Brain regulatory T cells suppress astrogliosis and potentiate neurological recovery.

In addition to maintaining immune tolerance, FOXP3+ regulatory T (Treg) cells perform specialized functions in tissue homeostasis and remodelling1,2. However, the characteristics and functions of brain Treg cells are not well understood because there is a low number of Treg cells in the brain under normal conditions. Here we show that there is massive accumulation of Treg cells in the mouse brain after ischaemic stroke, and this potentiates neurological recovery during the chronic phase of ischaemic brain injury. Although brain Treg cells are similar to Treg cells in other tissues such as visceral adipose tissue and muscle3-5, they are apparently distinct and express unique genes related to the nervous system including Htr7, which encodes the serotonin receptor 5-HT7. The amplification of brain Treg cells is dependent on interleukin (IL)-2, IL-33, serotonin and T cell receptor recognition, and infiltration into the brain is driven by the chemokines CCL1 and CCL20. Brain Treg cells suppress neurotoxic astrogliosis by producing amphiregulin, a low-affinity epidermal growth factor receptor (EGFR) ligand. Stroke is a leading cause of neurological disability, and there are currently few effective recovery methods other than rehabilitation during the chronic phase. Our findings suggest that Treg cells and their products may provide therapeutic opportunities for neuronal protection against stroke and neuroinflammatory diseases.

Systematic review and meta-analysis for 2023 clinical practice guidelines of the Japan Research Committee of the Ministry of Health, Labour, and Welfare for Intractable Vasculitis for the management of ANCA-associated vasculitis.

OBJECTIVES: The objective of this study is to provide evidence for the revision of clinical practice guidelines for the management of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis by the Japan Research Committee for Intractable Vasculitis. METHODS: PubMed, CENTRAL, and the Japan Medical Abstracts Society databases were searched for articles published between 2015 and 2020 to update the systematic review for existing clinical questions, while PubMed, CENTRAL, EMBASE, and the Japan Medical Abstracts Society were searched for articles published between 2000 and 2020 to conduct a systematic review for newly developed clinical questions. The certainty of evidence was assessed with the GRADE approach. RESULTS: For remission induction, when used in conjunction with cyclophosphamide or rituximab, reduced-dose glucocorticoid lowered the risk of serious adverse events compared to standard-dose glucocorticoid. Avacopan improved sustained remission at 12 months compared to high-dose glucocorticoid. Addition of plasma exchange to remission induction therapy did not reduce the risk of death, end-stage kidney disease, or relapse. For remission maintenance, rituximab reduced the risk of relapse compared to azathioprine. Long-term rituximab or azathioprine reduced the risk of relapse compared to short-term rituximab or azathioprine, respectively. CONCLUSIONS: This systematic review provided evidence required to develop the 2023 clinical practice guideline for the management of ANCA-associated vasculitis.

Theoretical study on the effect of applying an external static electric field on the singlet fission dynamics of pentacene dimer models.

We investigate the effect of applying an external static electric field on the singlet fission (SF) dynamics of pentacene dimer models using quantum chemical calculations and exciton dynamics simulations. It is found that the excitation energies of anion-cation (AC) and cation-anion (CA) pair exciton states in the SF process are significantly stabilized and destabilized, respectively, by applying an external static electric field (F) in the intermolecular direction. As a result, this change of excitation energies is found to accelerate the SF dynamics in pentacene dimer models. In particular, in the tilted- and parallel-type pentacene dimer models, SF rates at F = 0.001 a.u. are predicted to be about 2.3 and 3.0 times as large as those at F = 0.0 a.u. while keeping the TT yields large. The present result contributes to paving the way for novel physical and chemical controls, that is, an external static electric field application and donor/acceptor substitution on SF molecules, of SF dynamics.

Theoretical Study on Singlet Fission in Aromatic Diaza s-Indacene Dimers.

We theoretically show that diaza (N2)-substitution to s-indacene with 4n π-electrons, by which the number of π-electrons in N2-s-indacene amounts to 4n+2, is a new strategy to design efficient singlet fission (SF) molecules. By N2-substitution, the diradical character and the exchange integral are found to be tuned moderately, leading to satisfying the excitation energy level matching condition for SF with a large triplet excitation energy. On the basis of the effective electronic coupling related to the SF rate, we explore the optimal slip-stack dimer packings for fast SF. Their underlying mechanisms are well understood from the odd-electron density, resonance structure, and frontier orbital distribution, as the functions of the N2-substituted positions. Furthermore, aromaticities of N2-s-indacenes are evaluated explicitly on the basis of the magnetically induced current. Although N2-s-indacenes display strengths of aromaticities similar to that of anthracene, a local decrease in aromaticity is found to correlate to the spatial feature of diradical character, i.e., odd-electron density. The present findings not only newly propose N2-s-indacenes as feasible SF molecules but also contribute to comprehending the interplay between aromaticity and diradical electronic structures contributing to SF.

