In vivo and in vitro models demonstrate a role for caveolin-1 in the pathogenesis of ischaemic acute renal failure

Caveolae and their proteins, the caveolins, transport macromolecules; compartmentalize signalling molecules; and are involved in various repair processes. There is little information regarding their role in the pathogenesis of significant renal syndromes such as acute renal failure (ARF). In this study, an in vivo rat model of 30 min bilateral renal ischaemia followed by reperfusion times from 4 h to 1 week was used to map the temporal and spatial association between caveolin-1 and tubular epithelial damage (desquamation, apoptosis, necrosis). An in vitro model of ischaemic ARF was also studied, where cultured renal tubular epithelial cells or arterial endothelial cells were subjected to injury initiators modelled on ischaemia-reperfusion (hypoxia, serum deprivation, free radical damage or hypoxia-hyperoxia). Expression of caveolin proteins was investigated using immunohistochemistry, immunoelectron microscopy, and immunoblots of whole cell, membrane or cytosol protein extracts. In vivo, healthy kidney had abundant caveolin-1 in vascular endothelial cells and also some expression in membrane surfaces of distal tubular epithelium. In the kidneys of ARF animals, punctate cytoplasmic localization of caveolin-1 was identified, with high intensity expression in injured proximal tubules that were losing basement membrane adhesion or were apoptotic, 24 h to 4 days after ischaemia-reperfusion. Western immunoblots indicated a marked increase in caveolin-1 expression in the cortex where some proximal tubular injury was located. In vitro, the main treatment-induced change in both cell types was translocation of caveolin-1 from the original plasma membrane site into membrane-associated sites in the cytoplasm. Overall, expression levels did not alter for whole cell extracts and the protein remained membrane-bound, as indicated by cell fractionation analyses. Caveolin-1 was also found to localize intensely within apoptotic cells. The results are indicative of a role for caveolin-1 in ARF-induced renal injury. Whether it functions for cell repair or death remains to be elucidated.

Interleukin-1beta induces human proximal tubule cell injury, alpha-smooth muscle actin expression and fibronectin production

BACKGROUND Tubulointerstitial lesions, characterized by tubular injury, interstitial fibrosis and the appearance of myofibroblasts, are the strongest predictors of the degree and progression of chronic renal failure. These lesions are typically preceded by macrophage infiltration of the tubulointerstitium, raising the possibility that these inflammatory cells promote progressive renal disease through fibrogenic actions on resident tubulointerstitial cells. The aim of the present study, therefore, was to investigate the potentially fibrogenic mechanisms of interleukin-1beta (IL-1beta), a macrophage-derived pro-inflammatory cytokine, on human proximal tubule cells (PTC). METHODS Confluent, quiescent, passage 2 PTC were established in primary culture from histologically normal segments of human renal cortex (N = 11) and then incubated in serum- and hormone-free media supplemented with either IL-1beta (0 to 4 ng/mL) or vehicle (control). RESULTS IL-1beta significantly enhanced fibronectin secretion by up to fourfold in a time- and concentration-dependent fashion. This was accompanied by significant (2.5- to 6-fold) increases in alpha-smooth muscle actin (alpha-SMA) expression, transforming growth factor beta (TGF-beta1) secretion, nitric oxide (NO) production, NO synthase 2 (NOS2) mRNA and lactate dehydrogenase (LDH) release. Cell proliferation was dose-dependently suppressed by IL-1beta. NG-methyl-l-arginine (L-NMMA; 1 mmol/L), a specific inhibitor of NOS, blocked NO production but did not alter basal or IL-1beta-stimulated fibronectin secretion. In contrast, a pan-specific TGF-beta neutralizing antibody significantly blocked the effects of IL-1beta on PTC fibronectin secretion (IL-1beta, 268.1 +/- 30.6 vs. IL-1beta+alphaTGF-beta 157.9 +/- 14.4%, of control values, P < 0.001) and DNA synthesis (IL-1beta 81.0 +/- 6.7% vs. IL-1beta+alphaTGF-beta 93.4 +/- 2.1%, of control values, P < 0.01). CONCLUSION IL-1beta acts on human PTC to suppress cell proliferation, enhance fibronectin production and promote alpha-smooth muscle actin expression. These actions appear to be mediated by a TGF-beta1 dependent mechanism and are independent of nitric oxide release.

