Durable CD4+ T cell immunity: cherchez la stem.

Mammalian stem cells govern development, tissue homeostasis, and regeneration. Following years of study, their functions have been delineated with increasing precision. The past decade has witnessed heightened widespread use of stem cell terminology in association with durable T cell responses to infection, antitumor immunity, and autoimmunity. Interpreting this literature is complicated by the fact that descriptions are diverse and criteria for labeling 'stem-like' T cells are evolving. Working under the hypothesis that conceptual frameworks developed for actual stem cells can be used to better evaluate and organize T cells described to have stem-like features, we outline widely accepted properties of stem cells and compare these to different 'stem-like' CD4+ T cell populations.

OCA-B/Pou2af1 is sufficient to promote CD4+ T cell memory and prospectively identifies memory precursors.

The molecular mechanisms leading to the establishment of immunological memory are inadequately understood, limiting the development of effective vaccines and durable antitumor immune therapies. Here, we show that ectopic OCA-B expression is sufficient to improve antiviral memory recall responses, while having minimal effects on primary effector responses. At peak viral response, short-lived effector T cell populations are expanded but show increased Gadd45b and Socs2 expression, while memory precursor effector cells show increased expression of Bcl2, Il7r, and Tcf7 on a per-cell basis. Using an OCA-B mCherry reporter mouse line, we observe high OCA-B expression in CD4+ central memory T cells. We show that early in viral infection, endogenously elevated OCA-B expression prospectively identifies memory precursor cells with increased survival capability and memory recall potential. Cumulatively, the results demonstrate that OCA-B is both necessary and sufficient to promote CD4 T cell memory in vivo and can be used to prospectively identify memory precursor cells.

Biologically Analogous Calcium Phosphate Tubes from a Chemical Garden.

Calcium phosphate (CaPO4) tubes with features comparable to mineralized biological microstructures, such as Haversian canals, were grown from a calcium gel/phosphate solution chemical garden system. A significant difference in gel mass in response to high and low solute phosphate equivalent environments existed within 30 min of solution layering upon gel (p = 0.0067), suggesting that the nature of advective movement between gel and solution is dependent on the solution concentration. The transport of calcium cations (Ca2+) and phosphate anions (PO43-) was quantified and changes in pH were monitored to explain the preferential formation of tubes within a PO43- concentration range of 0.5-1.25 M. Ingress from the anionic solution phase into the gel followed by the liberation of Ca2+ ions from the gel was found to be essential for acquiring self-assembled tubular CaPO4 structures. Tube analysis by scanning electron microscopy (SEM), X-ray diffraction (XRD), and micro X-ray florescence (µ-XRF) revealed hydroxyapatite (HA, Ca10(PO4)6(OH)2) and dicalcium phosphate dihydrate (DCPD, CaHPO4·2H2O) phases organized in a hierarchical manner. Notably, the tubule diameters ranged from 100 to 150 µm, an ideal size for the permeation of vasculature in biological hard tissue.

OCA-B promotes autoimmune demyelination through control of stem-like CD4 + T cells.

Stem-like T cell populations can selectively promote autoimmunity, but the activities that sustain these populations are incompletely understood. Here, we show that T cell-intrinsic loss of the transcription cofactor OCA-B protects mice from experimental autoimmune encephalomyelitis (EAE) while preserving responses to infection. In EAE models driven by antigen re-encounter, OCA-B deletion eliminates CNS infiltration, proinflammatory cytokine production and clinical disease. OCA-B-expressing CD4 + T cells within the CNS of mice with EAE display a memory phenotype and preferentially confer disease. In a relapsing-remitting EAE model, OCA-B T cell-deficiency specifically protects mice from relapse. During remission, OCA-B promotes the expression of Tcf7 , Slamf6 , and Sell in proliferating T cell populations. At relapse, OCA-B loss results in both the accumulation of an immunomodulatory CD4 + T cell population expressing Ccr9 and Bach2 , and the loss of effector gene expression from Th17 cells. These results identify OCA-B as a driver of pathogenic stem-like T cells.

