Investigating age and ethnicity as novel high-risk phenotypes in mucinous ovarian cancer: retrospective study in a multi-ethnic population.

OBJECTIVES: Primary mucinous ovarian carcinoma represents 3% of ovarian cancers and is typically diagnosed early, yielding favorable outcomes. This study aims to identify risk factors, focussing on the impact of age and ethnicity on survival from primary mucinous ovarian cancer. METHODS: A retrospective observational study of patients treated at Sandwell and West Birmingham Hospitals NHS Trust and University Hospital Coventry and Warwickshire. Patients included were women aged ≥16 years, with primary mucinous ovarian cancer confirmed by specialist gynecological histopathologist and tumor immunohistochemistry, including cytokeratin-7, cytokeratin-20, and CDX2. Statistical analyses were performed using R integrated development environment, with survival assessed by Cox proportional hazards models and Kaplan-Meier plots. RESULTS: A total of 163 patients were analyzed; median age at diagnosis was 58 years (range 16-92), 145 (89%) were International Federation of Gynecology and Obstetrics stage I and 43 (26%) patients had infiltrative invasion. Women aged ≤45 years were more likely to have infiltrative invasion (RR=1.38, 95% CI 0.78 to 2.46), with increased risk of death associated with infiltrative invasion (HR=2.29, 95% CI 1.37 to 5.83). Compared with White counterparts, South Asian women were more likely to undergo fertility-sparing surgery (RR=3.52, 95% CI 1.48 to 8.32), and have infiltrative invasion (RR=1.25, 95% CI 0.60 to 2.58). South Asian women undergoing fertility-sparing surgery had worse prognosis than those undergoing traditional staging surgery (HR=2.20, 95% CI 0.39 to 13.14). In FIGO stage I disease, 59% South Asian and 37% White women received adjuvant chemotherapy (p=0.06). South Asian women exhibited a worse overall prognosis than White women (HR=2.07, 95% CI 0.86 to 4.36), particularly pronounced in those aged ≤45 years (HR=8.75, 95% CI 1.22 to 76.38). CONCLUSION: This study identified young age as a risk factor for diagnosis of infiltrative invasion. Fertility-sparing surgery in South Asian women is a risk factor for poorer prognosis. South Asian women exhibit poorer overall survival than their White counterparts.

Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches.

Anticytotoxic T lymphocyte-associated protein 4 (CTLA4) antibodies have shown potent antitumor activity, but systemic immune activation leads to severe immune-related adverse events, limiting clinical usage. We developed novel, conditionally active biologic (CAB) anti-CTLA4 antibodies that are active only in the acidic tumor microenvironment. In healthy tissue, this binding is reversibly inhibited by a novel mechanism using physiological chemicals as protein-associated chemical switches (PaCS). No enzymes or potentially immunogenic covalent modifications to the antibody are required for activation in the tumor. The novel anti-CTLA4 antibodies show similar efficacy in animal models compared to an analog of a marketed anti-CTLA4 biologic, but have markedly reduced toxicity in nonhuman primates (in combination with an anti-PD1 checkpoint inhibitor), indicating a widened therapeutic index (TI). The PaCS encompass mechanisms that are applicable to a wide array of antibody formats (e.g., ADC, bispecifics) and antigens. Examples shown here include antibodies to EpCAM, Her2, Nectin4, CD73, and CD3. Existing antibodies can be engineered readily to be made sensitive to PaCS, and the inhibitory activity can be optimized for each antigen's varying expression level and tissue distribution. PaCS can modulate diverse physiological molecular interactions and are applicable to various pathologic conditions, enabling differential CAB antibody activities in normal versus disease microenvironments.

DksA-dependent regulation of RpoS contributes to Borrelia burgdorferi tick-borne transmission and mammalian infectivity.

