Autoantibodies to nuclear valosin-containing protein-like protein: systemic sclerosis-specific antibodies revealed by in vitro human proteome.

OBJECTIVES: To identify and characterize undescribed systemic sclerosis (SSc)-specific autoantibodies targeting nucleolar antigens and to assess their clinical significance. METHODS: We conducted proteome-wide autoantibody screening (PWAS) against serum samples from SSc patients with nucleolar patterned anti-nuclear antibodies (NUC-ANAs) of specific antibodies (Abs) unknown, utilizing wet protein arrays fabricated from in vitro human proteome. Controls included SSc patients with already-known SSc-specific autoantibodies, patients with other connective tissue diseases, and healthy subjects. The selection of nucleolar antigens was performed by database search in the Human Protein Atlas. The Presence of autoantibodies was certified by immunoblots and immunoprecipitations. Indirect immunofluorescence assays on HEp-2 cells were also conducted. Clinical assessment was conducted by retrospective review of electric medical records. RESULTS: PWAS identified three candidate autoantibodies, including anti-nuclear valosin-containing protein-like (NVL) Ab. Additional measurements in disease controls revealed that only anti-NVL Abs are exclusively detected in SSc. Detection of anti-NVL Abs was reproduced by conventional assays such as immunoblotting and immunoprecipitation. Indirect immunofluorescence assays demonstrated homogeneous nucleolar patterns. Anti-NVL Ab-positive cases were characterized by significantly low prevalence of diffuse skin sclerosis and interstitial lung disease, compared with SSc cases with NUC-ANAs other than anti-NVL Abs, such as anti-U3-RNP and anti-Th/To Abs. CONCLUSION: Anti-NVL Ab is an SSc-specific autoantibody associated with a unique combination of clinical features, including limited skin sclerosis and lack of lung involvement.

Comprehensive autoantibody profiling in systemic autoimmunity by a highly-sensitive multiplex protein array.

Comprehensive autoantibody evaluation is essential for the management of autoimmune disorders. However, conventional methods suffer from poor sensitivity, low throughput, or limited availability. Here, using a proteome-wide human cDNA library, we developed a novel multiplex protein assay (autoantibody array assay; A-Cube) covering 65 antigens of 43 autoantibodies that are associated with systemic sclerosis (SSc) and polymyositis/dermatomyositis (PM/DM). The performance of A-Cube was validated against immunoprecipitation and established enzyme-linked immunosorbent assay. Further, through an evaluation of serum samples from 357 SSc and 172 PM/DM patients, A-Cube meticulously illustrated a diverse autoantibody landscape in these diseases. The wide coverage and high sensitivity of A-Cube also allowed the overlap and correlation analysis between multiple autoantibodies. Lastly, reviewing the cases with distinct autoantibody profiles by A-Cube underscored the importance of thorough autoantibody detection. Together, these data highlighted the utility of A-Cube as well as the clinical relevance of autoantibody profiles in SSc and PM/DM.

The Autoantibody Array Assay: A Novel Autoantibody Detection Method.

Systemic sclerosis (SSc) and dermatomyositis (DM) are autoimmune collagen diseases. Specific autoantibodies are known to be involved in their pathogeneses, each presenting with a different clinical manifestation. Although immunoprecipitation is the gold standard method for detecting autoantibodies, it is difficult to perform in all cases owing to the use of radioisotopes. In this study, we developed a new detection method for SSc and DM autoantibodies (A-cube) using cell-free protein synthesis and examined its validity. Proteins were synthesized using wheat germ cell-free protein synthesis. A total of 100 cases of SSc, 50 cases of DM, and 82 healthy controls were examined. The validity of the method was examined by a comparison with existing test results. Anti-centromere antibody, anti-topoisomerase I antibody, anti-RNA polymerase III antibody, anti-U1RNP anti-body, anti-Jo-1 antibody, anti-TIF1γ antibody, anti-Mi-2 antibody, and anti-ARS antibody were tested for. The results suggested that A-cube is comparable with existing testing methods or has a high sensitivity or specificity. In addition, there was a case in which the diagnosis was reconsidered using the A-cube. The quality of the A-cube was ensured, and its usefulness for a comprehensive analysis was demonstrated. The A-cube can therefore contribute to the clinical assessment and treatment of SSc and DM.

Corrigendum: Citrate synthase insufficiency leads to specific metabolic adaptations in the heart and skeletal muscles upon low-carbohydrate diet feeding in mice.

