Development of Indole Alkaloid-Type Dual Immune Checkpoint Inhibitors Against CTLA-4 and PD-L1 Based on Diversity-Enhanced Extracts.

Cancer immunotherapy involves the use of the immune system for cancer treatment. Recently, immune checkpoint-blocking antibodies have become integral for the treatment of some cancers. However, small molecules exhibit advantages over monoclonal antibody drugs, such as cell penetration, long half-life, and low manufacturing costs, and the possibility of oral administration. Thus, it is imperative to develop small-molecule immune checkpoint inhibitors. Previously, we have screened a library of synthetic indole-alkaloid-type compounds, which are produced by diversity-enhanced extracts of Japanese cornelian cherry, and reported that an unnatural pentacyclic compound inhibits CTLA-4 gene expression. In this study, immune checkpoint inhibitors with increased potency were developed by introducing substituents and conversion of functional groups based on the unnatural pentacyclic compound. The developed compounds suppressed not only CTLA-4 and PD-L1 gene expression but also protein expression on the cell surface. Their efficacy was not as potent as that of the existing small-molecule immune checkpoint inhibitors, but, to the best of our knowledge, the developed compounds are the first reported dual small-molecule inhibitors of CTLA-4 and PD-L1.

Downregulation of PD-L1 via amide analogues of brefelamide: Alternatives to antibody-based cancer immunotherapy.

The therapeutic blockade of immune checkpoint has emerged as an effective treatment option for a broad range of tumors. However, the objective tumor response is still limited to a small number of cases and tumor types. The full utility of monoclonal antibody (mAb)-based treatment is hindered by several inherent limitations. Thus, there is an urgent requirement to explore alternative modalities targeting the same pathways. In the present study, two amide analogues of brefelamide, TPFS-201 and TPFS-202, were identified as small molecular immune checkpoint inhibitors, as they downregulated PD-L1 expression in tumor cells. PD-L1 was suppressed in cancer cells treated with TPFD compounds at both mRNA and protein levels, as detected by reverse transcription quantitative PCR and flow cytometric analysis, respectively. Reporter assays using a PD-L1 promoter luciferase construct confirmed the transcriptional inhibition of PD-L1 by TPFS compunds. TPFS compound-mediated PD-L1 downregulation in cancer cells consequently restored T cell activity, as identified by the reduction of apoptosis and an increase in interleukin-2 promoter activity in Jurkat T cells, which were co-cultured with TPFS compound-treated A549 cells. TPFS compound-mediated PD-L1 inhibition was partially abolished by the disruption of the putative transcriptional co-activator with PDZ (TAZ)/TEA domain (TEAD)-binding motif in the PD-L1 promoter. The inhibitory effect of TPFS compounds on PD-L1 was markedly inhibited in mouse cell lines, which is consistent with previous research demonstrating that PD-L1 regulation by TAZ is not conserved in mice due to distinct promoter sequences flanking the TAZ/TEAD-binding motif. Together, the data of the current study indicated the potential utility of the brefelamide amide analogues as small molecule immune checkpoint inhibitors, thereby providing therapeutic alternatives, which could be used as monotherapy or in combination with mAbs-based treatment.

Long-term predictive factors of the morphology based outcome in bare platinum coiled intracranial aneurysms: Evaluation by pre- and post-contrast 3D time-of-flight MR angiography.

PURPOSE: Our aim was to identify long-term predictive factors of the morphology-based outcome (MBO) of bare platinum coiled intracranial aneurysms. MATERIALS AND METHODS: A retrospective analysis of 96 bare platinum coiled intracranial aneurysms followed up from 1997 to 2016 using pre- and post-contrast 3D time-of-flight MR angiography (MRA) was performed. Logistic regression analysis was used to identify factors associated with a positive history of surrounding coil mass enhancement (SCME) and poor MBO. Spearman's rank correlation test was used to analyze the relationship between the initial angiographic result (IAR) class, sequential change of the SCME category, and MBO grade. RESULTS: Factors independently associated with poor MBO were incomplete IAR (OR=14.94, 95%CI: 2.46, 289.21, P=0.002) and a history of SCME (OR=4.13, 95% CI: 1.05, 18.65, P=0.043). The MBO grade strongly correlated with the IAR class (correlation coefficient [r]=0.84, P<0.0001). MBO grade correlated with sequential change of the SCME category (r=0.56, P<0.0001). The sequential change of the SCME category correlated with IAR class (r=0.53, P<0.0001). CONCLUSION: Although IAR and its class were strong long-term predictive factors of MBO, a history of SCME and upgrading of sequential change of SCME category were also long-term predictive factors of the MBO of bare platinum coiled intracranial aneurysms.

