Complications after orthopaedic surgeries in patients with rheumatoid arthritis treated with Janus kinase inhibitors: A retrospective observational study.

OBJECTIVES: The current study compared the outcome after orthopaedic surgeries in patients with rheumatoid arthritis receiving Janus kinase inhibitors (JAKis) versus biologic disease-modifying anti-rheumatic drugs (bDMARDs). METHODS: This was a retrospective observational study of Japanese patients with rheumatoid arthritis. Sixty-two patients with rheumatoid arthritis using JAKi preoperatively underwent orthopaedic surgeries. Using propensity score matching, these 62 patients were matched with 62 patients using bDMARDs preoperatively. The number of adverse events was counted. We also examined whether the drug-withholding period in the JAKi-treated group was associated with the occurrence of major postoperative adverse events, namely inflammatory flares and delayed wound healing. RESULTS: JAKi-treated patients had a higher incidence of postoperative flares than bDMARD-treated patients (29% versus 12.1%, P = .01). The incidences of postoperative complications other than flares were not significantly different between the two groups. Among the JAKi-treated group, a longer perioperative drug-withholding period (≥11 days) was associated with a higher incidence of postoperative flares (P = .04). The incidences of delayed wound healing and surgical site infection were not associated with the duration of the JAKi-withholding period. CONCLUSIONS: JAKi-treated patients had a higher incidence of postoperative flares than bDMARD-treated patients. A total of ≥11 days of drug withdrawal was associated with postoperative flares.

Microglial inflammation after chronic spinal cord injury is enhanced by reactive astrocytes via the fibronectin/ß1 integrin pathway.

BACKGROUND: After spinal cord injury (SCI), glial scarring is mainly formed around the lesion and inhibits axon regeneration. Recently, we reported that anti-ß1 integrin antibody (ß1Ab) had a therapeutic effect on astrocytes by preventing the induction of glial scar formation. However, the cellular components within the glial scar are not only astrocytes but also microglia, and whether or not ß1Ab treatment has any influence on microglia within the glial scar remains unclear. METHODS: To evaluate the effects of ß1Ab treatment on microglia within the glial scar after SCI, we applied thoracic contusion SCI to C57BL/6N mice, administered ß1Ab in the sub-acute phase, and analyzed the injured spinal cords with immunohistochemistry in the chronic phase. To examine the gene expression in microglia and glial scars, we selectively collected microglia with fluorescence-activated cell sorting and isolated the glial scars using laser-captured microdissection (LMD). To examine the interaction between microglia and astrocytes within the glial scar, we stimulated BV-2 microglia with conditioned medium of reactive astrocytes (RACM) in vitro, and the gene expression of TNFα (pro-inflammatory M1 marker) was analyzed via quantitative polymerase chain reaction. We also isolated both naïve astrocytes (NAs) and reactive astrocytes (RAs) with LMD and examined their expression of the ligands for ß1 integrin receptors. Statistical analyses were performed using Wilcoxon's rank-sum test. RESULTS: After performing ß1Ab treatment, the microglia were scattered within the glial scar and the expression of TNFα in both the microglia and the glial scar were significantly suppressed after SCI. This in vivo alteration was attributed to fibronectin, a ligand of ß1 integrin receptors. Furthermore, the microglial expression of TNFα was shown to be regulated by RACM as well as fibronectin in vitro. We also confirmed that fibronectin was secreted by RAs both in vitro and in vivo. These results highlighted the interaction mediated by fibronectin between RAs and microglia within the glial scar. CONCLUSION: Microglial inflammation was enhanced by RAs via the fibronectin/ß1 integrin pathway within the glial scar after SCI. Our results suggested that ß1Ab administration had therapeutic potential for ameliorating both glial scar formation and persistent neuroinflammation in the chronic phase after SCI.

