Suppression of chloroplast triose phosphate isomerase evokes inorganic phosphate-limited photosynthesis in rice.

The availability of inorganic phosphate (Pi) for ATP synthesis is thought to limit photosynthesis at elevated [CO2] when Pi regeneration via sucrose or starch synthesis is limited. We report here another mechanism for the occurrence of Pi-limited photosynthesis caused by insufficient capacity of chloroplast triose phosphate isomerase (cpTPI). In cpTPI-antisense transgenic rice (Oryza sativa) plants with 55%-86% reductions in cpTPI content, CO2 sensitivity of the rate of CO2 assimilation (A) decreased and even reversed at elevated [CO2]. The pool sizes of the Calvin-Benson cycle metabolites from pentose phosphates to 3-phosphoglycerate increased at elevated [CO2], whereas those of ATP decreased. These phenomena are similar to the typical symptoms of Pi-limited photosynthesis, suggesting sufficient capacity of cpTPI is necessary to prevent the occurrence of Pi-limited photosynthesis and that cpTPI content moderately affects photosynthetic capacity at elevated [CO2]. As there tended to be slight variations in the amounts of total leaf-N depending on the genotypes, relationships between A and the amounts of cpTPI were examined after these parameters were expressed per unit amount of total leaf-N (A/N and cpTPI/N, respectively). A/N at elevated [CO2] decreased linearly as cpTPI/N decreased before A/N sharply decreased, owing to further decreases in cpTPI/N. Within this linear range, decreases in cpTPI/N by 80% led to decreases up to 27% in A/N at elevated [CO2]. Thus, cpTPI function is crucial for photosynthesis at elevated [CO2].

Overproduction of Chloroplast Glyceraldehyde-3-Phosphate Dehydrogenase Improves Photosynthesis Slightly under Elevated [CO2] Conditions in Rice.

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) limits the regeneration of ribulose 1,5-bisphosphate (RuBP) in the Calvin-Benson cycle. However, it does not always limit the rate of CO2 assimilation. In the present study, the effects of overproduction of GAPDH on the rate of CO2 assimilation under elevated [CO2] conditions, where the capacity for RuBP regeneration limits photosynthesis, were examined in transgenic rice (Oryza sativa). GAPDH activity was increased to 3.2- and 4.5-fold of the wild-type levels by co-overexpression of the GAPDH genes, GAPA and GAPB, respectively. In the transgenic rice plants, the rate of CO2 assimilation under elevated [CO2] conditions increased by approximately 10%, whereas that under normal and low [CO2] conditions was not affected. These results indicate that overproduction of GAPDH is effective in improving photosynthesis under elevated [CO2] conditions, although its magnitude is relatively small. By contrast, biomass production of the transgenic rice plants was not greater than that of wild-type plants under elevated [CO2] conditions, although starch content tended to increase marginally.

Overexpression of both Rubisco and Rubisco activase rescues rice photosynthesis and biomass under heat stress.

Global warming threatens food security by decreasing crop yields through damage to photosynthetic systems, especially Rubisco activation. We examined whether co-overexpression of Rubisco and Rubisco activase improves the photosynthetic and growth performance of rice under high temperatures. We grew three rice lines-the wild-type (WT), a Rubisco activase-overexpressing line (oxRCA) and a Rubisco- and Rubisco activase-co-overexpressing line (oxRCA-RBCS)-and analysed photosynthesis and biomass at 25 and 40°C. Compared with the WT, the Rubisco activase content was 153% higher in oxRCA and 138% higher in oxRCA-RBCS, and the Rubisco content was 27% lower in oxRCA and similar in oxRCA-RBCS. The CO2 assimilation rate (A) of WT was lower at 40°C than at 25°C, attributable to Rubisco deactivation by heat. On the other hand, that of oxRCA and oxRCA-RBCS was maintained at 40°C, resulting in higher A than WT. Notably, the dry weight of oxRCA-RBCS was 26% higher than that of WT at 40°C. These results show that increasing the Rubisco activase content without the reduction of Rubisco content could improve yield and sustainability in rice at high temperature.