Oct1 cooperates with the Smad family of transcription factors to promote mesodermal lineage specification.

The transition between pluripotent and tissue-specific states is a key aspect of development. Understanding the pathways driving these transitions will facilitate the engineering of properly differentiated cells for experimental and therapeutic uses. Here, we showed that during mesoderm differentiation, the transcription factor Oct1 activated developmental lineage-appropriate genes that were silent in pluripotent cells. Using mouse embryonic stem cells (ESCs) with an inducible knockout of Oct1, we showed that Oct1 deficiency resulted in poor induction of mesoderm-specific genes, leading to impaired mesodermal and terminal muscle differentiation. Oct1-deficient cells exhibited poor temporal coordination of the induction of lineage-specific genes and showed inappropriate developmental lineage branching, resulting in poorly differentiated cell states retaining epithelial characteristics. In ESCs, Oct1 localized with the pluripotency factor Oct4 at mesoderm-associated genes and remained bound to those loci during differentiation after the dissociation of Oct4. Binding events for Oct1 overlapped with those for the histone lysine demethylase Utx, and an interaction between Oct1 and Utx suggested that these two proteins cooperate to activate gene expression. The specificity of the ubiquitous Oct1 for the induction of mesodermal genes could be partially explained by the frequent coexistence of Smad and Oct binding sites at mesoderm-specific genes and the cooperative stimulation of mesodermal gene transcription by Oct1 and Smad3. Together, these results identify Oct1 as a key mediator of mesoderm lineage-specific gene induction.

The expression of 1-deoxy-D-xylulose synthase and geraniol-10-hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in Catharanthus roseus hairy roots.

The terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus produces two important anticancer drugs, vinblastine and vincristine, in very low yields. This study focuses on overexpressing several key genes in the upper part of the TIA pathway in order to increase flux toward downstream metabolites within hairy root cultures. Specifically, we constructed hairy root lines with inducible overexpression of 1-deoxy-D-xylulose synthase (DXS) or geraniol-10-hydroxylase (G10H). We also constructed hairy root lines with inducible expression of DXS and anthranilate synthase α subunit (ASA) or DXS and G10H. DXS overexpression resulted in a significant increase in ajmalicine by 67%, serpentine by 26% and lochnericine by 49% and a significant decrease in tabersonine by 66% and hörhammericine by 54%. Co-overexpression of DXS and G10H caused a significant increase in ajmalicine by 16%, lochnericine by 31% and tabersonine by 13%. Likewise, DXS and ASA overexpression displayed a significant increase in hörhammericine by 30%, lochnericine by 27% and tabersonine by 34%. These results point to the need for overexpressing multiple genes within the pathway to increase the flux toward vinblastine and vincristine.

Controlled self-assembly of chemical gardens enables fabrication of heterogeneous chemobrionic materials.

Chemical gardens are an example of a chemobrionic system that typically result in abiotic macro-, micro- and nano- material architectures, with formation driven by complex out-of-equilibrium reaction mechanisms. From a technological perspective, controlling chemobrionic processes may hold great promise for the creation of novel, compositionally diverse and ultimately, useful materials and devices. In this work, we engineer an innovative custom-built liquid exchange unit that enables us to control the formation of tubular chemical garden structures grown from the interface between calcium loaded hydrogel and phosphate solution. We show that systematic displacement of phosphate solution with water (H2O) can halt self-assembly, precisely control tube height and purify structures in situ. Furthermore, we demonstrate the fabrication of a heterogeneous chemobrionic composite material composed of aligned, high-aspect ratio calcium phosphate channels running through an otherwise dense matrix of poly(2-hydroxyethyl methacrylate) (pHEMA). Given that the principles we derive can be broadly applied to potentially control various chemobrionic systems, this work paves the way for fabricating multifunctional materials that may hold great potential in a variety of application areas, such as regenerative medicine, catalysis and microfluidics.

Trabecular bone organoids: a micron-scale 'humanised' prototype designed to study the effects of microgravity and degeneration.

