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Host-cell lipases can be present in monoclonal antibody drug products and can degrade polysorbates present in the formulations as stabilizers. We hypothesized that the in-use stability of the IV admixture prepared from such a drug product might be impacted by decreasing levels of polysorbate 20. Host-cell lipase activity has, in fact, been observed during development of one of our therapeutic monoclonal antibody drug products. Throughout the course of the product shelf life, polysorbate 20 levels decreased but no other quality attributes of the drug product were impacted. An experimental approach was developed to simulate how the prepared IV admixture in-use stability is affected as polysorbate 20 concentration in the drug product decreased over the shelf life, and from that a minimum level of polysorbate 20 required in the drug product was determined to estimate the in-use stability of the IV admixture as the polysorbate 20 in the drug product degrades. The results indicate that although the observed degradation of polysorbate 20 does not affect quality attributes of this drug product, in-use stability of the IV admixture as a function of polysorbate degradation can be impacted and should be assessed to ensure sufficient quality.

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AIMS: To evaluate the therapeutic effects of intradetrusor onabotulinum toxin A (BTX) injections in patients with adult neurogenic lower urinary tract dysfunction (ANLUTD) and medically refractory poorly compliant bladders. METHODS: We retrospectively evaluated patients with urodynamic studies (UDS)-proven, medically refractory impaired bladder compliance (≤20 mL/cm H2 O) secondary to spinal myelopathy treated with 300 units of BTX cystoscopically injected into the detrusor muscle. Objective improvement in compliance was defined as an increase ≥5 mL/cm H2 O on repeat urodynamics. Characteristics were compared between patients who demonstrated symptomatic and objective improvement following treatment versus those without. RESULTS: Seventy-one individuals were included in the final analysis. Mean patient age was 37.2 years (range: 18-78) and ANLUTD duration was 14.5 years (range: 1-34). Average pre-injection bladder compliance was 9.2 mL/cm H2 O (range: 3.0-16.7). After treatment with BTX, 37 of 71 (52%) patients reported subjective reductions in lower urinary tract symptoms. Repeat UDS demonstrated objective bladder compliance improvements in 22 of 71 (31%). Individuals with shorter time intervals since neurologic injury responded better to BTX than those with longer durations (P = .032). CONCLUSION: BTX injections significantly improved symptoms and bladder compliance in 31% of ANLUTD patients with medical refractory poorly compliant bladders.

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Site-specific conjugation technology frequently relies on antibody engineering to incorporate rare or non-natural amino acids into the primary sequence of the protein. However, when the primary sequence is unknown or when antibody engineering is not feasible, there are very limited options for site-specific protein modification. We have developed a transglutaminase-mediated conjugation that incorporates a thiol at a "privileged" location on deglycosylated antibodies (Q295). Perhaps surprisingly, this conjugation employs a reported transglutaminase inhibitor, cystamine, as the key enzyme substrate. The chemical incorporation of a thiol at the Q295 site allows for the site-specific attachment of a plethora of commonly used and commercially available payloads via maleimide chemistry. Herein, we demonstrate the utility of this method by comparing the conjugatability, plasma stability, and in vitro potency of these site-specific antibody-drug conjugates (ADCs) with analogous endogenous cysteine conjugates. Cytotoxic ADCs prepared using this methodology are shown to exhibit comparable in vitro efficacy to stochastic cysteine conjugates while displaying dramatically improved plasma stability and conjugatability. In particular, we note that this technique appears to be useful for the incorporation of highly hydrophobic linker payloads without the addition of PEG modifiers. We postulate a possible mechanism for this feature by probing the local environment of the Q295 site with two fluorescent probes that are known to be sensitive to the local hydrophobic environment. In summary, we describe a highly practical method for the site-specific conjugation of genetically nonengineered antibodies, which results in plasma-stable ADCs with low intrinsic hydrophobicity. We believe that this technology will find broad utility in the ADC community.