[This corrects the article DOI: 10.3389/fnut.2022.925908.].

Citrate Synthase Insufficiency Leads to Specific Metabolic Adaptations in the Heart and Skeletal Muscles Upon Low-Carbohydrate Diet Feeding in Mice.

A decrease in TCA cycle activity may lead to impaired nutrition metabolism and cellular energy shortage. Herein, we aimed to characterize the detailed metabolic changes that compensate for energy shortages in energy-consuming organs (heart and skeletal muscles) in mice with knockout of citrate synthase (CS), an important enzyme in the TCA cycle. CS hetero knockout (CS +/-) mice and wild-type mice were fed a low-carbohydrate ketogenic diet (LCKD) or high-fat, high-carbohydrate diet (HFHCD) to induce metabolic changes. Body weight, blood serum parameters, metabolic gene expression, and adenosine triphosphate (ATP) levels were measured in the heart and skeletal muscles. Glycogen content, anabolic and catabolic biomarkers, and morphological changes were also assessed in the skeletal muscles. After diet feeding, there were no differences observed in the body weight and blood serum parameters between wild-type and CS +/- mice. The cardiac expression of genes related to the utilization of fatty acids, monocarboxylates, and branched amino acids increased in LCKD-fed CS +/- mice. In contrast, no significant differences in gene expression were observed in the muscles of LCKD-fed mice or the heart and muscles of HFHCD-fed mice. ATP levels decreased only in the skeletal muscles of LCKD-fed CS +/- mice. Additionally, the decrease in glycogen content, suppression of p70 S6 kinase, and presence of type I fiber atrophy were observed in the muscles of LCKD-fed CS +/- mice. These results suggest that the energy-consuming organs with CS insufficiency may undergo tissue-specific adaption to compensate for energy shortages when the carbohydrate supply is limited.

Autoantibody Landscape Revealed by Wet Protein Array: Sum of Autoantibody Levels Reflects Disease Status.

Autoantibodies are found in various pathological conditions such as autoimmune diseases, infectious diseases, and malignant tumors. However their clinical implications have not yet been fully elucidated. Herein, we conducted proteome-wide autoantibody screening and quantification with wet protein arrays consisting of proteins synthesized from proteome-wide human cDNA library (HuPEX) maintaining their three-dimensional structure. A total of 565 autoantibodies were identified from the sera of three representative inflammatory disorders (systemic sclerosis, psoriasis, and cutaneous arteritis). Each autoantibody level either positively or negatively correlated with serum levels of C-reactive protein, the best-recognized indicator of inflammation. In particular, we discovered total levels of a subset of autoantibodies correlates with the severity of clinical symptoms. From the sera of malignant melanoma, 488 autoantibodies were detected. Notably, patients with metastases had increased overall autoantibody production compared to those with tumors limiting to the primary site. Collectively, proteome-wide screening of autoantibodies using the in vitro proteome can reveal the "autoantibody landscape" of human subjects and may provide novel clinical biomarkers.

Autologous Transplantation of the Internal Limiting Membrane for Refractory Macular Hole following Ruptured Retinal Arterial Macroaneurysm: A Case Report.

PURPOSE: To report a case of macular hole (MH) secondary to a retinal arterial macroaneurysm (RAMA) which was successfully treated with an autologous transplantation of internal limiting membrane (ILM). CASE REPORT: An 87-year-old female presented with a sudden decrease in central vision in the right eye. A fundus examination revealed a RAMA in the superonasal macular region, a subretinal hemorrhage (SRH), involving the macula, and a sub-ILM hemorrhage. A pars plana vitrectomy (PPV) was performed. Intraoperatively, an MH filled with coagulum was detected. We tried to blow off and drain the SRH with a current of BSS and a gentle suction with a 27-gauge vitreous cutter from the MH, but some amount of SRH at the bottom of the MH remained. The ILM was peeled off for 2 disc diameters around the MH. The vitreous cavity was filled with air at the end of the operation. Two weeks after the surgery, the MH was not closed. One month following the initial PPV, a second PPV was performed to achieve closure of the MH. RESULTS: An autologous transplantation of ILM was performed as second PPV. Six months after the final surgery, the MH was successfully closed and the best-corrected decimal visual acuity was 0.6. CONCLUSIONS: Autologous ILM transplantation can be an effective treatment option for MH closure following RAMA rupture.

Staphylococcus epidermidis is involved in a mechanism for female reproduction in mice.

