Single-cell transcriptomics reveals granzyme K-expressing cytotoxic Tfh cells in tertiary lymphoid structures in IgG4-RD.

BACKGROUND: Germinal center (GC) responses controlled by T follicular helper (Tfh) and T follicular regulatory (Tfr) cells are crucial for the generation of high-affinity antibodies. Acquired immune responses to tissue-released antigens might be mainly induced in tertiary lymphoid organs (TLOs) with GCs in affected tissues. IgG4-related disease (IgG4-RD) demonstrates polarized isotype switching and TLOs in affected tissues. We performed single-cell transcriptomics of tissue-infiltrating T cells from these TLOs to obtain a comprehensive, unbiased view of tissue-infiltrating GC-Tfh cells. OBJECTIVE: To identify GC-Tfh-cell subsets in TLOs in patients with IgG4-RD using single-cell transcriptomics. METHODS: Single-cell RNA sequencing of sorted CD3+ T cells and multicolor immunofluorescence analysis were used to investigate CD4+CXCR5+Bcl6+ GC-Tfh cells in affected lesions from patients with IgG4-RD. RESULTS: Infiltrating CD4+CXCR5+Bcl6+ Tfh cells were divided into 5 main clusters. We detected HLA+ granzyme K+ (GZMK+) Tfh cells with cytotoxicity-associated features in patients with IgG4-RD. We also observed abundant infiltrating Tfr cells with suppressor-associated features in patients with IgG4-RD. These GZMK+ Tfh cells and Tfr cells clustered together in affected tissues from patients with IgG4-RD. CONCLUSIONS: This single-cell data set revealed a novel subset of HLA+GZMK+ cytotoxic Tfh cells infiltrating affected organs in patients with IgG4-RD, suggesting that infiltrating Tfr cells might suppress cytotoxic Tfh cells.

Granzyme K- and amphiregulin-expressing cytotoxic T cells and activated extrafollicular B cells are potential drivers of IgG4-related disease.

BACKGROUND: IgG4-related disease (IgG4-RD), an example of a type I immune disease, is an immune-mediated fibrotic disorder characterized by dysregulated resolution of severe inflammation and wound healing. However, truly dominant or pathognomonic autoantibodies related to IgG4-RD are not identified. OBJECTIVE: We sought to perform single-cell RNA sequencing and T-cell receptor and B-cell receptor sequencing to obtain a comprehensive, unbiased view of tissue-infiltrating T and B cells. METHODS: We performed unbiased single-cell RNA-sequencing analysis for the transcriptome and T-cell receptor sequencing and B-cell receptor sequencing on sorted CD3+ T or CD19+ B cells from affected tissues of patients with IgG4-RD. We also conducted quantitative analyses of CD3+ T-cell and CD19+ B-cell subsets in 68 patients with IgG4-RD and 30 patients with Sjögren syndrome. RESULTS: Almost all clonally expanded T cells in these lesions were either Granzyme K (GZMK)-expressing CD4+ cytotoxic T cells or GZMK+CD8+ T cells. These GZMK-expressing cytotoxic T cells also expressed amphiregulin and TGF-ß but did not express immune checkpoints, and the tissue-infiltrating CD8+ T cells were phenotypically heterogeneous. MKI67+ B cells and IgD-CD27-CD11c-CXCR5- double-negative 3 B cells were clonally expanded and infiltrated affected tissue lesions. GZMK+CD4+ cytotoxic T cells colocalized with MKI67+ B cells in the extrafollicular area from affected tissue sites. CONCLUSIONS: The above-mentioned cells likely participate in T-B collaborative events, suggesting possible avenues for targeted therapies. Our findings were validated using orthogonal approaches, including multicolor immunofluorescence and the use of comparator disease groups, to support the central role of cytotoxic CD4+ and CD8+ T cells expressing GZMK, amphiregulin, and TGF-ß in the pathogenesis of inflammatory fibrotic disorders.

Whole body vibration accelerates the functional recovery of motor nerve components in sciatic nerve-crush injury model rats.

