Analysis of Mitochondrial Network Morphology in Cultured Myoblasts from Patients with Mitochondrial Disorders

Mitochondrial morphology was studied in cultivated myoblasts obtained from patients with mitochondrial disorders, including CPEO, MELAS and TMEM70 deficiency. Mitochondrial networks and ultrastructure were visualized by fluorescence microscopy and transmission electron microscopy, respectively. A heterogeneous picture of abnormally sized and shaped mitochondria with fragmentation, shortening, and aberrant cristae, lower density of mitochondria and an increased number of “megamitochondria” were found in patient myoblasts. Morphometric Fiji analyses revealed different mitochondrial network properties in myoblasts from patients and controls. The small number of cultivated myoblasts required for semiautomatic morphometric image analysis makes this tool useful for estimating mitochondrial disturbances in patients with mitochondrial disorders.     

Ultrastructural Changes of Mitochondria in the Cultivated Skin Fibroblasts of Patients with Point Mutations in Mitochondrial DNA

Mitochondrial disorders represent a heterogeneous group of multisystem diseases with extreme variability in clinical phenotype. The diagnosis of mitochondrial disorders relies heavily on extensive biochemical and molecular analyses combined with morphological studies including electron microscopy. Although muscle is the tissue of choice for electron microscopic studies, the authors investigated cultivated human skin fibroblasts (HSF) harboring 3 different pathologic mtDNA mutations: 3243A > G, 8344A > G, 8993T > G. They addressed to the possibility of whether mtDNA mutations influence mitochondrial morphology in HSF and if ultrastructural changes of mitochondria may be used for differential diagnostics of mitochondrial disorders caused by mtDNA mutations. Ultrastructural analysis of patients' HSF revealed a heterogeneous mixture of mainly abnormal, partially swelling mitochondria with unusual and sparse cristae. The most characteristic cristal abnormalities were heterogeneity in size and shapes or their absence. Typical filamentous and branched mitochondria with numerous cristae as appeared in control HSF were almost not observed. In all lines of cultured HSF with various mtDNA mutations, similar ultrastructural abnormalities and severely changed mitochondrial interior were found, although no alterations in function and amount of OXPHOS were detected by routinely used biochemical methods in two lines of cultured HSF. This highlights the importance of morphological analysis, even in cultured fibroblasts, in diagnostics of mitochondrial disorders.     

Mitochondrial DNA Aberrations of Bone Marrow Cells from Patients with Aplastic Anemia

This study was undertaken primarily to test the hypothesis that mitochondrial DNA (mtDNA) mutations may be associated with aplastic anemia. Complete mtDNA nucleotide sequence was analyzed in nine and eight bone marrow specimens from Korean patients with aplastic anemia and healthy individuals, respectively. We found a large number of polymorphisms as well as apparent new mutations in both patients and controls throughout the entire mtDNA genome; 12 mutations harbored amino acid changes in patients and none of the mutations in controls produced amino acid changes. There were heteroplasmic mutations and more nonsynonymous mtDNA changes observed in patients, so the mean number of mtDNA aberrations of bone marrow cells showed statistically significant difference overall between patients (mean=25.6) and controls (mean=12.8) (p=0.019). Our data may support an association of mtDNA aberrations with aplastic anemia.     

Clinical and Pathological Heterogeneity of Korean Patients with CAPN3 Mutations

Purpose

This study was designed to investigate the characteristics of Korean patients with calpainopathy.

Materials and Methods

Thirteen patients from ten unrelated families were diagnosed with calpainopathy via direct or targeted sequencing of the CAPN3 gene. Clinical, mutational, and pathological spectra were then analyzed.

Results

Nine different mutations, including four novel mutations ({"type":"entrez-nucleotide","attrs":{"text":"NM_000070","term_id":"27765081","term_text":"NM_000070"}}NM_000070: c.1524+1G>T, c.1789_1790inA, c.2184+1G>T, and c.2384C>T) were identified. The median age at symptom onset was 22 (interquartile range: 15-28). Common clinical findings were joint contracture in nine patients, winged scapula in four, and lordosis in one. However, we also found highly variable clinical features including early onset joint contractures, asymptomatic hyperCKemia, and heterogeneous clinical severity in three members of the same family. Four of nine muscle specimens revealed lobulated fibers, but three showed normal skeletal muscle histology.