CD151 is associated with prostate cancer cell invasion and lymphangiogenesis in vivo

CD151, a member of the tetraspanin family, is associated with regulation of migration of normal and tumour cells via cell surface microdomain formation. CD151 was found in our laboratory to have a prognostic value in prostate cancer and is a promoter of prostate cancer migration and invasion. These roles involve association with integrins on both cell-cell and cell-stroma levels. Furthermore, CD151 plays a role in endothelial cell motility. CD151 expression was examined in three commonly used prostate cancer cell lines. We investigated CD151 expression, angiogenesis (microvessel density; MVD) and lymphangiogenesis (lymphatic vessel density; LVD) in an orthotopic xenograft model of prostate cancer in matched tumours from primary and secondary sites. CD151 was found to be heterogeneously expressed across different prostate cancer cell lines and the levels of CD151 expression were significantly higher in the highly tumorigenic, androgen-insensitive cells PC-3 and DU-145 compared to the androgen-sensitive cell line LNCaP (P<0.05). The majority of in vivo xenografts developed pelvic lymph node metastases. Importantly, primary tumours that developed metastasis had significantly higher CD151 expression and MVD compared to those which did not develop metastasis (P<0.05). We identified, for the first time, that CD151 expression is associated with LVD in prostate cancer. These findings underscore the potential role of CD151 and angiogenesis in the metastatic potential of prostate cancer. CD151 has a prognostic value in this mouse model of prostate cancer and may play a role in lymphangiogenesis. CD151 is likely an important regulator of cancer cell communication with the surrounding microenvironment.

Assessment of freestanding membranes prepared from Antheraea pernyi silk fibroin as a potential vehicle for corneal epithelial cell transplantation

Freestanding membranes created from Bombyx mori silk fibroin (BMSF) offer a potential vehicle for corneal cell transplantation since they are transparent and support the growth of human corneal epithelial cells (HCE). Fibroin derived from the wild silkworm Antheraea pernyi (APSF) might provide a superior material by virtue of containing putative cell- attachment sites that are absent from BMSF. Thus we have investigated the feasibility of producing transparent, freestanding membranes from APSF and have analysed the behaviour of HCE cells on this material. No significant differences in cell numbers or phenotype were observed in short term HCE cell cultures established on either fibroin. Production of transparent freestanding APSF membranes, however, proved to be problematic as cast solutions of APSF were more prone to becoming opaque, displayed significantly lower permeability and were more brittle than BMSF-membranes. Cultures of HCE cells established on either membrane developed a normal stratified morphology with cytokeratin pair 3/12 being immuno-localized to the superficial layers. We conclude that while it is feasible to produce transparent freestanding membranes from APSF, the technical difficulties associated with this biomaterial, along with an absence of enhanced cell growth, currently favours the continued development of BMSF as a preferred vehicle for corneal cell transplantation. Nevertheless, it remains possible that refinement of techniques for processing APSF might yet lead to improvements in the handling properties and performance of this material.

Comparing methods for modelling spreading cell fronts

Spreading cell fronts play an essential role in many physiological processes. Classically, models of this process are based on the Fisher-Kolmogorov equation; however, such continuum representations are not always suitable as they do not explicitly represent behaviour at the level of individual cells. Additionally, many models examine only the large time asymptotic behaviour, where a travelling wave front with a constant speed has been established. Many experiments, such as a scratch assay, never display this asymptotic behaviour, and in these cases the transient behaviour must be taken into account. We examine the transient and asymptotic behaviour of moving cell fronts using techniques that go beyond the continuum approximation via a volume-excluding birth-migration process on a regular one-dimensional lattice. We approximate the averaged discrete results using three methods: (i) mean-field, (ii) pair-wise, and (iii) one-hole approximations. We discuss the performace of these methods, in comparison to the averaged discrete results, for a range of parameter space, examining both the transient and asymptotic behaviours. The one-hole approximation, based on techniques from statistical physics, is not capable of predicting transient behaviour but provides excellent agreement with the asymptotic behaviour of the averaged discrete results, provided that cells are proliferating fast enough relative to their rate of migration. The mean-field and pair-wise approximations give indistinguishable asymptotic results, which agree with the averaged discrete results when cells are migrating much more rapidly than they are proliferating. The pair-wise approximation performs better in the transient region than does the mean-field, despite having the same asymptotic behaviour. Our results show that each approximation only works in specific situations, thus we must be careful to use a suitable approximation for a given system, otherwise inaccurate predictions could be made.

