Biomaterial-Based Sustained-Release Drug Formulations for Localized Cancer Immunotherapy.

Cancer immunotherapy has revolutionized clinical cancer treatments by taking advantage of the immune system to selectively and effectively target and kill cancer cells. However, clinical cancer immunotherapy treatments often have limited efficacy and/or present severe adverse effects associated primarily with their systemic administration. Localized immunotherapy has emerged to overcome these limitations by directly targeting accessible tumors via local administration, reducing potential systemic drug distribution that hampers drug efficacy and safety. Sustained-release formulations can prolong drug activity at target sites, which maximizes the benefits of localized immunotherapy to increase the therapeutic window using smaller dosages than those used for systemic injection, avoiding complications of frequent dosing. The performance of sustained-release formulations for localized cancer immunotherapy has been validated preclinically using various implantable and injectable scaffold platforms. This review introduces the sustained-release formulations developed for localized cancer immunotherapy and highlights their biomaterial-based platforms for representative classes, including inorganic scaffolds, natural hydrogels, synthetic hydrogels, and microneedle patches. The design rationale and other considerations are summarized for further development of biomaterials for the construction of optimal sustained-release formulations.

Advances for the treatment of lower extremity arterial disease associated with diabetes mellitus.

Lower extremity arterial disease (LEAD) is a major vascular complication of diabetes. Vascular endothelial cells dysfunction can exacerbate local ischemia, leading to a significant increase in amputation, disability, and even mortality in patients with diabetes combined with LEAD. Therefore, it is of great clinical importance to explore proper and effective treatments. Conventional treatments of diabetic LEAD include lifestyle management, medication, open surgery, endovascular treatment, and amputation. As interdisciplinary research emerges, regenerative medicine strategies have provided new insights to treat chronic limb threatening ischemia (CLTI). Therapeutic angiogenesis strategies, such as delivering growth factors, stem cells, drugs to ischemic tissues, have also been proposed to treat LEAD by fundamentally stimulating multidimensional vascular regeneration. Recent years have seen the rapid growth of tissue engineering technology; tissue-engineered biomaterials have been used to study the treatment of LEAD, such as encapsulation of growth factors and drugs in hydrogel to facilitate the restoration of blood perfusion in ischemic tissues of animals. The primary purpose of this review is to introduce treatments and novel biomaterials development in LEAD. Firstly, the pathogenesis of LEAD is briefly described. Secondly, conventional therapies and therapeutic angiogenesis strategies of LEAD are discussed. Finally, recent research advances and future perspectives on biomaterials in LEAD are proposed.

Methacrylated gelatin shape-memorable cryogel subcutaneously delivers EPCs and aFGF for improved pressure ulcer repair in diabetic rat model.

Pressure ulcer (PU) in patients with diabetes mellitus (DM) is still a clinical intractable issue due to the complicated physiological characteristics by the prolonged high glucose level and impaired angiogenesis. The PU treatment includes surgical debridement, stem cell therapy and growth factors, leading to high cost and repeated professional involvement. Developing effective wound dressing combining the therapeutic cells and growth factors has become highly demanded. Herein, we reported the direct subcutaneous administration of endothelial progenitor cells (EPCs) and acid fibroblast growth factor (aFGF) with a shape-memorable methacrylated gelatin cryogel (EPCs/aFGF@GelMA) for the therapy of PU in rats with DM. This EPCs/aFGF@GelMA cryogel system presented microporous structure, elastic mechanical strength and enhanced cell migration property with controlled release of aFGF. Moreover, compared with EPCs/aFGF and GelMA alone, in vivo results showed that this EPCs/aFGF@GelMA system exhibited accelerated wound closure rate, enhanced granulation formation, collagen deposition as well as re-epithelization. Importantly, we found that the excellent positive performance of EPCs/aFGF@GelMA is due to its up-regulation of HIF-ɑ upon the wound site, modulating the microenvironment of wound site to initiate the impaired local angiogenesis. Collectively, this hybrid gelatin cryogels show great promise for biomedical applications, especially in tissue engineering and regenerative medicine.

Advances of Microneedles in Biomedical Applications.

