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Background: Despite immense progress in artificial intelligence (AI) models, there has been limited deployment in health care environments. The gap between potential and actual AI applications is likely due to the lack of translatability between controlled research environments (where these models are developed) and clinical environments for which the AI tools are ultimately intended., Objective: We previously developed the Translational Evaluation of Healthcare AI (TEHAI) framework to assess the translational value of AI models and to support successful transition to health care environments. In this study, we applied the TEHAI framework to the COVID-19 literature in order to assess how well translational topics are covered., Methods: A systematic literature search for COVID-19 AI studies published between December 2019 and December 2020 resulted in 3830 records. A subset of 102 (2.7%) papers that passed the inclusion criteria was sampled for full review. The papers were assessed for translational value and descriptive data collected by 9 reviewers (each study was assessed by 2 reviewers). Evaluation scores and extracted data were compared by a third reviewer for resolution of discrepancies. The review process was conducted on the Covidence software platform., Results: We observed a significant trend for studies to attain high scores for technical capability but low scores for the areas essential for clinical translatability. Specific questions regarding external model validation, safety, nonmaleficence, and service adoption received failed scores in most studies., Conclusions: Using TEHAI, we identified notable gaps in how well translational topics of AI models are covered in the COVID-19 clinical sphere. These gaps in areas crucial for clinical translatability could, and should, be considered already at the model development stage to increase translatability into real COVID-19 health care environments., Competing Interests: Conflicts of Interest: SR holds directorship in Medi-AI. The other authors have no conflicts of interest to declare., (©Aaron Edward Casey, Saba Ansari, Bahareh Nakisa, Blair Kelly, Pieta Brown, Paul Cooper, Imran Muhammad, Steven Livingstone, Sandeep Reddy, Ville-Petteri Makinen. Originally published in JMIR AI (https://ai.jmir.org), 06.07.2023.)

Autophagy is an intracellular recycling process that degrades harmful molecules and enables survival during starvation, with implications for diseases including dementia, cancer and atherosclerosis. Previous studies demonstrate how a limited number of transcription factors (TFs) can increase autophagy. However, this knowledge has not resulted in translation into therapy, thus, to gain understanding of more suitable targets, we utilized a systems biology approach. We induced autophagy by amino acid starvation and mTOR inhibition in HeLa, HEK 293 and SH-SY5Y cells and measured temporal gene expression using RNA-seq. We observed 456 differentially expressed genes due to starvation and 285 genes due to mTOR inhibition (P FDR  < 0.05 in every cell line). Pathway analyses implicated Alzheimer's and Parkinson's diseases (P FDR  ≤ 0.024 in SH-SY5Y and HeLa) and amyotrophic lateral sclerosis (ALS, P FDR  < 0.05 in mTOR inhibition experiments). Differential expression of the Senataxin (SETX) target gene set was predicted to activate multiple neurodegenerative pathways (P FDR  ≤ 0.04). In the SH-SY5Y cells of neuronal origin, the E2F transcription family was predicted to activate Alzheimer's disease pathway (P FDR  ≤ 0.0065). These exploratory analyses suggest that SETX and E2F may mediate transcriptional regulation of autophagy and further investigations into their possible role in neuro-degeneration are warranted., (© 2022. The Author(s).)

Cancer metabolism adapts the metabolic network of its tissue of origin. However, breast cancer is not a disease of a single origin. Multiple epithelial populations serve as the culprit cell of origin for specific breast cancer subtypes, yet our knowledge of the metabolic network of normal mammary epithelial cells is limited. Using a multi-omic approach, here we identify the diverse metabolic programmes operating in normal mammary populations. The proteomes of basal, luminal progenitor and mature luminal cell populations revealed enrichment of glycolysis in basal cells and of oxidative phosphorylation in luminal progenitors. Single-cell transcriptomes corroborated lineage-specific metabolic identities and additional intra-lineage heterogeneity. Mitochondrial form and function differed across lineages, with clonogenicity correlating with mitochondrial activity. Targeting oxidative phosphorylation and glycolysis with inhibitors exposed lineage-rooted metabolic vulnerabilities of mammary progenitors. Bioinformatics indicated breast cancer subtypes retain metabolic features of their putative cell of origin. Thus, lineage-rooted metabolic identities of normal mammary cells may underlie breast cancer metabolic heterogeneity and targeting these vulnerabilities could advance breast cancer therapy.