Selective regulation of p38β protein and signaling by integrin-linked kinase mediates bladder cancer cell migration

Integrin-linked kinase (ILK) and p38MAPK are protein kinases that transduce extracellular signals regulating cell migration and actin cytoskeletal organization. ILK-dependent regulation of p38MAPK is critical for mammalian kidney development and in smooth muscle cell migration, however, specific p38 isoforms has not been previously examined in ILK-regulated responses. Signaling by ILK and p38MAPK is often dysregulated in bladder cancer, and here we report a strong positive correlation between protein levels of ILK and p38β, which is the predominant isoform found in bladder cancer cells, as well as in patient-matched normal bladder and tumor samples. Knockdown by RNA interference of either p38β or ILK disrupts serum-induced, Rac1-dependent migration and actin cytoskeletal organization in bladder cancer cells. Surprisingly, ILK knockdown causes the selective reduction in p38β cellular protein level, without inhibiting p38β messenger RNA (mRNA) expression. The loss of p38β protein in ILK-depleted cells is partially rescued by the 26S proteasomal inhibitor MG132. Using co-precipitation and bimolecular fluorescent complementation assays, we find that ILK selectively forms cytoplasmic complexes with p38β. In situ proximity ligation assays further demonstrate that serum-stimulated assembly of endogenous ILK–p38β complexes is sensitive to QLT-0267, a small molecule ILK kinase inhibitor. Finally, inhibition of ILK reduces the amplitude and period of serum-induced activation of heat shock protein 27 (Hsp27), a target of p38β implicated in actin cytoskeletal reorganization. Our work identifies Hsp27 as a novel target of ILK–p38β signaling complexes, playing a key role in bladder cancer cell migration.

IL-23R is epigenetically regulated and modulated by chemotherapy in non-small cell lung cancer

The Interleukin-23 (IL-23)/IL-23R signaling axis is an important inflammatory pathway, involved in the stimulation and regulation of the T helper (Th) 17 lymphocytes, resulting in the production of IL-17. Aside from auto-immunity, this cytokine has also been linked to carcinogenesis and polymorphisms in the IL-23R gene are associated with an increased risk for the development of a number of different cancers. Activation of the IL-23 pathway results in the up-regulation of STAT3 and it is thought that the pathological consequences associated with this are in part due to the production of IL-17. We have previously identified IL-23A as pro-proliferative and epigenetically regulated in non-small cell lung cancer (NSCLC). The current study aims to evaluate IL-23R in greater detail in NSCLC. We demonstrate that IL-23R is expressed and epigenetically regulated in NSCLC through histone post-translation modifications and CpG island methylation. In addition, Gemcitabine treatment, a chemotherapy drug used in the treatment of NSCLC, resulted in the up-regulation of the IL-23R. Furthermore, Apilimod (STA 5326), a small molecule which blocks the expression of IL-23 and IL-12, reduced the proliferative capacity of NSCLC cells, particularly in the adenocarcinoma (A549) sub-type. Apilimod is currently undergoing investigation in a number of clinical trials for the treatment of auto-immune conditions such as Crohn's disease and Rheumatoid Arthritis. Our results may have implications for treating NSCLC patients with Gemcitabine or epigenetic targeted therapies. However, Apilimod may possibly provide a new treatment avenue for NSCLC patients. Work is currently ongoing to further delineate the IL-23/IL-23R axis in this disease.

