Anti-survival motor neuron complex antibodies as a novel biomarker for pulmonary arterial hypertension and interstitial lung disease in mixed connective tissue disease.

OBJECTIVE: The presence of anti-U1 RNP antibodies (Abs) is critical for diagnosing MCTD. The aim of this study is to evaluate the clinical relevance of anti-survival motor neuron (SMN) complex Abs, which often coexist with anti-U1 RNP Abs. METHODS: A total of 158 newly diagnosed consecutive cases of SLE, SSc or MCTD with anti-U1 RNP Abs were enrolled in this multicentre observational study between April 2014 and August 2022. Serum anti-SMN complex Abs were screened by immunoprecipitation of 35S-methionine-labelled cell extracts, and associations between anti-SMN complex Abs positivity and clinical characteristics were analysed. RESULTS: Anti-SMN complex Abs were detected in 36% of MCTD patients, which was significantly higher than that in SLE (8%) or SSc (12%). Among MCTD patients classified based on the combination of the clinical features of SLE, SSc and idiopathic inflammatory myopathies, anti-SMN complex Abs showed the highest prevalence in a subset with clinical features of all three components. Anti-SMN complex Abs-positive MCTD had a higher prevalence of pulmonary arterial hypertension (PAH) and interstitial lung disease (ILD), which are related to poor prognosis, than negative patients. Moreover, all three cases of death within 1 year of the treatment were positive for anti-SMN complex Abs. CONCLUSIONS: Anti-SMN complex Abs is the first biomarker of a typical subset of MCTD which bears organ damages such as PAH and ILD.

Nailfold microvascular abnormalities are associated with a higher prevalence of pulmonary arterial hypertension in patients with MCTD.

OBJECTIVE: MCTD manifests with microvasculopathy and overlapping clinical features of SLE, SSc and idiopathic inflammatory myopathies (IIM). The aim of this study was to investigate the clinical significance of microvasculopathy in patients with MCTD using nailfold videocapillaroscopy (NVC). METHODS: Fifty patients with newly diagnosed and untreated MCTD were enrolled in this multicentre, prospective and observational study. Clinical features and NVC findings were assessed at baseline and after 1 year post-intervention, along with disease controls [SLE (n = 40), SSc (n = 70) and IIM (n = 50)]. RESULTS: All MCTD patients presented Raynaud's phenomenon and were positive for anti-U1 RNP antibodies, and 22.0% (11/50) had pulmonary arterial hypertension (PAH). The prevalence of NVC scleroderma patterns in MCTD was 38.0%, which was lower than SSc (88.6%) but higher than SLE (10.0%). In addition, when we divided MCTD patients into two groups by presence or absence of NVC scleroderma patterns, we found a higher prevalence of PAH in patients with NVC scleroderma patterns. Namely, NVC scleroderma patterns were observed in all MCTD patients with PAH, and in 21.0% of those without PAH. After intensive immunosuppressive therapy, NVC scleroderma patterns disappeared in half of the MCTD patients but were not changed in SSc patients. CONCLUSIONS: MCTD differed from SLE, SSc and IIM in terms of the prevalence and responsiveness of NVC scleroderma patterns to immunosuppressive therapy. Detection of nailfold microvascular abnormalities in MCTD could contribute to predicting PAH and help us to understand further aspects of the pathogenesis of MCTD.

Eosinophilic granulomatosis with polyangiitis exhibits T cell activation and IgG4 immune response in the tissue; comparison with IgG4-related disease.

OBJECTIVE: To study the pathophysiological differences of EGPA and IgG4-related disease (RD) by clarifying their clinical, pathological and immunological features. METHODS: Clinical and pathological findings were compared in patients with EGPA and IgG4-RD. Peripheral blood mononuclear cells were used for comprehensive flow cytometric analysis. RESULTS: An elevation of the IgG4 level was found in all EGPA cases, with the accompanying pathological findings of lymphocytic infiltration and fibrosis observed in 30.8% patients, and the elevation of IgG4/IgG ratio in 61.5% patients. However, actual IgG4 levels, as well as the degree of the infiltration of IgG4-positive plasma cells, were still higher in patients with IgG4-RD than patients with EGPA. Examination by ACR/EULAR classification criteria showed only 13.6% of the EGPA patients met entry criteria, while all of them met the exclusion criteria. In regard to the immunophenotyping, EGPA patients had increases in activated CD4 and CD8 T cells compared with the healthy controls. However, no such similar changes occurred in IgG4-RD patients. On the other hand, both the EGPA and IgG4-RD patient groups had correlated increased plasmablasts and Tfh. These results indicate the presence of two axes: namely, the activation of T cells and that of B cells. Both axes are present in EGPA, but the T cell activation axis was not observed in IgG4-RD. CONCLUSIONS: The elevation of serum IgG4 as well as pathological IgG4 infiltration are not specific. Meanwhile, EGPA and IgG4-RD differ in immunological phenotypes, indicating the possible importance of the predominant activation of T cells in the development of vasculitis.