Tranexamic acid reduces heme cytotoxicity via the TLR4/TNF axis and ameliorates functional recovery after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is a catastrophic trauma accompanied by intralesional bleeding and neuroinflammation. Recently, there is increasing interest in tranexamic acid (TXA), an anti-fibrinolytic drug, which can reduce the bleeding volume after physical trauma. However, the efficacy of TXA on the pathology of SCI remains unknown. METHODS: After producing a contusion SCI at the thoracic level of mice, TXA was intraperitoneally administered and the bleeding volume in the lesion area was quantified. Tissue damage was evaluated by immunohistochemical and gene expression analyses. Since heme is one of the degraded products of red blood cells (RBCs) and damage-associated molecular pattern molecules (DAMPs), we examined the influence of heme on the pathology of SCI. Functional recovery was assessed using the open field motor score, a foot print analysis, a grid walk test, and a novel kinematic analysis system. Statistical analyses were performed using Wilcoxon's rank-sum test, Dunnett's test, and an ANOVA with the Tukey-Kramer post-hoc test. RESULTS: After SCI, the intralesional bleeding volume was correlated with the heme content and the demyelinated area at the lesion site, which were significantly reduced by the administration of TXA. In the injured spinal cord, toll-like receptor 4 (TLR4), which is a DAMP receptor, was predominantly expressed in microglial cells. Heme stimulation increased TLR4 and tumor necrosis factor (TNF) expression levels in primary microglial cells in a dose-dependent manner. Similarly to the in vitro experiments, the injection of non-lysed RBCs had little pathological influence on the spinal cord, whereas the injection of lysed RBCs or heme solution significantly upregulated the TLR4 and TNF expression in microglial cells. In TXA-treated SCI mice, the decreased expressions of TLR4 and TNF were observed at the lesion sites, accompanied by a significant reduction in the number of apoptotic cells and better functional recovery in comparison to saline-treated control mice. CONCLUSION: The administration of TXA ameliorated the intralesional cytotoxicity both by reducing the intralesional bleeding volume and preventing heme induction of the TLR4/TNF axis in the SCI lesion. Our findings suggest that TXA treatment may be a therapeutic option for acute-phase SCI.

Periostin Promotes Scar Formation through the Interaction between Pericytes and Infiltrating Monocytes/Macrophages after Spinal Cord Injury.

Scar formation is a prominent pathological feature of traumatic central nervous system (CNS) injury, which has long been implicated as a major impediment to the CNS regeneration. However, the factors affecting such scar formation remain to be elucidated. We herein demonstrate that the extracellular matrix protein periostin (POSTN) is a key player in scar formation after traumatic spinal cord injury (SCI). Using high-throughput RNA sequencing data sets, we found that the genes involved in the extracellular region, such as POSTN, were significantly expressed in the injured spinal cord. The expression of POSTN peaked at 7 days after SCI, predominantly in the scar-forming pericytes. Notably, we found that genetic deletion of POSTN in mice reduced scar formation at the lesion site by suppressing the proliferation of the pericytes. Conversely, we found that recombinant POSTN promoted the migration capacity of the monocytes/macrophages and increased the expression of tumor necrosis factor-α from the monocytes/macrophages in vitro, which facilitated the proliferation of pericytes. Furthermore, we revealed that the pharmacological blockade of POSTN suppressed scar formation and improved the long-term functional outcome after SCI. Our findings suggest a potential mechanism whereby POSTN regulates the scar formation after SCI and provide significant evidence that POSTN is a promising therapeutic target for CNS injury.

Macrophage Infiltration Is a Causative Factor for Ligamentum Flavum Hypertrophy through the Activation of Collagen Production in Fibroblasts.