Conclusion

We identified four novel CAPN3 mutations and demonstrated clinical and pathological heterogeneity in Korean patients with calpainopathy.     

Decreased CD8-p561ck Activity in Peripheral Blood T-Lymphocytes from Patients with Hereditary Haemochromatosis

Hereditary haemochromatosis (HH) is an autosomal recessive disease linked to certain MHC class-I specificities. The disease is characterized by increased iron absorption and, in some patients, abnormally low numbers of CDS8⁺ T cells in the periphery. We were interested in whether CD4- and CD8-associated p561ck kinase activities were altered in patients with HH. In a study of 18 patients with HH (with and without low numbers of CD8⁺ cells), the level of autophosphorylation of the CD8-associated p561ck as well as its phosphotransferase activity, as determined by phosphorylation of an exogenous substrate, was significantly reduced by two- to three-fold relative to a control population of 23 healthy blood donors (P < 6 × 10⁻⁷). CD8-p56 lck activity was decreased in 16 out of 18 patients (ranging from 1.5- to 10-fold decrease). By contrast, the level of CD4-p561ck activity did not show an overall decrease relative to controls. In addition to an occasional decrease in the amount of CD8-associated lck, HH patient-derived T cells showed a consistent decrease in the relative CD8-p561ck specific activity. Immunofiuorescence staining showed further that the difference could not be accounted by a discrepancy in the expression of CD8αα or CD8αβ complexes or MHC class I molecules. Decreased CD8-p561ck activity was seen both in patients undergoing intensive phlebotomy treatment and in patients in maintenance therapy (i. e. patients who had reached normal levels of iron stores), indicating that this abnormality does not appear to be corrected by iron depletion. To our knowledge, this is the first demonstration of an abnormality in a src-like receptor associated kinase in a human disease state linked to MHC class-I antigens.     

兔肺动脉高压时肺组织中内皮素和鸟苷环－磷酸含量的变化及意义

１１只兔进行从降主动脉至左肺动脉的分流手术，观察三个月后形成了左侧肺动脉高压。取左、右肺组织测定内皮素（ＥＴ）和ｃＧＭＰ，发现左侧肺组织中ＥＴ含量较右侧为高，而ｃＧＭＰ则较右侧为低，两侧比较差异显著。因组织中ｃＧＭＰ的含量间接反映了内皮舒缩血管因子（ＥＤＲＦ）的含量，故认为肺高压时肺组织中ＥＴ分泌增加和ＥＤＲＦ分泌减少。两者的变化在肺高压的形成过程中可能起重要作用。     

Novel CLCN1 Mutations and Clinical Features of Korean Patients with Myotonia Congenita

Myotonia congenita (MC) is a form of nondystrophic myotonia caused by a mutation of CLCN1, which encodes human skeletal muscle chloride channel (CLC-1). We performed sequence analysis of all coding regions of CLCN1 in patients clinically diagnosed with MC, and identified 10 unrelated Korean patients harboring mutations. Detailed clinical analysis was performed in these patients to identify their clinical characteristics in relation to their genotypes. The CLCN1 mutational analyses revealed nine different point mutations. Of these, six (p.M128I, p.S189C, p.M373L, p.P480S, p.G523D, and p.M609K) were novel and could be unique among Koreans. While some features including predominant lower extremity involvement and normal to slightly elevated creatine kinase levels were consistently observed, general clinical features were highly variable in terms of age of onset, clinical severity, aggravating factors, and response to treatment. Our study is the first systematic study of MC in Korea, and shows its expanding clinical and genetic spectrums.     