Bone is a highly responsive organ, which continuously adapts to the environment it is subjected to in order to withstand metabolic demands. These events are difficult to study in this particular tissue in vivo, due to its rigid, mineralised structure and inaccessibility of the cellular component located within. This manuscript presents the development of a micron-scale bone organoid prototype, a concept that can allow the study of bone processes at the cell-tissue interface. The model is constructed with a combination of primary female osteoblastic and osteoclastic cells, seeded onto femoral head micro-trabeculae, where they recapitulate relevant phenotypes and functions. Subsequently, constructs are inserted into a simulated microgravity bioreactor (NASA-Synthecon) to model a pathological state of reduced mechanical stimulation. In these constructs, we detected osteoclastic bone resorption sites, which were different in morphology in the simulated microgravity group compared to static controls. Once encapsulated in human fibrin and exposed to analogue microgravity for 5 days, masses of bone can be observed being lost from the initial structure, allowing to simulate the bone loss process further. Constructs can function as multicellular, organotypic units. Large osteocytic projections and tubular structures develop from the initial construct into the matrix at the millimetre scale. Micron-level fragments from the initial bone structure are detected travelling along these tubules and carried to sites distant from the native structure, where new matrix formation is initiated. We believe this model allows the study of fine-level physiological processes, which can shed light into pathological bone loss and imbalances in bone remodelling.

Formulation of a reactive oxygen producing calcium sulphate cement as an anti-bacterial hard tissue scaffold.

Prophylactic antibiotic bone cements are extensively used in orthopaedics. However, the development of antimicrobial resistance to antibiotics, demonstrates a need to find alternative treatments. Herein, an antimicrobial honey (SurgihoneyRO-SHRO) has been successfully incorporated into a calcium sulphate (CS) based cement to produce a hard tissue scaffold with the ability to inhibit bacterial growth. Antimicrobial properties elicited from SHRO are predominantly owed to the water-initiated production of reactive oxygen species (ROS). As an alternative to initially loading CS cement with SHRO, in order to prevent premature activation, SHRO was added into the already developing cement matrix, locking available water into the CS crystal structure before SHRO addition. Promisingly, this methodology produced > 2.5 times (715.0 ± 147.3 µM/mL/g) more ROS over 24 h and exhibited a compressive strength (32.2 ± 5.8 MPa) comparable to trabecular bone after 3 weeks of immersion. In-vitro the SHRO loaded CS scaffolds were shown to inhibit growth of clinically relevant organisms, Staphylococcus aureus and Pseudomonas aeruginosa, with comparable potency to equivalent doses of gentamicin. Encouragingly, formulations did not inhibit wound healing or induce an inflammatory response from osteoblasts. Overall this study highlights the prophylactic potential of CS-SHRO cements as an alternative to traditional antibiotics.

Formulation of an antimicrobial superabsorbent powder that gels in situ to produce reactive oxygen.

The enzymatic oxidation of glucose to produce reactive oxygen species (ROS) provides honey with antimicrobial efficacy. This mechanism offers an alternative to traditional antibiotics; however, topical use of honey is limited due to its adherent and highly viscous properties. This study aims to overcome these issues by engineering a powder-based system that eases delivery and offers in situ activation of ROS. Starch based drying agents were utilised to enable freeze drying of a medical honey, with methylated-ß-cyclodextrin (MCD) enabling the highest active incorporation (70%) while still producing a free-flowing powder. Addition of a superabsorbent, sodium polyacrylate (≤40%) was shown to facilitate in situ gelation of the powder, with an absorption capacity of up to 120.7 ± 4.5 mL g-1. Promisingly efficacy of the optimised superabsorbent powder was demonstrated in vitro against several clinically relevant Gram-negative and Gram-positive bacteria (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa). Alongside this no adverse effects were observed against human dermal fibroblasts. Application of the superabsorbent powder in an ex-vivo porcine wound model revealed capability to form a protective hydrogel barrier in less than 1 min. Overall, this novel ROS producing superabsorbent powder has potential to tackle topical infections without using traditional antibiotics.

