LOXL3 Function Beyond Amino Oxidase and Role in Pathologies, Including Cancer.

Lysyl oxidase like 3 (LOXL3) is a copper-dependent amine oxidase responsible for the crosslinking of collagen and elastin in the extracellular matrix. LOXL3 belongs to a family including other members: LOX, LOXL1, LOXL2, and LOXL4. Autosomal recessive mutations are rare and described in patients with Stickler syndrome, early-onset myopia and non-syndromic cleft palate. Along with an essential function in embryonic development, multiple biological functions have been attributed to LOXL3 in various pathologies related to amino oxidase activity. Additionally, various novel roles have been described for LOXL3, such as the oxidation of fibronectin in myotendinous junction formation, and of deacetylation and deacetylimination activities of STAT3 to control of inflammatory response. In tumors, three distinct roles were described: (1) LOXL3 interacts with SNAIL and contributes to proliferation and metastasis by inducing epithelial-mesenchymal transition in pancreatic ductal adenocarcinoma cells; (2) LOXL3 is localized predominantly in the nucleus associated with invasion and poor gastric cancer prognosis; (3) LOXL3 interacts with proteins involved in DNA stability and mitosis completion, contributing to melanoma progression and sustained proliferation. Here we review the structure, function and activity of LOXL3 in normal and pathological conditions and discuss the potential of LOXL3 as a therapeutic target in various diseases.

Treatment and outcomes in necrotising autoimmune myopathy: An Australian perspective.

Necrotising Autoimmune Myopathy (NAM) presents as a subacute proximal myopathy with high creatine kinase levels. It is associated with statin exposure, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) antibody, connective tissue diseases, signal recognition particle (SRP) antibody and malignancy. This case series presents our Western Australian NAM patient cohort: comparing the subgroup presentations, biopsy appearance and treatment outcomes. We retrospectively collected data on patients diagnosed with NAM at the Western Australian Neuroscience Research Institute between the years 2000 and 2015. We identified 20 patients with Necrotising Autoimmune Myopathy: 14 with anti-HMGCR antibodies; two with anti-SRP antibodies; three with connective tissue disease; two as yet unspecified. Median creatine kinase level was 6047units/L (range 1000-17000). The statin naïve patients with HMGCR antibodies and patients with SRP antibodies were the most severely affected subgroups, with higher creatine kinase levels, and were more resistant to immunotherapy. Two or more immunotherapy agents were required in 90%; eight patients required IVIG and rituximab. Steroid weaning commonly precipitated relapses. Four patients had complete remission, and the remaining patients still require immunotherapy. Necrotising Autoimmune Myopathy is a potentially treatable myopathy, which can be precipitated by statin therapy and requires early, aggressive immunotherapy, usually requiring multiple steroid sparing agents for successful steroid weaning.

The expression of matrix metalloproteinases in placental tissue depends on the severity of undifferentiated connective tissue dysplasia.

Immunohistochemical study of MMP-2 and MMP-9 expression in placental tissue of pregnant patients with undifferentiated connective tissue dysplasia of different severity showed that more severe condition was associated with higher expression of these MMP, this underlying the development of pregnancy and labor complications. The most pronounced elevation of the studied MMP levels was found in the basal plate decidual cells in women with undifferentiated connective tissue dysplasia of more than 18 score.

Relevance of the inner mitochondrial membrane enzyme F1F0-ATPase as an autoantigen in autoimmune liver disorders.

BACKGROUND AND AIMS: Recently, a non-M2-related mitochondrial 60 kDa protein found to be recognized by antimitochondrial antibody (AMA) negative sera from patients with primary biliary cirrhosis (PBC) has been shown to contain parts of the five F(1)-ATPase subunits α, ß, γ, δ and ε. In this study, we examined whether this enzyme is, indeed, a target antigen in PBC. METHODS: Analysed were 60 AMA-positive/anti-M2-negative and 103 anti-M2-positive PBC patients, 46 patients with autoimmune hepatitis (AIH), 35 patients with primary sclerosing cholangitis (PSC), 110 patients with viral hepatitis, 40 patients with inflammatory bowel diseases (IBD), 33 patients with connective tissue diseases (systemic lupus erythematosus, mixed connective tissue disease, Sjögren disease, systemic sclerosis) and 25 blood donors. The F(1)-ATPase-subunits α-δ were recombinantly expressed in Escherichia coli, purified and applied to ELISA and Western blotting. RESULTS: In all, 40 of the 60 AMA-positive/anti-M2-negative (67%) and 44 (43%) of the 103 anti-M2-positive PBC-sera reacted with at least one of the F(1)-subunits α-δ. The ß- and γ-subunits were preferentially recognized. However, also up to 57% of patients with AIH and 34% of patients with PSC had anti-ß- or γ-antibodies, while patients with viral hepatitis had these antibodies in up to 13%. Patients with IBD had anti-ß and anti-γ-antibodies in up to 20 and 5% respectively. None of the patients with connective tissue diseases had antibodies to the ß- and only 6% to the γ-subunit. Sera from healthy blood donors were negative. CONCLUSIONS: Antibodies to the ß- and γ-subunits of F(1)-ATPase are further AMAs in PBC but occur also in other autoimmune liver disorders; they may be, therefore, indicators for a general autoimmune process of the liver.

