Involvement of autophagy in the maintenance of rat intervertebral disc homeostasis: an in-vitro and in-vivo RNA interference study of Atg5.

OBJECTIVE: In the largest avascular low-nutrient intervertebral disc, resident cells would utilize autophagy, a stress-response survival mechanism by self-digestion and recycling wastes. Our goal was to elucidate the involvement of autophagy in disc homeostasis through RNA interference of autophagy-related gene 5 (Atg5). DESIGN: In vitro, small interfering RNAs (siRNAs) targeting autophagy-essential Atg5 were transfected into rat disc cells. Cell viability with levels of autophagy including Atg5 expression, apoptosis, and senescence was assessed under serum starvation and/or pro-inflammatory interleukin-1 beta (IL-1ß) stimulation. In vivo, time-course autophagic flux was monitored following Alexa Fluor® 555-labeled Atg5-siRNA injection into rat tail discs. Furthermore, 24-h temporary static compression-induced disruption of Atg5 siRNA-injected discs was observed by radiography, histomorphology, and immunofluorescence. RESULTS: In disc cells, three different Atg5 siRNAs consistently suppressed autophagy with Atg5 protein knockdown (mean 44.4% [95% confidence interval: -51.7, -37.1], 51.5% [-80.5, -22.5], 62.3% [-96.6, -28.2]). Then, Atg5 knockdown reduced cell viability through apoptosis and senescence not in serum-supplemented medium (93.6% [-0.8, 21.4]) but in serum-deprived medium (66.4% [-29.8, -8.6]) further with IL-1ß (44.5% [-36.9, -23.5]). In disc tissues, immunofluorescence detected intradiscal signals for the labeled siRNA even at 56-d post-injection. Immunoblotting found 56-d autophagy suppression with prolonged Atg5 knockdown (33.2% [-52.8, -5.3]). With compression, Atg5 siRNA-injected discs presented radiographic height loss ([-43.9, -0.8]), histological damage ([-5.5, -0.2]), and immunofluorescent apoptosis ([2.2, 22.2]) and senescence ([4.1, 19.9]) induction compared to control siRNA-injected discs at 56 d. CONCLUSIONS: This loss-of-function study suggests Atg5-dependent autophagy-mediated anti-apoptosis and anti-senescence. Autophagy could be a molecular therapeutic target for degenerative disc disease.

NF-kB decoy oligodeoxynucleotide preserves disc height in a rabbit anular-puncture model and reduces pain induction in a rat xenograft-radiculopathy model.

While it is known that the degenerated intervertebral disc (IVD) is one of the primary reasons for low-back pain and subsequent need for medical care, there are currently no established effective methods for direct treatment. Nuclear factor-κB (NF-κB) is a transcription factor that regulates various genes' expression, among which are inflammatory cytokines, in many tissues including the IVD. NF-κB decoy is an oligodeoxynucleotide containing the NF-κB binding site that entraps NF-κB subunits, resulting in suppression of NF-κB activity. In the present preclinical study, NF-κB decoy was injected into degenerated IVDs using the rabbit anular-puncture model. In terms of distribution, NF-κB decoy persisted in the IVDs up to at least 4 weeks after injection. The remaining amount of NF-κB decoy indicated that it fit a double-exponential-decay equation. Investigation of puncture-caused degeneration of IVDs showed that NF-κB decoy injection recovered, dose-dependently, the reduced disc height that was associated with reparative cell cloning and morphological changes, as assessed through histology. Gene expression, by quantitative real-time polymerase chain reaction (qRT-PCR), showed that NF-κB decoy attenuated inflammatory gene expression, such as that of interleukin-1 and tumor necrosis factor-α, in rabbit degenerated IVDs. NF-κB decoy also reduced the pain response as seen using the "pain sensor" nude rat xenograft-radiculopathy model. This is the first report demonstrating that NF-κB decoy suppresses the inflammatory response in degenerated IVDs and restores IVD disc height loss. Therefore, the intradiscal injection of NF-κB decoy may have the potential as an effective therapeutic strategy for discogenic pain associated with degenerated IVDs.

Respiratory Sarcopenia and Sarcopenic Respiratory Disability: Concepts, Diagnosis, and Treatment.