Tocilizumab monotherapy for large vessel vasculitis: results of 104-week treatment of a prospective, single-centre, open study.

OBJECTIVE: To evaluate the efficacy and safety of tocilizumab (TCZ) monotherapy for large vessel vasculitides (LVV), including Takayasu arteritis (TAK) and GCA. METHODS: Twelve patients with a newly diagnosed LVV (eight GCA, four TAK) were enrolled. One TAK patient withdrew consent, so 11 (eight GCA, three TAK) were analysed in a prospective, open-label study. TCZ (8 mg/kg) monotherapy, without glucocorticoids or immunosuppressants, was administered every 2 weeks for 2 months and then every 4 weeks for 10 months. Patients were followed for 1 year after the final TCZ dose. Complete and partial responses were defined as disappearance or improvement of all clinical symptoms and normalization of CRP. Relapse was defined as the worsening or recurrence of clinical symptoms, increase in CRP attributable to vasculitis, and/or the need for initiation of glucocorticoids and/or immunosuppressants. Poor clinical response described patients who did not fit the definition of complete response or partial response. RESULTS: Complete and partial responses rates were 75/66% and 25/0% in GCA/TAK patients, respectively, at week 24 and week 52. Five GCA patients and one TAK patient remained disease-free for 1 year after therapy. One GCA patient required TCZ discontinuation due to heart failure at week 24. CONCLUSION: TCZ monotherapy showed a high response rate for newly diagnosed LVV patients, and the majority of patients did not relapse for 1 year after TCZ cessation. Result of this study could help us to understand the crucial role of IL-6 in the pathogenesis of LVV.

Generation and application of human induced-stem cell memory T cells for adoptive immunotherapy.

Adoptive T-cell therapy is an effective strategy for cancer immunotherapy. However, infused T cells frequently become functionally exhausted, and consequently offer a poor prognosis after transplantation into patients. Adoptive transfer of tumor antigen-specific stem cell memory T (TSCM ) cells is expected to overcome this shortcoming as TSCM cells are close to naïve T cells, but are also highly proliferative, long-lived, and produce a large number of effector T cells in response to antigen stimulation. We previously reported that activated effector T cells can be converted into TSCM -like cells (iTSCM ) by coculturing with OP9 cells expressing Notch ligand, Delta-like 1 (OP9-hDLL1). Here we show the methodological parameters of human CD8+ iTSCM cell generation and their application to adoptive cancer immunotherapy. Regardless of the stimulation by anti-CD3/CD28 antibodies or by antigen-presenting cells, human iTSCM cells were more efficiently induced from central memory type T cells than from effector memory T cells. During the induction phase by coculture with OP9-hDLL1 cells, interleukin (IL)-7 and IL-15 (but not IL-2 or IL-21) could efficiently generate iTSCM cells. Epstein-Barr virus-specific iTSCM cells showed much stronger antitumor potentials than conventionally activated T cells in humanized Epstein-Barr virus transformed-tumor model mice. Thus, adoptive T-cell therapy with iTSCM offers a promising therapeutic strategy for cancer immunotherapy.

Efficacy and tolerability of six-week extended dosing interval with tocilizumab therapy in a prospective cohort as remission maintenance in patients with rheumatoid arthritis.

OBJECTIVES: To prospectively evaluate the efficacy and tolerability of a six-week extended dosing interval with tocilizumab (TCZ) in patients with rheumatoid arthritis (RA) in sustained remission. METHODS: Patients who received over six doses of intravenous TCZ in clinical remission (disease activity score [DAS] 28 - erythrocyte sedimentation rate [ESR] ≤ 2.6) maintained over 3 months between December 2013 and December 2015 were included. Flare was defined as DAS28-ESR >3.2 at two consecutive visits. RESULTS: Twenty-five patients were enrolled; 87.5% achieved clinical remission at week 54 after six-week extension and 95.5% achieved a van der Heijde modified total Sharp score (ΔmTSS) ≤0.5. The Health Assessment Questionnaire Disability Index (HAQ-DI) did not increase during 54 weeks. HAQ-DI at baseline and ΔDAS28-ESR at week six positively correlated with increase in DAS28-ESR at week 54. ΔSwollen joint count at week six positively correlated with ΔmTSS at week 54. A total of 12 adverse events occurring in 10 patients did not lead to cessation of TCZ except for one case of recurrent lymphoproliferative disorder at week five. CONCLUSION: A six-week extended dosing interval of TCZ for patients with RA in sustained remission is proposed as an acceptable treatment option for maintaining efficacy and tolerability.