Development of a small-scale rotary lobe-pump cell culture model for examining cell damage in large-scale N-1 seed perfusion process.

Perfusion technology has been identified as a process improvement capable of eliminating some of the constraints in cell culture and allows for high cell densities and viabilities. However, when implementing this N-1 seed perfusion platform in large-scale manufacturing, unexpected cell damage was observed as early as Day 1. Given that the shear rate within recirculation hollow fibers was normalized and aligned correctly across bench, pilot, and manufacture scale, the primary mitigation was placed on the rotary lobe pump. Lowering the pump rate in manufacture scale successfully alleviated the cell damage. To understand the source of cell damage within the pump, a small-scale rotary lobe-pump robustness model was developed. Testing different pump flow rates and back pressures, it was concluded that high back pressure can cause cell damage. The back pressure within the system can cause back flow and high shear within small clearances inside the pump, which lead to the primary cell damage observed at a large scale. This shear level can be significantly higher than the shear in the hollow fiber. This pump robustness model can be utilized to aid the perfusion skid design, including pump operation efficiency and cell shear sensitivity. Methods to reduce the back pressure and cell shearing were determined to better predict manufacturing performance in the future.

Chemobrionic structures in tissue engineering: self-assembling calcium phosphate tubes as cellular scaffolds.

A diverse range of complex patterns and mineralised hierarchical microstructures can be derived from chemobrionic systems, with formation driven by complex reaction-diffusion mechanisms far from thermodynamic equilibrium. In these experiments, self-assembling calcium phosphate tubes are generated using hydrogels made with 1 M calcium solutions layered with solutions of dibasic sodium phosphate over a range of concentrations between 0.2-1 M. Self-assembling structures prepared using 0.8 M dibasic sodium phosphate solutions were selected to assess cell-material interactions. Candidate chemobrionic scaffolds were characterised by micro-X-Ray fluorescence (µ-XRF) spectroscopy, Raman spectroscopy, powder X-ray diffraction (XRD), helium pycnometry and scanning electron microscopy (SEM). As prepared tubes were formed from non-stoichiometric hydroxyapatite (HA, Ca10-x(PO4)6-x(HPO4)x(OH)2-x (0 ≤x≤ 1)), which was confirmed as calcium deficient hydroxyapatite (CDHA, Ca9(PO4)5HPO4OH). Thermal treatment of tubes in air at 650 °C for 4 h converted the structures to beta tricalcium phosphate (ß-TCP, ß-Ca3(PO4)2). The potential of these scaffolds to support the attachment of bone marrow derived mesenchymal stem cells (BMSCs) was investigated for the first time, and we demonstrate cell attachment and elongation on the fabricated tubular structures.

Hexametaphosphate as a potential therapy for the dissolution and prevention of kidney stones.

The incidence of kidney stones is increasing worldwide, and recurrence is common (50% within 5 years). Citrate, the current gold standard therapy, which is usually given as potassium or sodium salts, is used because it raises urine pH and chelates calcium, the primary component of up to 94% of stones. In this study hexametaphosphate (HMP), a potent calcium chelator, was found to be 12 times more effective at dissolving calcium oxalate, the primary component of kidney stones, than citrate. HMP was also observed to be effective against other common kidney stone components, namely calcium phosphate and struvite (magnesium ammonium phosphate). Interestingly, HMP was capable of raising the zeta potential of calcium oxalate particles from -15.4 to -34.6 mV, which may prevent stone growth by aggregation, the most rapid growth mechanism, and thus avert occlusion. Notably, HMP was shown to be up to 16 times as effective as citrate at dissolving human kidney stones under simulated physiological conditions. It may thus be concluded that HMP is a promising potential therapy for calcium and struvite kidney stones.

The Quantification of Injectability by Mechanical Testing.