The heterogeneity of breast cancer makes current therapies challenging. Metformin, the anti-diabetic drug, has shown promising anti-cancer activities in epidemiological studies and breast cancer models. Yet, how metformin alters the normal adult breast tissue remains elusive. We demonstrate metformin intake at a clinically relevant dose impacts the hormone receptor positive (HR+) luminal cells in the normal murine mammary gland. Metformin decreases total cell number, progenitor capacity and specifically reduces DNA damage in normal HR+ luminal cells, decreases oxygen consumption rate and increases cell cycle length of luminal cells. HR+ luminal cells demonstrate the lowest levels of mitochondrial respiration and capacity to handle oxidative stress compared to the other fractions, suggesting their intrinsic susceptibility to long-term metformin exposure. Uncovering HR+ luminal cells in the normal mammary gland as the major cell target of metformin exposure could identify patients that would most benefit from repurposing this anti-diabetic drug for cancer prevention/therapy purposes., Competing Interests: Competing interestsThe authors declare no competing interests.

Late-onset Alzheimer's disease is the most common dementia type, yet no treatment exists to stop the neurodegeneration. Evidence from monogenic lysosomal diseases, neuronal pathology and experimental models suggest that autophagic and endolysosomal dysfunction may contribute to neurodegeneration by disrupting the degradation of potentially neurotoxic molecules such as amyloid-β and tau. However, it is uncertain how well the evidence from rare disorders and experimental models capture causal processes in common forms of dementia, including late-onset Alzheimer's disease. For this reason, we set out to investigate if autophagic and endolysosomal genes were enriched for genetic variants that convey increased risk of Alzheimer's disease; such a finding would provide population-based support for the endolysosomal hypothesis of neurodegeneration. We quantified the collective genetic associations between the endolysosomal system and Alzheimer's disease in three genome-wide associations studies (combined n = 62 415). We used the Mergeomics pathway enrichment algorithm that incorporates permutations of the full hierarchical cascade of SNP-gene-pathway to estimate enrichment. We used a previously published collection of 891 autophagic and endolysosomal genes (denoted as AphagEndoLyso, and derived from the Lysoplex sequencing platform) as a proxy for cellular processes related to autophagy, endocytosis and lysosomal function. We also investigated a subset of 142 genes of the 891 that have been implicated in Mendelian diseases (MenDisLyso). We found that both gene sets were enriched for genetic Alzheimer's associations: an enrichment score 3.67 standard deviations from the null model (P = 0.00012) was detected for AphagEndoLyso, and a score 3.36 standard deviations from the null model (P = 0.00039) was detected for MenDisLyso. The high enrichment score was specific to the AphagEndoLyso gene set (stronger than 99.7% of other tested pathways) and to Alzheimer's disease (stronger than all other tested diseases). The APOE locus explained most of the MenDisLyso signal (1.16 standard deviations after APOE removal, P = 0.12), but the AphagEndoLyso signal was less affected (3.35 standard deviations after APOE removal, P = 0.00040). Additional sensitivity analyses further indicated that the AphagEndoLyso Gene Set contained an aggregate genetic association that comprised a combination of subtle genetic signals in multiple genes. We also observed an enrichment of Parkinson's disease signals for MenDisLyso (3.25 standard deviations) and for AphagEndoLyso (3.95 standard deviations from the null model), and a brain-specific pattern of gene expression for AphagEndoLyso in the Gene Tissue Expression Project dataset. These results provide evidence that a diffuse aggregation of genetic perturbations to the autophagy and endolysosomal system may mediate late-onset Alzheimer's risk in human populations.