This study aimed to investigate the effect of whole body vibration (WBV) on the sensory and motor nerve components with sciatic nerve injury model rats. Surgery was performed on 21 female Wister rats (6-8 weeks) under intraperitoneal anesthesia. The nerve-crush injuries for the left sciatic nerve were inflicted using a Sugita aneurysm clip. The sciatic nerve model rats were randomly divided into two groups (n=9; control group, n=12; WBV group). The rats in the WBV group walked in the cage with a vibratory stimulus (frequency 50 Hz, 20 min/day, 5 times/wk), while those in the control group walked in the cage without any vibratory stimulus. We used heat stimulation-induced sensory threshold and lumbar magnetic stimulation-induced motor-evoked potentials (MEPs) to measure the sensory and motor nerve components, respectively. Further, morphological measurements, bilateral hind-limb dimension, bilateral gastrocnemius dimension, and weight were evaluated. Consequently, there were no significant differences in the sensory threshold at the injury side between the control and WBV groups. However, at 4 and 6 weeks postoperatively, MEPs latencies in the WBV group were significantly shorter than those in the control group. Furthermore, both sides of the hind-limb dimension at 6 weeks postoperatively, the left side of the gastrocnemius dimension, and both sides of the gastrocnemius weight significantly increased. In conclusion, WBV especially accelerates the functional recovery of motor nerve components in sciatic nerve-crush injury model rats.

Short-Term Collagen Nerve Wrapping Facilitates Motor and Sensory Recovery from Nerve Degeneration in a Sciatic Nerve Injury Rat Model.

Purpose: This study used a sciatic nerve injury rat model to investigate the short-term effects of a polyglycolic acid (PGA)-collagen tube for nerve injury in continuity. Materials and Methods: Sixteen female Wistar rats (6-8 weeks) were used, and the left sciatic nerve was crushed with a Sugita aneurysm clip. Sciatic nerve model rats were randomly categorized into two groups (n = 8; control group, n = 8; nerve wrapping group). Then, we measured four sensory thresholds, magnetically stimulated the lumbar region to induce motor-evoked potentials (MEPs), and evaluated the sciatic nerve histopathologically. Results: In the sensory thresholds, there were significant differences for the main effect in 250 and 2000 Hz stimulation (p = 0.048 and 0.006, respectively). Further, a significant difference was observed with 2000 Hz stimulation at 1 week (p = 0.003). In the heat stimulation, there were significant differences for the main effect in both weeks and groups (p = 0.0002 and 0.0185, respectively). The post-hoc test showed a significant difference between groups only in 2W (p = 0.0283). Three weeks after the surgery, both 2nd and 3rd MEPs waves-related latencies in the nerve wrapping group were significantly shorter than those in the control group (p = 0.0207 and 0.0271, respectively). Histological evaluation of the sciatic nerve revealed considerable differences in the number of axons between the two groups (p = 0.0352). Conclusion: The short-term PGA-collagen tube nerve wrapping facilitated motor and sensory recovery from nerve degeneration in the sciatic nerve injury rat model.

Distinct disease-specific Tfh cell populations in 2 different fibrotic diseases: IgG4-related disease and Kimura disease.

BACKGROUND: How T follicular (Tfh) cells contribute to many different B-cell class-switching events during T-cell-dependent immune responses has been unclear. Diseases with polarized isotype switching offer a unique opportunity for the exploration of Tfh subsets. Secondary and tertiary lymphoid organs in patients with elevated tissue expression levels of IgE (Kimura disease, KD) and those of IgG4 (IgG4-related disease, IgG4-RD) can provide important insights regarding cytokine expression by Tfh cells. OBJECTIVE: We sought to identify disease-specific Tfh cell subsets in secondary and tertiary lymphoid organs expressing IL-10 or IL-13 and thus identify different cellular drivers of class switching in 2 distinct types of fibrotic disorders: allergic fibrosis (driven by type 2 immune cells) and inflammatory fibrosis (driven by cytotoxic T lymphocytes). METHODS: Single-cell RNA sequencing, in situ sequencing, and multicolor immunofluorescence analysis were used to investigate B cells, Tfh cells, and infiltrating type 2 cells in lesion tissues from patients with KD or IgG4-RD. RESULTS: Infiltrating Tfh cells in tertiary lymphoid organs from IgG4-RD were divided into 6 main clusters. We encountered abundant infiltrating IL-10-expressing LAG3+ Tfh cells in patients with IgG4-RD. Furthermore, we found that infiltrating AICDA+CD19+ B cells expressing IL-4, IL-10, and IL-21 receptors correlated with IgG4 expression. In contrast, we found that infiltrating IL-13-expressing Tfh cells were abundant in affected tissues from patients with KD. Moreover, we observed few infiltrating IL-13-expressing Tfh cells in tissues from patients with IgG4-RD, despite high serum levels of IgE (but low IgE in the disease lesions). Cytotoxic T cells were abundant in IgG4-RD; in contrast, type 2 immune cells were abundant in KD. CONCLUSIONS: Our analysis revealed a novel subset of IL-10+LAG3+ Tfh cells infiltrating the affected organs of IgG4-RD patients. In contrast, IL-13+ Tfh cells and type 2 immune cells infiltrated those of KD patients.