Ligamentum flavum (LF) hypertrophy causes lumbar spinal canal stenosis, leading to leg pain and disability in activities of daily living in elderly individuals. Although previous studies have been performed on LF hypertrophy, its pathomechanisms have not been fully elucidated. In this study, we demonstrated that infiltrating macrophages were a causative factor for LF hypertrophy. Induction of macrophages into the mouse LF by applying a microinjury resulted in LF hypertrophy along with collagen accumulation and fibroblasts proliferation at the injured site, which were very similar to the characteristics observed in the severely hypertrophied LF of human. However, we found that macrophage depletion by injecting clodronate-containing liposomes counteracted LF hypertrophy even with microinjury. For identification of fibroblasts in the LF, we used collagen type I α2 linked to green fluorescent protein transgenic mice and selectively isolated green fluorescent protein-positive fibroblasts from the microinjured LF using laser microdissection. A quantitative RT-PCR on laser microdissection samples revealed that the gene expression of collagen markedly increased in the fibroblasts at the injured site with infiltrating macrophages compared with the uninjured location. These results suggested that macrophage infiltration was crucial for LF hypertrophy by stimulating collagen production in fibroblasts, providing better understanding of the pathophysiology of LF hypertrophy.

Zinc deficiency impairs axonal regeneration and functional recovery after spinal cord injury by modulating macrophage polarization via NF-κB pathway.

Background: Spinal cord injury (SCI) is a devastating disease that results in permanent paralysis. Currently, there is no effective treatment for SCI, and it is important to identify factors that can provide therapeutic intervention during the course of the disease. Zinc, an essential trace element, has attracted attention as a regulator of inflammatory responses. In this study, we investigated the effect of zinc status on the SCI pathology and whether or not zinc could be a potential therapeutic target. Methods: We created experimental mouse models with three different serum zinc concentration by changing the zinc content of the diet. After inducing contusion injury to the spinal cord of three mouse models, we assessed inflammation, apoptosis, demyelination, axonal regeneration, and the number of nuclear translocations of NF-κB in macrophages by using qPCR and immunostaining. In addition, macrophages in the injured spinal cord of these mouse models were isolated by flow cytometry, and their intracellular zinc concentration level and gene expression were examined. Functional recovery was assessed using the open field motor score, a foot print analysis, and a grid walk test. Statistical analysis was performed using Wilcoxon rank-sum test and ANOVA with the Tukey-Kramer test. Results: In macrophages after SCI, zinc deficiency promoted nuclear translocation of NF-κB, polarization to pro-inflammatory like phenotype and expression of pro-inflammatory cytokines. The inflammatory response exacerbated by zinc deficiency led to worsening motor function by inducing more apoptosis of oligodendrocytes and demyelination and inhibiting axonal regeneration in the lesion site compared to the normal zinc condition. Furthermore, zinc supplementation after SCI attenuated these zinc-deficiency-induced series of responses and improved motor function. Conclusion: We demonstrated that zinc affected axonal regeneration and motor functional recovery after SCI by negatively regulating NF-κB activity and the subsequent inflammatory response in macrophages. Our findings suggest that zinc supplementation after SCI may be a novel therapeutic strategy for SCI.

Fungal Arthritis of a Lumbar Facet Joint Because of Candida albicans: A Case Report.

CASE: A 73-year-old man who was treated in our hospital for pyelonephritis because of left ureteral stones and fungemia was referred to our orthopaedic department for persistent right lower-back pain. Magnetic resonance imaging and computed tomography findings showed pyogenic arthritis of the right L4/5 facet joint with a paraspinal muscle abscess. Based on cultures of samples aspirated from the lesions, we diagnosed the patient with fungal arthritis of the lumbar facet joint. We treated him conservatively with an antifungal agent for a year, and the infection resolved. CONCLUSION: This is the first report of hematogenous fungal arthritis of a lumbar facet joint.

[Promoting the "Revitalization of Healthy Life Expectancy" with the Town of Anamizu].