Breast cancer is the most common cancer in females. The number of years menstruating and length of an individual menstrual cycle have been implicated in increased breast cancer risk. At present, the proliferative changes within an individual reproductive cycle or variations in the estrous cycle in the normal mammary gland are poorly understood. Here we use Fucci2 reporter mice to demonstrate actively proliferating mammary epithelial cells have shorter G1 lengths, whereas more differentiated/non-proliferating cells have extended G1 lengths. We find that cells enter into the cell cycle mainly during diestrus, yet the expansion is erratic and does not take place every reproductive cycle. Single cell expression analyses feature expected proliferation markers ( Birc5, Top2a ), while HR+ luminal cells exhibit fluctuations of key differentiation genes ( ER, Gata3 ) during the cell cycle. We highlight the proliferative heterogeneity occurring within the normal mammary gland during a single-estrous cycle, indicating that the mammary gland undergoes continual dynamic proliferative changes., Competing Interests: The authors declare no competing interests.

The mammary epithelium depends on specific lineages and their stem and progenitor function to accommodate hormone-triggered physiological demands in the adult female. Perturbations of these lineages underpin breast cancer risk, yet our understanding of normal mammary cell composition is incomplete. Here, we build a multimodal resource for the adult gland through comprehensive profiling of primary cell epigenomes, transcriptomes, and proteomes. We define systems-level relationships between chromatin-DNA-RNA-protein states, identify lineage-specific DNA methylation of transcription factor binding sites, and pinpoint proteins underlying progesterone responsiveness. Comparative proteomics of estrogen and progesterone receptor-positive and -negative cell populations, extensive target validation, and drug testing lead to discovery of stem and progenitor cell vulnerabilities. Top epigenetic drugs exert cytostatic effects; prevent adult mammary cell expansion, clonogenicity, and mammopoiesis; and deplete stem cell frequency. Select drugs also abrogate human breast progenitor cell activity in normal and high-risk patient samples. This integrative computational and functional study provides fundamental insight into mammary lineage and stem cell biology., (© 2018 Casey et al.)

Anemia is a prevalent public health problem associated with nutritional and socio-economic factors that contribute to iron deficiency. To understand the complex interplay of risk factors, we investigated a prospective population sample from the Jiangsu province in China. At baseline, three-day food intake was measured for 2849 individuals (20 to 87 years of age, mean age 47 ± 14, range 20-87 years, 64% women). At a five-year follow-up, anemia status was re-assessed for 1262 individuals. The dataset was split and age-matched to accommodate cross-sectional ( n = 2526), prospective ( n = 837), and subgroup designs ( n = 1844). We applied a machine learning framework (self-organizing map) to define four subgroups. The first two subgroups were primarily from the less affluent North: the High Fibre subgroup had a higher iron intake (35 vs. 21 mg/day) and lower anemia incidence (10% vs. 25%) compared to the Low Vegetable subgroup. However, the predominantly Southern subgroups were surprising: the Low Fibre subgroup showed a lower anemia incidence (10% vs. 27%), yet also a lower iron intake (20 vs. 28 mg/day) compared to the High Rice subgroup. These results suggest that interventions and iron intake guidelines should be tailored to regional, nutritional, and socio-economic subgroups., Competing Interests: The authors declare no conflict of interest.

Cohesins are vital for chromosome organisation during meiosis and mitosis. In addition to the important function in sister chromatid cohesion, these complexes play key roles in meiotic recombination, DSB repair, homologous chromosome pairing and segregation. Egg-laying mammals (monotremes) feature an unusually complex sex chromosome system, which raises fundamental questions about organisation and segregation during meiosis. We discovered a dynamic and differential accumulation of cohesins on sex chromosomes during platypus prophase I and specific reorganisation of the sex chromosome complex around a large nucleolar body. Detailed analysis revealed a differential loading of SMC3 on the chromatin and chromosomal axis of XY shared regions compared with the chromatin and chromosomal axes of asynapsed X and Y regions during prophase I. At late prophase I, SMC3 accumulation is lost from both the chromatin and chromosome axes of the asynaptic regions of the chain and resolves into subnuclear compartments. This is the first report detailing unpaired DNA specific SMC3 accumulation during meiosis in any species and allows speculation on roles for cohesin in monotreme sex chromosome organisation and segregation.

