of GF-VI DEAE-II. The present study suggests the significant role of GF-VI DEAE-II in RVV-induced pathogenesis in victim/prey.

A Systemic Xenograft Model of Waldenström’s Macroglobulinemia Demonstrates the Potent Anti-Tumor Effect of Second Generation CD19 Directed Chimeric Antigen Receptor Modified T Cells in This Disease

Chimeric antigen receptor (CAR) modified T-cell therapy consists of ex-vivo genetic manipulation of autologous lymphocytes in order to establish robust T-cell mediated anti-tumor immunity. Our group was the first to design and evaluate a CAR targeted toward the B cell antigen CD19 in mice. Currently we utilize a second generation CAR comprised of a single-chain variable fragment (scFv) derived from an antibody against CD19 fused to the CD3 ζ chain and the CD28 intracellular signaling domain (19-28z) to provide the necessary signal 1 and signal 2 for enhanced T-cell activation and persistence. We have gone on to test the safety and efficacy of 19-28z CAR T-cells in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). We observed rapid complete molecular remissions in the first 14/16 patients treated with relapsed/refractory ALL. We hypothesize that contributing to the enhanced efficacy seen in ALL, when compared to solid tumors or extra-medullary CLL, is the fact that ALL is a bone marrow predominant disease, which may provide a microenvironment more amenable to T-cell therapy.Waldenström’s Macroglobulinemia (WM) is an ideal disease to test 19-28z CAR-modified T-cell therapy, as it is often bone marrow predominant, and WM cells from patient samples typically uniformly express high levels of CD19. Furthermore, despite recent progress made with novel BCR-directed therapy, complete eradication of the WM clone from the bone marrow niche remains elusive, therefore providing an ideal clinical scenario for treatment consolidation via alternative cytotoxic methods such as cellular immunotherapy.Using the human WM cell line, BCWM.1, we evaluated the in vitro efficacy of 19-28z CAR-modified T-cells when compared to mock transduced T cells or T cells transduced with an irrelevant second generation CAR directed towards the ovarian antigen MUC16. In a 4 hour co-culture assay, we observed significant cytotoxicity, even at low effector:target ratios (52% lysis at 1:1; 92% lysis at 10:1; p<0.01). This corresponded to increased secretion of INFγ and IL-2, markers of T-cell activation (p<0.01).We then conducted in vivo studies using sublethally irradiated SCID/beige mice to generate a systemic model of WM via tail vein injection of 1x106 luciferase transduced BCWM.1 cells. This model is characterized by tumor growth in the bone marrow followed by rapid spread to the liver, lungs, kidney, and CNS. Mice were monitored by weekly bioluminescent imaging (BLI) and ultimately were sacrificed when they developed hind leg paralysis. 19-28z CAR modified T-cells administered at day 7, after tumor establishment, when compared to non-treated and irrelevant CAR-modified T cell controls, delayed the progression of disease and doubled the median survival time of the mice after treatment (p=0.001).Taken together, the pre-clinical efficacy demonstrated in this abstract and the clinical features of WM, listed above, provide the rational for testing 19-28z CAR modified T cells clinically for WM. We have now opened a clinical trial for patients with relapsed or refractory WM, in which chemotherapy preconditioning is followed by a single dose of 19-28z CAR modified autologous T-cells (NCT00466531). DisclosuresBrentjens:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.

Antimyeloma activity of NK012, a micelle‐forming macromolecular prodrug of SN‐38, in an orthotopic model

NK012 is a micelle-forming macromolecular prodrug of 7-ethyl-10-hydroxy camptothecin (SN-38), an active metabolite of irinotecan. It is accumulated and retained in tumor tissues and gradually releases SN-38 in an enzyme-independent manner. NK012 was previously demonstrated to have stronger antitumor activity than irinotecan in a broad range of human solid-tumor xenograft models. In our study, we used an orthotopic multiple myeloma (MM) model created by injecting CD138-positive U266B1, a myeloma cell line that produces human IgE lambda light chain (monoclonal protein, M protein), into immunodeficient NOD/Shi-scid, IL-2Rγc (null) mice. This model shows typical bone marrow infiltration by the human myeloma cells. We evaluated the antimyeloma activity of intravenously administered NK012 in this model and showed that it suppressed the M protein concentration in the plasma and proliferation of myeloma cells in the bone marrow in a dose-dependent manner. NK012 suppressed the progression of hind-leg paralysis and prolonged the survival time of the mice compared to the untreated control group. In combination with bortezomib (BTZ), NK012 increased the median survival time compared to that with BTZ alone. In conclusion, these results suggest that NK012 is a potential candidate for use-alone and in combination-in the treatment of MM in humans.