Continuous Monitoring of Specific mRNA Expression Responses with a Fluorescence Resonance Energy Transfer-Based DNA Nano-tweezer Technique That Does Not Require Gene Recombination.

This letter discusses the feasibility of continuously monitoring specific mRNA expression responses in a living cell with a probe structured as a fluorescence resonance energy transfer (FRET)-based DNA nano-tweezer (DNA-NT). The FRET-based DNA-NT, self-assembled from three single-stranded DNAs, alters its structure from an open state to a closed state in recognition of a target mRNA, resulting in the closing of the distal relation of previously modified FRET-paired fluorescent dyes and generating a FRET signal. The expressions of glucose transporters (GLUT) 1 and 4 in a mouse hepato-carcinoma (Hepa 1-6 cells) were selected as the target model. Live-cell imaging analysis of Hepa 1-6 cells with both FRET-based DNA-NTs indicated that the behaviors of the FRET signals integrated in each individual cell were similar to those measured with the conventional mass analysis technique of semiquantitative real-time (RT) polymerase chain reaction (PCR). From these results, it is concluded that continuous monitoring of gene expression response without gene recombination is feasible with a FRET-based DNA-NT, even in a single cell manner.

A split G-quadruplex-based DNA nano-tweezers structure as a signal-transducing molecule for the homogeneous detection of specific nucleic acids.

A portable method of specific nucleic acid detection would be very useful for monitoring public health in a variety of settings for point-of-care and point-of-need testing. However, conventional methods for the detection of nucleic acids are not ideal for use in the field, as they require skilled operators and complex equipment. Here, we constructed a method for specific nucleic acid detection using a split G-quadruplex (Gq) structure that can recognize target nucleic acids without competitive reactions in a bimolecular reaction and directly produce a detectable signal based on peroxidase activity. We developed a single signal-transducing molecule with a split Gq-based DNA-nano tweezers (NT) structure that self-assembles from three single-stranded DNAs through simple mixing, and detects its target without requiring any washing steps. A model target, a partial norovirus mRNA (NV-RNA), was specifically recognized by the split Gq-based DNA-NT, causing it to undergo a structural change that restored its peroxidase activity. The peroxidase activity was measured by following the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), which gave a greenish colorimetric response, and was proportional to the NV-RNA concentration. The lower detection limit was 4 nM. Our results demonstrated the feasibility of detecting specific nucleic acids with a split Gq-based DNA-NT structure as a nucleic acid signal-transducing molecule in a homogenous assay format. Also the target recognition sites of split Gq-based DNA-NT can easily be designed without delicate optimization of tweezers structure. Thus a split Gq-based DNA-NT technique is readily applicable to a basic platform for the development of a portable device.

A FRET-based DNA nano-tweezer technique for the imaging analysis of specific mRNA.

A DNA nano-tweezer (DNA-NT) structure-based target mRNA detection probe, which uses fluorescence resonance energy transfer (FRET) as a detection signal and works as a single molecule, has been developed. This FRET-paired fluorescent dye-modified DNA-NT, self-assembled from three single-stranded DNAs, alters its structure from open to closed states and produces a FRET signal in response to in vitro transcripts of Hes-1 mRNA. Our results showed that the FRET-based DNA-NT detected both GLUT1 mRNA as a pre-fixed target mRNA model and Hes-1 mRNA as a model expressed inside a living cell. These results confirm the feasibility of using the FRET-based DNA-NT for imaging analysis of target mRNA.

Overcoming treatment unresponsiveness mediated by P-glycoprotein overexpression on lymphocytes in refractory active systemic lupus erythematosus.