Effects of Overproduction of Rubisco Activase on Rubisco Content in Transgenic Rice Grown at Different N Levels.

It has been reported that overproduction of Rubisco activase (RCA) in rice (Oryza sativa L.) decreased Rubisco content, resulting in declining photosynthesis. We examined the effects of RCA levels on Rubisco content using transgenic rice with overexpressed or suppressed RCA under the control of different promoters of the RCA and Rubisco small subunit (RBCS) genes. All plants were grown hydroponically with different N concentrations (0.5, 2.0 and 8.0 mM-N). In RCA overproduced plants with > 2-fold RCA content (RCA-HI lines), a 10%-20% decrease in Rubisco content was observed at 0.5 and 2.0 mM-N. In contrast, at 8.0 mM-N, Rubisco content did not change in RCA-HI lines. Conversely, in plants with 50%-60% increased RCA content (RCA-MI lines), Rubisco levels remained unchanged, regardless of N concentration. Such effects on Rubisco content were independent of the promoter that was used. In plants with RCA suppression to < 10% of the wild-type RCA content, Rubisco levels were increased at 0.5 mM-N, but were unchanged at 2.0 and 8.0 mM-N. Thus, the effects of the changes in RCA levels on Rubisco content depended on N supply. Moreover, RCA overproduction was feasible without a decrease in Rubisco content, depending on the degree of RCA production.

Effects of co-overexpression of the genes of Rubisco and transketolase on photosynthesis in rice.

Metabolome analyses have indicated an accumulation of sedoheptulose 7-phosphate in transgenic rice plants with overproduction of Rubisco (Suzuki et al. in Plant Cell Environ 35:1369-1379, 2012. doi: 10.1111/j.1365-3040.2012.02494.x ). Since Rubisco overproduction did not quantitatively enhance photosynthesis even under CO2-limited conditions, it is suspected that such an accumulation of sedoheptulose 7-phosphate hampers the improvement of photosynthetic capacity. In the present study, the gene of transketolase, which is involved in the metabolism of sedoheptulose 7-phosphate, was co-overexpressed with the Rubisco small subunit gene in rice. Rubisco and transketolase were successfully overproduced in comparison with those in wild-type plants by 35-53 and 39-84 %, respectively. These changes in the amounts of the proteins were associated with those of the mRNA levels. However, the rate of CO2 assimilation under high irradiance and different [CO2] did not differ between co-overexpressed plants and wild-type plants. Thus, co-overproduction of Rubisco and transketolase did not improve photosynthesis in rice. Transketolase was probably not a limiting factor of photosynthesis as overproduction of transketolase alone by 80-94 % did not affect photosynthesis.

Target resequencing of neuromuscular disease-related genes using next-generation sequencing for patients with undiagnosed early-onset neuromuscular disorders.

Neuromuscular disorders are clinically and genetically heterogeneous diseases with broadly overlapping clinical features. Progress in molecular genetics has led to the identification of numerous causative genes for neuromuscular disorders, but Sanger sequencing-based diagnosis remains labor-intensive and expensive because the genes are large, the genotypes and phenotypes of neuromuscular disorders overlap and multiple genes related to a single phenotype exist. Recently, the advent of next-generation sequencing (NGS) has enabled efficient, concurrent examination of several related genes. Thus, we used NGS for target resequencing of neuromuscular disease-related genes from 42 patients in whom undiagnosed early-onset neuromuscular disorders. Causative genes were identified in 19/42 (45.2%) patients (six, congenital muscular dystrophy; two, Becker muscular dystrophy (BMD); three, limb-girdle muscular dystrophy; one, concurrent BMD and Fukuyama congenital muscular dystrophy; three, nemaline myopathy; one, centronuclear myopathy; one, congenital fiber-type disproportion; one, myosin storage myopathy; and one, congenital myasthenic syndrome). We detected variants of uncertain significance in two patients. In 6/19 patients who received a definitive diagnosis, the diagnosis did not require muscle biopsy. Thus, for patients with suspected neuromuscular disorders not identified using conventional genetic testing alone, NGS-based target resequencing has the potential to serve as a powerful tool that allows definitive diagnosis.