A microneedle (MN) is a painless and minimally invasive drug delivery device initially developed in 1976. As microneedle technology evolves, microneedles with different shapes (cone and pyramid) and forms (solid, drug-coated, hollow, dissolvable and hydrogel-based microneedles) have been developed. The main objective of this review is the applications of microneedles in biomedical areas. Firstly, the classifications and manufacturing of microneedle are briefly introduced so that we can learn the advantages and fabrications of different MNs. Secondly, research of microneedles in biomedical therapy such as drug delivery systems, diagnoses of disease, as well as wound repair and cancer therapy are overviewed. Finally, the safety and the vision of the future of MNs are discussed.

PRMT7 targets of Foxm1 controls alveolar myofibroblast proliferation and differentiation during alveologenesis.

Although aberrant alveolar myofibroblasts (AMYFs) proliferation and differentiation are often associated with abnormal lung development and diseases, such as bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF), epigenetic mechanisms regulating proliferation and differentiation of AMYFs remain poorly understood. Protein arginine methyltransferase 7 (PRMT7) is the only reported type III enzyme responsible for monomethylation of arginine residue on both histone and nonhistone substrates. Here we provide evidence for PRMT7's function in regulating AMYFs proliferation and differentiation during lung alveologenesis. In PRMT7-deficient mice, we found reduced AMYFs proliferation and differentiation, abnormal elastin deposition, and failure of alveolar septum formation. We further shown that oncogene forkhead box M1 (Foxm1) is a direct target of PRMT7 and that PRMT7-catalyzed monomethylation at histone H4 arginine 3 (H4R3me1) directly associate with chromatin of Foxm1 to activate its transcription, and thereby regulate of cell cycle-related genes to inhibit AMYFs proliferation and differentiation. Overexpression of Foxm1 in isolated myofibroblasts (MYFs) significantly rescued PRMT7-deficiency-induced cell proliferation and differentiation defects. Thus, our results reveal a novel epigenetic mechanism through which PRMT7-mediated histone arginine monomethylation activates Foxm1 transcriptional expression to regulate AMYFs proliferation and differentiation during lung alveologenesis and may represent a potential target for intervention in pulmonary diseases.

Antimicrobial α-defensins as multi-target inhibitors against amyloid formation and microbial infection.

Amyloid aggregation and microbial infection are considered as pathological risk factors for developing amyloid diseases, including Alzheimer's disease (AD), type II diabetes (T2D), Parkinson's disease (PD), and medullary thyroid carcinoma (MTC). Due to the multifactorial nature of amyloid diseases, single-target drugs and treatments have mostly failed to inhibit amyloid aggregation and microbial infection simultaneously, thus leading to marginal benefits for amyloid inhibition and medical treatments. Herein, we proposed and demonstrated a new "anti-amyloid and antimicrobial hypothesis" to discover two host-defense antimicrobial peptides of α-defensins containing ß-rich structures (human neutrophil peptide of HNP-1 and rabbit neutrophil peptide of NP-3A), which have demonstrated multi-target, sequence-independent functions to (i) prevent the aggregation and misfolding of different amyloid proteins of amyloid-ß (Aß, associated with AD), human islet amyloid polypeptide (hIAPP, associated with T2D), and human calcitonin (hCT, associated with MTC) at sub-stoichiometric concentrations, (ii) reduce amyloid-induced cell toxicity, and (iii) retain their original antimicrobial activity upon the formation of complexes with amyloid peptides. Further structural analysis showed that the sequence-independent amyloid inhibition function of α-defensins mainly stems from their cross-interactions with amyloid proteins via ß-structure interactions. The discovery of antimicrobial peptides containing ß-structures to inhibit both microbial infection and amyloid aggregation greatly expands the new therapeutic potential of antimicrobial peptides as multi-target amyloid inhibitors for better understanding pathological causes and treatments of amyloid diseases.

Freeze-Thawing Chitosan/Ions Hydrogel Coated Gauzes Releasing Multiple Metal Ions on Demand for Improved Infected Wound Healing.