Background: In therian mammals heteromorphic sex chromosomes are subject to meiotic sex chromosome inactivation (MSCI) during meiotic prophase I while the autosomes maintain transcriptional activity. The evolution of this sex chromosome silencing is thought to result in retroposition of genes required in spermatogenesis from the sex chromosomes to autosomes. In birds sex chromosome specific silencing appears to be absent and global transcriptional reductions occur through pachytene and sex chromosome-derived autosomal retrogenes are lacking. Egg laying monotremes are the most basal mammalian lineage, feature a complex and highly differentiated XY sex chromosome system with homology to the avian sex chromosomes, and also lack autosomal retrogenes. In order to delineate the point of origin of sex chromosome specific silencing in mammals we investigated whether MSCI exists in platypus., Results: Our results show that platypus sex chromosomes display only partial or transient colocalisation with a repressive histone variant linked to therian sex chromosome silencing and surprisingly lack a hallmark MSCI epigenetic signature present in other mammals. Remarkably, platypus instead feature an avian like period of general low level transcription through prophase I with the sex chromosomes and the future mammalian X maintaining association with a nucleolus-like structure., Conclusions: Our work demonstrates for the first time that in mammals meiotic silencing of sex chromosomes evolved after the divergence of monotremes presumably as a result of the differentiation of the therian XY sex chromosomes. We provide a novel evolutionary scenario on how the future therian X chromosome commenced the trajectory toward MSCI.

The platypus and echidna are the only extant species belonging to the clade of monotremata, the most basal mammalian lineage. The platypus is particularly well known for its mix of mammalian and reptilian characteristics and work in recent years has revealed this also extends to the genetic level. Amongst the monotreme specific features is the unique multiple sex chromosome system (5X4Y in the echidna and 5X5Y in the platypus), which forms a chain in meiosis. This raises questions about sex chromosome organisation at meiosis, including whether there has been changes in genes coding for synaptonemal complex proteins which are involved in homologous synapsis. Here we investigate the key structural components of the synaptonemal complex in platypus and echidna, synaptonemal complex proteins 1, 2 and 3 (SYCP1, SYCP2 and SYCP3). SYCP1 and SYCP2 orthologues are present, conserved and expressed in platypus testis. SYCP3 in contrast is highly diverged, but key residues required for self-association are conserved, while those required for tetramer stabilisation and DNA binding are missing. We also discovered a second SYCP3-like gene (SYCP3-like) in the same region. Comparison with the recently published Y-borne SYCP3 amino acid sequences revealed that SYCP3Y is more similar to SYCP3 in other mammals than the monotreme autosomal SYCP3. It is currently unclear if these changes in the SYCP3 gene repertoire are related to meiotic organisation of the extraordinary monotreme sex chromosome system., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Progesterone drives mammary stem and progenitor cell dynamics through paracrine mechanisms that are currently not well understood. Here, we demonstrate that CXCR4, the receptor for stromal-derived factor 1 (SDF-1; CXC12), is a crucial instructor of hormone-induced mammary stem and progenitor cell function. Progesterone elicits specific changes in the transcriptome of basal and luminal mammary epithelial populations, where CXCL12 and CXCR4 represent a putative ligand-receptor pair. In situ, CXCL12 localizes to progesterone-receptor-positive luminal cells, whereas CXCR4 is induced in both basal and luminal compartments in a progesterone-dependent manner. Pharmacological inhibition of CXCR4 signaling abrogates progesterone-directed expansion of basal (CD24 + CD49f hi ) and luminal (CD24 + CD49f lo ) subsets. This is accompanied by a marked reduction in CD49b + SCA-1 - luminal progenitors, their functional capacity, and lobuloalveologenesis. These findings uncover CXCL12 and CXCR4 as novel paracrine effectors of hormone signaling in the adult mammary gland, and present a new avenue for potentially targeting progenitor cell growth and malignant transformation in breast cancer., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Stored red blood cells (RBCs) release hemoglobin (Hb) that leads to oxidative damage, which may contribute to thrombosis in susceptible transfusion recipients. Oxidative stress stimulates the generation of a new class of lipid mediators called F2 -isoprostanes (F2 -IsoPs) and isofurans (IsoFs) that influence cellular behavior. This study investigated RBC-derived F2 -IsoPs and IsoFs during storage and their influence on human platelets (PLTs)., Study Design and Methods: F2 -IsoP and IsoF levels in RBC supernatants were measured by mass spectrometry during storage and after washing. The effects of stored supernatants, cell-free Hb, or a key F2 -IsoP, 8-iso-prostaglandin F2α (PGF2α ), on PLT function were examined in vitro., Results: F2 -IsoPs, IsoFs, and Hb accumulated in stored RBC supernatants. Prestorage leukoreduction reduced supernatant F2 -IsoPs and IsoFs levels, which increased again over storage time. Stored RBC supernatants and 8-iso-PGF2α induced PLT activation marker CD62P (P-selectin) expression and prothrombotic thromboxane A2 release. Cell-free Hb did not alter PLT mediator release, but did inhibit PLT spreading. Poststorage RBC washing reduced F2 -IsoP and IsoF levels up to 24 hours., Conclusions: F2 -IsoPs and IsoFs are produced by stored RBCs and induce adverse effects on PLT function in vitro, supporting a potential novel role for bioactive lipids in adverse transfusion outcomes. F2 -IsoP and IsoF levels could be useful biomarkers for determining the suitability of blood components for transfusion. A novel finding is that cell-free Hb inhibits PLT spreading and could adversely influence wound healing. Poststorage RBC washing minimizes harmful lipid mediators, and its use could potentially reduce transfusion complications., (© 2013 American Association of Blood Banks.)