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more viscous than traditional aqueous injections and may be semi-solid, therefore, their injectability cannot be assumed. This protocol describes a method to objectively assess the injectability of these materials using a standard mechanical tester. The syringe plunger is compressed by the crosshead at a set rate, and the force is measured. The maximum or plateau force value can then be used for comparison between samples, or to an absolute force limit. This protocol can be used with any material, and any syringe and needle size or geometry. The results obtained may be used to make decisions about formulations, syringe and needle sizes early in the translational process. Further, the effects of altering formulations on injectability may be quantified, and the optimum time to inject temporally changing materials determined. This method is also suitable as a reproducible way to examine the effects of injection on a material, to study phenomena such as self-healing and filter pressing or study the effects of injection on cells. This protocol is faster and more directly applicable to injectability than rotational rheology, and requires minimal post processing to obtain key values for direct comparisons.

Modulation of transmembrane pressure in manufacturing scale tangential flow filtration N-1 perfusion seed culture.

Mammalian cells were grown to high density in a 3,000 L culture using perfusion with hollow fibers operated in a tangential flow filtration mode. The high-density culture was used to inoculate the production stage of a biomanufacturing process. At constant permeate flux operation, increased transmembrane pressures (TMPs) were observed on the final day of the manufacturing batches. Small scale studies suggested that the filters were not irreversibly fouled, but rather exposed to membrane concentration polarization that could be relieved by tangential sweeping of the hollow fibers. Studies were undertaken to analyze parameters that influence the hydrodynamic profile within hollow fibers; including filter area, cell density, recirculation flow rate, and permeate flow rate. Results indicated that permeate flow rate had the greatest influence on modulating TMP. Further evaluation showed a significant decrease in TMP when permeate flow was reduced, and this occurred without any negative effect on cell growth or viability. Hence, a 30% reduction of permeate flow rate was implemented at manufacturing scale. A stable operation was achieved as TMP was successfully reduced by 75% while preserving all critical factors for performance in the perfusion bioreactor.

Filling the Gap: A Correlation between Objective and Subjective Measures of Injectability.

Various injectable biomaterials are developed for the minimally invasive delivery of therapeutics. Typically, a mechanical tester is used to ascertain the force required to inject these biomaterials through a given syringe-needle system. However, currently there is no method to correlate the force measured in the laboratory to the perceived effort required to perform that injection by the end user. In this article, the injection force (F) for a variety of biomaterials, displaying a range of rheological properties, is compared with the effort scores from a 50 person panel study. The maximum injection force measured at crosshead speed 1 mm s-1 is a good proxy for injection effort, with an R2 of 0.89. This correlation leads to the following conclusions: participants can easily inject 5 mL of substance for F < 12 N; considerable effort is required to inject 5 mL for 12 N < F < 38 N; great effort is required and <5 mL can be injected for 38 N < F < 64 N; and materials are entirely non-injectable for F > 64 N. These values may be used by developers of injectable biomaterials to make decisions about formulations and needle sizes early in the translational process.

Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time.

Jasmonic acid (JA) activates the transcriptional regulator ORCA3, which has a role in regulating the terpenoid indole alkaloid (TIA) pathway within Catharanthus roseus. The TIA pathway leads to the production of the anticancer drugs vinblastine and vincristine. This work explores the transient effects of overexpressing ORCA3 under the control of a glucocorticoid-inducible promoter system in C. roseus hairy roots along with the simultaneous feeding of JA. The changes in TIA metabolites and in mRNA transcripts of pathway genes and regulators were tracked for 72h. Upon induction of ORCA3 expression and elicitation with JA, ORCA3 transcripts increased 170-fold whereas ORCA3 expression caused an 89-fold increase and JA elicitation caused a 5-fold increase in ORCA3 transcripts. JA treatment caused the largest increase in TIA metabolites and transcripts of pathway genes. These transcripts displayed a transient response with the maximum expression reached between 12 and 24h. In the samples overexpressing ORCA3, the largest increase in the transcripts of ZCT1 and ZCT2 (ZCT-zinc finger-binding protein), TIA transcriptional repressors, coincided with the largest increase in ORCA3 transcripts. This counter response of transcriptional repressors may explain why the large increase in ORCA3 transcripts do not correspond with larger increases in transcripts of TIA pathway genes.