Establishment of monitoring methods for autophagy in rice reveals autophagic recycling of chloroplasts and root plastids during energy limitation.

Autophagy is an intracellular process leading to vacuolar or lysosomal degradation of cytoplasmic components in eukaryotes. Establishment of proper methods to monitor autophagy was a key step in uncovering its role in organisms, such as yeast (Saccharomyces cerevisiae), mammals, and Arabidopsis (Arabidopsis thaliana), in which chloroplastic proteins were found to be recycled by autophagy. Chloroplast recycling has been predicted to function in nutrient remobilization for growing organs or grain filling in cereal crops. Here, to develop our understanding of autophagy in cereals, we established monitoring methods for chloroplast autophagy in rice (Oryza sativa). We generated transgenic rice-expressing fluorescent protein (FP) OsAuTophaGy8 (OsATG8) fusions as autophagy markers. FP-ATG8 signals were delivered into the vacuolar lumen in living cells of roots and leaves mainly as vesicles corresponding to autophagic bodies. This phenomenon was not observed upon the addition of wortmannin, an inhibitor of autophagy, or in an ATG7 knockout mutant. Markers for the chloroplast stroma, stromal FP, and FP-labeled Rubisco were delivered by a type of autophagic body called the Rubisco-containing body (RCB) in the same manner. RCB production in excised leaves was suppressed by supply of external sucrose or light. The release of free FP caused by autophagy-dependent breakdown of FP-labeled Rubisco was induced during accelerated senescence in individually darkened leaves. In roots, nongreen plastids underwent both RCB-mediated and entire organelle types of autophagy. Therefore, our newly developed methods to monitor autophagy directly showed autophagic degradation of leaf chloroplasts and root plastids in rice plants and its induction during energy limitation.

Enhanced leaf photosynthesis as a target to increase grain yield: insights from transgenic rice lines with variable Rieske FeS protein content in the cytochrome b6 /f complex.

Although photosynthesis is the most important source for biomass and grain yield, a lack of correlation between photosynthesis and plant yield among different genotypes of various crop species has been frequently observed. Such observations contribute to the ongoing debate whether enhancing leaf photosynthesis can improve yield potential. Here, transgenic rice plants that contain variable amounts of the Rieske FeS protein in the cytochrome (cyt) b6 /f complex between 10 and 100% of wild-type levels have been used to investigate the effect of reductions of these proteins on photosynthesis, plant growth and yield. Reductions of the cyt b6 /f complex did not affect the electron transport rates through photosystem I but decreased electron transport rates through photosystem II, leading to concomitant decreases in CO2 assimilation rates. There was a strong control of plant growth and grain yield by the rate of leaf photosynthesis, leading to the conclusion that enhancing photosynthesis at the single-leaf level would be a useful target for improving crop productivity and yield both via conventional breeding and biotechnology. The data here also suggest that changing photosynthetic electron transport rates via manipulation of the cyt b6 /f complex could be a potential target for enhancing photosynthetic capacity in higher plants.

A Case of Fungal Keratitis Treated Surgically.

Purpose: Here we report a patient who underwent removal of a retrocorneal plaque and anterior chamber irrigation for acute-stage fungal keratitis. Case report: A 56-year-old woman was referred to the Baptist Yamasaki Eye Clinic, Kyoto, Japan due to refractory infectious keratitis. A white plume infiltration from the superficial to deep corneal stroma was present at the central cornea, and a white giant plaque was present on the posterior surface of the cornea. For diagnostic purposes and to reduce inflammation, the retrocorneal plaque was surgically removed and the anterior chamber was irrigated. Findings obtained from the surgically removed plaque revealed many neutrophils coiled with fibrin and filamentous fungus positive to Fungiflora Y staining. At 1-day postoperative, the amount of inflammation and infiltration were drastically decreased and the infection focus became gradually becoming smaller, finally disappearing at 6-weeks postoperative and with no signs of recurrence. Conclusion: The findings of this study show that surgical intervention can be an effective treatment option for cases of fungal keratitis with retrocorneal plaque.