Imbalance of metal ions in the wound microenvironment is a key factor that leads to delayed wound healing. However, single metal administration to enhance wound repair is usually not enough due to the overlapping nature of the wound healing phases. Herein, a facile freeze-thawing strategy is developed to incorporate chitosan/ions hydrogel into medical gauzes to realize on-demand release of multiple ions to accelerate wound healing. In vitro study reveals that the gauzes can temporally release multiple metal ions on demand, and the released metal ions show effectiveness in killing bacteria and expediting cell migration. In vivo studies demonstrate that the metal ions loaded gauzes can efficiently enhance infected wound healing. Further histological analysis find that these metal ion-loaded gauzes accelerate wound healing by promoting granulation formation, collagen deposition and maturation, re-epithelization, angiogenesis, and inhibiting inflammation via regulating the expression of inflammatory factors (e.g., tumor necrosis factor-α) and polarization of macrophages. Thus, this novel metal ions delivery system has great potential in infected tissue repair and antibacterial applications.

Zwitterionic hydrogel for sustained release of growth factors to enhance wound healing.

Growth factors (GFs) have been well known for their therapeutic effects on wound healing. Due to their vulnerable biostability, biomaterial carriers are usually used to deliver GFs to maintain their bioactivity. Among the carriers, PEG hydrogels are the most widely applied. But the uncontrolled release of GFs and their immunogenicity dramatically retard the application of PEG hydrogels as carriers of GFs. Herein, FGF2 loaded zwitterionic sulfobetaine methacrylate (SBMA) hydrogels were developed, and it was revealed that these hydrogels were more effective in delivering FGF2 for wound healing than were PEG hydrogels. In vitro studies demonstrated that SBMA hydrogels could successfully prolong the release of FGF2, which effectively maintained the bioactivity of FGF2. Further in vivo investigation showed that SBMA hydrogels could efficiently accelerate wound regeneration by promoting granulation tissue formation, collagen deposition, cell proliferation and migration, reepithelialization and angiogenesis. All results validated that SBMA hydrogels were promising substituents of PEG hydrogels for delivering FGF2 for wound regeneration.

Molecular Dynamics Simulations of Cholesterol Effects on the Interaction of hIAPP with Lipid Bilayer.

Fundamental understanding of specific interactions of human islet amyloid polypeptide (hIAPP) with cell membrane is critical for elucidating the underlying pathogenesis of type II diabetes mellitus (T2DM). Membrane cholesterol is known to regulate membrane functions and properties, but its exact role in driving hIAPP-membrane interactions still remains controversial. In this work, we computationally investigated the concentration effect of cholesterol on the adsorption, orientation, and surface interaction of hIAPP oligomers on POPC bilayers containing different amounts of cholesterol (χ = 0, 20, and 40 mol %). Collective MD simulations consistently showed that an increased cholesterol level modulated the structure and dynamics of POPC bilayer, leading to an increase of bilayer thickness, lipid packing order, and surface hydrophobicity but a decrease of lipid mobility. Cholesterol-induced bilayer changes further caused hIAPP oligomer to more preferentially bind to POPC bilayer in the presence of cholesterol via C-terminal residues, in contrast to weak or no binding of hIAPP oligomer on pure POPC bilayers. The cholesterol-enhanced hIAPP-membrane binding is mainly contributed by electrostatic interactions between C-terminal residues and lipid head groups, which may explain the rapid adsorption and aggregation of hIAPP in the presence of cholesterol in cell membranes. This computational work provides some insights into drug development and therapeutic strategies for T2DM by considering cholesterol effects.

A bioactivatable self-quenched nanogel for targeted photodynamic therapy.

Photodynamic therapy has attracted significant attention due to its localized treatment advantage. However, the non-specific distribution of photosensitizers and the subsequent potential toxicity caused by sunshine exposure hinder its wide adoption in cancer treatment. To minimize these unwanted effects and improve its efficacy, we developed a bioactivatable self-quenched nanogel, which remains in its inactive state in healthy tissues. Anti-EGFR Affibody decorated nanogels can effectively target head and neck cancer and release activated pheophorbide A in a reducing environment, such as in the tumor stroma and cytoplasm. Consequently, the EGFR targeted nanogel coupled with NIR irradiation alleviates tumor burden by 94.5% while not inducing systemic toxicity.

Novel H2S-Releasing hydrogel for wound repair via in situ polarization of M2 macrophages.