P-glycoprotein (P-gp) expels various drugs from cells, resulting in multidrug resistance, including against glucocorticoids. Here, we present a case of systemic lupus erythematosus (SLE) that suggests the importance of initial intensive treatment in overcoming unresponsiveness due to P-gp overexpression on activated lymphocytes. A 28-year-old woman had been diagnosed with highly active SLE including severe pericarditis, hemolytic anemia, lupus nephritis, and retinopathy. The disease activity of SLE progressed despite 1 mg/kg per day oral prednisolone. At the time, P-gp expression was extremely high, as evaluated by flow cytometric analysis on peripheral lymphocytes. After intensive treatment with three courses of methylprednisolone pulse therapy and plasmapheresis, we succeeded in controlling disease activity in association with marked reduction of P-gp overexpression; namely, the clinical symptoms immediately improved along with the reduction of P-gp expression. These results imply that patients with highly active SLE might have drug unresponsiveness that is mediated by P-gp overexpression on lymphocytes. Therefore, downregulation of P-gp by initial intensive immunosuppressive therapy might be important for overcoming glucocorticoid resistance. We also propose that measurement of P-gp on lymphocytes is a useful test for prediction of drug resistance and may assist in the selection of appropriate initial treatment.

[Successful treatment of intravenous cyclophosphamide pulse therapy for lupus myelitis with urinary disturbance and acute confusional state].

A 18-year-old female had low grade fever, butterfly rush, proteinuria, leukocytopenia and hypocomplimentemia in 1988, and she was diagnosed as systemic lupus erythematosus (SLE) with lupus nephritis (WHOIIb). Treatments with prednisolone and mizoribine resulted in the remission for three years. In May 2001, she presented neurosis and polakisuria despite of the increase of prednisolone to 20 mg/day. Finally, she admitted in our hospital because of manic and repressive state and disorientaion. A brain MRI revealed high intensity lesions in bilateral basal ganglia in T2 weighted images, and cerebrospinal fluid showed elevated protein and IFN-alpha (421 IU/ml). In addition, she manifested neurogenic bladder, muscle weakness and hyperactive deep tendon reflex of bilateral lower limbs due to both supranuclear disorder and hypesthesia under the Th 10 level. Spinal MRI revealed marked atrophy and high intensity signals at the middle to lower thoracic spinal cord in T2 weighted images, indicating complication of lupus myelitis as well as cerebral involvement. Although the symptoms of CNS lupus did not respond to prednisolone, twelve monthly cyclophosphamide pulse therapy (IV-CY) has resolved urinary disturbance, muscle weakness and sensory loss, along with the improvement of both cerebral and spinal MRI images. Lupus myelitis and neurogenic bladder are the rare, but very refractory manifestation among CNS involvement of SLE. We here propose IV-CY as an invaluable choice for the treatment of not only active lupus myelitis but also neurogenic bladder resisted for steroid.

[Active arthritis due to cryoglobulinemia based on HCV infection in a patient with RA improvement of arthritis with IFN-alpha].

A 49-year-old Japanese woman was diagnosed with rheumatoid arthritis (RA) based on ACR criteria in May 1999. She developed liver injury after initiation of disease-modifying antirheumatic drugs (DMARDs) and was found to have contracted HCV infection. RA disease activity worsened following restriction of medication due to liver dysfunction. However, 3 mg/day of prednisolone (PSL) resulted in a temporary but marked improvement of RA in December 2001; but arthritis recurred along with Raynaud's phenomenon and palpable purpura. Differential diagnosis between arthritis caused by cryoglobulinemia and exacerbation of RA was important for the selection of appropriate treatment. She manifested non-erosive arthritis on medium and large joints with proteinuria, hematuria and hypocomplementemia. In addition, type III cryoglobulin was detected and chronic active hepatitis was observed on liver biopsy in March 2002. We considered that the main cause for the arthritis was HCV-related mixed cryoglobulinemia. Administration of IFN-alpha resulted in the disappearance of HCV-RNA and cryoglobulin followed by amelioration of arthritis without exacerbation of RA.

[Successful treatment of intravenous cyclophosphamide pulse therapy for systemic lupus erythematosus complicated with steroid-resistant hemolytic anemia].