Monoterpene Indole Alkaloid-Like Compounds Based on Diversity-Enhanced Extracts of Iridoid-Containing Plants and Their Immune Checkpoint Inhibitory Activity.

A library of iridoid-conjugated indole alkaloid-like compounds was constructed from diversity-enhanced extracts, which constitutes an approach for increasing the chemical diversity of natural-product-like compounds by combining natural product chemistry and diversity-oriented synthesis. Pharmacological screening of the library revealed a seed compound that can be used for the development of small molecular immune checkpoint inhibitors.

Identification of brefelamide as a novel inhibitor of osteopontin that suppresses invasion of A549 lung cancer cells.

The contribution of aberrant osteopontin (OPN) expression to tumor progression and metastasis has been documented in a wide spectrum of malignancies, and targeted inhibition of OPN has therefore emerged as an attractive strategy for cancer therapy. Transcription of OPN is regulated by various transcription factors, and our recently published study demonstrated that downregulation of OPN is an important event in the TGF­ß cytostatic program. We report here that brefelamide exerts an inhibitory effect on OPN expression and function in A549 human lung carcinoma cells. The promoter, RNA, and protein levels of OPN were decreased in brefelamide­treated A549 cells, which was accompanied by reduced invasive ability in vitro. OPN inhibition by brefelamide was largely abrogated by disruption of a putative TGF­ß inhibitory element in the OPN promoter. Treatment with brefelamide induced Smad4 expression, and knockdown of Smad4 by RNA interference partially diminished the inhibitory effect of brefelamide on OPN. These results indicate that brefelamide inhibited OPN­mediated cell invasion through restoration of the OPN repression by TGF­ß/Smad signaling. Together with the reported antiproliferative property, our findings suggest that brefelamide might serve as a potential candidate for the development of a new antitumor and antimetastatic agent.

Down-regulation of osteopontin mediates a novel mechanism underlying the cytostatic activity of TGF-ß.

PURPOSE: Loss of a cytostatic response to TGF-ß has been implicated in multiple hyper-proliferative disorders, including cancer. Although several key genes involved in the cytostatic activity of TGF-ß have in the past been identified, its exact mode of action is yet to be elucidated. A comprehensive understanding of the mechanisms underlying the cytostatic activity of TGF-ß may open up new avenues for the development of therapeutic strategies. METHODS: Quantitative real-time RT-PCR was used to assess osteopontin (OPN) gene expression in human hepatoma-derived Huh-7 and lung adenocarcinoma-derived A549 cells. Reporter assays using an OPN promoter-luciferase construct and its mutated counterparts were performed to assess its transcriptional activity. Binding of Smad4 to the OPN gene promoter was investigated using chromatin immunoprecipitation (CHIP). The putative role of Smad4 in OPN gene expression down-regulation was also assessed using a shRNA-mediated knockdown strategy. The anti-proliferative effect of TGF-ß on different cancer-derived cell lines was determined using the cell proliferation reagent WST-1. RESULTS: We found that the OPN expression levels dose-dependently decreased in TGF-ß-treated Huh-7 and A549 cells. Our reporter assays indicated that this TGF-ß-induced repression occurred at the transcriptional level, and could largely be abrogated by disruption of an element (TIE2) similar to the TGF-ß inhibitory element found in other TGF-ß-repressed genes. Our CHIP assay revealed that the Smad protein complex specifically binds to the OPN gene promoter, and that the TGF-ß-mediated inhibition of OPN was lost upon shRNA-mediated knockdown of Smad4. Moreover, we found that the deregulation of OPN gene expression by TGF-ß occurred concomitantly with loss of the TGF-ß anti-proliferative response, whereas a neutralizing anti-OPN antibody partially restored this response. CONCLUSIONS: Our results indicate that the OPN gene is a direct target of Smad-mediated TGF-ß signaling, implying that OPN expression inhibition serves as a novel mechanism underlying the cytostatic activity of TGF-ß.

[New findings and concepts on production and absorption of cerebrospinal fluid: reconsiderations and revisions of an unquestioningly accepted dogma of 100 years].