Hydrogen sulfide (H2S), as a gaseous messenger, exhibits potential therapeutic effects in biological and clinical applications. Herein, an in situ forming biomimetic hyaluronic acid (HA) hydrogel was used as a matrix to dope a pH-controllable H2S donor, JK1, to form a novel HA-JK1 hybrid system. This HA-JK1 hydrogel was designed as an ideal delivery scaffold for JK1 with pH-dependent prolonged H2S releasing profile. In vitro study suggested that JK1 could induce the polarization of M2 phenotype indicating a higher pro-healing efficiency of macrophages. The in vivo studies on dermal wounds showed that the HA-JK1 hybrid hydrogel significantly accelerated the wound regeneration process through enhanced re-epithelialization, collagen deposition, angiogenesis and cell proliferation. Furthermore, the in vivo results also demonstrated a higher level of M2 polarization in HA-JK1 treated group with reduced inflammation and improved wound remodeling effects, which was consistent with the in vitro results. These observations could be considered as a key to the efficient wound treatment. Therefore, we suggest that HA-JK1 can be used as a novel wound dressing material toward cutaneous wound model in vivo. This system should significantly enhance wound regeneration through the release of H2S that induces the expression of M2 macrophage phenotype.

Zwitterionic poly(sulfobetaine methacrylate) hydrogels with optimal mechanical properties for improving wound healing in vivo.

Zwitterionic hydrogels, as highly hydrated and soft materials, have been considered as promising materials for wound dressing, due to their unique antifouling and mechanical properties. While the viscoelasticity and softness of zwitterionic hydrogels are hypothetically essential for creating adaptive cellular niches, the underlying mechanically regulated wound healing mechanism still remains elusive. To test this hypothesis, we fabricated zwitterionic poly(sulfobetaine methacrylate) (polySBMA) hydrogels with different elastic moduli prepared at different crosslinker contents, and then applied the hydrogels to full-thickness cutaneous wounds in mice. In vivo wound healing studies compared the mechanical cue-induced effects of soft and stiff polySBMA hydrogels on wound closure rates, granulation tissue formation and collagen deposition. Collective results showed that the softer and more viscoelastic hydrogels facilitated cell proliferation, granulation formation, collagen aggregation, and chondrogenic ECM deposition. Such high wound healing efficiency by the softer hydrogels is likely attributed to stress dissipation by expanding the cell proliferation, the up-regulation of blood vessel formation, and the enhanced polarization of M2/M1 macrophages, both of which would provide more oxygen and nutrients for cell proliferation and migration, leading to enhanced wound repair. This work not only reveals a mechanical property-wound healing relationship of zwitterionic polySBMA hydrogels, but also provides a promising candidate and strategy for the next-generation of wound dressings.

Loureirin B Promotes Axon Regeneration by Inhibiting Endoplasmic Reticulum Stress: Induced Mitochondrial Dysfunction and Regulating the Akt/GSK-3ß Pathway after Spinal Cord Injury.

Axon retraction greatly limits functional recovery after spinal cord injury (SCI) and neuron polarization, which affects processes including axon formation and development, is a promising target for promoting axon regeneration. Increasing microtubule stability has been demonstrated to improve intrinsic axon regeneration processes and is critically related to endoplasmic reticulum (ER)-mitochondria interactions. We used real-time polymerase chain reaction, Western blotting, and immunofluorescence to screen a variety of natural compounds, and found that Loureirin B (LrB) effectively promoted neuron polarization and axon regeneration in vitro and in vivo. LrB significantly inhibited ER stress and thereby promoted mitochondrial functions by regulating mitochondrial fusion. Further, LrB reactivated the Akt/GSK-3ß pathway, which plays critical roles in cell survival and microtubule stabilization. Taken together, our results suggest that the effects of LrB on neuron regeneration involve the inhibition of ER stress-induced mitochondrial dysfunction and activation of the Akt/GSK-3ß pathway, which further promotes microtubule stabilization. LrB may therefore be a promising candidate for facilitating recovery following SCI.

Repurposing disulfiram for cancer therapy via targeted nanotechnology through enhanced tumor mass penetration and disassembly.