Throughout its enzootic cycle, the Lyme disease spirochete Borreliella (Borrelia) burgdorferi, senses and responds to changes in its environment using a small repertoire of transcription factors that coordinate the expression of genes required for infection of Ixodes ticks and various mammalian hosts. Among these transcription factors, the DnaK suppressor protein (DksA) plays a pivotal role in regulating gene expression in B. burgdorferi during periods of nutrient limitation and is required for mammalian infectivity. In many pathogenic bacteria, the gene regulatory activity of DksA, along with the alarmone guanosine penta- and tetra-phosphate ((p)ppGpp), coordinate the stringent response to various environmental stresses, including nutrient limitation. In this study, we sought to characterize the role of DksA in regulating the transcriptional activity of RNA polymerase and its role in the regulation of RpoS-dependent gene expression required for B. burgdorferi infectivity. Using in vitro transcription assays, we observed recombinant DksA inhibits RpoD-dependent transcription by B. burgdorferi RNA polymerase independent of ppGpp. Additionally, we determined the pH-inducible expression of RpoS-dependent genes relies on DksA, but this relationship is independent of (p)ppGpp produced by Relbbu. Subsequent transcriptomic and western blot assays indicate DksA regulates the expression of BBD18, a protein previously implicated in the post-transcriptional regulation of RpoS. Moreover, we observed DksA was required for infection of mice following intraperitoneal inoculation or for transmission of B. burgdorferi by Ixodes scapularis nymphs. Together, these data suggest DksA plays a central role in coordinating transcriptional responses in B. burgdorferi required for infectivity through DksA's interactions with RNA polymerase and post-transcriptional control of RpoS.

Nonsquamous Malignancies of Vagina and Vulva: 23-Year Experience at a Tertiary Center in the United Kingdom.

Under 10% of gynaecological cancers are diagnosed in the vulva and vagina, mostly squamous cell carcinomas. Melanoma, Paget disease, basal cell carcinomas, and other cancers can present with vulval/vaginal symptoms. The pathology information system of a tertiary referral center for vulvo-vaginal cancers was searched for cancers of the vulva and vagina from 1996 to 2019. Squamous carcinomas were excluded, and the remaining entities were catalogued. A total of 221 nonsquamous cancers were found, including 135 vaginal and 86 vulval cases. One hundred eight cases of metastatic carcinomas from the endometrium, cervix, ovary, bowel, bladder, kidney, and breast formed the largest category. Basal cell carcinomas constituted the second largest category. Others included melanomas, Paget disease, and adenoid cystic carcinomas. Primary adenocarcinomas included porocarcinoma, mammary type carcinoma, enteric type carcinoma, clear cell carcinoma, Bartholin gland adenocarcinoma and malignant transformation of hidradenoma papilliferum. The vulva and vagina can harbor a wide range of nonsquamous malignancies. The most challenging of these are adenocarcinomas which can be metastatic from other sites. The dominance of metastatic carcinomas in this series is likely to reflect consultation practice of specialist pathologists.

Uterine Neurotrophic Tyrosine Receptor Kinase Rearranged Spindle Cell Neoplasms: Three Cases of an Emerging Entity.

Uterine sarcomas are rare; most are either smooth muscle or endometrial stromal in origin. Recent molecular advances have identified several, genetically defined entities with specific morphologic, clinicopathological associations, and therapeutic options. We report 3 cases of uterine neurotrophic tyrosine receptor kinase (NTRK)-rearranged spindle cell neoplasms," currently classified as "emerging entities" in the WHO Classification of Female Genital Tract Tumors, 2020, Fifth Edition. The affected patients were 32, 34, and 42 years of age. Two patients presented with vaginal bleeding; the third presented with a cervical mass found incidentally during laparoscopy for an ectopic gestation. All 3 tumors were polypoid masses that distorted the cervix. Microscopically, they comprised cellular, fascicular, and storiform, plump spindle cells, with occasional rounded cells, and frequent mitoses (4-48/10 high power fields) in a myxoid stroma. All 3 cases showed entrapment of benign cervical glands. Inflammatory cell infiltrates, including plasma cells, were noted in all 3 tumors. One case had tumor cell necrosis, osteoid-like material, and osteoclast-like giant cells and showed lymphovascular invasion. Immunohistochemically, our cases showed patchy S100 (2/3) and CD34 (3/3) positivity. CD10 was positive in 2/3 cases. 3/3 cases showed pan-tropomyosin receptor kinase positivity (cytoplasmic). The NTRK-translocations demonstrated were: NTRK1::TMP3, NTRK1::TPR, and NTRK3::SPECC1L. Two of the patients had extensive disease and underwent chemotherapy. Larotrectinib was approved for one patient who demonstrated a striking reduction in tumor volume upon initiation of this treatment.