Antinucleosome Antibodies and Decreased Deoxyribonuclease Activity in Sera of Patients with Systemic Lupus Erythematosus

Nucleosomes are the dominant autoantigens in patients with systemic lupus erythematosus (SLE), and immune complexes involving nucleosomes are the major cause of tissue damage. The activity of DNase I, the enzyme responsible for nucleosome degradation, has been found to be decreased in patients with SLE. However, it is not known whether DNase activity is a clinically useful parameter. The aim of our study was to assess DNase activity in a prospective study of 113 patients with SLE in relation to disease activity and organ involvement. We included two control groups: 9 patients with undifferentiated connective tissue disease (UCTD) and 14 healthy individuals. DNase activity was found to be lower in patients with SLE (63.75% ± 12.1%) than in the controls (81.3% ± 9.25%) (P < 0.001). DNase activity in patients with UCTD (64.9% ± 18.2%; P = 0.854) did not differ from that in patients with SLE. Patients with SLE had higher antinucleosome antibody titers (356.3 ± 851) than the controls (1.44 ± 2.77; P < 0.01) or UCTD patients (39.9 ± 57.7; P < 0.01). In addition, samples positive for antinucleosome antibodies displayed low levels of DNase activity. Within the SLE group, the presence of renal disease had no impact on DNase activity or antinucleosome antibody titers. Also, the SLE disease activity index showed no correlation with DNase activity. In a longitudinal study of six SLE patients, DNase activity did not follow disease activity or autoantibody titers. Our results confirm that serum DNase activity is decreased in patients with SLE, but we conclude that it is not a clinically useful parameter for the prediction of flare-ups of disease or renal involvement.     

High Prevalence of Dihydropteroate Synthase Mutations in Pneumocystis jirovecii Isolated from Patients with Pneumocystis Pneumonia in South Africa

Pneumocystis jirovecii pneumonia (PCP) is an important cause of morbidity and mortality in immunocompromised patients. Sulfa-containing drugs are used for the treatment and prophylaxis of PCP. Mutations in the P. jirovecii fas gene, which encodes dihydropteroate synthase (DHPS), are associated with prior exposure to sulfa drugs, and their appearance suggests the emergence of variants with reduced sulfa susceptibility. The present study examined the prevalence of DHPS mutations in P. jirovecii strains isolated from South African patients with PCP. P. jirovecii infection was investigated by immunofluorescence microscopy and quantitative real-time PCR with respiratory specimens from 712 patients (93% of whom were >15 years of age) with suspected PCP consecutively received for the detection of P. jirovecii over 1 year. PCR amplification and sequencing of the DHPS fas gene was attempted with DNA from the P. jirovecii-positive samples. P. jirovecii infection was confirmed by immunofluorescence microscopy in 168/712 (24%) of the patients. Carriage of the fungus was revealed by real-time PCR in 17% of the patients with negative microscopy results. The P. jirovecii fas gene was successfully amplified from specimens from 151 patients and sequenced. Mutations resulting in the Thr55Ala and/or Pro57Ser amino acid substitution were detected in P. jirovecii strains from 85/151 (56%) patients. The high frequency of PCP episodes with P. jirovecii harboring DHPS mutations in South Africa indicates that populations of this fungus are evolving under the considerable selective pressure exerted by sulfa-containing antibiotics. These results, similar to previous observations of sulfa drug resistance in bacterial populations, underscore the importance of the rational use of sulfa medications either prophylactically against PCP or for the treatment of other infections.Pneumocystis pneumonia (PCP), a major opportunistic infection in immunocompromised patients, is caused by the fungus Pneumocystis jirovecii. The incidence of PCP, which increased dramatically with the advent of the HIV/AIDS pandemic, has decreased in the industrialized world owing to the widespread use of sulfa drug prophylaxis and the introduction of highly active antiretroviral therapy (HAART). However, PCP remains an important cause of morbidity and mortality in HIV/AIDS patients, as well as in immunocompromised non-HIV-infected patients, in whom its incidence is increasing (17, 35). In South Africa, which has a population of 48.5 million, an estimated 5.7 million people were living with HIV in 2007, and 350,000 deaths were attributed to AIDS during the same year (20). The South African government initiated the provision of HAART to the public sector in April 2004, but prior to that, the HIV epidemic was largely untreated. By the end of 2006, the rate of HAART coverage was estimated to be 21% among those needing antiretroviral therapy (20). Studies from African countries report variable incidences of PCP in adult patients with HIV/AIDS and generally higher rates in children (1, 3, 27, 28, 43, 47, 49). In South Africa, where a limited number of laboratories offer testing for P. jirovecii, the vast majority of PCP cases are diagnosed clinically and radiologically.Sulfonamides, usually combined with trimethoprim, as in trimethoprim-sulfamethoxazole (TMP-SMX), and dapsone are used for the treatment and prophylaxis of PCP. There are few alternative drugs for the treatment of this infection. Sulfonamides inhibit the enzyme dihydropteroate synthase (DHPS), an essential component of the folate synthesis pathway (36). In P. jirovecii, two nonsynonymous point mutations in the fas gene, which encodes the DHPS enzyme, are associated with prior exposure to sulfa drugs (5, 15, 16, 22, 26, 32), and concerns have been raised about the possible emergence of resistance to sulfa drugs (38). These mutations, at nucleotide positions 165 and 171, cause the amino acid substitutions Thr55Ala and Pro57Ser in the DHPS protein, respectively. Point mutations in the DHPS-encoding genes of microorganisms such as Plasmodium falciparum, Staphylococcus aureus, Mycobacterium leprae, and Escherichia coli have been shown to confer resistance to sulfonamides (6, 14, 21, 45). As P. jirovecii cannot yet be cultured, conventional in vitro susceptibility tests cannot be utilized; therefore, studies of drug resistance in this organism rely on the use of genetic markers and suitable models. Functional complementation of either DHPS-disrupted E. coli with a mutant P. jirovecii fas gene or FOL1-disrupted Saccharomyces cerevisiae with the fol1 gene mutated at positions analogous to positions 165 and 171 in fas results in the loss of susceptibility to sulfamethoxazole and other sulfa-containing drugs (19, 29).The prevalence of P. jirovecii DHPS mutations reported from countries in the developed world ranges widely, from 4% to 81% (2, 5, 15, 18, 32, 39, 41, 42). In South Africa, a few studies that screened specimens from limited numbers of adults and children have reported mostly low mutation rates (8, 12, 34, 48). Here we present the results of a large laboratory-based study aimed at ascertaining the prevalence in South Africa of P. jirovecii strains harboring mutations at positions 165 and 171 in the fas gene.     