AMP-Activated Protein Kinase Signaling Protects Oligodendrocytes that Restore Central Nervous System Functions in an Experimental Autoimmune Encephalomyelitis Model

AMP-activated protein kinase (AMPK) signaling is reported to protect neurons under pathologic conditions; however, its effect on oligodendrocytes (OLs) remains to be elucidated. We investigated whether AMPK signaling protects OLs to restore central nervous system (CNS) functions in an experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. Increased inflammation and demyelination in the CNS and peripheral immune responses were consistent with the observed clinical impairments in EAE animals, which were attenuated by treatment with metformin compared with vehicle. In addition, expressions of neurotrophic factors and of signatory genes of OL lineages were increased in the CNS of metformin-treated EAE animals. Likewise, metformin attenuated inflammatory response and enhanced expressions of neurotrophic factors, thereby protecting OLs via AMPK activation in mixed glial cultures stimulated with lipopolysaccharide/interferon γ invitro, as evidenced by analysis of the expression of signatory genes of O1(+)/MBP(+) OLs and their cellular populations. Metformin also attenuated oxidative stress and malondialdehyde-containing protein levels, with corresponding induction of antioxidative defenses in OLs exposed to cytokines via AMPK activation. These effects of metformin were evident in the CNS of EAE animals. These data provide evidence that AMPK signaling is crucial to protect OLs and, thus, CNS functions in EAE animals. We conclude that AMPK activators, including metformin, have the potential to limit neurologic deficits in multiple sclerosis and related neurodegenerative disorders.

Intraperitoneal administration of Shiga toxin 2 induced neuronal alterations and reduced the expression levels of aquaporin 1 and aquaporin 4 in rat brain

Shiga toxin-producing Escherichia coli produces watery and hemorrhagic diarrhea, and hemolytic uremic syndrome (HUS) characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure. Central nervous system (CNS) complications are observed in around 30% of infant population with HUS. Common signs of severe CNS involvement leading to death include seizures, alteration of consciousness, hemiparesis, visual disturbances, and brain stem symptoms. The purpose of the present work was to study the effects of Shiga toxin 2 (Stx2) in the brain of rats intraperitoneally (i.p.) injected with a supernatant from recombinant E. coli expressing Stx2 (sStx2). Neurological alterations such as postural and motor abnormalities including lethargy, abnormal walking, and paralysis of hind legs, were observed in this experimental model of HUS in rats. Neuronal damage, as well as significant decrease in aquaporin 1 (AQP1) and aquaporin 4 (AQP4) expression levels were observed in the brain of rats, 2 days after sStx2 injection, compared to controls. Downregulation of aquaporin protein levels, and neuronal alterations, observed in brain of rats injected with sStx2, may be involved in edema formation and in neurological manifestations characteristic of HUS.

Quantifying subtle but persistent peri-spine inflammation in vivo to submicron cobalt–chromium alloy particles

We evaluated the consequences of cobalt-chromium alloy (CoCr) wear debris challenge in the peri-spine region to determine the inflammation and toxicity associated with submicron particulates of CoCr-alloy and nickel on the peri-spine. The lumbar epidural spaces of (n = 50) New Zealand white rabbits were challenged with: 2.5 mg CoCr, 5.0 mg CoCr, 10.0 mg CoCr, a positive control (20.0 mg of nickel) and a negative control (ISOVUE-M-300). The CoCr-alloy and Ni particles had a mean diameter of 0.2 and 0.6 μm, respectively. Five rabbits per dose group were studied at 12 and 24 weeks. Local and distant tissues were analyzed histologically and quantitatively analyzed immunohistochemically (TNF-α and IL-6). Histologically, wear particles were observed in all animals. There was no evidence of toxicity or local irritation noted during macroscopic observations in any CoCr-dosed animals. However, Ni-treated control animals experienced bilateral hind leg paralysis and were euthanized at Day 2. Histopathology of the Ni particle-treated group revealed severe neuropathy. Quantitative immunohistochemistry demonstrated a CoCr-alloy dose-dependent increase in cytokines (IL-6, TNF-α, p < 0.05) at 12 and 24 weeks. Subtle peri-spine inflammation associated with CoCr-alloy implant particles was dose dependent and persistent. Neuropathy can be induced by highly reactive Ni particles. This suggests peri-spine challenge with CoCr-alloy implant debris (e.g., TDA) is consistent with past reports using titanium alloy particles, i.e., mild persistent inflammation.