Gastrointestinal Stromal Tumors (GISTs) as Incidental Findings in Gynecological Surgery.

Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors of the gastrointestinal tract that may be diagnosed incidentally as a part of intra-abdominal surgery for other diseases. This is a single center review to document the incidental finding of GIST at surgery for gynecological malignancies during a 10-yr period. Sixteen cases of incidental GISTs were identified in women ranging in age from 39 to 82 yr. GISTs presented as incidental secondary lesions in women undergoing surgery for other indications, typically primary debulking surgery for tubo-ovarian high-grade serous carcinoma. The GIST was located in the stomach wall in 9 cases. Other sites were cecum, omentum, and mesentery. Diagnosis of GIST was supported by immunohistochemistry in all cases and by molecular studies in 3 cases. Seventy-five percent of cases were micro-GISTs, measuring <2 cm in diameter and, where Miettinen and Lasota criteria could be applied, fitted into "no risk," "very low risk" or "low risk" prognostic groups. Seventy-five percent of women for whom survival data was available, showed disease-free survival at follow-up. The 2 women who died had concurrent high stage or high-grade gynecological malignancy at initial diagnosis.

In vitro physicochemical characterization of five root canal sealers and their influence on an ex vivo oral multi-species biofilm community.

AIM: To evaluate the physicochemical properties of five root canal sealers and assess their effect on an ex vivo dental plaque-derived polymicrobial community. METHODOLOGY: Dental plaque-derived microbial communities were exposed to the sealers (AH Plus [AHP], GuttaFlow Bioseal [GFB], Endoseal MTA [ESM], Bio-C sealer [BCS] and BioRoot RCS [BRR]) for 3, 6 and 18 h. The sealers' effect on the biofilm biomass and metabolic activity was quantified using crystal violet (CV) staining and MTT assay, respectively. Biofilm community composition and morphology were assessed by denaturing gradient gel electrophoresis (DGGE), 16S rRNA sequencing and scanning electron microscopy. The ISO6876:2012 specifications were followed to determine the setting time, radiopacity, flowability and solubility. Obturated acrylic teeth were used to assess the sealers' effect on pH. Surface chemical characterization was performed using SEM with coupled energy-dispersive spectroscopy. Data normality was assessed using the Shapiro-Wilk test. One-way anova and Tukey's tests were used to analyze data from setting time, radiopacity, flowability and solubility. Two-way anova and Dunnett's tests were used for the data analysis from CV, MTT and pH. 16S rRNA sequencing data were analyzed for alpha (Shannon index and Chao analysis) and beta diversity (Bray-Curtis dissimilarities). Differences in community composition were evaluated by analysis of similarity (p < .05). RESULTS: The sealers significantly influenced microbial community composition and morphology. All sealers complied with ISO6876:2012 requirements for setting time, radiopacity and flowability. Although only AHP effectively reduced the biofilm biomass, all sealers, except BRR, reduced biofilm metabolic activity. CONCLUSION: Despite adequate physical properties, none of the sealers tested prevented biofilm growth. Significant changes in community composition were observed. If observed in vivo, these changes could affect intracanal microbial survival, pathogenicity and treatment outcomes.

Calprotectin (S100A8/A9) Is an Innate Immune Effector in Experimental Periodontitis.

Upregulated in inflammation, calprotectin (complexed S100A8 and S100A9; S100A8/A9) functions as an innate immune effector molecule, promoting inflammation, and also as an antimicrobial protein. We hypothesized that antimicrobial S100A8/A9 would mitigate change to the local microbial community and promote resistance to experimental periodontitis in vivo. To test this hypothesis, S100A9-/- and wild-type (WT; S100A9+/+) C57BL/6 mice were compared using a model of ligature-induced periodontitis. On day 2, WT mice showed fewer infiltrating innate immune cells than S100A9-/- mice; by day 5, the immune cell numbers were similar. At 5 days post ligature placement, oral microbial communities sampled with swabs differed significantly in beta diversity between the mouse genotypes. Ligatures recovered from molar teeth of S100A9-/- and WT mice contained significantly dissimilar microbial genera from each other and the overall oral communities from swabs. Concomitantly, the S100A9-/- mice had significantly greater alveolar bone loss than WT mice around molar teeth in ligated sites. When the oral microflora was ablated by antibiotic pretreatment, differences disappeared between WT and S100A9-/- mice in their immune cell infiltrates and alveolar bone loss. Calprotectin, therefore, suppresses emergence of a dysbiotic, proinflammatory oral microbial community, which reduces innate immune effector activity, including early recruitment of innate immune cells, mitigating subsequent alveolar bone loss and protecting against experimental periodontitis.