Corticosteroid-free treatment of tocilizumab monotherapy for microscopic polyangiitis: a single-arm, single-center, clinical trial.

OBJECTIVES: To assess the efficacy of tocilizumab (TCZ) monotherapy for the remission induction of microscopic polyangiitis (MPA) in a prospective single-arm, single-center, cohort, pilot study. METHODS: Eligible patients were aged between 20 and 80 years and were newly diagnosed with MPA according to Watts' classification algorithm. Seven patients received 8 mg/kg of intravenous TCZ fortnightly for the first 2 months (5 courses), and monthly for the next 10 months (10 courses). One year after TCZ monotherapy, the patients were followed-up without any treatment. The protocol did not permit the use corticosteroids or any other immunosuppressants. Complete remission (CR) was defined as the Birmingham Vasculitis Activity Score of 0 at two consecutive visits made at least a month apart. RESULTS: CR was achieved in two of six patients (33.3%) at 6 months and three patients (50.0%) at 12 months. Two patients were withdrawn: one because of inefficacy at 6 weeks and the other because of flare at 6 months. One patient voluntarily withdrew after CR at 3 months. Four patients (66.7%) could be kept drug-free after 1 year of TCZ without relapse for 6-15 months at the last visit. CONCLUSION: TCZ monotherapy may be an alternative treatment strategy in some patients with MPA.

A novel JAK-STAT inhibitor, 2-[(3-Carbamoyl-2-thienyl)amino]-2-oxoethyl(2,6-dichlorophenyl)acetate, suppresses helper T cell differentiation in vitro and collagen-induced arthritis in vivo.

Th17 cells, which have been implicated in autoimmune diseases including rheumatoid arthritis (RA), require the JAK-STAT3 pathway for their differentiation and functions. Recently, JAK inhibitors have been developed as a therapeutic drug for RA. However, the current JAK inhibitors are not optimized to STAT3 compared with other STATs. In this study, we found a new lead compound of a small molecule JAK-STAT inhibitor, 2-[(3-Carbamoyl-2-thienyl)amino]-2-oxoethyl (2,6-dichlorophenyl)acetate, which inhibits STAT3 as efficiently as other STATs. This compound, named JI069, was selected by STAT3 reporter assay in combination with an in silico docking model. JI069 inhibited gp130 signaling by inducing dissociation between gp130 and JAK1. In HEK293T cells and primary T cells, JI069 suppressed STAT3 activation as efficiently as other STATs, including STAT1, STAT5, and STAT6. JI069 effectively suppressed Th1, Th2, and Th17 differentiation while strongly promoted iTreg differentiation. JI069 suppressed symptoms of the collagen-induced arthritis (CIA) model in mice, and inhibited the cytokine production from T cells as well as the STAT3 phosphorylation of synovial cells. These data suggest that JI069 is a new type of JAK inhibitor which has potential for the treatment of immunological disorders.

Designed construction of recombinant DNA at the ura3Δ0 locus in the yeast Saccharomyces cerevisiae.

Recombinant DNAs are traditionally constructed using Escherichia coli plasmids. In the yeast Saccharomyces cerevisiae, chromosomal gene targeting is a common technique, implying that the yeast homologous recombination system could be applied for recombinant DNA construction. In an attempt to use a S. cerevisiae chromosome for recombinant DNA construction, we selected the single ura3Δ0 locus as a gene targeting site. By selecting this single locus, repeated recombination using the surrounding URA3 sequences can be performed. The recombination system described here has several advantages over the conventional plasmid system, as it provides a method to confirm the selection of correct recombinants because transformation of the same locus replaces the pre-existing selection marker, resulting in the loss of the marker in successful recombinations. In addition, the constructed strains can serve as both PCR templates and hosts for preparing subsequent recombinant strains. Using this method, several yeast strains that contained selection markers, promoters, terminators and target genes at the ura3Δ0 locus were successfully generated. The system described here can potentially be applied for the construction of any recombinant DNA without the requirement for manipulations in E. coli. Interestingly, we unexpectedly found that several G/C-rich sequences used for fusion PCR lowered gene expression when located adjacent to the start codon.