The condition of muscle fiber atrophy and weakness that occurs in respiratory muscles along with systemic skeletal muscle with age is known as respiratory sarcopenia. The Japanese Working Group of Respiratory Sarcopenia of the Japanese Association of Rehabilitation Nutrition narratively reviews these areas, and proposes the concept and diagnostic criteria. We have defined respiratory sarcopenia as "whole-body sarcopenia and low respiratory muscle mass followed by low respiratory muscle strength and/or low respiratory function." Respiratory sarcopenia can be caused by various factors such as aging, decreased activity, undernutrition, disease, cachexia, and iatrogenic causes. We have also created an algorithm for diagnosing respiratory sarcopenia. Respiratory function decreases with age in healthy older people, along with low respiratory muscle mass and strength. We have created a new term, "Presbypnea," meaning a decline in respiratory function with aging. Minor functional respiratory disability due to aging, such as that indicated by a modified Medical Research Council level 1 (troubled by shortness of breath when hurrying or walking straight up hill), is an indicator of presbypnea. We also define sarcopenic respiratory disability as "a disability with deteriorated respiratory function that results from respiratory sarcopenia." Sarcopenic respiratory disability is diagnosed if respiratory sarcopenia is present with functional disability. Cases of respiratory sarcopenia without functional disability are diagnosed as "at risk of sarcopenic respiratory disability." Functional disability is defined as a modified Medical Research Council grade of 2 or more. Rehabilitation nutrition, treatment that combines rehabilitation and nutritional management, may be adequate to prevent and treat respiratory sarcopenia and sarcopenic respiratory disability.

Expressions of conventional vitellogenin and vitellogenin-like A in worker brains are associated with a nursing task in a ponerine ant.

In eusocial insect colonies, non-reproductive workers often perform different tasks. Tasks of an individual worker are shifted depending on various factors, e.g., age and colony demography. Although a vitellogenin (Vg) gene play regulatory roles in both reproductive and non-reproductive division of labours in a honeybee, it has been shown that the insect Vg underwent multiple gene duplications and sub-functionalisation, especially in apical ant lineages. The regulatory roles of duplicated Vgs were suggested to change evolutionarily among ants, whereas such roles in phylogenetically basal ants remain unclear. Here, we examined the expression patterns of conventional Vg (CVg), Vg-like A, Vg-like B and Vg-like C, as well as Vg receptor, during the task shift in an age-dependent manner and under experimental manipulation of colony demography in a primitive ant Diacamma sp. Expressions of CVg and Vg-like A in a brain were associated with a nursing task. It is suggested that associations of brain expressions of these Vgs with worker tasks were acquired in the basal ant lineage, and that such Vg functions could have sub-functionalised in the derived ant lineage.

Amyloid Signature Proteins in Feline Amyloidosis.

In human amyloidoses, amyloid signature proteins (ASPs), such as serum amyloid P component (SAP) and apolipoprotein E (ApoE), are deposited in tissues together with amyloid fibrils and are implicated in the pathogenesis of amyloidosis. Few reports describe ASPs in animals. In this study, we examined feline amyloidosis and performed immunohistochemical and proteomic analyses of SAP, ApoE, apolipoprotein A-I (ApoAI) and apolipoprotein A-IV (ApoAIV). Ten cases of systemic amyloidosis, three cases of amyloid-producing odontogenic tumour and three cases of islet amyloidosis were used for immunohistochemistry (IHC) and/or proteomic analyses. IHC showed that ApoE was present in amyloid deposits in all samples. ApoAI and ApoAIV differed in the degree of co-deposition with amyloid depending on the type of amyloid and the affected organ. SAP was negative in all amyloid deposits. Proteomic analysis showed that ApoE was present in all samples, but ApoAI and ApoAIV were detected only in some samples and SAP was not detected in any samples. The observation that ApoE was detected in all types of amyloid suggests the involvement of ApoE in the development of feline amyloidosis. ASPs in feline amyloidosis are significantly different from those in human amyloidosis, suggesting that the involvement of ASPs in the pathological condition differs between animal species.

Pharmacological inhibition of mTORC1 but not mTORC2 protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism through Akt and autophagy induction.