Gene Regulation and Transcriptomics.

Borrelia (Borreliella) burgdorferi, along with closely related species, is the etiologic agent of Lyme disease. The spirochete subsists in an enzootic cycle that encompasses acquisition from a vertebrate host to a tick vector and transmission from a tick vector to a vertebrate host. To adapt to its environment and persist in each phase of its enzootic cycle, B. burgdorferi wields three systems to regulate the expression of genes: the RpoN-RpoS alternative sigma factor cascade, the Hk1/Rrp1 two-component system and its product c-di-GMP, and the stringent response mediated by RelBbu and DksA. These regulatory systems respond to enzootic phase-specific signals and are controlled or fine- tuned by transcription factors, including BosR and BadR, as well as small RNAs, including DsrABb and Bb6S RNA. In addition, several other DNA-binding and RNA-binding proteins have been identified, although their functions have not all been defined. Global changes in gene expression revealed by high-throughput transcriptomic studies have elucidated various regulons, albeit technical obstacles have mostly limited this experimental approach to cultivated spirochetes. Regardless, we know that the spirochete, which carries a relatively small genome, regulates the expression of a considerable number of genes required for the transitions between the tick vector and the vertebrate host as well as the adaptation to each.

The relapsing fever spirochete Borrelia turicatae persists in the highly oxidative environment of its soft-bodied tick vector.

The relapsing fever spirochete Borrelia turicatae possesses a complex life cycle in its soft-bodied tick vector, Ornithodoros turicata. Spirochetes enter the tick midgut during a blood meal, and, during the following weeks, spirochetes disseminate throughout O. turicata. A population persists in the salivary glands allowing for rapid transmission to the mammalian hosts during tick feeding. Little is known about the physiological environment within the salivary glands acini in which B. turicatae persists. In this study, we examined the salivary gland transcriptome of O. turicata ticks and detected the expression of 57 genes involved in oxidant metabolism or antioxidant defences. We confirmed the expression of five of the most highly expressed genes, including glutathione peroxidase (gpx), thioredoxin peroxidase (tpx), manganese superoxide dismutase (sod-1), copper-zinc superoxide dismutase (sod-2), and catalase (cat) by reverse-transcriptase droplet digital polymerase chain reaction (RT-ddPCR). We also found distinct differences in the expression of these genes when comparing the salivary glands and midguts of unfed O. turicata ticks. Our results indicate that the salivary glands of unfed O. turicata nymphs are highly oxidative environments where reactive oxygen species (ROS) predominate, whereas midgut tissues comprise a primarily nitrosative environment where nitric oxide synthase is highly expressed. Additionally, B. turicatae was found to be hyperresistant to ROS compared with the Lyme disease spirochete Borrelia burgdorferi, suggesting it is uniquely adapted to the highly oxidative environment of O. turicata salivary gland acini.

DksA Controls the Response of the Lyme Disease Spirochete Borrelia burgdorferi to Starvation.