Kinematic study of root elongation in Arabidopsis thaliana with a novel image-analysis program.

The measurement of the spatial profile of root elongation needs to determine matching points between time-lapse images and calculate their displacement. These data have been obtained by laborious manual methods in the past. Some computer-based programs have been developed to improve the measurement, but they require many time-series digital images or sprinkling graphite particles on the root prior to image capture. Here, we have developed GrowthTracer, a new image-analysis program for the kinematic study of root elongation. GrowthTracer employs a multiresolution image matching method with a nonlinear filter called the critical point filter (CPF), which extracts critical points from images at each resolution and can determine precise matching points by analysis of only two intact images, without pre-marking by graphite particles. This program calculates the displacement of each matching point and determines the displacement velocity profile along the medial axis of the root. In addition, a manual input of distinct matching points increases the matching accuracy. We show a successful application of this novel program for the kinematic analysis of root growth in Arabidopsis thaliana.

Skeletal analysis of the long bone abnormality (lbab/lbab) mouse, a novel chondrodysplastic C-type natriuretic peptide mutant.

Long bone abnormality (lbab/lbab) is a strain of dwarf mice. Recent studies revealed that the phenotype is caused by a spontaneous mutation in the Nppc gene, which encodes mouse C-type natriuretic peptide (CNP). In this study, we analyzed the chondrodysplastic skeletal phenotype of lbab/lbab mice. At birth, lbab/lbab mice are only slightly shorter than their wild-type littermates. Nevertheless, lbab/lbab mice do not undergo a growth spurt, and their final body and bone lengths are only ~60% of those of wild-type mice. Histological analysis revealed that the growth plate in lbab/lbab mice, especially the hypertrophic chondrocyte layer, was significantly thinner than in wild-type mice. Overexpression of CNP in the cartilage of lbab/lbab mice restored their thinned growth plate, followed by the complete rescue of their impaired endochondral bone growth. Furthermore, the bone volume in lbab/lbab mouse was severely decreased and was recovered by CNP overexpression. On the other hand, the thickness of the growth plate of lbab/+ mice was not different from that of wild-type mice; accordingly, impaired endochondral bone growth was not observed in lbab/+ mice. In organ culture experiments, tibial explants from fetal lbab/lbab mice were significantly shorter than those from lbab/+ mice and elongated by addition of 10(-7) M CNP to the same extent as lbab/+ tibiae treated with the same dose of CNP. These results demonstrate that lbab/lbab is a novel mouse model of chondrodysplasia caused by insufficient CNP action on endochondral ossification.

Recessive RYR1 mutations in a patient with severe congenital nemaline myopathy with ophthalomoplegia identified through massively parallel sequencing.

Nemaline myopathy (NM) is a group of congenital myopathies, characterized by the presence of distinct rod-like inclusions "nemaline bodies" in the sarcoplasm of skeletal muscle fibers. To date, ACTA1, NEB, TPM3, TPM2, TNNT1, and CFL2 have been found to cause NM. We have identified recessive RYR1 mutations in a patient with severe congenital NM, through high-throughput screening of congenital myopathy/muscular dystrophy-related genes using massively parallel sequencing with target gene capture. The patient manifested fetal akinesia, neonatal severe hypotonia with muscle weakness, respiratory insufficiency, swallowing disturbance, and ophthalomoplegia. Skeletal muscle histology demonstrated nemaline bodies and small type 1 fibers, but without central cores or minicores. Congenital myopathies, a molecularly, histopathologically, and clinically heterogeneous group of disorders are considered to be a good candidate for massively parallel sequencing.

High yielding ability of a large-grain rice cultivar, Akita 63.