A novel perfusion culture system for screening mitochondrial toxicity in primary mouse hepatocytes.

The liver microphysiological system (MPS) model is an in-vitro culture method that mimics physiological blood flow, which enhances basal cellular functions. However, the liver MPS model has not been tested in the preclinical stage because of its obscure utility. It can overcome the major problem of conventional systems-rapid loss of mitochondrial activity in cultured hepatocytes due to limited oxygen supply-by supplying oxygen to cultured hepatocytes using a perfusion device. In this study, we developed a new perfusion culture system that can detect mitochondrial toxicity. Primary mouse hepatocytes were cultured under perfusion condition for 48 hr. The hepatocytes showed increased oxygen consumption and reduced lactate release. These results indicated that the ATP-production pathway was switched from glycolysis to mitochondrial oxidative phosphorylation in the perfusion culture system. Furthermore, ATP levels were considerably reduced in the perfusion culture system after exposure to phenformin, a mitochondrial complex I inhibitor. To summarize, the perfusion culture system could improve the mitochondrial activity in primary mouse hepatocytes, and thus, has potential implications in the detection of mitochondrial toxicity.

A successful case of "temporary endoluminal bypass technique" using a guide extension catheter during thrombolysis for acute limb ischemia in the non-stenting zone.

It is still difficult to treat acute limb ischemia (ALI) in the non-stenting zone such as the popliteal artery. We describe a temporary endoluminal bypass technique for ALI in the non-stenting zone using a guide extension catheter. An 83-year-old female was admitted and diagnosed with ALI in her left leg. The angiogram showed a thrombotic obstruction of the left popliteal artery. Aspiration and dilation by angioplasty could not revascularize. Although Fogarty thrombectomy can be applicable, we avoided it because of its risk of complications and performed a temporary endoluminal bypass technique. After evaluating the occluded lesion by intravascular ultrasound, we delivered a guide extension catheter to fully cover it. Because it played the role of an endoluminal bypass, the blood flow to the distal tibial arteries could be confirmed in the angiogram. A thrombolytic drug was administered intra-arterially for the whole day, and the angiogram showed a reduction of the thrombus on postoperative day (POD) 1. On POD 2, the blood flow was maintained without flow limitation even after removing the catheter. Finally, she was discharged without any complications. This technique might be an alternative in cases of failed conventional treatments for ALI although further investigation needs to be undertaken. .

Identification of hub molecules of FUS-ALS by Bayesian gene regulatory network analysis of iPSC model: iBRN.

Fused in sarcoma/translated in liposarcoma (FUS) is a causative gene of amyotrophic lateral sclerosis (ALS). Mutated FUS causes accumulation of DNA damage and cytosolic stress granule (SG) formation, thereby motor neuron (MN) death. However, key molecular aetiology remains unclear. Here, we applied a novel platform technology, iBRN, "Non- biased" Bayesian gene regulatory network analysis based on induced pluripotent stem cell (iPSC)-derived cell model, to elucidate the molecular aetiology using transcriptome of iPSC-derived MNs harboring FUSH517D. iBRN revealed "hub molecules", which strongly influenced transcriptome network, such as miR-125b-5p-TIMELESS axis and PRKDC for the molecular aetiology. Next, we confirmed miR-125b-5p-TIMELESS axis in FUSH517D MNs such that miR-125b-5p regulated several DNA repair-related genes including TIMELESS. In addition, we validated both introduction of miR-125b-5p and knocking down of TIMELESS caused DNA damage in the cell culture model. Furthermore, PRKDC was strongly associated with FUS mis-localization into SGs by DNA damage under impaired DNA-PK activity. Collectively, our iBRN strategy provides the first compelling evidence to elucidate molecular aetiology in neurodegenerative diseases.