The pathogenic spirochete Borrelia burgdorferi senses and responds to changes in the environment, including changes in nutrient availability, throughout its enzootic cycle in Ixodes ticks and vertebrate hosts. This study examined the role of DnaK suppressor protein (DksA) in the transcriptional response of B. burgdorferi to starvation. Wild-type and dksA mutant B. burgdorferi strains were subjected to starvation by shifting cultures grown in rich complete medium, Barbour-Stoenner-Kelly II (BSK II) medium, to a defined mammalian tissue culture medium, RPMI 1640, for 6 h under microaerobic conditions (5% CO2, 3% O2). Microarray analyses of wild-type B. burgdorferi revealed that genes encoding flagellar components, ribosomal proteins, and DNA replication machinery were downregulated in response to starvation. DksA mediated transcriptomic responses to starvation in B. burgdorferi, as the dksA-deficient strain differentially expressed only 47 genes in response to starvation compared to the 500 genes differentially expressed in wild-type strains. Consistent with a role for DksA in the starvation response of B. burgdorferi, fewer CFU of dksA mutants were observed after prolonged starvation in RPMI 1640 medium than CFU of wild-type B. burgdorferi spirochetes. Transcriptomic analyses revealed a partial overlap between the DksA regulon and the regulon of RelBbu, the guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp] synthetase that controls the stringent response; the DksA regulon also included many plasmid-borne genes. Additionally, the dksA mutant exhibited constitutively elevated (p)ppGpp levels compared to those of the wild-type strain, implying a regulatory relationship between DksA and (p)ppGpp. Together, these data indicate that DksA, along with (p)ppGpp, directs the stringent response to effect B. burgdorferi adaptation to its environment.IMPORTANCE The Lyme disease bacterium Borrelia burgdorferi survives diverse environmental challenges as it cycles between its tick vectors and various vertebrate hosts. B. burgdorferi must withstand prolonged periods of starvation while it resides in unfed Ixodes ticks. In this study, the regulatory protein DksA is shown to play a pivotal role controlling the transcriptional responses of B. burgdorferi to starvation. The results suggest that DksA gene regulatory activity impacts B. burgdorferi metabolism, virulence gene expression, and the ability of this bacterium to complete its natural life cycle.

Molecular Additives Significantly Enhance Glycopolymer-Mediated Transfection of Large Plasmids and Functional CRISPR-Cas9 Transcription Activation Ex Vivo in Primary Human Fibroblasts and Induced Pluripotent Stem Cells.

Fast, efficient, and inexpensive methods for delivering functional nucleic acids to primary human cell types are needed to advance regenerative medicine and cell therapies. Plasmid-based gene editing (such as with CRISPR-Cas9) can require the delivery of plasmids that are large (∼9.5-13 kbp) in comparison to common reporter plasmids (∼5-8 kbp). To develop more efficient plasmid delivery vehicles, we investigated the effect of plasmid size on the transfection of primary human dermal fibroblasts (HDFs) and induced pluripotent stem cells (iPSCs) using a heparin-treated trehalose-containing polycation (Tr4-heparin). Transfections with 4.7 kbp to 10 kbp plasmids exhibited high rates of polyplex internalization with both plasmid sizes. However, transfection with the large plasmid was nearly eliminated in HDFs and significantly reduced in iPSCs. Molecular additives were used to probe intracellular barriers to transfection. Chloroquine treatments were used to destabilize endosomes, and dexamethasone and thymidine were used to destabilize the nuclear envelope. Destabilizing the nuclear envelope resulted in significantly increased large-plasmid-transfection, indicating that nuclear localization may be more difficult for large plasmids. To demonstrate the potential clinical utility of this formulation, HDFs and iPSCs were treated with to dexamethasone-Tr4-heparin polyplexes encoding dCas9-VP64, synthetic transcription activator, targeted to collagen type VII. These transfections enhanced collagen expression in HDFs and iPSCs by 5- and 20-fold, respectively, compared to an untransfected control and were the more effective than the Lipofectamine 2000 control. Functional plasmid transfection efficiency can be significantly improved by nuclear destabilization, which could lead to improved development of nonviral vehicles for ex vivo CRISPR-Cas9 gene editing.

Aggregated Solution Morphology of Poly(acrylic acid)-Poly(styrene) Block Copolymers Improves Drug Supersaturation Maintenance and Caco-2 Cell Membrane Permeation.