OBJECTIVE: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth. We hypothesized that mTOR is influential in the intervertebral disc-largest avascular, low-nutrient organ. Our objective was to identify the optimal mTOR inhibitor for treating human degenerative disc disease. DESIGN: mTOR complex 1 (mTORC1) regulates p70/ribosomal S6 kinase (p70/S6K), negatively regulates autophagy, and is controlled by Akt. Akt is controlled by phosphatidylinositol 3-kinase (PI3K) and mTOR complex 2 (mTORC2). mTORC1 inhibitors-rapamycin, temsirolimus, everolimus, and curcumin, mTORC1&mTORC2 inhibitor-INK-128, PI3K&mTOR inhibitor-NVP-BEZ235, and Akt inhibitor-MK-2206-were applied to human disc nucleus pulposus (NP) cells. mTOR signaling, autophagy, apoptosis, senescence, and matrix metabolism were evaluated. RESULTS: mTORC1 inhibitors decreased p70/S6K but increased Akt phosphorylation, promoted autophagy with light chain 3 (LC3)-II increases and p62/sequestosome 1 (p62/SQSTM1) decreases, and suppressed pro-inflammatory interleukin-1 beta (IL-1ß)-induced apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity (versus rapamycin, 95% confidence interval (CI) -0.431 to -0.194; temsirolimus, 95% CI -0.529 to -0.292; everolimus, 95% CI -0.477 to -0.241; curcumin, 95% CI -0.248 to -0.011) and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage, senescent senescence-associated beta-galactosidase (SA-ß-gal) positivity (versus rapamycin, 95% CI -0.437 to -0.230; temsirolimus, 95% CI -0.534 to -0.327; everolimus, 95% CI -0.485 to -0.278; curcumin, 95% CI -0.210 to -0.003) and p16/INK4A expression, and catabolic matrix metalloproteinase (MMP) release and activation. Meanwhile, dual mTOR inhibitors decreased p70/S6K and Akt phosphorylation without enhanced autophagy and suppressed apoptosis, senescence, and matrix catabolism. MK-2206 counteracted protective effects of temsirolimus. Additional disc-tissue analysis found relevance of mTOR signaling to degeneration grades. CONCLUSION: mTORC1 inhibitors-notably temsirolimus with an improved water solubility-but not dual mTOR inhibitors protect against inflammation-induced apoptosis, senescence, and matrix catabolism in human disc cells, which depends on Akt and autophagy induction.

Selective interference of mTORC1/RAPTOR protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism with Akt and autophagy induction.

OBJECTIVE: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth and protein synthesis. We hypothesized that mTOR is essential for the intervertebral disc, the largest avascular, low-nutrient organ. Our objective was to elucidate roles of mTOR signaling in human disc cells. DESIGN: The mTOR exists in two complexes: mTORC1 containing the regulatory-associated protein of mTOR (RAPTOR) and mTORC2 containing the rapamycin-insensitive companion of mTOR (RICTOR). To analyze their functions in human disc nucleus pulposus cells, RNA interference (RNAi) of mTOR targeting mTORC1 and mTORC2, RAPTOR targeting mTORC1, or RICTOR targeting mTORC2 or rapamycin, a pharmacological mTORC1 inhibitor, was applied. First, mTOR signaling including Akt, p70/ribosomal S6 kinase (p70/S6K), and autophagy were assessed. Then, apoptosis, senescence, and matrix metabolism were evaluated under pro-inflammatory interleukin-1 beta (IL-1ß) stimulation. RESULTS: Western blotting showed significant decreases in specific proteins by each RNAi (all P < 0.0001). In mTOR signaling, RNAi of mTOR and RICTOR decreased p70/S6K and Akt phosphorylation, whereas RAPTOR RNAi decreased p70/S6K but increased Akt phosphorylation. All RNAi treatments increased light chain 3 (LC3)-II and decreased p62/sequestosome 1 (p62/SQSTM1), indicating enhanced autophagy. In apoptosis, IL-1ß-induced terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage decreased by RAPTOR RNAi. In senescence, IL-1ß-induced senescence-associated beta-galactosidase (SA-ß-gal)-positive cells and p16/INK4A expression also decreased by RAPTOR RNAi. In matrix metabolism, RAPTOR RNAi reduced IL-1ß-induced catabolic matrix metalloproteinase (MMP) release and activation and up-regulated anabolic gene expression. These findings were all consistent with rapamycin administration. Additional disc-tissue analysis detected expression and phosphorylation of mTOR-signaling molecules in varying ages. CONCLUSION: Selective interference of mTORC1/RAPTOR protects against inflammation-induced apoptosis, senescence, and matrix catabolism possibly through Akt and autophagy induction in human disc cells.