Missense mutations that cause Bruck syndrome affect enzymatic activity, folding, and oligomerization of lysyl hydroxylase 2.

Bruck syndrome is a rare autosomal recessive connective tissue disorder characterized by fragile bones, joint contractures, scoliosis, and osteoporosis. The telopeptides of bone collagen I are underhydroxylated in these patients, leading to abnormal collagen cross-linking. Three point mutations in lysyl hydroxylase (LH) 2, the enzyme responsible for the hydroxylation of collagen telopeptides, have been identified in Bruck syndrome. As none of them affects the residues known to be critical for LH activity, we studied their consequences at the molecular level by analyzing the folding and catalytic properties of the corresponding mutant recombinant polypeptides. Folding and oligomerization of the R594H and G597V mutants were abnormal, and their activity was reduced by >95% relative to the wild type. The T604I mutation did not affect the folding properties, although the mutant retained only approximately 8% activity under standard assay conditions. As the reduced activity was caused by a 10-fold increase in the K(m) for 2-oxoglutarate, the mutation interferes with binding of this cosubstrate. In the presence of a saturating 2-oxoglutarate concentration, the activity of the T604I mutant was approximately 30% of that of the wild type. However, the T604I mutant did not generate detectable amounts of hydroxylysine in the N-terminal telopeptide of a recombinant procollagen I chain when coexpressed in insect cells. The low activity of the mutant LH2 polypeptides is in accordance with the markedly reduced extent of collagen telopeptide hydroxylation in Bruck syndrome, with consequent changes in the cross-linking of collagen fibrils and severe abnormalities in the skeletal structures.

Will Jill come tumbling after? The case for a JAK2-type mutation as a prequel to the connective tissue disorders.

The JAK2 [V617F] mutation has recently been recognised as critical to the pathogenesis of the myeloproliferative disorders (MPDs). Thus, a common mutation affecting a haematopoietic precursor stem cell is capable of giving rise to diverse clinical phenotypes. In this hypothesis paper, we propose that a similar mutation affecting a stem cell precursor, most likely of the B cell lineage, could underlie the development of the connective tissue disorders which could be regarded as "lymphoproliferative" disorders. Consistent with this hypothesis is the observation that there are similarities between the myeloproliferative disorders and the connective tissue disorders in terms of their biological behaviour. Diseases within each family can transform into each other and sometimes into haematological malignancies (most often B cell origin non-Hodgkins lymphoma for the connective tissue disorders and acute myeloid leukemia for the myeloproliferative disorders). The timecourse for development of the connective tissue disorders involves a long latent period when autoantibodies are present (anti-CCP and ANA) possibly reflecting production by a B cell clone. A similar time-dependent increase in clonal dominance has been described in erythroblastic clones taken from the bone marrow of polycythemia vera patients, long before the onset of clinical disease. Evidence of B cell clonality has been described in bone marrow samples from rheumatoid arthritis patients and from glandular biopsies from those with Sjogren's syndrome. Moreover, pseudofollicles containing activated B cells are features of rheumatoid synovial membrane and have also recently been described in subchondral bone where they are associated with macrophages, T cells and osteoclasts. The success of B cell depletion therapy in rheumatoid arthritis and systemic lupus erythematosus is also strong circumstantial evidence for this hypothesis.

Telomerase activity in connective tissue diseases: elevated in rheumatoid arthritis, but markedly decreased in systemic sclerosis.

Telomerase is a reverse transcriptase enzyme contributing to the maintenance of the telomeric structure by adding telomere repeat sequences to chromosomal ends, thus compensating for its shortening. Telomerase activity which is common in cancers and human germ line tissue, may also be increased, although to a lesser extent, in systemic autoimmune diseases. We aimed to evaluate telomerase activity in a group of Turkish patients with various connective tissue diseases. In this cross sectional study, 19 patients with systemic sclerosis (SSc), 15 with systemic lupus erythematosus (SLE), 10 with rheumatoid arthritis (RA) and 14 with primary Sjögren's syndrome (pSjS) were studied. As the control group, 29 healthy subjects were also included. Human telomerase-specific reverse transcriptase (hTERT) was measured in peripheral blood lymphocytes, using online real-time reverse-transcriptase polymerase chain reaction (PCR). We also investigated if hTERT values in each patient group were correlated with clinical parameters and disease activity. Highest hTERT values were observed in RA group (21.24 +/- 28.54), followed by SLE (13.38 +/- 26.05) and pSjS (11.73 +/- 10.59) groups. Only hTERT values in RA group was significantly higher than the healthy control group (7.62 +/- 4.21) (p < 0.05). Interestingly, hTERT values in SSc were very low (2.09 +/- 3.18), even less than the healthy control group. In consistent with previous studies, telomerase activity was increased in SLE and RA. Very low telomerase activity in SSc group was rather surprising. Since existing telomerase data in SSc was limited and telomere shortening was previously reported in SSc, our finding of low telomerase activity in SSc group deserves relevant discussion and further studies.