Amorphous solid dispersions of polymers and drugs have been shown to improve supersaturation maintenance of poorly water-soluble drugs. Herein, amorphous spray-dried dispersions (SDDs) of poly(acrylic acid)-polystyrene (PS-b-PAA) diblock copolymers with differing degrees of polymerization were prepared in aggregated and nonaggregated states with the Biopharmaceutical Classification System Class II drug, probucol (PBC). Specifically, PS90-b-PAA15, PS90-b-PAA80, PS38-b-PAA220, and PS38-b-PAA320 amphiphilic block polymers that covered a compositional range in the area of oral drug delivery were prepared to examine the role of molecular weight and controlled aggregation in promoting drug supersaturation and maintenance. In addition, hydrophilic homopolymers PAA20, PAA96, PAA226, and PAA392 were prepared as controls to evaluate the role of the block copolymer-based SDDs in PBC solubilization. Characterization such as powder X-ray diffraction, scanning electron microscopy, and dissolution tests under nonsink conditions were then performed to evaluate the SDDs. When comparing the block copolymer systems, polymers that were preaggregated into micellular structures prior to spray drying with the drug promoted higher drug solubility and maintenance than when the drug was formulated with molecularly dissolved PS-PAA block polymer. Interestingly, the aggregated PS90-b-PAA80 SDD with 25 wt % PBC achieved 100% burst release and maintained full supersaturation of PBC at pH 6.5 (physiological pH in the small intestine). Dissolution studies conducted at the pH of the stomach (pH = 1.2) show that a minimal amount of drug (∼10 µg/mL) was released, which could be used for protecting drugs from acidic environments (stomach) before reaching the small intestine. To evaluate drug bioavailability, in vitro Caco-2 cell assays were performed, which reveal that PAA-based excipients do not hinder drug permeation across the epithelial membrane and that PS90-b-PAA80 SDD with 25 wt % PBC achieved the highest membrane permeability coefficient. This work demonstrates that block copolymer-based SDDs capable of preaggregating into nanostructures may be a tunable drug-delivery platform that can improve solubility and supersaturation maintenance of Class II pharmaceutics while also not prohibiting bioavailability through model intestinal membranes. Indeed, this concept may be extended to accommodate a myriad of pharmaceutical and excipient structures.

Imaging of Borrelia turicatae Producing the Green Fluorescent Protein Reveals Persistent Colonization of the Ornithodoros turicata Midgut and Salivary Glands from Nymphal Acquisition through Transmission.

Relapsing fever (RF) spirochetes colonize and are transmitted to mammals primarily by Ornithodoros ticks, and little is known regarding the pathogen's life cycle in the vector. To further understand vector colonization and transmission of RF spirochetes, Borrelia turicatae expressing a green fluorescent protein (GFP) marker (B. turicatae-gfp) was generated. The transformants were evaluated during the tick-mammal infectious cycle, from the third nymphal instar to adult stage. B. turicatae-gfp remained viable for at least 18 months in starved fourth-stage nymphal ticks, and the studies indicated that spirochete populations persistently colonized the tick midgut and salivary glands. Our generation of B. turicatae-gfp also revealed that within the salivary glands, spirochetes are localized in the ducts and lumen of acini, and after tick feeding, the tissues remained populated with spirochetes. The B. turicatae-gfp generated in this study is an important tool to further understand and define the mechanisms of vector colonization and transmission.IMPORTANCE In order to interrupt the infectious cycle of tick-borne relapsing fever spirochetes, it is important to enhance our understanding of vector colonization and transmission. Toward this, we generated a strain of Borrelia turicatae that constitutively produced the green fluorescent protein, and we evaluated fluorescing spirochetes during the entire infectious cycle. We determined that the midgut and salivary glands of Ornithodoros turicata ticks maintain the pathogens throughout the vector's life cycle and remain colonized with the spirochetes for at least 18 months. We also determined that the tick's salivary glands were not depleted after a transmission blood feeding. These findings set the framework to further understand the mechanisms of midgut and salivary gland colonization.

Heparin Enhances Transfection in Concert with a Trehalose-Based Polycation with Challenging Cell Types.