To increase the yield potential while limiting the environmental impact of N management practices is an important issue in rice cultivation. The large-grain rice cultivar Akita 63 showed higher N-use efficiency for grain production. To elucidate this, we analyzed yield characteristics of Akita 63 in comparison with those of a maternal cultivar, Oochikara with a large grain, a paternal cultivar, Akita 39 with a normal grain, and a Japanese leading cultivar, Akitakomachi. The yields of Akita 63 were 20% higher than those of Oochikara and Akita 39, and 50% higher than those of Akitakomachi for the same N application. Akita 63 showed superior N uptake capacity. Whereas a trade-off between single grain weight and grain number was found for Oochikara, Akita 63 did not show such a relationship. The success in Akita 63 breeding was due to overcoming such a trade-off. Akita 63 had the large-grain alleles of GS3 and qSW5. Thus, an enlargement of grain size can have a great impact on an increase in yield with improved N-use efficiency. However, an enlargement of sink capacity led to source limitation. Thus, both sink and source improvements are essential for a further increase in the yield of today's high-yielding cultivars.

Ascending Colon Schwannoma Surgically Treated after Accurate Preoperative Diagnosis.

Colorectal schwannomas are rare and usually benign gastrointestinal mesenchymal tumors. However, these tumors are often overtreated, possibly owing to misleading malignant potential. To our knowledge, there have been no previous reports of ascending colon schwannoma preoperatively diagnosed as benign schwannoma. Herein, we report a case of ascending colon schwannoma accurately diagnosed by endoscopic biopsy and successfully treated by wedge resection. The patient was a 76-year-old woman with complaints of bloody stool. She had no relevant past medical history. Radiological findings revealed a protruded mass in the ascending colon, and colonoscopy revealed a submucosal tumor measuring approximately 3 cm in diameter with a reddish and uneven surface. Histological and immunohistochemical analysis for vimentin and S100 protein of the specimen obtained by endoscopic biopsy confirmed the diagnosis of schwannoma. Thus, we performed laparoscopy-assisted endoscopic full-thickness resection of the ascending colon wall, as appropriate for a benign soft tissue tumor. The postoperative course has been uneventful for 2 years. This case demonstrates that colonic schwannoma can be successfully treated with adequate resection if an accurate preoperative diagnosis is made, thereby avoiding overtreatment, such as surgery for colorectal tumor including lymph node dissection. Preoperatively diagnosed schwannomas should be treated by wedge resection, with postoperative pathological findings confirming the presence or absence of malignancy. Additional resection should be considered for very rare cases of coexisting malignant tissue.

Chronically elevated plasma C-type natriuretic peptide level stimulates skeletal growth in transgenic mice.

C-type natriuretic peptide (CNP) plays a critical role in endochondral ossification through guanylyl cyclase-B (GC-B), a natriuretic peptide receptor subtype. Cartilage-specific overexpression of CNP enhances skeletal growth and rescues the dwarfism in a transgenic achondroplasia model with constitutive active mutation of fibroblast growth factor receptor-3. For future clinical application, the efficacy of CNP administration on skeletal growth must be evaluated. Due to the high clearance of CNP, maintaining a high concentration is technically difficult. However, to model high blood CNP concentration, we established a liver-targeted CNP-overexpressing transgenic mouse (SAP-CNP tgm). SAP-CNP tgm exhibited skeletal overgrowth in proportion to the blood CNP concentration and revealed phenotypes of systemic stimulation of cartilage bones, including limbs, paws, costal bones, spine, and skull. Furthermore, in SAP-CNP tgm, the size of the foramen magnum, the insufficient formation of which results in cervico-medullary compression in achondroplasia, also showed significant increase. CNP primarily activates GC-B, but under high concentrations it cross-reacts with guanylyl cyclase-A (GC-A), a natriuretic peptide receptor subtype of atrial natriuretic peptides (ANP) and brain natriuretic peptides (BNP). Although activation of GC-A could alter cardiovascular homeostasis, leading to hypotension and heart weight reduction, the skeletal overgrowth phenotype in the line of SAP-CNP tgm with mild overexpression of CNP did not accompany decrease of systolic blood pressure or heart weight. These results suggest that CNP administration stimulates skeletal growth without adverse cardiovascular effect, and thus CNP could be a promising remedy targeting achondroplasia.