Efficacy of weekly mizoribine pulse therapy in refractory lupus nephritis.

OBJECTIVE: We investigated the efficacy of a high-dose intermittent dosing treatment method (weekly mizoribine pulse therapy) conceived in the hope of achieving better efficacy by increasing the peak blood levels of mizoribine in patients with refractory lupus nephritis. METHODS: Seventeen patients with lupus nephritis who had been resistant to corticosteroid and immunosuppressant therapy received weekly mizoribine pulse therapy. Mizoribine (350 mg) was administered three times at 12 h intervals over 2 consecutive days (700 mg for day 1 and 350 mg for day 2), followed by a washout period from day 3 to day 7. RESULTS: This therapeutic strategy enabled the peak blood levels of mizoribine to be increased to more than 3 µg/mL in most of the patients. Although SLEDAI, anti-ds-DNA antibody titer, CH-50, and serum albumin level did not significantly improve, urinary protein levels decreased, and it was possible to taper the dose of concomitant steroids. Using our definition of clinical response, 10 of the 17 patients were responders and 4 of them were nonresponders. The average peak serum mizoribine concentration of the responders was as high as 3.5 µg/mL. Elevation of serum liver enzymes was seen in 1 patient, and hyperuricemia occurred in 4 cases, but none of these adverse events were serious. CONCLUSION: Intermittent administration of mizoribine can increase blood levels and may be effective for refractory lupus nephritis.

Concurrent Takayasu Arteritis and Vascular Ehlers-Danlos Syndrome: A Case Report.

Takayasu arteritis (TAK) is a rare primary systemic inflammatory vasculopathy. It is classified as a large-vessel vasculitis and is known to cause inflammatory aneurysms and vascular stenosis. Vascular Ehlers-Danlos syndrome (vEDS) is an autosomal dominant condition known to cause multiple aneurysms and arterial dissection at a young age owing to a mutation in the gene for type III collagen, COL3A1. Here, we present a case of TAK associated with vEDS with the development of multi-organ infarction of the brain, kidney, and spleen owing to multiple arterial aneurysms and stenosis of the internal carotid artery. The patient was successfully treated using anti-inflammatory agents, glucocorticoids, and tocilizumab with the addition of interventional radiology. In our case, a high inflammatory response led to vasculitis being the main cause of the disease with concurrent vEDS. When patients develop multiple aneurysms, stenosis, and dissections leading to multiple organ infarctions, a systemic differential diagnosis to consider concurrent vasculitis syndrome and non-inflammatory vasculopathy, including hereditary disorders, is important even with time constraints.

Potential Triggers for Thrombocytopenia and/or Hemorrhage by the BNT162b2 Vaccine, Pfizer-BioNTech.

Immune thrombocytopenia is an autoimmune disease that can cause bleeding in severe cases. Although available published data do not associate the BNT162b2 vaccine (Pfizer-BioNTech) with the risk of developing thrombocytopenia, the ChAdOx1 nCov-19 vaccine has raised concerns about its potential link with thrombosis and thrombocytopenia. We would like to clarify whether the BNT162b2 vaccine administration may interfere with pre-existing conditions and whether it may cause a risk of thrombocytopenia. Herein, we report three cases of post-vaccine thrombocytopenia among patients with rheumatoid arthritis (RA); one case in which a causal relationship cannot be ruled out with the BNT162b2 vaccine was officially announced. Furthermore, we reviewed reports of adverse events and death cases with a focus on thrombocytopenia and hemorrhages, following vaccination with BNT162b2 in Japan between February 17, 2021 and July 16, 2021, as reported by the Ministry of Health, Labour, and Welfare within the general population. The three cases in this report share the common features of old age, RA, chronic renal failure or hypertension, and pre-existing mild thrombocytopenia at baseline. A total of 746 death cases were reported during this time period, with death by bleeding accounting for 8.8% of the total deaths, of which 84.8% were cranial and statistically higher in young women than among elderly women. The risk-benefit ratio of the vaccine needs to be reconsidered based on high- and low-risk population types and ethnicity. To do so, the expansion of the pharmacovigilance system for BNT162b2 vaccination is urgently required worldwide.

High-grade B-cell lymphoma developed during the treatment of chronic myeloid leukemia with bosutinib.