Improving the delivery of nucleic acids to diverse tissue types in culture is important for translating genome editing for regenerative cell therapies. Herein, we examine the effect of transfection media additives, such as the sulfated glycosaminoglycan heparin, in dramatically increasing pDNA delivery efficiency and transgene expression in a wide variety of cell types. Polyplexes formed by combining pDNA and Tr4, a cationic glycopolymer containing repeated trehalose and pentaethylenetetramine groups, were treated with low concentrations of heparin prior to in vitro transfection with plasmid DNA. Polyplex formulations were found to be stable and form ternary complexes upon heparin addition according to dynamic light scattering and ethidium bromide dye exclusion assays. Heparin-coated polyplexes offer significant increases (approximately 4-fold) in GFP expression compared to polyplexes prepared with Tr4 only in primary fibroblasts, U87, and HepG2 cells. Heparin was also shown to increase GFP expression in a linear, dose-dependent manner. The heparin-treated Tr4 polyplexes exhibited more than 50% higher cellular internalization with HepG2 cells while showing minimal increases with U87 and primary fibroblasts. Pharmacological inhibition was used to further understand the endocytic pathways taken during transfection in the presence and absence of heparin. It was found that heparin-treated polyplexes are endocytosed primarily through macropinocytosis and clathrin-mediated pathways, while Tr4 polyplexes without heparin appear to be internalized primarily via caveolae. Heparin appears to also modify the nuclear localization behavior of Tr4 polyplexes, which likely contributes to increased efficiency and transgene expression.

Coupling mammalian cell surface display with somatic hypermutation for the discovery and maturation of human antibodies.

A novel approach has been developed for the isolation and maturation of human antibodies that replicates key features of the adaptive immune system by coupling in vitro somatic hypermutation (SHM) with mammalian cell display. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID), and can be replicated in non-B cells through expression of recombinant AID. A library of human antibodies, based on germline V-gene segments with recombined human regions was used to isolate low-affinity antibodies to human ß nerve growth factor (hßNGF). These antibodies, initially naïve to SHM, were subjected to AID-directed SHM in vitro and selected using the same mammalian cell display system, as illustrated by the maturation of one of the antibodies to low pM K(D). This approach overcomes many of the previous limitations of mammalian cell display, enabling direct selection and maturation of antibodies as full-length, glycosylated IgGs.

A novel conditional active biologic anti-EpCAM x anti-CD3 bispecific antibody with synergistic tumor selectivity for cancer immunotherapy.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that plays several roles in cancer biology. EpCAM is an attractive therapeutic target because of its expression in most solid tumors. However, targeting EpCAM has been challenging because it is also highly expressed in normal epithelial tissues. Initial attempts to develop EpCAM-specific T-cell engagers were unsuccessful due to severe cytokine release effects, as well as serious on-target, off-tumor drug-related toxicities. We developed novel, conditionally active biological (CAB) bispecific antibodies that bind to both EpCAM and CD3 in an acidic tumor microenvironment. In healthy tissues, binding to EpCAM and CD3 is greatly reduced by a novel, dual CAB selection, where each binding domain is independently blocked by the presence of physiological chemicals known as Protein-associated Chemical Switches (PaCS). The CAB anti-EpCAM T-cell engagers displayed the anticipated bispecific binding properties and mediated the potent lysis of EpCAM-positive cancer cell lines through the recruitment of T cells in the tumor microenvironment. Xenograft studies showed that the efficacy of CAB bispecific antibodies is similar to that of a non-CAB anti-EpCAM bispecific antibody, but they have markedly reduced toxicity in non-human primates, indicating an unprecedentedly widened therapeutic index of over 100-fold. These preclinical results indicate that the dual CAB bispecific antibody is potentially both a powerful and safe therapeutic platform and a promising T cell-engaging treatment for patients with EpCAM-expressing tumors.

Development of a novel conditionally active EpCAM-specific T-cell engager with enhanced safety and tolerability for treatment of solid tumors.

Intracellular calprotectin (S100A8/A9) facilitates DNA damage responses and promotes apoptosis in head and neck squamous cell carcinoma.