Induction of a Protective Response in Mice by the Dengue Virus NS3 Protein Using DNA Vaccines

The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection.

Abstract 4435: Anti-multiple myeloma activity of NK012, a micelle-forming macromolecular prodrug of SN-38, in an orthotopic model

Abstract NK012 is a micelle-forming macromolecular prodrug of SN-38, an active metabolite of irinotecan (CPT-11). It accumulates in tumor tissue and gradually releases SN-38 in an enzyme-independent manner. We previously reported that NK012 showed stronger antitumor activity in a broad range of human solid tumor xenograft models than CPT-11 (2006 AACR Annual Meeting, #3062). Currently, phase II clinical trials of NK012 in patients with triple-negative breast cancer, small cell lung cancer, and colorectal cancer are ongoing in the United States and Japan. In this study, we evaluated the antitumor activity of NK012 in an orthotopic multiple myeloma model that shows typical infiltration of human myeloma cells into murine bone marrow tissue. A suspension of 2 × 106 cells of CD138 positive U266 human myeloma cells was injected into immunodeficient NOG (NOD/Shi-scid, IL-2Rγnull) mice via tail vein. The transplanted mice produced plasma M protein (human IgE) and developed lytic bone lesions. In addition, hind-leg paralysis caused by myeloma progression in spinal cord was observed. These symptoms were similar to complications observed in the patients with multiple myeloma. The intravenous administration of NK012 at 3.75, 7.5, and 15 mg/kg/day by q4d × 3 suppressed M protein concentration in plasma and proliferation of myeloma cells in bone marrow in a dose-dependent manner. Also observed were decrease in the area of osteolytic lesions and recovery of decreased total bone mineral density in the tibias. To examine the efficacy of NK012 in survival time, the myeloma-transplanted mice were treated with 9.4 mg/kg/day (28.2 mg/m2/day), q7d × 6, equivalent to the clinical recommended dose of 28 mg/m2/day as a single dose. NK012 significantly prolonged survival time of the mice compared with control group (78.5 days vs. 40 days, P&amp;lt;0.001). We next examined the combination effect of NK012 with bortezomib, which is commonly used for multiple myeloma treatment. The combined treatment of NK012 (9.4 mg/kg/day, q7d × 3) with bortezomib (0.5 mg/kg/day, 2 times/week × 2, i.v.) exhibited increase in median survival time over bortezomib alone (83.5 days vs. 68 days). In conclusion, these results suggested NK012 is a promising candidate for human multiple myeloma treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4435. doi:10.1158/1538-7445.AM2011-4435

Abstract 3304: Conditional Pten loss in Olig2 expressing neural stem/progenitor cells results in massive myelination and disruption of the neuronal differentiation in the absence of neoplasia