It has been recently pointed out that there are fallacies in the bulk flow theory, a dogmatic idea of cerebrospinal fluid (CSF) physiology, which assumes that CSF is produced by the choroid plexus and circulates unidirectionally from the ventricles to the subarachnoid space to be absorbed by the arachnoid villi in the parietal region. Therefore, CSF physiology now needs to be reconsidered. Since there is free exchange of water between the CSF and the brain interstitial fluid (ISF), when considering the dynamics of water in the brain, we should regard these two fluid compartments collectively as brain extracellular fluid. In this review, I discuss the contentious issues in the traditional theory of CSF physiology, and then introduce new understandings of the production and absorption of the brain extracellular fluid, such as the capillary theory asserting that brain ISF is produced and absorbed mainly at the cerebral capillaries, the brain lymphatic drainage pathway, and the non-existence of unidirectional CSF circulation.

Pathological examination of a ruptured fusiform aneurysm of the middle cerebral artery.

BACKGROUND: Little is known about the pathogenesis and clinical course of fusiform compared with saccular aneurysms. The case of a ruptured fusiform aneurysm accompanied by dissection at the M2 portion of the middle cerebral artery (MCA) is reported, along with pathological findings. CASE DESCRIPTION: A 41-year-old female presenting with subarachnoid hemorrhage was revealed to have a ruptured fusiform aneurysm at the M2 portion of the right MCA on angiography. She was treated with superficial temporal artery-MCA anastomosis and trapping of the aneurysm. The aneurysm consisted of a whitish fusiform dilatation with a thickened wall of the MCA and two red protrusions on it. Pathological examinations revealed disruption and fragmentation of the internal elastic lamina and intimal thickening in the fusiform lesion. There were two aneurysmal protrusions on the main fusiform dilatation. In one protruded lesion, a dissection of the intima was observed. CONCLUSION: We propose that a dissection and saccular aneurysm additionally developed on the wall of a preexisting segmental ectasia of the MCA in our case. In this report, we discuss the etiology of fusiform aneurysms of the MCA.

[Progress in diagnosis of and therapy for idiopathic normal-pressure hydrocephalus--Lymphatic drainage of CSF and ISF from the brain: recent concept and hypothesis].

Cerebrospinal fluid (CSF) drains via the cribriform plate and nasal mucosa to cervical lymph nodes. There are no conventional lymphatics in the brain but physiological studies have revealed a substantial and immunologically significant lymphatic drainage from brain to cervical lymph nodes. Interstitial fluid (ISF) and solutes from brain parenchyma drain along capillary and perivascular space of artery, and path through the skull base, then reach to the cervical lymph nodes. CSF and ISF appear to drain by separate routes from the brain, especially in humans. However, there are interrelationships between the two fluid compartments that become more significant when drainage of CSF or ISF is impaired by disease processes. Vessel pulsations appear to be the driving force for the perivascular lymphatic drainage along artery walls, and as vessels stiffen with age, amyloid peptides (Aß) deposit in the drainage pathways as cerebral amyloid angiopathy (CAA). Blockage of lymphatic drainage of ISF and solutes from the brain by CAA may result in loss of homeostasis of the neuronal environment that may contribute to neuronal malfunction and dementia. Such failure of perivascular drainage may associated with the pathoetiology of Alzheimer's disease, cerebral small artery disease and idiopathic normal pressure hydrocephalus (iNPH).

Spindle cell oncocytoma of the adenohypophysis with marked hypervascularity. Case report.

A 68-year-old male presented with a very rare case of spindle cell oncocytoma (SCO), a recently identified very rare neoplasm of the anterior pituitary, manifesting as panhypopituitarism and visual field defect. The pituitary tumor with suprasellar extension was only partially resected via transsphenoidal surgery because of the tumor consistency and bleeding. Histological diagnosis was consistent with schwannoma. The tumor regrew and angiography revealed hypervascularity, so a transcranial approach was employed for the re-operation which only achieved partial resection because of intraoperative extensive bleeding. The tumor cells showed similar histological and immunohistochemical profiles to the previous specimen, but electron microscopy demonstrated that cytoplasm abundantly filled with mitochondria. The final diagnosis of SCO was established and the patient received postoperative conventional radiation therapy of 50 Gy. Only 15 cases of SCO have been reported, and the diagnosis was mistaken in many cases as schwannoma, oncocytic pituitary adenoma, or craniopharyngioma, and multiple surgeries followed by radiation therapy were required.

Central nervous system primitive neuroectodermal tumor of spinal cord developing 20 years after curative treatment of pineal tumor.