Tyrosine kinase inhibitor (TKI) can help to increase the survival time in chronic myeloid leukemia (CML) patients; however, the risk of secondary malignancies due to TKIs is a growing concern. Only few reports showed clinical course of patients who developed lymphoma during TKI therapies. Herein, we report a case of high-grade B-cell lymphoma diagnosed in the course of CML treatment with bosutinib. The 75-year-old male patient had been diagnosed with CML 25 years ago. After receiving TKIs (imatinib, nilotinib, and bosutinib), he achieved a major molecular response. Over 3 years after starting bosutinib, he was diagnosed with a high-grade B-cell lymphoma. A total of six courses of DA-EPOCH-R therapy brought complete remission of the lymphoma. Moreover, BCR-ABL1 transcript copies remained undetectable by RT-PCR, 8 months after stopping bosutinib. The risk of secondary malignancy due to TKI has been controversial. It is reported that TKI induces irreversible chromosomal abnormalities or chromosome aberrations and inhibits the proliferation or function of T cells, B cells, and NK cells. These mechanisms of TKI may contribute to the development of secondary malignancy. There remains no consensus on the management of secondary lymphoma during TKI therapies. At present, the only alternative is to observe patients receiving TKI treatment cautiously and to treat secondary lymphoma in the same manner as de novo lymphoma.

Kidney GATA3+ regulatory T cells play roles in the convalescence stage after antibody-mediated renal injury.

FoxP3+ regulatory T cells (Tregs) play crucial roles in peripheral immune tolerance. In addition, Tregs that reside or accumulate in nonlymphoid tissues, called tissue Tregs, exhibit tissue-specific functions and contribute to the maintenance of tissue homeostasis and repair. In an experimental mouse model of crescentic glomerulonephritis induced by an anti-glomerular basement membrane antibody, Tregs started to accumulate in the kidney on day 10 of disease onset and remained at high levels (~30-35% of CD4+ T cells) during the late stage (days 21-90), which correlated with stable disease control. Treg depletion on day 21 resulted in the relapse of renal dysfunction and an increase in Th1 cells, suggesting that Tregs are essential for disease control during the convalescence stage. The Tregs that accumulated in the kidney showed tissue Treg phenotypes, including high expression of GATA3, ST2 (the IL33 receptor subunit), amphiregulin (Areg), and PPARγ. Although T-bet+ Tregs and RORγt+ Tregs were observed in the kidney, GATA3+ Tregs were predominant during the convalescence stage, and a PPARγ agonist enhanced the accumulation of GATA3+ Tregs in the kidney. To understand the function of specific genes in kidney Tregs, we developed a novel T cell transfer system to T cell-deficient mice. This experiment demonstrates that ST2, Areg, and CCR4 in Tregs play important roles in the accumulation of GATA3+ Tregs in the kidney and in the amelioration of renal injury. Our data suggest that GATA3 is important for the recruitment of Tregs into the kidney, which is necessary for convalescence after renal tissue destruction.

Regulatory T Cells: Pathophysiological Roles and Clinical Applications.

Inflammation and immune responses after tissue injury play pivotal roles in the resolution of inflammation, tissue recovery, fibrosis, and remodeling. Regulatory T cells (Tregs) are responsible for immune tolerance and are usually activated in secondary lymphatic tissues. Activated Tregs subsequently regulate effector T cell and dendritic cell activation. For clinical applications such as the suppression of both autoimmune diseases and the rejection of transplanted organs, methods to generate stabilized antigen-specific Tregs are required. For this purpose, transcriptional and epigenetic regulation of Foxp3 expression has been investigated. In addition to conventional Tregs, there are some Tregs that reside in tissues and are called tissue Tregs. Tissue Tregs exhibit tissue-specific functions that contribute to the maintenance of tissue homeostasis and repair. Such tissue Tregs could also be useful for Treg-based cell therapy. We recently discovered brain Tregs that accumulate in the brain during the chronic phase of ischemic brain injury. Brain Tregs resemble other tissue Tregs, but are unique in expressing neural cell-specific genes such as the serotonin receptor (Htr7); consequently, brain Tregs respond to serotonin. Here, we describe our experiences in the use of Tregs to suppress graft-versus-host disease and to promote neural recovery after stroke.

Tocilizumab monotherapy uncovered the role of the CCL22/17-CCR4+ Treg axis during remission of crescentic glomerulonephritis.