Both external and internal surfaces of organs (e.g., skin, mouth, gut, and intestine) are covered with bacteria, which often contribute to physiological events in host animals. Despite externally opened organs, the presence of bacteria in the mammalian female reproductive tract is uncertain. Here we assessed this problem using wild-type strains of mice, C57BL/6N and ICR. We first demonstrated that bacterial colonies were formed from the oviductal fluid in the C57BL/6N mice with birth experience ("parous"), but not in the mice without birth experience ("non-parous"). Sequence analysis of 16S ribosomal RNA (rRNA) revealed that Staphylococcus epidermidis existed in the oviductal fluid of the parous mice, confirmed by immunohistochemical analysis. Furthermore, extinction of bacterial population with intraperitoneal injection of antibiotics, penicillin G and streptomycin, disturbed the regularly implanted pattern of embryos in ICR mice. Our results indicate that symbiotic S. epidermidis plays a role in interaction between embryo and uterus upon implantation in mice.

CD81 and CD9 work independently as extracellular components upon fusion of sperm and oocyte.

When a sperm and oocyte unite into one cell upon fertilization, membranous fusion between the sperm and oocyte occurs. In mice, Izumo1 and a tetraspanin molecule CD9 are required for sperm-oocyte fusion as one of the oocyte factors, and another tetraspanin molecule CD81 is also thought to involve in this process. Since these two tetraspanins often form a complex upon cell-cell interaction, it is probable that such a complex is also formed in sperm-oocyte interaction; however, this possibility is still under debate among researchers. Here we assessed this problem using mouse oocytes. Immunocytochemical analysis demonstrated that both CD9 and CD81 were widely distributed outside the oocyte cell membrane, but these molecules were separate, forming bilayers, confirmed by immunobiochemical analysis. Electron-microscopic analysis revealed the presence of CD9- or CD81-incorporated extracellular structures in those bilayers. Finally, microinjection of in vitro-synthesized RNA showed that CD9 reversed a fusion defect in CD81-deficient oocytes in addition to CD9-deficient oocytes, but CD81 failed in both oocytes. These results suggest that both CD9 and CD81 independently work upon sperm-oocyte fusion as extracellular components.

Innate immune system still works at diapause, a physiological state of dormancy in insects.

Diapause is most often observed in insects and is a physiologically dormant state different from other types of dormancy, such as hibernation. It allows insects to survive in harsh environments or extend longevity. In general, larval, pupal, or adult non-diapausing insects possess an innate immune system preventing the invasion of microorganisms into their bodies; however, it is unclear whether this system works under the dormant condition of diapause. We here report the occurrence of innate cellular reactions during diapause using pupae of a giant silkmoth, Samia cynthia pryeri. Scanning electron microscopic analysis demonstrated the presence of two major types of cells in the body fluid isolated from the thoracic region of a pupa. Phagocytosis and encapsulation, characteristics of innate cellular reactions, by these cells were observed when latex beads as foreign targets were microinjected into the internal portion of a pupa. Such behavior by these cells was still observed even when pupae were continuously chilled at 4°C. Our results indicate that innate cellular reactions can work in diapausing insects in a dormant state.

Discontinuous rectal cancer spread in the mesorectum and the optimal distal clearance margin in situ.

PURPOSE: We examined the frequency, mode, and extent of discontinuous spread of rectal cancer in the mesorectum to determine the optimal distal clearance margin in situ. METHODS: Forty consecutive patients with rectal cancer undergoing locally curative resection were studied prospectively. Discontinuous cancer spread in the mesorectum and the extent of distal spread was examined microscopically. A tissue shrinkage ratio comparing the distal clearance margin measured before transection to that measured after fixation in each case, was used to convert microscopically measured extent of distal spread to extent in situ. RESULTS: Discontinuous cancer spread in the mesorectum was observed in 17 cases (43 percent); lymph node metastasis in 15 cases (38 percent) and small deposits other than nodal metastases in 8 cases (20 percent). Distal cancer spread (either intramural or mesorectal) was observed in 6 cases (15 percent). The mean distal clearance margin before transection and after fixation was 3.2 cm and 2 cm, respectively. The mean tissue shrinkage ratio was 60 percent. The maximum extent of microscopic distal spread and adjusted distal spread in situ were 20 and 24 mm, respectively. CONCLUSIONS: Excising the mesorectum with fascia propria circumferentially intact is essential for rectal surgery. The optimal distal clearance margin for the rectal wall as well as the mesorectum in situ can be reduced to 3 cm with a right angle.