Associations Between Fundus Types and Clinical Manifestations in Patients with RDH12 Gene Mutations

Brain Topography - To study the associations between RDH12 gene mutations, fundus types, and clinical manifestations. In total, 46 patients with inherited eye diseases caused by RDH12 gene...     

Decreased Progesterone Receptor B/A Ratio in Endometrial Cells by Tumor Necrosis Factor-Alpha and Peritoneal Fluid from Patients with Endometriosis

PurposeProgesterone resistance is thought to be a major factor that contributes to progression of endometriosis. However, it is not clear what causes progesterone resistance in endometriosis. This study aimed to assess whether cytokines or peritoneal fluid can affect progesterone receptor (PR) expression in endometrial cells and to verify whether PR expression is reduced in endometriosis.ResultsThe PR-B/A ratio was significantly decreased by treatment with either TNF-α (p=0.011) or peritoneal fluid from women with advanced-stage endometriosis (p=0.027). Immunoreactivity of PR-B expression was significantly lower during the secretory phase than during the proliferative phase in endometrial tissues from control subjects (p<0.001). PR-B expression was significantly reduced in the eutopic endometrium (p=0.031) and ovarian endometrioma (p=0.036) from women with advanced-stage endometriosis compared with eutopic endometrium tissues from control subjects.ConclusionProgesterone resistance in endometriosis may be caused by proinflammatory conditions in the pelvic peritoneal microenvironment.     

Decreased IDO Activity and Increased TTS Expression Break Immune Tolerance in Patients with Immune Thrombocytopenia

Introduction  

Indoleamine 2,3-dioxygenase (IDO) can promote peripheral immune tolerance and control autoimmune responses through tryptophan catabolism. Tryptophanyl-tRNA synthetase (TTS) can protect T cells from IDO-mediated cell injury. Impaired IDO-mediated tryptophan catabolism has been observed in some autoimmune diseases.     