Disulfiram (DSF), an FDA approved drug for the treatment of alcoholism, degrades to therapeutically active diethyldithiocarbamate (DDTC) in the body by reduction. Hereby, we developed a redox sensitive DDTC-polymer conjugate for targeted cancer therapy. It was found that the DDTC-polymer conjugate modified with a ß-d-galactose receptor targeting ligand can self-assemble into LDNP nanoparticle and efficiently enter cancer cells by receptor-mediated endocytosis. Upon cellular uptake, the LDNP nanoparticle degrades and releases DDTC due to the cleavage of disulfide bonds, and subsequently forms copper (II) DDTC complex to kill a broad spectrum of cancer cells. 3D cell culture revealed that this nanoparticle shows much stronger tumor mass penetrating and destructive capacity. Furthermore, LDNP nanoparticles exhibited much greater potency in inhibiting tumor growth in a peritoneal metastatic ovarian tumor model. STATEMENT OF SIGNIFICANCE: The ß-d-galactose receptor targeted disulfiram loaded nanoparticle (LDNP) is novel in the following aspects.

Mussel-inspired PLGA/polydopamine core-shell nanoparticle for light induced cancer thermochemotherapy.

Most photothermal converting systems are not biodegradable, which bring the uneasiness when they are administered into human body due to the uncertainty of their fate. Hereby, we developed a mussel-inspired PLGA/polydopamine core-shell nanoparticle for cancer photothermal and chemotherapy. With the help of an anti-EGFR antibody, the nanoparticle could effectively enter head and neck cancer cells and convert near-infrared light to heat to trigger drug release from PLGA core for chemotherapy as well as ablate tumors by the elevated temperature. Due to the unique nanoparticle concentration dependent peak working-temperature nature, an overheating or overburn situation can be easily prevented. Since the nanoparticle was retained in the tumor tissue and subsequently released its payload inside the cancer cells, no any doxorubicin-associated side effects were detected. Thus, the developed mussel-inspired PLGA/polydopamine core-shell nanoparticle could be a safe and effective tool for the treatment of head and neck cancer. STATEMENT OF SIGNIFICANCE: The described EGFR targeted PLGA/polydopamine core-shell nanoparticle (PLGA/PD NP) is novel in the following aspects: Different from most photothermal converting nanomaterials, PLGA/PD NP is biodegradable, which eliminates the long-term safety concerns thwarting the clinical application of photothermal therapy. Different from most photothermal nanomaterials, upon NIR irradiation, PLGA/PD NP quickly heats its surrounding environment to a NP concentration dependent peak working temperature and uniquely keeps that temperature constant through the duration of light irradiation. Due to this unique property an overheating or overburn situation for the adjacent healthy tissue can be easily avoided. The PLGA/PD NP releases its payload through detaching PD shell under NIR laser irradiation. The EGFR-targeted doxorubicin-loaded PLGA/PD NP effectively eradicate head and neck tumor in vivo through the synergism of photothermal therapy and chemotherapy while not introducing doxorubicin associated cardiotoxicity.

Gold Nanosphere Gated Mesoporous Silica Nanoparticle Responsive to Near-Infrared Light and Redox Potential as a Theranostic Platform for Cancer Therapy.

A gold/mesoporous silica hybrid nanoparticle (GoMe), which possesses the best of both conventional gold nanoparticles and mesoporous silica nanoparticles, such as excellent photothermal converting ability as well as high drug loading capacity and triggerable drug release, has been developed. In contrast to gold nanorod and other heat generating gold nanoparticles, GoMe is photothermal stable and can be repetitively activated through NIR irradiation. Doxorubicin loaded GoMe (DOX@GoMe) is sensitive to both NIR irradiation and intracellularly elevated redox potential. DOX@GoMe coupled with NIR irradiation exhibits a synergistic effect of photothermal therapy and chemotherapy in killing cancer cells. Furthermore, 64Cu-labeled GoMe can successfully detect the existence of clinically relevant spontaneous lung tumors in a urethane-induced lung cancer mouse model through PET imaging. Altogether, GoMe can be utilized as an effective theranostic platform for cancer therapy.

Cancer cell-selective killing polymer/copper combination.

Chemotherapy has been adopted for cancer treatment for decades. However, its efficacy and safety are frequently compromised by the multidrug-resistance of cancer cells and the poor cancer cell selectivity of anticancer drugs. Hereby, we report a combination of a pyridine-2-thiol containing polymer and copper which can effectively kill a wide spectrum of cancer cells, including drug resistant cancer cells, while sparing normal cells. The polymer nanoparticle enters cells via an exofacial thiol facilitated route, and releases active pyridine-2-thiol with the help of intracellularly elevated glutathione (GSH). Due to their high GSH level, cancer cells are more vulnerable to the polymer/copper combination. In addition, RNA microarray analysis revealed that the treatment can reverse cancer cells' upregulated oncogenes (CIRBP and STMN1) and downregulated tumor suppressor genes (CDKN1C and GADD45B) to further enhance the selectivity for cancer cells.