The Japanese Postmarketing Adverse Event Relief System: A Confluence of Regulatory Science, the Legal System, and Clinical Pharmacology.

The Japanese Postmarketing Relief System provides for compensation to patients with adverse reactions, based on the acknowledgment that unpredicted adverse events occur inevitably once a drug is marketed. The system also provides new knowledge about the benefit-risk profile of a drug that may be incorporated into product labeling. The system relies on causality assessments that are based on sound clinical pharmacology principles. The system may serve as a model for other countries' healthcare systems.

In-situ straining and time-resolved electron tomography data acquisition in a transmission electron microscope.

This paper reports the preliminary results of a new in-situ three-dimensional (3D) imaging system for observing plastic deformation behavior in a transmission electron microscope (TEM) as a directly relevant development of the recently reported straining-and-tomography holder [Sato K et al. (2015) Development of a novel straining holder for transmission electron microscopy compatible with single tilt-axis electron tomography. Microsc. 64: 369-375]. We designed an integrated system using the holder and newly developed straining and image-acquisition software and then developed an experimental procedure for in-situ straining and time-resolved electron tomography (ET) data acquisition. The software for image acquisition and 3D visualization was developed based on the commercially available ET software TEMographyTM. We achieved time-resolved 3D visualization of nanometer-scale plastic deformation behavior in a Pb-Sn alloy sample, thus demonstrating the capability of this system for potential applications in materials science.

Effect of Antihypertensive Drugs on Uric Acid Metabolism in Patients with Hypertension: Cross-Sectional Cohort Study.

Background: Hypertension is a common complication in patients with gout and/or hyperuricemia. Besides, hyperuricemia is a risk factor of gout as well as ischemic heart disease in hypertensive patients. Moreover, the risk of gout is modified by antihypertensive drugs. However, it remains unclear how antihypertensive agents affect uric acid metabolism. Purpose: In the present study, we investigated the uric acid metabolism in treated hypertensive patients to find out whether any of them would influence serum levels of uric acid. Patients and methods: 751 hypertensive patients (313 men and 438 women) under antihypertensive treatment were selected. Blood pressure (BP), serum uric acid (SUA) and serum creatinine (Scr) were measured and evaluated statistically. Results: In patients treated with diuretics, beta-blockers and/or alpha-1 blockers SUA levels were significantly higher than in patients who were not taking these drugs. Besides, the estimated glomerular filtration rate (eGFR) in patients treated with diuretics, beta-blockers and/or alpha-1 blockers was negatively correlated with SUA level. There were gender differences in the effects of beta-blockers and alpha-1 blockers. Multiple regression analysis indicated that both diuretics and beta-blockers significantly contributed to hyperuricemia in patients with medication for hypertension. Conclusion: Diuretics, beta-blockers and alpha-1 blockers reduced glomerular filtration rate and raised SUA levels. Calcium channel blockers, ACE inhibitors and angiotensin receptor blockers, including losartan, did not increase SUA levels.

Optimum rolling ratio for obtaining {001}<110> recrystallization texture in Ti-Nb-Al biomedical shape memory alloy.

The rolling rate (r) dependence of textures was investigated in the Ti-26Nb-3Al (mol%) alloy to reveal the conditions required to form the {001}<110> recrystallization texture, which is a desirable orientation for the ß-titanium shape memory alloy. {001}<110> was the dominant cold-rolling texture when r=90% and it was transferred to the recrystallization texture without forming {112}<110>, which is detrimental for the isotropic mechanical properties of the rolled sheet. A further increase in r resulted in the formation of {112}<110> in both rolling and recrystallization textures. Therefore, r should be controlled to form only the {001}<110> rolling texture, because the {112}<110> texture can overwhelm the {001}<110> texture during recrystallization.

Effects of Uric Acid on the NO Production of HUVECs and its Restoration by Urate Lowering Agents.