Hypomorphic mutation in mouse Nppc gene causes retarded bone growth due to impaired endochondral ossification.

Long bone abnormality (lbab/lbab) is a spontaneous mutant mouse characterized by dwarfism with shorter long bones. A missense mutation was reported in the Nppc gene, which encodes C-type natriuretic peptide (CNP), but it has not been confirmed whether this mutation is responsible for the dwarf phenotype. To verify that the mutation causes the dwarfism of lbab/lbab mice, we first investigated the effect of CNP in lbab/lbab mice. By transgenic rescue with chondrocyte-specific expression of CNP, the dwarf phenotype in lbab/lbab mice was completely compensated. Next, we revealed that CNP derived from the lbab allele retained only slight activity to induce cGMP production through its receptor. Histological analysis showed that both proliferative and hypertrophic zones of chondrocytes in the growth plate of lbab/lbab mice were markedly reduced. Our results demonstrate that lbab/lbab mice have a hypomorphic mutation in the Nppc gene that is responsible for dwarfism caused by impaired endochondral ossification.

A case of cortisol producing adrenal adenoma without phenotype of Cushing's syndrome due to impaired 11beta-hydroxysteroid dehydrogenase 1 activity.

This report concerns a case of cortisol-producing adrenocortical adenoma without the phenotype of Cushing's syndrome. A left adrenal tumor was incidentally detected in this patient. A diagnosis of adrenal Cushing's syndrome was based on the results of endocrinological and radiological examinations, although she showed none of the physical signs of Cushing's syndrome, glucose intolerance, hypertension or dyslipidermia. After a successful laparoscopic left adrenalectomy, the pathological diagnosis was adrenocortical adenoma. Slow tapering of glucocorticoids was needed to prevent adrenal insufficiency after surgery, and the plasma ACTH level remained high even though the serum cortisol level had reached the upper limit of the normal range. Further examination showed a urinary THF + allo-THF/THE ratio of 0.63, which was lower than that of control (0.90 +/- 0.13, mean +/- SD). Serum cortisol/cortisone ratios after the cortisone acetate administration were also decreased, and the serum half-life of cortisol was shorter than the normal range which has been reported. These findings indicated a partial defect in 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) activity, which converts cortisone to cortisol. Our case suggests that a change in 11beta-HSD1 activity results in inter-individual differences in glucocorticoid efficacy.

Effects of growth hormone on thyroid function are mediated by type 2 iodothyronine deiodinase in humans.

PURPOSE: Growth hormone (GH) therapy in adults alters thyroid function, and acromegaly often involves thyroid disease. The present study aimed to elucidate roles and mechanisms of GH in regulating thyroid function. METHODS: We performed two retrospective observational studies, which focused on consecutive patients with severe adult GH deficiency who received recombinant human GH (rhGH) therapy (n = 20) and consecutive patients with acromegaly who underwent transsphenoidal surgery (TSS) (n = 25). In both studies, serum free triiodothyronine (fT3), free thyroxine (fT4), and fT3/fT4 ratio were examined before and after the interventions. We subsequently administered GH to four human cell lines (HepG2, TSA201, MCF7, and HTC/C3) in vitro, and examined changes in mRNA levels of iodothyronine deiodinases (D1, D2, and D3). RESULTS: Median serum fT3 level significantly increased after rhGH therapy from 2.38 to 2.78 pg/mL (p < 0.001), and fT4 decreased from 1.115 to 1.065 ng/dL (p = 0.081). TSS significantly decreased median serum fT3 from 3.03 to 2.53 pg/mL (p < 0.001), and increased fT4 from 1.230 to 1.370 ng/dL (p < 0.001). In vitro, GH significantly increased D2 expression at the mRNA level in HTC/C3 cells (p < 0.01), as well as D2 protein and its activity. CONCLUSIONS: GH increased serum fT3 level and decreased serum fT4 level in humans. Our results suggest that its mechanism involves D2 upregulation. Considering this GH effect on thyroid hormone metabolism, data on thyroid function could be useful in the management of GH deficiency and acromegaly.