A 65-year-old male who had previously received curative treatment for a pineal tumor presented with an extremely rare case of primary central nervous system (CNS) primitive neuroectodermal tumor (PNET) of the spinal cord manifesting as progressive tetraparesis. Although the histology was not verified, highly radiosensitive tumor was suspected because of the benign clinical course for over 20 years after only radiation therapy. Magnetic resonance imaging demonstrated an intramedullary tumor extending from C5 to T1. He underwent partial resection and histological examination revealed blue tumor with undifferentiated small round cells. Immunohistochemically, c-kit was negative but CD99 was strongly and diffusely positive. Therefore, rearrangement of the Ewing sarcoma gene was examined to determine the presence of peripheral type of PNET. The results were negative and systemic workup revealed no other disease. These findings led to the diagnosis of primary intramedullary CNS PNET of the spinal cord, and suggested that the spinal cord tumor occurred independently of the prior pineal disease. The residual tumor was controlled by postoperative local radiation therapy.

Identification of CD44 as a downstream target of noncanonical NF-κB pathway activated by human T-cell leukemia virus type 1-encoded Tax protein.

Our previous study showed Human T-cell leukemia virus type 1 Tax induces osteopontin (OPN) expression by transactivating its promoter. As an extension, we investigated here the possible influence of Tax on CD44, an important receptor for OPN. Co-expression of Tax, but not its NF-κB-defective mutant, significantly increased the reporter gene expression directed by CD44 promoter. Tax-mediated CD44 activation was largely diminished by disrupting an element similar to the noncanonical κß site found in other IKKα target genes, and further, co-transfection of RelB siRNA abolished CD44 induction by Tax, suggesting an involvement of noncanonical NF-κB pathway in Tax-mediated transactivation. Consistently, chromatin immunoprecipitation revealed a specific interaction of CD44 promoter with RelB-containing complex. Together, these results indicate that D44 gene is one of the downstream target genes of aberrantly activated noncanonical NF-κB signaling by Tax, providing an additional line of evidence explaining how Tax-induced NF-κB signaling is integrated into a fate-determining cellular program.

Evaluation of synthetic cell-penetrating peptides, Pro-rich peptide and octaargine derivatives, as adenovirus vector carrier.

Two cell-penetrating peptides, a Pro-rich peptide derivative, acetyl-(Val-Arg-Leu-Pro-Pro-Pro)(3)-Gly-Cys amide, and an octaarginine derivative, acetyl-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Cys amide, were prepared by the solid phase method. Each peptide was coupled to the heterobifunctional cross-linking reagent, 6-maleimidohexanoic acid N-hydroxysuccinimide ester, and then conjugated to the Adenovirus vector containing luciferase gene. Peptide-modified Ad, as compared with wild-type Ad, exhibited excellent luciferase activity in B16BL6 cells.

Transduction of adenovirus vectors modified with cell-penetrating peptides.

Adenovirus vectors (Advs) are widely used for basic and clinical research because of their high transduction efficiency. However, they are poorly transduced into cells lacking the primary adenovirus receptor, the coxsackievirus and adenovirus receptor (CAR). Here, we generated Adv conjugated with cell-penetrating peptides (CPPs), such as Tat, octaarginine (R8) or proline-rich (Pro) peptides, and compared the transduction properties of these constructs. We constructed the Advs conjugated to the CPPs (CPP-Adv) by chemical conjugation. The CPP-conjugated Advs created with optimal modification ratios led to gene expression 1-2log orders higher than unmodified Adv in CAR-negative cells. Tat-Adv and R8-Adv were taken up into the cells mainly through macropinocytosis, independently of the CAR. In addition, the cellular uptake of Tat-Adv was highly dependent on heparan sulfate on the cell surface, whereas that of R8-Adv was dependent on chondroitin sulfate B. These data suggest that the use of CPP-Advs with different cellular uptake pathways might create new methods for the delivery of Adv. The results obtained in this research encourage the use of CPP-peptide-modified Advs as an attractive tool for transducing cells and as useful platform vectors for gene therapy and basic research.

Tat conjugation of adenovirus vector broadens tropism and enhances transduction efficiency.

AIMS: Adenovirus vectors (Advs) have been very useful for basic research and clinical gene therapy because they propagate to high titers and efficiently transduce cells and tissues regardless of the mitotic status. However, poor transduction of cells that lack the coxsackievirus and adenovirus receptor (CAR), the primary receptor for Advs, has limited Adv application. In this study, we attempted to generate novel Tat-Advs (Advs conjugated with the HIV Tat-derived peptide, a protein-transduction domain (PTD)) to broaden Adv tropism and enhance transduction efficiency. MAIN METHODS: We constructed Tat-Advs by chemically conjugating Tat peptide to the surface-exposed lysine residues on Advs. We compared the gene transfer activity of Tat-Advs with that of unmodified Advs by measuring the luciferase expression in several types of cell lines. KEY FINDINGS: Tat-Advs showed gene expression 1 to 3 log orders higher than unmodified Advs in CAR-negative adherent cells and blood cells, which are refractory to conventional Advs. The inhibition of Tat-Adv-mediated gene expression by heparin and macropinocytosis inhibitor confirms that binding of Tat-Adv to cellular HSPGs and macropinocytosis are essential for efficient CAR-independent transduction. We also demonstrated that Adv modified with another PTD (R8) had the same high transduction efficiency as Tat-Adv. SIGNIFICANCE: These data suggest that Tat-Advs are important tools for transducing cells and will be useful as platform vectors for gene therapy.