Background: ABO-mismatched platelets (PLTs) are commonly transfused despite reported complications. We hypothesized that because PLTs possess A and B antigens on their surface, ABO-mismatched transfused or recipient PLTs could become activated and/or dysfunctional after exposure to anti-A or -B in the transfused or recipient plasma. We present here in vitro modeling data on the functional effects of exposure of PLTs to ABO antibodies., Study Design and Methods: PLT functions of normal PLTs of all ABO types were assessed before and after incubation with normal saline, ABO-identical plasma samples, or O plasma samples with varying titers of anti-A and anti-B (anti-A/B). Assays used for this assessment include PLT aggregation, clot kinetics, thrombin generation, PLT cytoskeletal function, and mediator release., Results: Exposure of antigen-bearing PLTs to O plasma with moderate to high titers of anti-A/B significantly inhibits aggregation, prolongs PFA-100 epinephrine closure time, disrupts clot formation kinetics, accelerates thrombin generation, reduces total thrombin production, alters PLT cytoskeletal function, and influences proinflammatory and prothrombotic mediator release., Conclusions: Our findings demonstrate a wide range of effects that anti-A/B have on PLT function, clot formation, thrombin generation, PLT cytoskeletal function, and mediator release. These data provide potential explanations for clinical observations of increased red blood cell utilization in trauma and surgical patients receiving ABO-nonidentical blood products. Impaired hemostasis caused by anti-A/B interacting with A and B antigens on PLTs, soluble proteins, and perhaps even endothelial cells is a potential contributing factor to hemorrhage in patients receiving larger volumes of ABO-nonidentical transfusions., (© 2012 American Association of Blood Banks.)

Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor-β1 (TGFβ1). In this study, we demonstrate that TGFβ1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGFβ1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGFβ1-driven myofibroblast differentiation in AhR(-/-) fibroblasts: Its ability to inhibit TGFβ1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the presence and role of NF-kappaB proteins in megakaryocytes and platelets. The nuclear factor-kappaB (NF-kappaB) transcription factor family is well known for its role in eliciting inflammation and promoting cell survival. We discovered that human megakaryocytes and platelets express the majority of NF-kappaB family members, including the regulatory inhibitor-kappaB (I-kappaB) and I-kappa kinase (IKK) molecules., Methods and Results: Anucleate platelets exposed to NF-kappaB inhibitors demonstrated impaired fundamental functions involved in repairing vascular injury and thrombus formation. Specifically, NF-kappaB inhibition diminished lamellapodia formation, decreased clot retraction times, and reduced thrombus stability. Moreover, inhibition of I-kappaB-alpha phosphorylation (BAY-11-7082) reverted fully spread platelets back to a spheroid morphology. Addition of recombinant IKK-beta or I-kappaB-alpha protein to BAY inhibitor-treated platelets partially restored platelet spreading in I-kappaB-alpha inhibited platelets, and addition of active IKK-beta increased endogenous I-kappaB-alpha phosphorylation levels., Conclusions: These novel findings support a crucial and nonclassical role for the NF-kappaB family in modulating platelet function and reveal that platelets are sensitive to NF-kappaB inhibitors. As NF-kappaB inhibitors are being developed as antiinflammatory and anticancer agents, they may have unintended effects on platelets. On the basis of these data, NF-kappaB is also identified as a new target to dampen unwanted platelet activation.

Human platelets play a key role in hemostasis and thrombosis and have recently emerged as key regulators of inflammation. Platelets stored for transfusion produce pro-thrombotic and pro-inflammatory mediators implicated in adverse transfusion reactions. Correspondingly, these mediators are central players in pathological conditions including cardiovascular disease, the major cause of death in diabetics. In view of this, a mass spectrometry based proteomics study was performed on platelets collected from healthy and type-2 diabetics stored for transfusion. Strikingly, our innovative and sensitive proteomic approach identified 122 proteins that were either up- or down-regulated in type-2 diabetics relative to nondiabetic controls and 117 proteins whose abundances changed during a 5-day storage period. Notably, our studies are the first to characterize the proteome of platelets from diabetics before and after storage for transfusion. These identified differences allow us to formulate new hypotheses and experimentation to improve clinical outcomes by targeting "high risk platelets" that render platelet transfusion less effective or even unsafe.

Molecular methods are a necessary tool for sexing monomorphic birds. These molecular approaches are usually reliable, but sexing protocols should be evaluated carefully because biochemical interactions may lead to errors. We optimized laboratory protocols for genetic sexing of a monomorphic shorebird, the upland sandpiper (Bartramia longicauda), using two independent sets of primers, P2/P8 and 2550F/2718R, to amplify regions of the sex-linked CHD-Z and CHD-W genes. We discovered polymorphisms in the region of the CHD-Z intron amplified by the primers P2/P8 which caused four males to be misidentified as females (n = 90 mated pairs). We cloned and sequenced one CHD-W allele (370 bp) and three CHD-Z alleles in our population: Z° (335 bp), Z' (331 bp) and Z″ (330 bp). Normal (Z°Z°) males showed one band in agarose gel analysis and were easily differentiated from females (Z°W), which showed two bands. However, males heterozygous for CHD-Z alleles (Z'Z″) unexpectedly showed two bands in a pattern similar to females. While the Z' and Z″ fragments contained only short deletions, they annealed together during the polymerase chain reaction (PCR) process and formed heteroduplex molecules that were similar in size to the W fragment. Errors previously reported for molecular sex-assignment have usually been due to allelic dropout, causing females to be misidentified as males. Here, we report evidence that events in PCRs can lead to the opposite error, with males misidentified as females. We recommend use of multiple primer sets and large samples of known-sex birds for validation when designing protocols for molecular sex analysis., (© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.)

The herpes simplex virus (HSV)-derived amplicon vector has evolved into a promising gene transfer platform for widespread DNA delivery in gene replacement strategies and vaccine development given its ease of molecular manipulation, large transgene capacity, and transduction efficiencies of numerous cell types in vivo. The recent development of helper virus-free packaging methodologies bodes well for this vector system in its eventual implementation as a clinically viable therapeutic modality. For realization of clinical application, efforts have been made to enhance yields and quality of helper-free amplicon stocks. Hexamethylene bisacetamide (HMBA), a hybrid polar compound that exhibits stimulatory activity of HSV-1 immediate-early gene expression, has been employed as a standard reagent in helper virus-free packaging given its purported mode of action on virus gene expression kinetics. Unexpectedly, we have found that HMBA exhibits no titer-enhancing activity; in contrast, the compound enhances the proportion of amplicon virions that are non-expressive. Omission of HMBA during vector packaging led to a marked reduction in the ratios of vector genome-transducing to transgene-expressing virions. This effect was neither packaging-cell-specific nor amplicon-promoter-dependent. Analysis of resultant vector stocks indicated amplicon genome replication/concatenation was unaffected, but the level of particle-associated ICP0 was reduced in stocks packaged in the presence of HMBA. Inclusion of a co-transfected, ICP0-expressing plasmid into the packaging process led to significant rescue of amplicon expression titers, indicating that regulation of ICP0 concentrations is critical for maintenance of the amplicon genome expressive state.