The town of Anamizu in Housu District, Ishikawa Prefecture, in the northern part of the Noto Peninsula, has 8600 residents of whom 45.4% are 65 years or older, making it a region with an aging population. A project was launched to "make Anamizu a town known for the good health and long life" of its citizens. The project involved, among other things, health classes run by health outreach workers. The local government of Anamizu enlisted the participation of local pharmacists, by requesting that the Anamizu group of the northern Noto chapter of the Ishikawa Prefecture Pharmacists Association (comprising seven pharmacies) conduct health classes. The pharmacies within the specified group collaborated to hold 17 classes in 17 areas within six months, starting from October 2016. They conducted the health classes and held periodic meetings to confirm class topics and to standardize the responses to questions from the participants. In all, 258 residents joined the health classes, and the Anamizu pharmacist group received requests to hold more classes in 2017, based on positive feedback. The strong relationship among the pharmacies in the Anamizu group was one factor in the success of this project, executed in cooperation with the Department of Health Promotion. The shared goals of the pharmacies while tackling various projects-for example, in running a booth representing the Anamizu group at health festivals-enhanced their collaborative activities. The Anamizu group plans to conduct various other community activities in the future to improve the QOL of local residents.

Locomotor Training Increases Synaptic Structure With High NGL-2 Expression After Spinal Cord Hemisection.

BACKGROUND: We previously demonstrated that step training leads to reorganization of neuronal networks in the lumbar spinal cord of rodents after a hemisection (HX) injury and step training, including increases excitability of spinally evoked potentials in hindlimb motor neurons. METHODS: In this study, we investigated changes in RNA expression and synapse number using RNA-Seq and immunohistochemistry of the lumbar spinal cord 23 days after a mid-thoracic HX in rats with and without post-HX step training. RESULTS: Gene Ontology (GO) term clustering demonstrated that expression levels of 36 synapse-related genes were increased in trained compared with nontrained rats. Many synaptic genes were upregulated in trained rats, but Lrrc4 (coding NGL-2) was the most highly expressed in the lumbar spinal cord caudal to the HX lesion. Trained rats also had a higher number of NGL-2/synaptophysin synaptic puncta in the lumbar ventral horn. CONCLUSIONS: Our findings demonstrate clear activity-dependent regulation of synapse-related gene expression post-HX. This effect is consistent with the concept that activity-dependent phenomena can provide a mechanistic drive for epigenetic neuronal group selection in the shaping of the reorganization of synaptic networks to learn the locomotion task being trained after spinal cord injury.

The acute phase serum zinc concentration is a reliable biomarker for predicting the functional outcome after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is a devastating disorder for which the accurate prediction of the functional prognosis is urgently needed. Due to the lack of reliable prediction methods, the acute evaluation of SCI severity and therapeutic intervention efficacy is extremely difficult, presenting major obstacles to the development of acute SCI treatment. We herein report a novel method for accurately predicting the functional prognosis using the acute-phase serum zinc concentration after SCI. METHODS: We produced experimental animal SCI models with different prognoses and examined the relationship among the SCI severity, functional outcome, and acute-phase serum zinc concentration. We also examined whether we could predict the functional prognosis by evaluating the serum zinc concentration within 72 h after SCI in a human prospective study. FINDINGS: In a mouse model, the acute serum zinc concentrations decreased in proportion to SCI severity and the serum zinc concentrations at 12 h after SCI accurately predicted the functional prognosis. We clarified the mechanism underlying this serum zinc proportional decrease, showing that activated monocytes took up zinc from blood-serum and then infiltrated the lesion area in a severity-dependent manner. A non-linear regression analysis of 38 SCI patients showed that the serum zinc concentrations in the acute-phase accurately predicted the long-term functional outcome (R2 = 0·84) more accurately than any other previously reported acute-phase biomarkers. INTERPRETATION: The acute-phase serum zinc concentration could be a useful biomarker for predicting the functional prognosis. This simple method will allow for more objective clinical trials and the development of patient-tailored treatment for SCI.

Pathological changes of distal motor neurons after complete spinal cord injury.