Thromboxane synthase expression and correlation with VEGF and angiogenesis in non-small cell lung cancer

Background Thromboxane synthase (TXS) metabolizes prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with angiogenesis and poor outcome. TXS has been identified as a potential therapeutic target in NSCLC. This study examines a link between TXS expression, angiogenesis, and survival in NSCLC. Methods TXS and VEGF metabolite levels were measured in NSCLC serum samples (n=46) by EIA. TXB2 levels were correlated with VEGF. A 204-patient TMA was stained for TXS, VEGF, and CD-31 expression. Expression was correlated with a range of clinical parameters, including overall survival. TXS expression was correlated with VEGF and CD-31. Stable TXS clones were generated and the effect of overexpression on tumor growth and angiogenesis markers was examined in-vitro and in-vivo (xenograft mouse model). Results Serum TXB2 levels were correlated with VEGF (p<0.05). TXS and VEGF were expressed to a varying degree in NSCLC tissue. TXS was associated with VEGF (p<0.0001) and microvessel density (CD-31; p<0.05). TXS and VEGF expression levels were higher in adenocarcinoma (p<0.0001) and female patients (p<0.05). Stable overexpression of TXS increased VEGF secretion in-vitro. While no significant association with patient survival was observed for either TXS or VEGF in our patient cohort, TXS overexpression significantly (p<0.05) increased tumor growth in-vivo. TXS overexpression was also associated with higher levels of VEGF, microvessel density, and reduced apoptosis in xenograft tumors. Conclusion TXS promotes tumor growth in-vivo in NSCLC, an effect which is at least partly mediated through increased tumor angiogenesis.

High coexpression of both EGFR and IGF1R correlates with poor patient prognosis in resected non-small-cell lung cancer

Background Recent experimental and biomarker evidence indicates that the epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor 1 (IGF1R) interact in the pathogenesis of malignant epithelial tumors, including lung cancer. This study examines the expression of both receptors and their prognostic significance in surgically resected non-small-cell lung cancer (NSCLC). Methods EGFR and IGF1R expression were evaluated in 184 patients with NSCLC (83 squamous cell carcinomas [SCCs], 83 adenocarcinomas [ADCs], and 18 other types) using immunohistochemical (IHC) analysis. Expression of both receptors was examined in matched fresh frozen normal and tumor tissues from 40 patients with NSCLC (20 SCCs and 20 ADCs) by Western blot analysis. Results High EGFR expression was detected in 51% of patients, and SCCs had higher EGFR expression than did non-SCCs (57.4% vs. 42.5%; P =.028). High IGF1R expression was observed in 53.8% of patients, with SCC having higher expression than non-SCC (62.6% vs. 37.3%; P =.0004). A significant association was shown between EGFR and IGF1R protein overexpression (P <.005). Patients with high expression of both receptors had a poorer overall survival (OS) (P =.04). Higher EGFR and IGF1R expression was detected in resected tumors relative to matched normal tissues (P =.0004 and P =.0009), with SCC having higher expression levels than ADC. Conclusion Our findings indicate a close interrelationship between EGFR and IGF1R. Coexpression of both receptors correlates with poor survival. This subset of patients may benefit from treatments cotargeting EGFR and IGF1R. © 2014 Elsevier Inc. All rights reserved.

Targeted therapies in non-small cell lung cancer

The majority of non-small cell lung cancer (NSCLC) patients present with advanced disease and with a 5 year survival rate of <15% for these patients, treatment outcomes are considered extremely disappointing. Standard chemotherapy regimens provide some improvement to ~40% of patients. However, intrinsic and acquired chemoresistance are a significant problem and hinder sustained long term benefits of such treatments. Advances in proteomic and genomic profiling have increased our understanding of the aberrant molecular mechanisms that are driving an individual's tumour. The increased sensitivity of these technologies has enabled molecular profiling at the stage of initial biopsy thus paving the way for a more personalised approach to the treatment of cancer patients. Improvements in diagnostics together with a wave of new targeted small molecule inhibitors and monoclonal antibodies have revolutionised the treatment of cancer. To date there are essentially three targeted agents approved for clinical use in NSCLC. The tyrosine kinase inhibitor (TKI) erlotinib, which targets the epidermal growth factor receptor (EGFR) TK domain, has proven to be an effective treatment strategy in patients who harbour activating mutations in the EGFR TK domain. Bevacizumab a monoclonal antibody targeting the vascular endothelial growth factor (VEGF) can improve survival, response rates, and progression-free survival when used in combination with chemotherapy. Crizotinib, a small-molecule drug, inhibits the tyrosine kinase activity of the echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-ALK) fusion protein, resulting in decreased tumour cell growth, migration, and invasiveness in patients with locally advanced or metastatic NSCLC. The clinical relevance of several other targeted agents are under investigation in distinct molecular subsets of patients with key "driver" mutations including: KRAS, HER2, BRAF, MET, PIK3CA, AKT1,MAP2K1, ROS1 and RET. Often several pathways are activated simultaneously and crosstalk between pathways allows tumour cells to escape the inhibition of a single targeted agent. This chapter will explore the clinical development of currently available targeted therapies for NSCLC as well as those in clinical trials and will examine the synergy between cytotoxic therapies.