Critical and diverse roles of phosphates in human bone formation.

Humans utilise biomineralisation in the formation of bone and teeth. Human biomineralisation processes are defined by the transformation of an amorphous phosphate-based precursor to highly organised nanocrystals. Interestingly, ionic phosphate species not only provide a fundamental building block of biological mineral, but rather exhibit several diverse roles in mediating mineral formation in the physiological milieu. In this review, we focus on elucidating the complex roles of phosphate ions and molecules within human biomineralisation pathways, primarily referring to the nucleation and crystallisation of bone mineral.

Formulation of a covalently bonded hydroxyapatite and poly(ether ether ketone) composite.

Spinal fusion devices can be fabricated from composites based on combining hydroxyapatite and poly(ether ether ketone) phases. These implants serve as load-bearing scaffolds for the formation of new bone tissue between adjacent vertebrae. In this work, we report a novel approach to covalently bond hydroxyapatite and poly(ether ether ketone) to produce a novel composite formulation with enhanced interfacial adhesion between phases. Compared to non-linked composites (HA_PEEK), covalently linked composites (HA_L_PEEK), loaded with 1.25 vol% hydroxyapatite, possessed a greater mean flexural strength (170 ± 5.4 vs 171.7 ± 14.8 MPa (mean ± SD)) and modulus (4.8 ± 0.2 vs 5.0 ± 0.3 GPa (mean ± SD)). Although the mechanical properties were not found to be significantly different (p > 0.05), PEEK_L_HA contained substantially larger hydroxyapatite inclusions (100-1000 µm) compared to HA_PEEK (50-200 µm), due to the inherently agglomerative nature of the covalently bonded hydroxyapatite and poly(ether ether ketone) additive. Larger inclusions would expectedly weaken the HA_L_PEEK composite; however, there is no significant difference between the flexural modulus of poly(ether ether ketone) with respect to HA_L_PEEK (p = 0.13). In addition, the flexural modulus of HA_PEEK is significantly lower compared to poly(ether ether ketone) (p = 0.03). Ultimately, covalent linking reduces hydroxyapatite particulate de-bonding from the polymeric matrix and inhibits micro-crack development, culminating in enhanced transfer of stiffness between hydroxyapatite and poly(ether ether ketone) under loading.

Formulation and viscoelasticity of mineralised hydrogels for use in bone-cartilage interfacial reconstruction.

Articular cartilage is a viscoelastic tissue whose structural integrity is important in maintaining joint health. To restore the functionality of osteoarthritic joints it is vital that regenerative strategies mimic the dynamic loading response of cartilage and bone. Here, a rotating simplex model was employed to optimise the composition of agarose and gellan hydrogel constructs structured with hydroxyapatite (HA) with the aim of obtaining composites mechanically comparable to human cartilage in terms of their ability to dissipate energy. Addition of ceramic particles was found to reinforce both matrices up to a critical concentration (< 3w/v%). Beyond this, larger agglomerates were formed, as evidenced by micro computed tomography data, which acted as stress risers and reduced the ability of composites to dissipate energy demonstrated by a reduction in tan δ values. A maximum compressive modulus of 450.7±24.9 kPa was achieved with a composition of 5.8w/v% agarose and 0.5w/v% HA. Interestingly, when loaded dynamically (1-20Hz) this optimised formulation did not exhibit the highest complex modulus instead a sample with a higher concentration of mineral was identified (5.8w/v% agarose and 25w/v% HA). Thus, demonstrating the importance of examining the mechanical behaviour of biomaterials under conditions representative of physiological environments. While the complex moduli of the optimised gellan (1.0 ± 0.2MPa at 1Hz) and agarose (1.7 ± 0.2MPa at 1Hz) constructs did not match the complex moduli of healthy human cartilage samples (26.3 ± 6.5MPa at 1Hz), similar tan δ values were observed between 1 and 5Hz. This is promising since these frequencies represent the typical heel strike time of the general population. In summary, this study demonstrates the importance of considering more than just the strength of biomaterials since tissues like cartilage play a more complex role.