Amplification of Full-Length Hepatitis B Virus Genomes from Samples from Patients with Low Levels of Viremia: Frequency and Functional Consequences of PCR-Introduced Mutations

To facilitate the investigation of hepatitis B virus (HBV) sequence variation, we recently established a method for functional analysis of PCR-amplified full-length HBV genomes. This study aimed at estimating the number of mutations introduced during amplification of genomes from samples from patients with low levels of viremia and their influence on replication and antigen expression. Wild-type HBV DNA template molecules in concentrations like those present in samples from patients with very low levels of viremia were amplified, sequenced (30 kb total), and functionally tested. We found that Taq polymerase and a Taq-Pwo polymerase mixture introduced an average of 5.7 and 3.1 mutations per genome, respectively, corresponding to polymerase error rates of 12.1 × 10⁻⁵ and 6.0 × 10⁻⁵. One of 8 genomes (12%) amplified with Taq polymerase, but 7 of 17 genomes amplified with Taq-Pwo polymerases (41%), remained replication competent. All replication-competent genomes expressed HBs and HBe antigens and had an average of only 0.9 mutations per genome. In contrast, replication-defective genomes had an average of 5.4 mutations, which frequently also disturbed viral antigen expression. From these data we conclude that many of the replication-competent HBV genomes from a clinical specimen will retain their replication and antigen expression phenotypes even after extensive amplification with Taq-Pwo polymerases. Because replication competence is highly sensitive to random mutations, it is the best marker for the identification of HBV genomes with few or no PCR-introduced mutations.     

Correction to: Clinical Manifestations,Mutational Analysis,and Immunological Phenotype in Patients with RAG1/2 Mutations: First Cases Series from Mexico and Description of Two Novel Mutations

Journal of Clinical Immunology - A Correction to this paper has been published: https://doi.org/10.1007/s10875-021-01075-7     

Relationship between European Mitochondrial Haplogroups and Chronic Renal Allograft Rejection in Patients with Kidney Transplant

Mitochondrial DNA variants may contribute to differences in mitochondrial function, leading to an altered immune system. The aim of this study was to analyze the relationship between mtDNA haplogroups and the development of chronic allograft dysfunction in patients with kidney transplant. A retrospective observational study was carried out on 261 patients who received kidney transplant (114 had stable transplant and 147 patients developed chronic allograft dysfunction). DNA samples were genotyped for 14 mtDNA polymorphisms by using Sequenom''s MassARRAY platform (San Diego, CA, USA). Only European white patients within the N macro-cluster were included. Patients with haplogroups V (odds ratio (OR)=0.32; p=0.037) and J (OR=0.36; p=0.038) showed lower odds for developing CRAD than patients with haplogroup H. After adjusting for the most significant variables, haplogroups V and J tended to statistical significance (p=0.091 and p=0.067 respectively). This is a preliminary study in which mtDNA haplogroups seem to be implicated in susceptibility or protection for developing chronic allograft dysfunction.     

Expression of G protein αq Subunit is Decreased in Lymphocytes from Patients with Rheumatoid Arthritis and is Correlated with Disease Activity

Gαq, the alpha subunit of Gq, a member of the Gq/11 sub‐family, was reported to inhibit phosphatidylinositol‐3‐Kinase (PI3K) activation and prevent the activation of Akt. Previous studies demonstrated that mice losing Gαq in their immune system could spontaneously develop inflammatory arthritis. In this study, we showed that the Gαq expressions at mRNA and protein levels in the peripheral blood lymphocytes (PBLs) from patients with rheumatoid arthritis (RA) were significantly decreased in comparison of which in healthy individuals. The expression levels of Gαq mRNA in PBLs from patients with RA were correlated with RA disease activity (DAS28), anti‐cyclic citrullinated protein antibodies, C‐reactive protein and rheumatoid factor. We also demonstrated that Gαq controlled the apoptosis of RA PBLs through regulating the activity of Mcl‐1 and caspase‐3. These data suggested that Gαq might be involved in the pathogenesis of RA by regulating PBLs apoptosis.