BACKGROUND: Although urate impaired the endothelial function, its underlying mechanism remains unknown. We hypothesized that urate impaired nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs) via activation of uric acid transporters (UATs). PURPOSE AND METHOD: In the present study, we studied effects of urate on NO production and eNOS protein expression in HUVEC cells in the presence and absence of urate lowering agents using molecular biological and biochemical assays. RESULTS: HUVECs expressed the 4 kinds of UATs, URATv1, ABCG2, MRP4 and MCT9. Exposure to urate at 7 mg/dl for 24 h significantly reduced production of NO. Pretreatment with benzbromarone, losartan or irbesartan normalized NO production. The same exposure resulted in dephosphorylation of endothelial NO synthase (eNOS) in HUVECs. Again pretreatment with benzbromarone, losartan or irbesartan abolished this effect. CONCLUSION: Urate reduced NO production by impaired phosphorylation of eNOS in HUVEC via activation of UATs, which could be normalized by urate lowering agents.

Development of a novel straining holder for transmission electron microscopy compatible with single tilt-axis electron tomography.

We have developed a newly designed straining specimen holder for in situ transmission electron microscopy (TEM) compatible with high-angle single tilt-axis electron tomography. The holder can deform a TEM specimen under tensile stress with the strain rate between 1.5 × 10(-6) and 5.2 × 10(-3) s(-1). We have also confirmed that the maximum tilt angle of the specimen holder reaches ±60° with a rectangular shape aluminum specimen. The new specimen holder, termed as 'straining and tomography holder', will have wide range potential applications in materials science.

General tissue reactions and implications for radiation protection.

Non-cancer effects and risks at low doses from ionising radiation are controversial topics within the field of radiation protection. These issues are discussed in International Commission on Radiological Protection (ICRP) Publication 118, 'ICRP statement on tissue reactions'. Both non-cancer effects and risks are expected to become increasingly important to the system of radiation protection. Before this can happen, several factors must be considered: thorough characterisation of the relationship between dose and risk; verification of the biological mechanisms for any noted excess risk; and adjustment of noted excess risks through the use of a detriment factor. It is difficult to differentiate the relatively small risks associated with radiation from other risk factors in the low-dose region of the dose-response curve. Several recent papers have indicated the possibility of a non-linear dose-response relationship for non-cancer effects. In addition, there are still many uncertainties associated with the biological mechanisms for non-cancer effects. Finally, it is essential to consider the incorporation of detriment into a well-defined system of radiological protection. Given the recent interest in non-cancer effects, it is essential to facilitate discussions in order to define dose limits more clearly within the existing system of radiation protection for both cancer and non-cancer effects.

Impact of different fluoride concentrations and pH of dentifrices on tooth erosion/abrasion in vitro.

BACKGROUND: This in vitro study analysed the effect of different fluoride concentrations in acidic or neutral liquid dentifrices in protecting enamel and dentine from erosive and abrasive wear. METHODS: Bovine enamel and dentine specimens (n = 132) were randomly allocated to 11 groups (each n = 12): experimental liquid dentifrices with 550 ppm F, 1100 ppm F, 5000 ppm F or 0 ppm F/placebo (each at pH 4.5 and pH 7.0); and commercial dentifrices with 550 ppm F (Colgate Baby, pH 7.0), 1100 ppm F (Crest, pH 7.0) and 5000 ppm F (Duraphat, pH 7.0). The specimens were subjected to erosion for 90 seconds, 4 times/day, over 7 days. Immediately after the first and last erosion, the specimens were brushed for 15 seconds using one of the dentifrices. Tooth wear was measured profilometrically (µm) and analysed by ANOVA (p < 0.05). RESULTS: All fluoridated liquid dentifrices significantly reduced enamel wear compared to the placebo and commercial dentifrices. Only liquid dentifrices with 1100 and 5000 ppm F significantly reduced dentine wear compared to placebo dentifrice. The pH had no effect, but the consistency had a significant impact on the effect of dentifrices. CONCLUSIONS: Liquid dentifrices with high F concentration appear to be a good option to prevent tooth wear.

Which are the most reliable predictors of recurrence of atrial fibrillation after transcatheter ablation?: a meta-analysis.