Rats deficient C-type natriuretic peptide suffer from impaired skeletal growth without early death.

We have previously investigated the physiological role of C-type natriuretic peptide (CNP) on endochondral bone growth, mainly with mutant mouse models deficient in CNP, and reported that CNP is indispensable for physiological endochondral bone growth in mice. However, the survival rate of CNP knockout (KO) mice fell to as low as about 70% until 10 weeks after birth, and we could not sufficiently analyze the phenotype at the adult stage. Herein, we generated CNP KO rats by using zinc-finger nuclease-mediated genome editing technology. We established two lines of mutant rats completely deficient in CNP (CNP KO rats) that exhibited a phenotype identical to that observed in mice deficient in CNP, namely, a short stature with severely impaired endochondral bone growth. Histological analysis revealed that the width of the growth plate, especially that of the hypertrophic chondrocyte layer, was markedly lower and the proliferation of growth plate chondrocytes tended to be reduced in CNP KO rats. Notably, CNP KO rats did not have malocclusions and survived for over one year after birth. At 33 weeks of age, CNP KO rats persisted significantly shorter than wild-type rats, with closed growth plates of the femur in all samples, which were not observed in wild-type rats. Histologically, CNP deficiency affected only bones among all body tissues studied. Thus, CNP KO rats survive over one year, and exhibit a deficit in endochondral bone growth and growth retardation throughout life.

Clinical Features of Nivolumab-Induced Thyroiditis: A Case Series Study.

BACKGROUND: The programmed cell death-1 (PD-1) pathway is a novel therapeutic target in immune checkpoint therapy for cancer. It consists of the PD-1 receptor and its two ligands, programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2). Nivolumab is an anti-PD-1 monoclonal antibody approved for malignant melanoma, advanced non-small cell lung cancer, and advanced renal cell carcinoma in Japan. Thyrotoxicosis and hypothyroidism have both been reported in international Phase 3 studies and national post-marketing surveillance of nivolumab in Japan. METHODS: This study analyzed five consecutive cases with thyroid dysfunction associated with nivolumab therapy. Second, it examined the mRNA and protein expressions of PD-L1 and PD-L2 by reverse transcription polymerase chain reaction and Western blotting. RESULTS: All patients were diagnosed with painless thyroiditis. Thyrotoxicosis developed within four weeks from the first administration of nivolumab and normalized within four weeks of onset in three of the five patients. Hypothyroidism after transient thyrotoxicosis developed in two patients, and preexisting hypothyroidism persisted in one patient. The other two patients were treated with glucocorticoids and discontinued nivolumab therapy for comorbid adverse events. One did not develop hypothyroidism, and the other developed mild, transient hypothyroidism. In addition, it was verified that normal thyroid tissue expresses PD-L1 and PD-L2 mRNA and those proteins. CONCLUSIONS: In the present cases, nivolumab-induced thyrotoxicosis seemed to be associated with painless thyroiditis, while no patient with Graves' disease was observed. A transient and rapid course with subsequent hypothyroidism was observed in nivolumab-induced thyroiditis. In addition, it was verified that PD-L1 and PD-L2 are expressed in normal thyroid tissue. This suggests that nivolumab therapy reduces immune tolerance, even in normal thyroid tissue, and leads to the development of thyroiditis. Treating thyrotoxicosis with only supportive care and considering levothyroxine replacement therapy once subsequent hypothyroidism occurs is proposed. Further investigations are required to confirm whether glucocorticoid therapy and discontinuation of nivolumab therapy prevent subsequent hypothyroidism.