OBJECTIVES: Tocilizumab (TCZ) is a humanised anti-interleukin (IL)-6 receptor (IL-6R) monoclonal antibody that is a promising agent to treat various autoimmune diseases. However, the mechanism of TCZ efficacy is unclear. This study aims to elucidate the relationship between Tregs and IL-6R blockade in autoimmunity-mediated renal disease based on a TCZ-treated cohort of patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and in an experimental model of crescentic glomerulonephritis (cGN). METHODS: We examined multiple serum levels of cytokines and chemokines and peripheral blood mononuclear cells in patients with AAV who received TCZ monotherapy and achieved drug-free remission. Moreover, we investigated the mechanistic role of IL-6R blockade in accelerated cGN model to analyse the local sites of inflammation. RESULTS: Serum chemokines CCL22 and CCL17, in addition to the CCR4+Foxp3+ Treg population, increased in patients who demonstrated drug-free remission after the cessation of TCZ. In the cGN model, IL-6R blockade ameliorated the disease, elevated CCL22/17 in CD206+CD11b+CD11c+ kidney M2-like type macrophages, and increased the migration of Tregs into the kidney and regional lymph nodes. The local administration of CCL22 in the kidney facilitated Treg accumulation and reduced glomerular crescent formation. CONCLUSIONS: This study revealed a new mechanism whereby effector Tregs migrate into the inflammatory kidney via the CCL22/17-CCR4 axis that is facilitated by M2-like type macrophages that are induced by IL-6R blockade.

Tocilizumab monotherapy for polymyalgia rheumatica: A prospective, single-center, open-label study.

OBJECTIVES: Polymyalgia rheumatica (PMR) is a systemic inflammatory disease in the elderly of unknown etiology. While glucocorticoids are the mainstay of treatment for PMR, various glucocorticoid-related adverse events are common. Recently, several studies have reported the efficacy of tocilizumab (TCZ), an anti-interleukin-6 receptor antibody, for PMR treatment in addition to an accompanying reduction, or even tapering-off, of glucocorticoids in some cases. The objective of this study was to elucidate the efficacy of TCZ monotherapy in the absence of glucocorticoids for PMR. METHOD: We conducted a 2-year, prospective, single-center, open-label pilot study of TCZ monotherapy in patients with PMR. TCZ (8 mg/kg) was administered at fortnightly intervals for the first 2 months and monthly over the next 10 months. Subsequently, patients were observed for another year without any treatment. The primary endpoints were the remission rates at weeks 12 and 52, and the secondary endpoints were the relapse rate and safety over the total 104 weeks. RESULTS: Thirteen patients were included in this study. Four of these patients achieved remission at week 12 (remission rate 31%). Four patients withdrew from the study due to adverse events (n = 2) and inefficacy (n = 2). At week 52, all 9 patients who had completed the first year achieved remission. Eight patients completed the drug-free second year, with 7 maintaining remission. CONCLUSIONS: TCZ monotherapy is well tolerated and can lead to remission in most patients with PMR in the absence of glucocorticoids.

Mechanism for oxidation of high-molecular-weight substrates by a fungal versatile peroxidase, MnP2.

Unlike general peroxidases, Pleurotus ostreatus MnP2 was reported to have a unique property of direct oxidization of high-molecular-weight compounds, such as Poly R-478 and RNase A. To elucidate the mechanism for oxidation of polymeric substrates by MnP2, a series of mutant enzymes were produced by using a homologous gene expression system, and their reactivities were characterized. A mutant enzyme with an Ala substituting for an exposing Trp (W170A) drastically lost oxidation activity for veratryl alcohol (VA), Poly R-478, and RNase A, whereas the kinetic properties for Mn(2+) and H(2)O(2) were substantially unchanged. These results demonstrated that, in addition to VA, the high-molecular-weight substrates are directly oxidized by MnP2 at W170. Moreover, in the mutants Q266F and V166/168L, amino acid substitution(s) around W170 resulted in a decreased activity only for the high-molecular-weight substrates. These results, along with the three-dimensional modeling of the mutants, suggested that the mutations caused a steric hindrance to access of the polymeric substrates to W170. Another mutant, R263N, contained a newly generated N glycosylation site and showed a higher molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Interestingly, the R263N mutant exhibited an increased reactivity with VA and high-molecular-weight substrates. The existence of an additional carbohydrate modification and the catalytic properties in this mutant are discussed. This is the first study of a direct mechanism for oxidation of high-molecular-weight substrates by a fungal peroxidase using a homologous gene expression system.