OBJECTIVES: In head and neck squamous cell carcinoma (HNSCC), poor prognosis and low survival rates are associated with downregulated calprotectin. Calprotectin (S100A8/A9) inhibits cancer cell migration and invasion and facilitates G2/M cell cycle arrest. We investigated whether S100A8/A9 regulates DNA damage responses (DDR) and apoptosis in HNSCC after chemoradiation. MATERIALS AND METHODS: Human HNSCC cases in TCGA were analyzed for relationships between S100A8/A9 and expression of apoptosis-related genes. Next, S100A8/A9-expressing and non-expressing carcinoma lines (two different lineages) were exposed to genotoxic agents and assessed for 53BP1 and γH2AX expression and percent of viable/dead cells. Finally, S100A8/A9-wild-type and S100A8/A9null C57BL/6j mice were treated with 4-NQO to induce oral dysplastic and carcinomatous lesions, which were compared for levels of 53BP1. RESULTS: In S100A8/A9-high HNSCC tumors, apoptosis-related caspase family member genes were upregulated, whereas genes limiting apoptosis were significantly downregulated based on TCGA analyses. After X-irradiation or camptothecin treatment, S100A8/A9-expressing carcinoma cells (i.e., TR146 and KB-S100A8/A9) showed significantly higher 53BP1 and γH2AX expression, DNA fragmentation, proportions of dead cells, and greater sensitivity to cisplatin than wild-type KB or TR146-S100A8/A9-KD cells. Interestingly, KB-S100A8/A9Δ113-114 cells showed similar 53BP1 and γH2AX levels to S100A8/A9-negative KB and KB-EGFP cells. After 4-NQO treatment, 53BP1 expression in oral lesions was significantly greater in calprotectin+/+ than S100A8/A9null mice. CONCLUSIONS: In HNSCC cells, intracellular calprotectin is strongly suggested to potentiate DDR and promote apoptosis in response to genotoxic agents. Hence, patients with S100A8/A9-high HNSCC may encounter more favorable outcomes because more tumor cells enter apoptosis with increased sensitivity to chemoradiation therapy.

Essential Components of Borreliella (Borrelia) burgdorferi In Vitro Transcription Assays.

Borreliella burgdorferi is a bacterial pathogen with limited metabolic and genomic repertoires. B. burgdorferi transits extracellularly between vertebrates and ticks and dramatically remodels its transcriptional profile to survive in disparate environments during infection. A focus of B. burgdorferi studies is to clearly understand how the bacteria responds to its environment through transcriptional changes. In vitro transcription assays allow for the basic mechanisms of transcriptional regulation to be biochemically dissected. Here, we present a detailed protocol describing B. burgdorferi RNA polymerase purification and storage, sigma factor purification, DNA template generation, and in vitro transcription assays. The protocol describes the use of RNA polymerase purified from B. burgdorferi 5A4 RpoC-His (5A4-RpoC). 5A4-RpoC is a previously published strain harboring a 10XHis-tag on the rpoC gene encoding the largest subunit of the RNA polymerase. In vitro transcription assays consist of the RNA polymerase purified from strain 5A4-RpoC, a recombinant version of the housekeeping sigma factor RpoD, and a PCR-generated double-stranded DNA template. While the protein purification techniques and approaches to assembling in vitro transcription assays are conceptually well understood and relatively common, handling considerations for RNA polymerases often differ from organism to organism. The protocol presented here is designed for enzymatic studies on the B. burgdorferi RNA polymerase. The method can be adapted to test the role of transcription factors, promoters, and post-translational modifications on the activity of the RNA polymerase.

TMP3-NTRK1 rearranged uterine sarcoma: A case report.

INTRODUCTION: Uterine sarcomas are a group of rare tumours with heterogeneous morphological and genetic features. Recent advances in the molecular characterisation of these tumours have identified a novel clinicopathological category underpinned by NTRK gene fusions. CASE REPORT: We present the case of a 42-year-old woman with a polypoid cervical lesion formed of densely cellular, short, haphazard fascicles of monomorphic spindle cells that lacked coagulative necrosis and which showed high mitotic activity. On immunohistochemistry, the tumour was diffusely positive for pan-Trk and weakly positive for CD34 but was negative for a range of other markers, including cytokeratins, smooth muscle markers, hormone receptors and S100. FISH analysis using a NTRK1 break-apart probe was above the threshold for translocation positivity and subsequent next-generation sequencing (NGS) identified a TPM3-NTRK1 fusion. DISCUSSION: NTRK-rearranged uterine sarcomas are a novel subset of gynaecological mesenchymal neoplasms characterised by cytological isomorphism and fibrosarcoma-like morphology. Although distinction from more common mesenchymal neoplasms is possible on the basis of morphology and immunohistochemistry, exclusion of rare differential diagnoses, such as malignant peripheral nerve sheath tumour or the recently described COL1A1-PDGFB fusion sarcoma, requires molecular work-up with FISH or NGS. Identification of these rare tumours is clinically relevant because of their cervical location and the possible role for tropomyosin receptor kinase inhibitors in their treatment.