CONTEXT: Transcatheter ablation of atrial fibrillation (AF) has undergone important development, with acceptable midterm results in terms of the safety and recurrence. A meta-analysis was performed to identify the periprocedural complications, midterm success rates and predictors of recurrence after AF ablation. METHODS AND RESULTS: 4357 patients with paroxysmal AF, 1083 with persistent AF and 1777 with long standing AF were included. The pooled analysis showed that there was an in-hospital complication rate of tamponade requiring drainage of 0.99% (0.44-1.54; CI 99%), stroke with neurological persistent impairment of 0.22% (0.04-0.47; CI 99%), and stroke without of 0.36% (0.03-0.70; CI 99%) After a follow up of 22 (13-28) months and 1.23 (1.19-1.5; CI 99%) procedures per patient, the AF recurrence rate was 31.20% (24.87-34.81; CI 99%). The persistent AF patients exhibited a greater risk of recurrence after the first ablation (OR 1.78 [1.14, 2.77] CI 99%), but a trend towards non significance was present in the patients with more than one procedure (OR 1.69 [0.95, 3.00] CI 99%). The most powerful predictors of an AF ablation failure in the overall population were a recurrence within 30-days (OR 4.30; 2.00-10.80), valvular AF (OR 5.20; 2.22-9.50) and a left atrium diameter of more than 50mm (OR 5.10 2.00-12.90; all CI 95%). CONCLUSIONS: Persistent AF remains burdened from higher recurrence rates, however not so following redo-procedures. Three predictors, valvular AF, a left atrium diameter longer than 50mm and recurrence within 30 days, could be appraised to drive selection of patients and therapeutic strategy.

A vasodilating ß1 blocker celiprolol inhibits muscular release of uric acid precursor in patients with essential hypertension.

Although nonvasodilating ß1 blockers increase the levels of uric acid in serum, it is not known whether vasodilating ß1 blockers have a similar effect. In the present study, we evaluated the effect of celiprolol on the release of hypoxanthine, a uric acid precursor, from muscles after an exercise. We used the semi-ischemic forearm test to examine the release of lactate (ΔLAC), ammonia (ΔAmm), and hypoxanthine (ΔHX) before and 4, 10, and 60 min after an exercise in 18 hypertensive patients as well as 4 normotensive subjects. Before celiprolol treatment, all the levels of ΔHX and ΔAmm, and ΔLAC were increased by semi-ischemic exercise in hypertensive patients, and the increases were remarkably larger than those in normotensive subjects. Celiprolol decreased both systolic and diastolic pressure. It also decreased the levels of ΔHX and ΔAmm without changes in ΔLAC after an exercise. These findings also were confirmed by summation of each metabolite (ΣΔMetabolites). Celiprolol caused a marginal decrease of serum uric acid, but the difference was not statistically significant. On the other hand, nonvasodilating ß1 blockers did not suppress the levels of ΔHX and ΔAmm, whereas they significantly increased ΔLAC after an exercise. Celiprolol improved energy metabolism in skeletal muscles. It suppressed HX production and consequently did not adversely affect serum uric acid levels.

Quantitative evaluation of cryptococcal pathogenesis and antifungal drugs using a silkworm infection model with Cryptococcus neoformans.

AIMS: To develop an in vivo system that could quantitatively evaluate the therapeutic effects of antifungal drugs using a silkworm infection model with Cryptococcus neoformans. METHODS AND RESULTS: Silkworms reared at 37°C died after an injection of viable serotype A C. neoformans fungus into the haemolymph. The serotype A C. neoformans, which is known to have higher mammal pathogenicity than the serotype D, was also more virulent against the silkworm. Furthermore, the deletion mutants of genes gpa1, pka1 and cna1, which are genes known to be necessary for the pathogenesis in mammals, showed an increase in the number of fungal cells necessary to kill half of the silkworm population (LD(50) value). Antifungal drugs, amphotericin B, flucytosine, fluconazole and ketoconazole, showed therapeutic effects in silkworms infected with C. neoformans. However, amphotericin B was not therapeutically effective when injected into the silkworm intestine, comparable to the fact that amphotericin B is not absorbed by the intestine in mammals. CONCLUSIONS: The silkworm-C. neoformans infection model is useful for evaluating the therapeutic effects of antifungal drugs. SIGNIFICANCE AND IMPACT OF THE STUDY: The silkworm infection model has various advantages for screening antifungal drug candidates. We can also elucidate the cryptococcal pathogenesis and evaluate the in vivo pharmacokinetics and toxicity of each drug.