Herpes simplex virus type-1 (HSV-1) amplicon vectors are being explored for a wide range of potential applications, including vaccine delivery and immunotherapy of cancer. While extensive effort has been directed towards the improvement of the amplicon "payload" in these vectors, relatively little attention has been paid to the effect of the packaging HSV-1 strains on the biological properties of co-packaged amplicon vectors. We therefore compared the biological properties of amplicon stocks prepared using a panel of primary HSV-1 isolates, a molecularly cloned strain used to package helper-free amplicons (designated here as F5), and two laboratory isolates (KOS and strain 17, which is the parent of the F5 clone). This analysis revealed considerable inter-strain variability in the ability of amplicon stocks packaged by different primary HSV-1 isolates to efficiently transduce established cell lines and primary human dendritic cells (DC). Amplicons packaged by both the F5 molecularly cloned virus and its laboratory-adapted parent (strain 17) were very inefficient at transducing DC, when compared to amplicons packaged by KOS or by several of the primary virus isolates. These finding have important implications for the future development of improved amplicon-based vaccine delivery systems and suggest that DC tropism may be an instrinsic property of some HSV-1 strains, independent of passage history or molecular cloning.

Given the participation of amyloid beta (Abeta) in Alzheimer's disease (AD) pathogenesis the derivation of experimental therapeutics to prevent Abeta fibrillogenesis and/or enhance removal of parenchymal amyloid deposits represent viable disease-modifying approaches. Active Abeta-based immunotherapies have shown promise in mouse AD models, but application in human trials was accompanied by moderate brain inflammation in a subset of patients. Immune-shaping vaccine platforms may mitigate adverse effects. Herein, we describe the use of herpes simplex virus (HSV)-derived amplicons to elicit distinctive immune responses against Abeta. Two vaccine vectors were constructed: one expressing Abeta1-42 alone (HSVAbeta), and a second expressing Abeta1-42 fused with the molecular adjuvant tetanus toxin Fragment C (HSVAbeta/TtxFC). Peripheral administration of these vaccines augmented humoral responses to Abeta and reduced CNS Abeta deposition in Tg2576 AD mice. Interestingly and unexpectedly, HSVAbeta vaccination was uniquely toxic and incited the expression of pro-inflammatory molecule transcripts within the hippocampi of Tg2576 mice, suggesting that this paradigm may serve as a relevant model to study Abeta vaccine-elicited CNS inflammatory syndromes.

Chronic lymphocytic leukemia (CLL) is a B lymphocyte malignancy that remains a largely incurable disease. CLL B cells possess the ability to process and present tumor antigens but lack expression of costimulatory molecules, rendering them inefficient effectors of T-cell activation. We previously demonstrated that helper virus-free preparations of herpes simplex virus (HSV) amplicon vectors encoding CD40L efficiently transduce CLL B cells and render them capable of eliciting specific anti-tumor T-cell responses. LIGHT (TNFSF14), a member of the tumor necrosis factor (TNF) superfamily, efficiently activates both T cells and antigen-presenting cells (APCs). We employed an HSV amplicon vector expressing human LIGHT (hf-HSV-LIGHT) to transduce CLL B cells and compared the immunomodulatory function and T-cell activation induced by hf-HSV-LIGHT transduction to that observed with a CD40L-expressing HSV amplicon (hf-HSV-CD40L). hf-HSV-LIGHT transduction induced expression of endogenous B7.1, B7.2, and ICAM.1 on CLL cells, albeit to a lesser degree than that observed in response to transduction with hf-HSV-CD40L. hf-HSV-LIGHT enhanced the antigen-presenting capacity of CLL B cells, as measured by induction of T-cell proliferation in an allogeneic mixed lymphocyte tumor reaction. Finally, hf-HSV-LIGHT-transduced CLL B cells successfully stimulated the outgrowth of autologous cytotoxic T-lymphocytes in vitro. In aggregate, these data suggest that hf-HSV-LIGHT transduction may be useful for induction of immune responses to CLL and other B-cell lymphoid malignancies.