Traumatic spinal cord injury (SCI) causes serious disruption of neuronal circuits that leads to motor functional deficits. Regeneration of disrupted circuits back to their original target is necessary for the restoration of function after SCI, but the pathophysiological condition of the caudal spinal cord has not been sufficiently studied. Here we investigated the histological and biological changes in the distal part of the injured spinal cord, using a mice model of complete thoracic SCI in the chronic stage (3 months after injury). Atrophic changes were widely observed in the injured spinal cord both rostral and caudal to the lesion, but the decrease in area was mainly in the white matter in the rostral spinal cord while both the white and gray matter decreased in the caudal spinal cord. The number of the motor neurons was maintained in the chronic phase of injury, but the number of presynaptic boutons decreased in the lumbar motor neurons caudal to the lesion. Using laser microdissection, to investigate gene expressions in motor neurons caudal to the lesion, we observed a decrease in the expressions of neuronal activity markers. However, we found that the synaptogenic potential of postsynapse molecules was maintained in the motor neurons after SCI with the expression of acetylcholine-related molecules actually higher after SCI. Collectively, our results show that the potential of synaptogenesis is maintained in the motor neurons caudal to the lesion, even though presynaptic input is decreased. Although researches into SCI concentrate their effort on the lesion epicenter, our findings suggest that the area caudal to the lesion could be an original therapeutic target for the chronically injured spinal cord.

Macrophage centripetal migration drives spontaneous healing process after spinal cord injury.

Traumatic spinal cord injury (SCI) brings numerous inflammatory cells, including macrophages, from the circulating blood to lesions, but pathophysiological impact resulting from spatiotemporal dynamics of macrophages is unknown. Here, we show that macrophages centripetally migrate toward the lesion epicenter after infiltrating into the wide range of spinal cord, depending on the gradient of chemoattractant C5a. However, macrophages lacking interferon regulatory factor 8 (IRF8) cannot migrate toward the epicenter and remain widely scattered in the injured cord with profound axonal loss and little remyelination, resulting in a poor functional outcome after SCI. Time-lapse imaging and P2X/YRs blockade revealed that macrophage migration via IRF8 was caused by purinergic receptors involved in the C5a-directed migration. Conversely, pharmacological promotion of IRF8 activation facilitated macrophage centripetal movement, thereby improving the SCI recovery. Our findings reveal the importance of macrophage centripetal migration via IRF8, providing a novel therapeutic target for central nervous system injury.

Astrocyte reactivity and astrogliosis after spinal cord injury.

After traumatic injuries of the central nervous system (CNS), including spinal cord injury (SCI), astrocytes surrounding the lesion become reactive and typically undergo hypertrophy and process extension. These reactive astrocytes migrate centripetally to the lesion epicenter and aid in the tissue repair process, however, they eventually become scar-forming astrocytes and form a glial scar which produces axonal growth inhibitors and prevents axonal regeneration. This sequential phenotypic change has long been considered to be unidirectional and irreversible; thus glial scarring is one of the main causes of the limited regenerative capability of the CNS. We recently demonstrated that the process of glial scar formation is regulated by environmental cues, such as fibrotic extracellular matrix material. In this review, we discuss the role and mechanism underlying glial scar formation after SCI as well as plasticity of astrogliosis, which helps to foster axonal regeneration and functional recovery after CNS injury.

Periostin Promotes Fibroblast Migration and Inhibits Muscle Repair After Skeletal Muscle Injury.