Abstract PTEN represents one of the most frequently lost tumor suppressor genes in human cancers with inactivation (deletion or mutation) being particularly frequent in glioblastoma, endometrial cancer, and prostate cancer. Recent work has shown that the functional consequences of Pten loss leading to PI3K pathway activation are highly dependent on cell lineages in which pathway activation occurs. The bHLH transcription factor Olig2 is a critical regulator of stem/progenitor cells in the oligodendroglial lineage and gliomas. In addition, cells of this lineage are likely cells of origin for human GBM. Given these findings, we hypothesized that Pten loss in this lineage might lead to novel insights into stem cell development and glioma formation. To create mice with Pten loss in Olig2+ cells at all stages of development we crossed Olig2-tva-cre (aka Olig2-cre) mice to mice containing a conditional Pten allele. Olig2-Cre:PtenloxP/loxP mice were viable, able to reproduce and grossly normal until 10 months of age when they developed progressive ataxia and hind leg paralysis leading to death. Pathologic exam showed no evidence of CNS tumor or gross developmental abnormalities but instead revealed massively expanded white matter tracts throughout all regions of the CNS associated with severe neurodegenerative changes, inflammation and axonal degeneration. Most interestingly PTEN deletion in the Olig2+ cells revealed an unexpected requirement of Pten in stem cells maintenance and neuronal differentiation. In vitro assay highlighted the increased proliferation and self-renewal capacity of adult and embryonic Olig2-Cre:PtenloxP/loxP neurospheres. Moreover, when cultured with either serum or growth factor, Pten deleted neurospheres harbor a higher number of differentiated inhibitory class neurons leading to more doublecortin positive neuroblasts in the SVZ/RMS as well as post-mitotic calretinin positive neurons in the SVZ and corpus callosum. To explain this phenotype we postulate that either Pten deleted neural stem cells underwent abnormal neuronal differentiation during development leading to excessive production of neurons or that extra neuroblasts born during development that failed to undergo apoptosis due to their Pten deletion. Overall these findings demonstrate that alterations of PTEN/PI3K signaling have unique and dramatic cell type specific effects on oligodendrocytes and neural stem cell within the Olig2 lineage. These findings are of particular importance given the development of PI3K pathway inhibitors destined for use in glioma clinical trials and suggest potential for effects on myelination within treated patients. Further studies will also address the secondary events necessary to promote tumorigenesis by using the Olig2-Cre:PtenloxP/loxP mice as a platform. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3304. doi:10.1158/1538-7445.AM2011-3304

Abstract LB-157: A murine model of Fanconi anemia-associated head and neck cancer

Abstract Fanconi anemia (FA) predisposes patients to aggressive head-and-neck-squamous-cell-carcinomas (HNSCCs) early in life. These tumors are particularly difficult to treat because of the underlying hypersensitivity of all somatic cells from these patients to cross-linking agents. An in vivo model is thus badly needed to delineate the unique molecular mechanisms involved in tumor formation and to identify opportunities to exploit this mechanism therapeutically. Although mice with inactivating mutations of FA genes have been developed, these mice do not spontaneously develop HNSCCs. Therefore, we sought to develop our own model by treating wild-type (WT) and Fancc-deficient (FA-C) mice with the carcinogen 4-nitroquinoline 1-oxide for 45 weeks. Although tumor incidence, histological type and grade, and anatomical location were similar in WT and FA-C mice, cells isolated from FA-C tumors displayed higher levels of endogenous chromosomal instability compared to cells from WT tumors. None of the mice developed metastases, most likely because they required early euthanasia as tumor formation inhibited the ability to eat and drink. To compare the metastatic potential of WT and FA-C HNSCC cells, we subcutaneously injected these cells lines into the flanks of immunocompetent (C57Bl/6J) and immunodeficient (NOD/SCID) mice. Interestingly, tumors only formed in NOD/SCID mice indicating immune-mediated suppression of tumor growth in the immunocompetent mice. In NOD/SCID mice, 4 out of 6 WT and 3 out of 4 FA-C cell lines induced tumor formation. However, all of the tumor-forming FA-C lines produced aggressive tumors that penetrated into the retro-peritoneal space and/or produced hind-leg paralysis, while only 1 out of 4 WT lines invaded to this extent (a phenomenon that occurred in fewer than 25% of mice injected with this line). In addition, we found that tumor forming ability of FA-C HNSCC cell lines did not require loss of Fancc because FA-C lines transduced with wild-type Fancc remained fully tumorigenic in vivo. In conclusion, we have developed a model of FA related HNSCCs utilizing FA and WT cell lines. The model permits the outgrowth of tumors that are histologically, genomically, and biologically similar to corresponding tumors in humans. Currently we are utilizing this model to test the efficacy of small molecules in inhibiting FA tumor progression and are testing the role of the tumor microenvironment and the immune system in modulating the development of FA-related HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-157.