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Targeted cancer immunotherapy with irradiated, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting, allogeneic cancer cell lines has been an effective approach to reduce tumor burden in several patients. It is generally assumed that to be effective, these cell lines need to express immunogenic antigens coexpressed in patient tumor cells, and antigen-presenting cells need to take up such antigens then present them to patient T cells. We have previously reported that, in a phase I pilot study (ClinicalTrials.gov NCT00095862), a subject with stage IV breast cancer experienced substantial regression of breast, lung, and brain lesions following inoculation with clinical formulations of SV-BR-1-GM, a GM-CSF-secreting breast tumor cell line. To identify diagnostic features permitting the prospective identification of patients likely to benefit from SV-BR-1-GM, we conducted a molecular analysis of the SV-BR-1-GM cell line and of patient-derived blood, as well as a tumor specimen. Compared to normal human breast cells, SV-BR-1-GM cells overexpress genes encoding tumor-associated antigens (TAAs) such as PRAME, a cancer/testis antigen. Curiously, despite its presumptive breast epithelial origin, the cell line expresses major histocompatibility complex (MHC) class II genes (HLA-DRA, HLA-DRB3, HLA-DMA, HLA-DMB), in addition to several other factors known to play immunostimulatory roles. These factors include MHC class I components (B2M, HLA-A, HLA-B), ADA (encoding adenosine deaminase), ADGRE5 (CD97), CD58 (LFA3), CD74 (encoding invariant chain and CLIP), CD83, CXCL8 (IL8), CXCL16, HLA-F, IL6, IL18, and KITLG. Moreover, both SV-BR-1-GM cells and the responding study subject carried an HLA-DRB3*02:02 allele, raising the question of whether SV-BR-1-GM cells can directly present endogenous antigens to T cells, thereby inducing a tumor-directed immune response. In support of this, SV-BR-1-GM cells (which also carry the HLA-DRB3*01:01 allele) treated with yellow fever virus (YFV) envelope (Env) 43-59 peptides reactivated YFV-DRB3*01:01-specific CD4+ T cells. Thus, the partial HLA allele match between SV-BR-1-GM and the clinical responder might have enabled patient T lymphocytes to directly recognize SV-BR-1-GM TAAs as presented on SV-BR-1-GM MHCs. Taken together, our findings are consistent with a potentially unique mechanism of action by which SV-BR-1-GM cells can act as APCs for previously primed CD4+ T cells.

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Pluripotent stem cells are a promising source of endothelial cells (ECs) for the treatment of vascular diseases. We have developed a robust protocol to differentiate human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) into ECs with high purities (94%-97% CD31+ and 78%-83% VE-cadherin+ ) in 8 days without cell sorting. Passaging of these cells yielded a nearly pure population of ECs (99% of CD31+ and 96.8% VE-cadherin+ ). These ECs also expressed other endothelial markers vWF, Tie2, NOS3, and exhibited functions of ECs such as uptake of Dil-acetylated low-density lipoprotein and formation of tubes in vitro or vessels in vivo on matrigel. We found that FGF2, VEGF, and BMP4 synergistically induced early vascular progenitors (VPs) from hiPSC-derived mesodermal cells. The MAPK and PI3K pathways are crucial not only for the initial commitment to vascular lineages but also for the differentiation of vascular progenitors to ECs, most likely through regulation of the ETS family transcription factors, ERG and FLI1. We revealed novel roles of the p38 and JNK MAPK pathways on EC differentiation. Furthermore, inhibition of the ERK pathway markedly promoted the differentiation of smooth muscle cells. Finally, we demonstrate that pluripotent stem cell-derived ECs are capable of forming patent blood vessels that were connected to the host vasculature in the ischemic limbs of immune deficient mice. Thus, we demonstrate that ECs can be efficiently derived from hiPSCs and hESCs, and have great potential for vascular therapy as well as for mechanistic studies of EC differentiation. Stem Cells 2017;35:909-919.

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The halteres of flies are mechanosensory organs that provide information about body rotations during flight. We measured haltere movements in a range of fly taxa during free walking and tethered flight. We find a diversity of wing-haltere phase relationships in flight, with higher variability in more ancient families and less in more derived families. Diverse haltere movements were observed during free walking and were correlated with phylogeny. We predicted that haltere removal might decrease behavioural performance in those flies that move them during walking and provide evidence that this is the case. Our comparative approach reveals previously unknown diversity in haltere movements and opens the possibility of multiple functional roles for halteres in different fly behaviours.

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Porcine epidemic diarrhea virus (PEDV) spread rapidly after being diagnosed in the USA in April 2013. In this study we assessed whether PEDV could become airborne and if so, whether the virus was infectious. Air samples were collected both from a room containing experimentally infected pigs and at various distances from the outside of swine farms experiencing acute PEDV outbreaks. Results indicated presence of infectious PEDV in the air from experimentally infected pigs and genetic material of PEDV was detected up to 10 miles downwind from naturally infected farms. Airborne transmission should be considered as a potential route for PEDV dissemination.

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PURPOSE: We examined the degree of exclusion bias that may occur due to missing data when grouping prostate cancer cases from the SEER (Surveillance, Epidemiology and End Results) database into D'Amico clinical risk groups. Exclusion bias may occur since D'Amico staging requires all 3 variables to be known and data may not be missing at random. MATERIALS AND METHODS: From the SEER database we identified 132,606 men with incident prostate cancer from 2004 to 2006. We documented age, race, Gleason score, clinical T stage, PSA and geographic region. Men were categorized into D'Amico risk groups. Those with 1 or more unknown tumor variables (prostate specific antigen, T stage and/or Gleason score) were labeled unclassified. We compared the value of the other 2 known clinical variables for men with known vs unknown prostate specific antigen, Gleason score and T stage. Demographics were compared for those with and without missing data. Results were compared using chi-square and logistic regression. RESULTS: Of the men 33% had 1 or more unknown tumor variables with T stage the most commonly missing variable. There was no clinically significant difference in the value of the other 2 known tumor variables when T stage or prostate specific antigen was missing. Men older than 75 years were more likely to have unknown variables than younger men. There was significant geographic variation in the frequency of unclassified D'Amico data. CONCLUSIONS: In studies in which the data set is limited to men who can be classified into a D'Amico risk group 33% of eligible patients are excluded from analysis. Such men are older and from certain SEER registries but they have tumor characteristics similar to those with complete data.