BACKGROUND: Skeletal muscle injury (SMI) can cause physical disability due to insufficient recovery of the muscle. The development of muscle fibrosis after SMI has been widely regarded as a principal cause of this failure to recover. Periostin (Postn) exacerbates tissue fibrosis in various organs. We investigated whether Postn is involved in the pathophysiology after SMI. METHODS: Partial laceration injuries of the gastrocnemius were created in wild-type (WT) and Postn knockout (Postn) mice. We examined the expression of the Postn gene before and after SMI. Regeneration and fibrosis of skeletal muscle were evaluated by histological analyses, and recovery of muscle strength was measured by physiological testing. Immunohistochemistry was used to examine the number and proliferative potential of infiltrating fibroblasts in injured muscle. A trans-well migration assay was used to assess the migration capability of fibroblasts. Control immunoglobulin G (IgG) or Postn-neutralizing antibody (Postn-nAb) was injected into injured muscle at 7 and 14 days after injury (dpi). We evaluated the effects of Postn-nAb on muscle repair after SMI. RESULTS: The expression of Postn was dramatically upregulated after SMI. Compared with WT mice, Postn mice had improved muscle recovery and attenuated fibrosis as well as a significantly reduced number of infiltrating fibroblasts. The proliferative potential of these fibroblasts in WT and Postn mice was comparable at 14 dpi; however, the migration capability of fibroblasts was significantly enhanced in the presence of Postn (mean, 258%; 95% confidence interval, 183% to 334%). Moreover, the administration of Postn-nAb inhibited fibroblast infiltration and promoted muscle repair after SMI. CONCLUSIONS: Postn exacerbates fibrotic scar formation through the promotion of fibroblast migration into injured muscle after SMI. Treatment with Postn-nAb is effective for attenuating fibrosis and improving muscle recovery after SMI. CLINICAL RELEVANCE: Our findings may provide a potential therapeutic strategy to enhance muscle repair and functional recovery after SMI.

Interaction of reactive astrocytes with type I collagen induces astrocytic scar formation through the integrin-N-cadherin pathway after spinal cord injury.

Central nervous system (CNS) injury transforms naive astrocytes into reactive astrocytes, which eventually become scar-forming astrocytes that can impair axonal regeneration and functional recovery. This sequential phenotypic change, known as reactive astrogliosis, has long been considered unidirectional and irreversible. However, we report here that reactive astrocytes isolated from injured spinal cord reverted in retrograde to naive astrocytes when transplanted into a naive spinal cord, whereas they formed astrocytic scars when transplanted into injured spinal cord, indicating the environment-dependent plasticity of reactive astrogliosis. We also found that type I collagen was highly expressed in the spinal cord during the scar-forming phase and induced astrocytic scar formation via the integrin-N-cadherin pathway. In a mouse model of spinal cord injury, pharmacological blockade of reactive astrocyte-type I collagen interaction prevented astrocytic scar formation, thereby leading to improved axonal regrowth and better functional outcomes. Our findings reveal environmental cues regulating astrocytic fate decisions, thereby providing a potential therapeutic target for CNS injury.

Experimental Mouse Model of Lumbar Ligamentum Flavum Hypertrophy.

Lumbar spinal canal stenosis (LSCS) is one of the most common spinal disorders in elderly people, with the number of LSCS patients increasing due to the aging of the population. The ligamentum flavum (LF) is a spinal ligament located in the interior of the vertebral canal, and hypertrophy of the LF, which causes the direct compression of the nerve roots and/or cauda equine, is a major cause of LSCS. Although there have been previous studies on LF hypertrophy, its pathomechanism remains unclear. The purpose of this study is to establish a relevant mouse model of LF hypertrophy and to examine disease-related factors. First, we focused on mechanical stress and developed a loading device for applying consecutive mechanical flexion-extension stress to the mouse LF. After 12 weeks of mechanical stress loading, we found that the LF thickness in the stress group was significantly increased in comparison to the control group. In addition, there were significant increases in the area of collagen fibers, the number of LF cells, and the gene expression of several fibrosis-related factors. However, in this mecnanical stress model, there was no macrophage infiltration, angiogenesis, or increase in the expression of transforming growth factor-ß1 (TGF-ß1), which are characteristic features of LF hypertrophy in LSCS patients. We therefore examined the influence of infiltrating macrophages on LF hypertrophy. After inducing macrophage infiltration by micro-injury to the mouse LF, we found excessive collagen synthesis in the injured site with the increased TGF-ß1 expression at 2 weeks after injury, and further confirmed LF hypertrophy at 6 weeks after injury. Our findings demonstrate that mechanical stress is a causative factor for LF hypertrophy and strongly suggest the importance of macrophage infiltration in the progression of LF hypertrophy via the stimulation of collagen production.

The feasibility of in vivo imaging of infiltrating blood cells for predicting the functional prognosis after spinal cord injury.