(Case 1) A 13-years-old female had multiple arthralgia and butterfly rush, when she admitted in our hospital in May 2001. Nephropathy, hemolytic anemia (Hb 6.3 g/dl and direct Coombs 3+) and high titers of antinuclear antibodies and anti-ds-DNA antibody were disclosed and she was diagnosed as systemic lupus erythematosus (SLE). Although combination therapy of PSL 60 mg/day with a steroid pulse therapy, cyclosporine or an immunosorbent treatment, severe hemolytic anemia remained. However, monthly cyclophosphamide pulse therapy (IV-CY), which was started for the steroid-resistant hemolytic anemia, has gradually become effective and Hb improved up to 11.4 g/dl after 6 courses of IV-CY. (Case 2) A 53-years-old woman diagnosed as SLE in 1978 and she had PSL 5 mg for over 10 years. Severe anemia (Hb 5.9 g/dl) was disclosed with a slight fever in June 2001, and she admitted in our hospital for further examinations. Progressive hemolytic anemia was revealed with marked decrease of Hb (3.4 g/dl) and high titer of direct Coombs (3+). Neither PSL (50 mg/day) nor steroid pulse therapy were effective against hemolytic anemia. In contrast, 3 courses of monthly IV-CY (500 mg/day) resulted in the resolution of hemolysis. It is well known that the steroid-resistant hemolytic anemia is extremely hard to treat and leads to miserable prognosis, but we here propose IV-CY as an alternative and invaluable choice for the treatment of refractory hemolytic anemia complicated with SLE.

Efficacy of mizoribine treatment in patients with Sjögren's syndrome: an open pilot trial.

Abstract The aim of this study was to evaluate the efficacy and safety of mizoribine in patients with Sjögren's syndrome. Forty patients with sicca syndrome, whose conditions were definitely diagnosed as Sjögren's syndrome, were given mizoribine orally at a dosage of 150 mg/day for 12 months. The percentage change in salivary secretion after 3, 6, and 12 months of the therapy increased to +112.2% (P < 0.001), +119.9% (P < 0.01), and +147.3% (P < 0.001), respectively, compared with the baseline. Serum IgG levels decreased significantly throughout the study, and the level was 1969.4 ± 620.0 mg/dl after treatment for 12 months compared with the pretreatment value of 2094.3 ± 746.6 mg/dl (P < 0.05). The patient's assessment of clinical signs and symptoms on a 10-cm visual analog scale improved significantly from 7.2 ± 2.3 cm at the start of the treatment to 5.0 ± 1.9 cm after 12 months (P < 0.001). There was a similar improvement in the physician's assessment using the 10-cm visual analog scale: 7.1 ± 1.6 cm at the start of the treatment and 5.2 ± 1.9 cm after 12 months (P < 0.001). With regard to safety, no serious adverse reactions were observed. Although a controlled study would be required to clarify the efficacy of mizoribine, these preliminary observations indicate its efficacy for ameliorating glandular symptoms through improvements in immune abnormalities in patients with Sjögren's syndrome.

Severe skin ulceration associated with Wegener's granulomatosis: successful treatment with hyperbaric oxygen and prostaglandin E1.

Abstract A 36-year-old woman patient with intractable skin ulcers associated with Wegener's granulomatosis was successfully cured by combination therapy with hyperbaric oxygenation therapy (HBO) and intravenous prostaglandin E1 (PGE1). Treatment for 4 months with prednisolone, intravenous PGE1, and cyclophosphamide did not result in healing of the multiple skin lesions on her legs. Repeated HBO with intravenous PGE1 therapy over 6 months resulted in a complete healing of the skin ulcers. We recommend the use of HBO combined with PGE1 for severe cutaneous lesions associated with generalized vasculitis.

[A case of rheumatoid arthritis with secondary amyloidosis in urinary bladder].

We document a case of 61-year-old woman with a 24 year history of rheumatoid arthritis (RA), who developed severe polyarthralgia, dry cough, paresthesia in the legs, frequent micturition, and severe macrohematuria. We diagnosed as severe RA with extraarticular manifestations based on interstitial pneumonia, mononeuritis multiplex, subcutaneous nodules, and high titer of rheumatoid factor. Ultrasonography demonstrated an intravesical mass lesion. A histological examination of the urinary bladder by endoscopic biopsy revealed marked deposition of AA amyloid. The diagnosis of secondary amyloidosis and bacterial cystitis were made based on histological findings and urine culture. At first, we administered antibiotics by intravenous infusion, which resulted in cure of cystitis and partial improvement of macrohematuria. Then combination therapy of corticosteroids and cyclophosphamide successfully reduced the disease activity of RA. There have only been a few reports published so far on the vesical amyloidosis in patients with RA. However, 5 of 10 patients (50%) in vesical amyloidosis died because of continuous massive hematuria, which induced disseminated intravascular coagulation and multiple organ failure. In conclusion, secondary amyloidosis of the urinary bladder should be considered as a possible cause of hematuria in patients with long-term RA and as an important prognosis factor of RA.