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Numerous investigators have found that serum prolactin levels increase after tonic-clonic and partial complex seizures, but the effect of syncope on prolactin levels has been studied little. Serum prolactin levels were measured following unexpected syncopal attacks in patients seeking emergency treatment in a community hospital. Levels sampled 18 to 60 minutes after syncopal episodes were increased in 8 of 11 cases. Follow-up prolactin levels, measured 17 to 222 days later, were normal in all eight cases in which they were initially increased. Most subjects had concurrent illness. Although the current study does not clarify whether it was the syncope, the concurrent illness, or both that caused the prolactin elevations, it implies that measurement of this hormone will not help the clinician in distinguishing between seizures and syncopal attacks.

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A fibrous band of extracellular materials on the chick embryo area pellucida/area opaca border is a preferential migratory pathway for chick embryo primordial germ cells (PGC). This band contains fibronectin, collagen Type I and sulphated glycosaminoglycans. It is known that PGCs from Xenopus laevis interact with fibronectin as they undergo migration in the embryo from their site of origin to the gonads. To establish whether this pathway is species specific in chick embryos it was decided to transplant PGC from Xenopus laevis embryos stage 48 on to chick embryos stage 4 fibrous band. Their rapid migration on this extracellular matrix and their subsequent re-orientation of the basement membrane has been studied by scanning electron microscopy.

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Intercellularly aligned microtubule arrays are present in cell layers associated with the growth and secretion of scales in the zebra fish Brachydanio rerio and the neon tetra fish Hyphessobrycon innesi. The layers in question are: the osteoblast layer that covers the ossified outer surface of a scale, and the layer of fibroblasts that is situated immediately underneath the inner collagenous surface of a scale's fibrillary plate. In certain osteoblasts, the proximal portions of microtubules (with respect to centrosomes) run closely alongside the anterior margin of each cell where it flanks one of a scale's ridge-shaped circuli. These osteoblasts and microtubule portions are arranged in aligned rows that are parallel to circuli. However, the distal portions of the microtubules curve into an orientation that is approximately at right angles to circuli and they are aligned with each other and similar microtubule portions in adjacent osteoblasts. Such microtubule alignments only occur in osteoblasts that are associated with circuli. In Hyphessobrycon osteoblasts situated elsewhere on a scale's surface, microtubules radiate from cell centres but their distal portions curve into alignment with each other and are oriented alongside cell margins. The proximal portions of fibroblast microtubules radiate from centrally positioned centrosomes but the distal portions curve into alignment with each other and distal microtubule portions in neighbouring fibroblasts. The overall pattern of microtubule alignment is similar to that of collagen fibres, which these fibroblasts are secreting onto the fibrillary plate. The immunofluorescence protocol that was used to demonstrate the microtubule alignments described above did not reveal such alignments in the osteoblast and fibroblast layers associated with scales of the brown trout Salmo trutta fario. These findings are assessed in terms of intra-and inter-cellular control of microtubule alignment, and decentralized reorientation of microtubules at distances of several micrometres from centrosomal microtubule-organizing centres. The functional significance of the relationships between microtubule alignment and supracellular patterns of alignment that take place as collagen deposition and ossification proceed during scale formation is also considered.

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Distinct changes in epidermal cell shaping largely define the overall pattern of growth and form during generation of the ectodermal ridge and early stages of fin fold morphogenesis. The epidermal portion of the ridge and early fin fold are formed from a strip of epidermal cells that is only six to nine cells wide. There is apparently no increase in the number of these cells during initial formation of the ridge and its subsequent conversion into a fin fold which contains extracellular matrix fibres. Epidermal cells adopt a wedge-shaped morphology during ridge production. Distinct changes in the shaping and contact relationships between basal portions of these cells generate intercellular spaces at several discrete loci within the ridge. These spaces become continuous with each other to form a subepidermal space. Hence, the subepidermal space is not produced by straight-forward folding of an epidermal sheet. Cells flanking the sides of the ridge start to flatten as it is converted into a fin fold. A continuous row of distinctive cells is positioned along the apex of the developing fold. The term 'cleft cells' is suggested for these apical cells. Each cleft cell retains a wedge-shaped form during fold formation and develops a basal cleft-shaped invagination. Invaginations are aligned in neighbouring cleft cells so that these cells cap the distal boundary of the subepidermal space where collagenous extracellular fibres called actinotrichia run anteroposteriorly along the length of the fin fold. This orientation is in direct contrast to the proximodistal orientation of actinotrichia within the remainder of the subepidermal space. During early stages of fold production a temporary set of previously unreported extracellular cross fibres spans the subepidermal space at right angles to actinotrichia. These configurations of extracellular fibres could be advantageous for maintaining the structural integrity of the early fin fold.

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