Histone Arginine Methylation by PRMT7 Controls Germinal Center Formation via Regulating Bcl6 Transcription.

B cells are the center of humoral immunity and produce Abs to protect against foreign Ags. B cell defects lead to diseases such as leukemia and lymphomas. Histone arginine methylation is important for regulating gene activation and silencing in cells. Although the process commonly exists in mammalian cells, its roles in B cells are unknown. To explore the effects of aberrant histone arginine methylation on B cells, we generated mice with a B cell-specific knockout of PRMT7, a member of the methyltransferases that mediate arginine methylation of histones. In this article, we showed that the loss of PRMT7 led to decreased mature marginal zone B cells and increased follicular B cells and promoted germinal center formation after immunization. Furthermore, mice lacking PRMT7 expression in B cells secreted low levels of IgG1 and IgA. Abnormal expression of germinal center genes (i.e., Bcl6, Prdm1, and Irf4) was detected in conditional knockout mice. By overexpressing PRMT7 in the Raji and A20 cell lines derived from B cell lymphomas, we validated the fact that PRMT7 negatively regulated Bcl6 expression. Using chromatin immunoprecipitation-PCR, we found that PRMT7 could recruit H4R3me1 and symmetric H4R3me2 to the Bcl6 promoter. These results provide evidence for the important roles played by PRMT7 in germinal center formation.

Triple-responsive expansile nanogel for tumor and mitochondria targeted photosensitizer delivery.

A pH, thermal, and redox potential triple-responsive expansile nanogel system (TRN), which swells at acidic pH, temperature higher than its transition temperature, and reducing environment, has been developed. TRN quickly expands from 108 nm to over 1200 nm (in diameter), achieving more than 1000-fold size enlargement (in volume), within 2 h in a reducing environment at body temperature. Sigma-2 receptor targeting-ligand functionalized TRN can effectively target head and neck tumor, and help Pc 4 targeting mitochondria inside cancer cells to achieve enhanced photodynamic therapy efficacy.

NF-kappa B mediated up-regulation of CCCTC-binding factor in pediatric acute lymphoblastic leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most frequently occurring malignant neoplasm in children. Despite advances in treatment and outcomes for ALL patients, the pathogenesis of the disease remains unclear. Microarray analysis of samples from 100 Chinese children with ALL revealed the up-regulation of CTCF (CCCTC binding factor). CTCF is a highly conserved 11-zinc finger protein that is involved in many human cancers; however, the biological function of CTCF in pediatric ALL is unknown. METHODS: The expression patterns of CTCF were evaluated in matched newly diagnosed (ND), complete remission (CR), and relapsed (RE) bone marrow samples from 28 patients. The potential oncogenic mechanism of CTCF and related pathways in leukemogenesis were investigated in leukemia cell lines. RESULTS: We identified significant up-regulation of CTCF in the ND samples. Importantly, the expression of CTCF returned to normal levels after CR but rebounded in the RE samples. In the pre-B ALL cell line Nalm-6, siRNA-mediated silencing of CTCF expression promoted cell apoptosis and reduced cell proliferation; accordingly, over-expression of a cDNA encoding full-length CTCF protected cells from apoptosis and enhanced cell proliferation. Furthermore, inhibition or activation of the nuclear factor-kappa B (NF-κB) pathway resulted in marked variations in the levels of CTCF mRNA and protein in leukemic cells, indicating that CTCF may be involved downstream of the NF-κB pathway. Moreover, inhibition of the NF-κB pathway increased cell apoptosis, which was partially rescued by ectopic over-expression of CTCF, suggesting that CTCF may play a significant role in the anti-apoptotic pathway mediated by NF-κB. CONCLUSIONS: Our results indicate that CTCF serves as both an anti-apoptotic factor and a proliferative factor in leukemic cells. It potentially contributes to leukemogenesis through the NF-κB pathway in pediatric ALL patients.