Unusual angiographic changes in an internal carotid artery pseudoaneurysm after infection in the deep neck space.

A 48-year-old man presented with a pseudoaneurysm at the cervical portion of the left internal carotid artery (ICA) secondary to infection in the deep neck space. Magnetic resonance (MR) imaging demonstrated enhancement of the wall of the ICA and a pseudoaneurysm, considered to be sequelae of infection spread. ICA occlusion occurred on the next day resulting in sudden onset of right hemiparesis and motor aphasia. The ICA pseudoaneurysm shrank gradually and his neurological deficits improved with conservative therapy. One month later, he presented with aneurysm regrowth. The common carotid artery was occluded with Guglielmi detachable coils to block arterial flow into the pseudoaneurysm. There were no neurological complications. Marked enhancement of the ICA wall on computed tomography and MR imaging may indicate the possibility of vascular complications such as rupture, pseudoaneurysm development, or ICA occlusion, and consequent neurological deficits. ICA occlusion caused by spread of infection in the deep neck space may cause accelerated coagulopathy due to ICA wall inflammation.

Preparation of a Tat-related transporter peptide for carrying the adenovirus vector into cells.

We synthesized a Tat-related peptide acetyl-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Gly-Cys amide, Ac-Tat(48-60)-Gly-Cys-NH(2), having high intracellular permeability, and conjugated this peptide to adenovirus vector to enhance gene transfer efficiency of adenovirus vector into cells. The peptide was prepared by the solid-phase peptide synthesis method and a bifunctional crosslinker 6-maleimidohexanoic acid N-hydroxysuccinimide ester was used to conjugate the peptide to adenovirus vector containing luciferase gene. The novel conjugate of adenovirus vector and Ac-Tat(48-60)-Gly-Cys-NH(2) peptide exhibited excellent gene transfer efficacy in B16BL6 cells.

Solid-phase peptide synthesis using nanoparticulate amino acids in water.

Solid-phase peptide synthesis has many advantages compared with solution peptide synthesis. However, this procedure requires a large amount of organic solvents. Since safe organic solvent waste disposal is an important environmental problem, a technology based on coupling reaction of suspended nanoparticle reactants in water was studied. Fmoc-amino acids are used widely, but most of them show low solubility in water. We prepared well-dispersible Fmoc-amino acid nanoparticles in water by pulverization using a planetary ball mill in the presence of poly(ethylene glycol). Leu-enkephalin amide was prepared successfully using the nanoparticulate Fmoc-amino acid on a poly(ethylene glycol)-grafted Rink amide resin in water.

Studies on heterobifunctional cross-linking reagents, 6-maleimidohexanoic acid active esters.

6-maleimidohexanoic acid N-hydroxysuccinimide ester has been used widely for preparation of enzyme immunoconjugates as a unique heterobifunctional cross-linking reagent. Its heterobifunctional reactivity is good, but its ester portion hydrolyzes easily in the presence of water. Several 6-maleimidohexanoic acid active esters (6-maleimidohexanoic acid 4-nitrophenyl ester, 6-maleimidohexanoic acid N-hydroxy-5-norbornene-endo-2,3-dicarboximide ester, and 6-maleimidohexanoic acid pentafluorophenyl ester) were prepared and their reactivity and stability in an aqueous media were tested. Of the synthetic esters, the pentafluorophenyl ester exhibited the highest reactivity and stability in aqueous media.

Usefulness of grasping a guiding catheter with a loop-snare wire for stent placement into the vertebrobasilar artery.

In cases in which the subclavian artery is severely tortuous or branches with an extremely angulated origin, stent placement in the vertebrobasilar artery on the approach from the femoral artery is often technically difficult. We report two cases in which a stent placement procedure for the vertebrobasilar artery was successfully performed by grasping a guiding catheter with a loop-snare wire. This technique is useful for tortuous arteries or arteries branching with an extremely angulated origin.