After a spinal cord injury (SCI), a reliable prediction of the potential functional outcome is essential for determining the optimal treatment strategy. Despite recent advances in the field of neurological assessment, there is still no satisfactory methodology for predicting the functional outcome after SCI. We herein describe a novel method to predict the functional outcome at 12 hours after SCI using in vivo bioluminescence imaging. We produced three groups of SCI mice with different functional prognoses: 50 kdyn (mild), 70 kdyn (moderate) and 90 kdyn (severe). Only the locomotor function within 24 hours after SCI was unable to predict subsequent functional recovery. However, both the number of infiltrating neutrophils and the bioluminescence signal intensity from infiltrating blood cells were found to correlate with the severity of the injury at 12 hours after SCI. Furthermore, a strong linear relationship was observed among the number of infiltrating neutrophils, the bioluminescence signal intensity, and the severity of the injury. Our findings thus indicate that in vivo bioluminescence imaging is able to accurately predict the long-term functional outcome in the hyperacute phase of SCI, thereby providing evidence that this imaging modality could positively contribute to the future development of tailored therapeutic approaches for SCI.

The NtAMI1 gene functions in cell division of tobacco BY-2 cells in the presence of indole-3-acetamide.

Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells can be grown in medium containing indole-3-acetamide (IAM). Based on this finding, the NtAMI1 gene, whose product is functionally equivalent to the AtAMI1 gene of Arabidopsis thaliana and the aux2 gene of Agrobacterium rhizogenes, was isolated from BY-2 cells. Overexpression of the NtAMI1 gene allowed BY-2 cells to proliferate at lower concentrations of IAM, whereas suppression of the NtAMI1 gene by RNA interference (RNAi) caused severe growth inhibition in the medium containing IAM. These results suggest that IAM is incorporated into plant cells and converted to the auxin, indole-3-acetic acid, by NtAMI1.

Function of the aux and rol genes of the Ri plasmid in plant cell division in vitro.

Auxin-autonomous growth in vitro may be related to the integration and expression of the aux and rol genes from the root-inducing (Ri) plasmid in plant cells infected by agropine-type Agrobacterium rhizogenes. To elucidate the functions of the aux and rol genes in plant cell division, plant cell lines transformed with the aux1 and aux2 genes or with the rolABCD genes were established using tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells. The introduction of the aux1 and aux2 genes enabled the auxin-autonomous growth of BY-2 cells, but the introduction of the rolABCD genes did not affect the auxin requirement of the BY-2 cells. The results clearly show that the aux genes are necessary for auxinautotrophic cell division, and that the rolABCD genes are irrelevant in auxin autotrophy.

The aux1 gene of the Ri plasmid is sufficient to confer auxin autotrophy in tobacco BY-2 cells.

Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells are rapidly proliferating meristematic cells that require auxin for culture in vitro. We have established several transgenic BY-2 cell lines that carry the T-DNA of Agrobacterium rhizogenes 15834, which harbors an agropine-type root-inducing (Ri) plasmid. Two of these lines, BYHR-3 and BYHR-7, were used to test the role of auxin in the proliferation of plant cells. The lines grew rapidly in Linsmaier-Skoog (LS) medium lacking auxin and other phytohormones. The TR-DNA, containing the aux1 (tryptophan monooxygenase) and aux2 (indoleacetamide hydrolase) genes, was present in the genomes of both transgenic lines, whereas the TL-DNA, containing the rolA, B, C and D genes, was present in the genome of BYHR-7 but not BYHR-3. Since the introduction of the rolABCD genes alone did not affect the auxin requirement of BY-2 cells, the aux1 and aux2 genes, but not the rolABCD genes, appear to be relevant to the auxin autotrophy of these transgenic lines. Furthermore, the overexpression of aux1 allowed BY-2 cells to grow rapidly in the absence of auxin, suggesting the existence in plant cells of an unidentified gene whose product is functionally equivalent or similar to that of aux2 of the Ri plasmid.