Impact of a poor functional capacity on the clinical outcomes in patients with a pacemaker implantation -Results from the Japanese Heart Rhythm Society Registry.

BACKGROUND: Functional capacity (FC) correlates with mortality in various cardiovascular diseases. The aim of this study was to examine whether cardiac pacemaker implantations improve the FC and affect the prognosis. METHODS AND RESULTS: We prospectively enrolled 621 de novo pacemaker recipients (age 76 ± 9 years, 50.7% male). The FC was assessed by metabolic equivalents (METs) during the implantation and periodically thereafter. The patients were a priori classified into poor FC (<2 METs, n = 40), moderate FC (2 ≤ METs < 4, n = 239), and good FC (≥4 METs, n = 342). Three months after the pacemaker implantation, poor FC or moderate FC patients improved to a good FC by 43%. The distribution of the three FCs remained at those levels until after 1 year of follow-up (P = .18). During a median follow-up of 2.4 years, 71 patients (11%) had cardiovascular hospitalizations and 35 (5.6%) all-cause death. A multivariate Cox analysis revealed that a poor FC at baseline was an independent predictor of both cardiovascular hospitalization (hazard ratio [HR] 2.494, P = .012) and all-cause death (HR 3.338, P = .016). One year after the pacemaker implantation, the eight who remained with a poor FC had a high mortality rate of 37.5% (P < .01). CONCLUSION: Approximately half of the poor or moderate FC patients improved to good FC 3 months after the pacemaker implantation. The baseline FC predicted the prognosis, and patients with an improved FC after the pacemaker implantation had a better prognosis.

Successful wound healing of refractory digital ulcer in patient with systemic sclerosis by Waon therapy.

The treatment of digital ulcer (DU) in systemic sclerosis (SSc) has not been established. A 77-year-old female with a refractory DU in SSc on the right foot was transferred to our hospital. Wound healing had not been achieved despite several endovascular treatments (EVT) and minor amputations. We started Waon therapy 5 days per week as an adjunct therapy. She was placed in a far-infrared-ray dry sauna maintained at 45 °C for 15 min, and was subsequently kept to rest with soothing warmth using a blanket for additional 30 min outside the room. Gradually, the wound had become smaller and the skin perfusion pressure (SPP) had increased. The increase of the blood flow to the wound could be observed in the angiogram on day 109. An additional EVT on day 109 also accelerated wound healing. Finally, wound healing was completely achieved without additional amputations on day 173. In this presented case, Waon therapy contributed to increase of the blood flow to the wound, evidenced by SPP value. Waon therapy may serve as an effective adjunct therapy of DU in SSc. .

Free gait in a shallow pool accelerates recovery after exercise in model mice with fibromyalgia.

This study aimed to determine the effect of pool gait exercise using fibromyalgia-induced model mice. The sensory threshold, locomotive behavior, electrocardiogram, and onset time after the gait test in shallow water using male C57BL/6J mice (weight, 30-35 g; n=21) were investigated. To induce fibromyalgia in model mice, reserpine was injected intraperitoneally into wild-type mice once a day for 3 days. Subsequently, the fibromyalgia-induced model mice were randomly classified into two groups as follows: the control group (n=11) and the pool gait group (n=10). The mice in the pool gait group walked in the same cage containing shallow warm water 5 times per week. Both groups underwent sensory thresholds and video recordings to determine locomotive behaviors weekly. Further, both heart rate and video recordings for observation of a recovery after the gait test in shallow water were undertaken (control group; n=5, pool gait group; n=5). The pool gait did not affect sensory thresholds and locomotive behavior; however, in the pool gait group, both the recovery after the test, such as onset time and gait distance, were considerably better than those of the control group. Furthermore, changes in heart rate and heart rate irregularity after the test were more apparent in the control group than in the pool gait group. The free gait in a shallow pool accelerated recovery after exercise, unlike the sensory threshold.

Comparison of Immunohistochemical Staining for Large T Antigen and Capsid Protein VP1 in BK Polyomavirus-Associated Nephropathy.