On routine hospital admission, 23,714 patients received a 28-test serum metabolic profile. The 33 most common diseases (4,132 patients) of liver, pancreas, and gallbladder (LPG) had unique chemical templates averaging 15 significant serum deviations. Each LPG disease differed from all others by elevations of both leucine-aminopeptidase (LAP) and alkaline phosphatase (AP) levels. LAP level was low or normal and serum glutamic oxaloacetic transaminase (SGOT) and AP levels were elevated in 43 non-LPG diseases. Patients with acute and chronic pancreatitis had elevated amylase levels. The four nonmalignant diseases of the gallbladder were associated with normal levels of amylase and lactic dehydrogenase (LDH); except for silent cholelithiasis, each showed elevated total bilirubin (BIL) levels. Patients with solitary or scattered lesions of the liver had normal bilirubin levels (2,115 patients), and those with diffuse interstitial or parencymal disease had elevated BIL levels. Cancer patients had elevated LDH and alpha1 globulin (A1G) levels, but low albumin levels. The importance of comprehensive liver profiles in the treatment of psychoses is emphasized by significant liver damage in a number of these patients. A1G was normal and LDH was elevated in patients having mononucleosis, hepatitis, lupus erythematosus, alcoholism, and alcoholic cirrhosis.

1. Statistical analyses have been made of the weekly variations in the blood counts of groups of normal rabbits to find whether there exists any relationship between the numerical changes occurring in the various cell types. Consecutive blood counts and differential white cell determinations on five groups of normal male rabbits comprising 45 animals in all were made at weekly intervals from October, 1927, to June, 1929, the number of observations on each group varying from eight to thirty-five. 2. The following relationships between the varying group means were found to be consistent and significant:-The number of the red blood cells varied with the amount of hemoglobin per cubic millimeter and with the number of lymphocytes. There was an inverse relationship between the amount of hemoglobin and the number of monocytes. The neutrophiles varied in number with the monocytes; the basophiles with the eosinophiles; and the eosinophiles with the monocytes. Other associations not always similar but of high significance as far as the combined values were concerned, were the relations of the red blood cells with the basophiles and the monocytes. The relations of the neutrophiles with the red blood cells and the hemoglobin were very irregular. 3. Significant association of the white blood cells with variations in the red blood cells and the hemoglobin content were observed. The numerical variations in the group means of the total white cells were associated with similar variations in the group means of the neutrophiles, the lymphocytes, the monocytes, the basophiles, and the eosinophiles almost to the degree of their numerical occurrence in the peripheral blood. 4. With the exception of the total white cells, approximately only half the variations in the group levels of the various cells and of the hemoglobin content can be accounted for on the basis of simultaneous associations with each other. 5. The red blood cells, the lymphocytes, and the basophiles as one group, the eosinophiles and the monocytes as another group, and the hemoglobin content and the neutrophiles as a third group, described a definite shift from a high to a low numerical value during the 2 year observation period. From the standpoint of the magnitude of the shift, the basophiles, the eosinophiles, the monocytes, the lymphocytes, and the red blood cells participated in the order mentioned. The neutrophiles were only slightly affected and the hemoglobin content relatively not at all. 6. No significant relationship was ever found, even in the component groups, between the weekly mean values of the following: the hemoglobin with the basophiles, the eosinophiles, or the lymphocytes; the neutrophiles with the basophiles or the eosinophiles; and the lymphocytes with the eosinophiles or the monocytes.