Estrogen receptor β activation impairs mitochondrial oxidative metabolism and affects malignant mesothelioma cell growth in vitro and in vivo

Estrogen receptor (ER)-β has been shown to possess a tumor suppressive effect, and is a potential target for cancer therapy. Using gene-expression meta-analysis of human malignant pleural mesothelioma, we identified an ESR2 (ERβ coding gene) signature. High ESR2 expression was strongly associated with low succinate dehydrogenase B (SDHB) (which encodes a mitochondrial respiratory chain complex II subunit) expression. We demonstrate that SDHB loss induced ESR2 expression, and that activated ERβ, by over-expression or by selective agonist stimulation, negatively affected oxidative phosphorylation compromising mitochondrial complex II and IV activity. This resulted in reduced mitochondrial ATP production, increased glycolysis dependence and impaired cell proliferation. The observed in vitro effects were phenocopied in vivo using a selective ERβ agonist in a mesothelioma mouse model. On the whole, our data highlight an unforeseen interaction between ERβ-mediated tumor suppression and energy metabolism that may be exploited to improve on the therapy for clinical management of malignant mesothelioma.

Weekly cisplatin concurrently with radiotherapy in head and neck squamous cell cancer : a retrospective analysis of a tertiary institute experience

Radiotherapy combined with three weekly 100 mg/m2 of cisplatin is the accepted standard of care in head and neck squamous cell carcinoma. However, this regimen is associated with severe toxicities with devastating effects on patients. Alternative protocols like weekly 40 mg/m2 have been used in an attempt to reduce toxicities. The main objective of the present study is to identify the dose intensities and toxicities of weekly cisplatin in patients treated in a tertiary centre over a 12 month period. Included patients had squamous cell carcinoma arising in the oral cavity, oropharynx, larynx, or hypopharynx. Patients were excluded if they had nasopharyngeal squamous cell carcinoma, distant metastasis or if they had prior treatment for head and neck cancer excluding neck dissection. During the study period, 52 patients met the inclusion criteria and their data were retrospectively obtained from the patients' database of St James hospital, Dublin. The median age of the study cohort was 54 years (range 33-73). Of the patients, 40 (76.9 %) were male and 12 (20.1 %) were female. The primary tumour sites were as follows: oral cavity and oropharynx in 38 (73 %), larynx in 10 (19 %), and hypopharynx in 4 (8 %). In total, 33 (63.5 %) patients had stage IV disease, while 19 (36.5 %) had stage III disease. Treatment was definitive in 35 (67 %) patients and adjuvant in 17 (35 %). Full-dose radiotherapy was achieved in 50 (96 %) patients. Only 22 (42.3 %) patients completed the intended six cycles of chemotherapy. Cumulative dose of 200 mg/m2 or more was reached in 37 (71 %) patients. The acute adverse effects included grades 3 and 4 mucositis, which occurred in 22 (43.3 %) and 6 patients (12 %), respectively. Grade 3 and 4 neutropenia occurred in six (11.5 %) and three (5.7 %) patients, respectively. The only other haematological toxicity was grade 3 anaemia in 20 (38.4 %) patients. There was no grade 3 or 4 renal toxicity among the study cohort, although grade 2 was observed in six (11.5 %) patients. Death occurred in one patient due to neutropenic septicaemia. In conclusion, weekly cisplatin is associated with moderate to severe toxicities and might lead to suboptimal chemotherapy delivery. More prospective clinical studies are required to determine the optimal chemoradiation regimen in head and neck squamous cell carcinoma.