AIM: Most transplant centres use SV40 large T antigen (TAg) staining for the diagnosis and assessment of BK polyomavirus-associated nephropathy (BKPyVAN). This study was performed to evaluate the significance of capsid protein VP1 expression in BKPyVAN. METHODS: We performed immunohistochemical staining using anti-SV40 TAg and anti-BKPyV VP1 antibodies in 16 index biopsies and 12 re-biopsies of BKPyVAN and compared the patterns of positivity and the percentage of positive tubules by counting whole specimens. We investigated the correlation between serum creatinine increase from baseline and the percentage of positive tubules for both markers in 16 index biopsies. RESULTS: In VP1 staining, positive findings were observed not only in the nuclei of tubular epithelial cells but also in the cytoplasm, cells shedding into the lumen, intra-tubular casts, and in the interstitium. Two of 28 biopsies (7.1%) showed TAg-positive and VP1-negative results, in which TAg-positive cells were detected only in a single tubule. The median (interquartile range) percentage of positive tubules was 2.8% (0.7-9.8%) for TAg and 1.4% (0.5-3.9%) for VP1 staining (p = 0.2). In 16 index biopsies, serum creatinine increases significantly correlated with the percentage of VP1-positive tubules (r = 0.49, p = 0.02), while this correlation revealed borderline significance with TAg-positive tubules. CONCLUSIONS: VP1 expression showed various patterns, but was detected in half as many tubules as TAg staining, which might lead to false negatives in the samples with minimal viral replication. However, increased VP1-positive tubules indicate advanced tubular damage and possible association with graft dysfunction.

Excessive exercise induces cardiac arrhythmia in a young fibromyalgia mouse model.

BACKGROUND: Fibromyalgia patients experience cardiovascular complications in addition to musculoskeletal pain. This study aimed to investigate the cardiac effects of a prolonged shallow water gait in a fibromyalgia-induced young mouse model. METHODS: To produce a fibromyalgia mouse model, wild-type mice were administered an intraperitoneal injection of reserpine once a day for three days, and two primary experiments were performed. First, three types of gait tests were performed before and after the reserpine injections as follows: (i) 5 minutes of free gait outside the water, (ii) 1 minute of free gait in shallow warm water, and (iii) 5 minutes of free gait in shallow warm water. Second, electrocardiogram recordings were taken before and after the three gait tests. The average heart rate and heart rate irregularity scores were analyzed. RESULTS: Exercise-induced cardiac arrhythmia was observed at 1-minute gait in shallow water during the acute stage of induced FM in young mice. Further, both cardiac arrhythmia and a decrease in HR have occurred at 5-minute gait in shallow water at the same mice. However, this phenomenon was not observed in the wild-type mice under any test conditions. CONCLUSION: Although a short-term free gait in shallow warm water may be advantageous for increasing the motor activity of FM-model mice, we should be aware of the risk of prolonged and excessive exercise-induced cardiac arrhythmia. For gait exercises in shallow water as a treatment in FM patients. We suggest a gradual increase in exercise duration may be warranted.

Absence of shock therapy related to improper sensing of noise on the defibrillation test during subcutaneous implantable cardioverter-defibrillator implantation: a case report.

BACKGROUND: Subcutaneous implantable cardioverter-defibrillator (S-ICD) represents an efficient alternative to transvenous ICD in patients who do not require pacing. The intraoperative defibrillation test (DFT) is recommended during S-ICD implantation to confirm appropriate sensing and successful 65-J termination of induced ventricular fibrillation (VF). However, few cases of oversensing of noise inhibiting therapies have been reported. CASE SUMMARY: We report the case of a 50-year-old man who underwent S-ICD implantation for secondary prevention of sudden cardiac death. Immediately after S-ICD implantation, VF was induced using a 50-Hz burst; however, shock was not delivered owing to sustained noise on the electrogram in the primary vector. Therefore, an external rescue shock was needed at 150 J. We changed the sensing vector from primary to secondary and performed a second DFT. The S-ICD could deliver an appropriate shock and was able to successfully terminate VF without noise markers in the secondary vector. During the second DFT, one back-up pacing was delivered after the shock; the sensing vector then automatically switched from the secondary to the alternate vector. However, noise was observed in the alternate vector despite sinus rhythm restoration. DISCUSSION: The present case demonstrated that noise was recorded in two different vectors during DFT, possibly supporting the hypothesis that the muscle spasm of the diaphragm induced by the 50-Hz burst causes oversensing of noise by the S-ICD.