Acquired differential regulation of caspase-8 in cisplatin-resistant non-small-cell lung cancer

Failure to efficiently induce apoptosis contributes to cisplatin resistance in non-small-cell lung cancer (NSCLC). Although BCL-2-associated X protein (BAX) and BCL-2 antagonist killer (BAK) are critical regulators of the mitochondrial apoptosis pathway, their requirement has not been robustly established in relation to cisplatin. Here, we show that cisplatin can efficiently bypass mitochondrial apoptosis block caused by loss of BAX and BAK, via activation of the extrinsic death receptor pathway in some model cell lines. Apoptosis resistance following cisplatin can only be observed when both extrinsic and intrinsic pathways are blocked, consistent with redundancy between mitochondrial and death receptor pathways in cisplatin-induced apoptosis. In H460 NSCLC cells, caspase-8 cleavage was shown to be induced by cisplatin and is dependent on death receptor 4, death receptor 5, Fas-associated protein with death domain, acid sphingomyelinase and ceramide synthesis. In contrast, cisplatin-resistant cells fail to activate caspase-8 via this pathway despite conserving sensitivity to death ligand-driven activation. Accordingly, caspase-8 activation block acquired during cisplatin resistance, can be bypassed by death receptor agonism.

Meta-analysis of individual patient data from randomized trials of chemotherapy plus cetuximab as first-line treatment for advanced non-small cell lung cancer

OBJECTIVES: Four randomized phase II/III trials investigated the addition of cetuximab to platinum-based, first-line chemotherapy in patients with advanced non-small cell lung cancer (NSCLC). A meta-analysis was performed to examine the benefit/risk ratio for the addition of cetuximab to chemotherapy. MATERIALS AND METHODS: The meta-analysis included individual patient efficacy data from 2018 patients and individual patient safety data from 1970 patients comprising respectively the combined intention-to-treat and safety populations of the four trials. The effect of adding cetuximab to chemotherapy was measured by hazard ratios (HRs) obtained using a Cox proportional hazards model and odds ratios calculated by logistic regression. Survival rates at 1 year were calculated. All applied models were stratified by trial. Tests on heterogeneity of treatment effects across the trials and sensitivity analyses were performed for all endpoints. RESULTS: The meta-analysis demonstrated that the addition of cetuximab to chemotherapy significantly improved overall survival (HR 0.88, p=0.009, median 10.3 vs 9.4 months), progression-free survival (HR 0.90, p=0.045, median 4.7 vs 4.5 months) and response (odds ratio 1.46, p<0.001, overall response rate 32.2% vs 24.4%) compared with chemotherapy alone. The safety profile of chemotherapy plus cetuximab in the meta-analysis population was confirmed as manageable. Neither trials nor patient subgroups defined by key baseline characteristics showed significant heterogeneity for any endpoint. CONCLUSION: The addition of cetuximab to platinum-based, first-line chemotherapy for advanced NSCLC significantly improved outcome for all efficacy endpoints with an acceptable safety profile, indicating a favorable benefit/risk ratio.

Prognostic and therapeutic relevance of FLIP and procaspase-8 overexpression in non-small cell lung cancer

Non-small cell lung carcinoma remains by far the leading cause of cancer-related deaths worldwide. Overexpression of FLIP, which blocks the extrinsic apoptotic pathway by inhibiting caspase-8 activation, has been identified in various cancers. We investigated FLIP and procaspase-8 expression in NSCLC and the effect of HDAC inhibitors on FLIP expression, activation of caspase-8 and drug resistance in NSCLC and normal lung cell line models. Immunohistochemical analysis of cytoplasmic and nuclear FLIP and procaspase-8 protein expression was carried out using a novel digital pathology approach. Both FLIP and procaspase-8 were found to be significantly overexpressed in tumours, and importantly, high cytoplasmic expression of FLIP significantly correlated with shorter overall survival. Treatment with HDAC inhibitors targeting HDAC1-3 downregulated FLIP expression predominantly via post-transcriptional mechanisms, and this resulted in death receptor- and caspase-8-dependent apoptosis in NSCLC cells, but not normal lung cells. In addition, HDAC inhibitors synergized with TRAIL and cisplatin in NSCLC cells in a FLIP- and caspase-8-dependent manner. Thus, FLIP and procaspase-8 are overexpressed in NSCLC, and high cytoplasmic FLIP expression is indicative of poor prognosis. Targeting high FLIP expression using HDAC1-3 selective inhibitors such as entinostat to exploit high procaspase-8 expression in NSCLC has promising therapeutic potential, particularly when used in combination with TRAIL receptor-targeted agents.