Tumour necrosis factor alpha, lipid peroxidation and NO* are increased and associated with decreased free-radical scavenging enzymes in patients with Weill-Marchesani syndrome.

AIM: Weill-Marchesani syndrome (WMS) is a rare systemic disorder with both autosomal recessive and dominant inheritances. Accumulation of reactive oxygen species such as O2*-, H2O2 and OH* causes lipid peroxidation (LPO), whereas antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GSHPx)) mediate defence against oxidative stress. Excess tumour necrosis factor (TNF)-alpha and NO* react with O2*- and cause further antioxidant depletion with an increase in mutation frequency by H2O2. This study investigated the levels of SOD, GSHPx, catalase (CAT), TNF-alpha, NO and LPO in patients with WMS. METHODS: A group of 10 WMS patients (four males, six females; age, 26.5+/-19.0 years) and 10 age-matched and sex-matched controls (five males, five females; age, 27.3+/-18.2 years) were included. Serum TNF-alpha levels were determined by a spectrophotometer technique using immulite chemiluminescent immunometric assay. Malondialdehyde (MDA) was determined in plasma; CAT in red blood cells (RBCs), and SOD and GSHPx in both plasma and RBCs. Total serum NO* levels were evaluated by Griess reaction. RESULTS: Mean levels of TNF-alpha (8.3+/-0.6 pg/ml) in WMS patients were significantly (p<0.001) higher than controls (4.3+/-0.2 pg/ml). Plasma MDA levels in patients and controls were 5.4+/-0.8 and 1.8+/-0.6 micromol/l, respectively, and the difference was significant (p=0.0002). SOD and GSHPx activities were significantly lower in both RBCs and plasma of WMS than in controls (RBC-SOD, 3981.9+/-626.6 versus 5261.6+/-523.0 U/g haemoglobin (Hb), p=0.0005; plasma-SOD, 529.4+/-49.3 versus 713.4+/-55.7 U/g protein, p=0.0002; RBC-GSHPx, 682.7+/-42.0 versus 756.5+/-47.6 U/g Hb, p=0.0011; plasma-GSHPx, 107.3+/-15.0 versus 131.4+/-19.7 U/g protein, p=0.0113). In addition, serum NO (NO*-2 + NO*-3) levels were also significantly (p = 0.0002) increased in WMS patients (54.4+/-5.7 versus 26.9+/-6.7 micromol/l). RBC-CAT levels were similar between groups (125.6+/-21.3 versus 131.0+/-21.5 k/g Hb, p = 0.8798). CONCLUSIONS: The elevated LPO, TNF-alpha and NO* with decreased antioxidant enzyme activities indicated impaired antioxidative defence mechanisms with an oxidative injury and cell toxicity in WMS patients. The use of multiple antioxidants and free radical scavengers might be helpful in this genetic disorder.

Telomerase activity in peripheral blood mononuclear cells of systemic connective tissue diseases.

OBJECTIVE: Telomerase activity has been detected in a large number of cancers, as well as human germline tissue, but is absent in most normal somatic tissue. It has been reported that telomerase is also expressed at a low level in normal peripheral blood lymphocytes and that its activity is increased by antigen processing. We investigated if the telomerase activity in peripheral blood mononuclear cells (PBMC) from patients with systemic connective tissue diseases reflects disease activity. METHODS: We examined the enzyme activity of PBMC from patients with systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), primary Sjögren's syndrome (SS), and systemic sclerosis (SSc) using the telomeric repeat amplification protocol. In SLE and SS patients, the telomerase activity level was assessed for correlations with clinical disease activity. RESULTS: Telomerase activity was detected in 64.7% (11/17) of SLE patients, 63.6% (7/11) of MCTD, 54.5% (6/11) of SS, and 44.4% (4/9) of SSc. There was a significant correlation between SLE Disease Activity Index scores and telomerase activity in patients with SLE (p < 0.01). In patients with SS, telomerase activity was detected predominantly in patients with extraglandular manifestations, but was less detectable in patients without extraglandular manifestations (p < 0.05). CONCLUSION: Our data show that telomerase activity of PBMC is an indicator of disease activity, and may play a role in pathogenesis of systemic connective tissue diseases.

Interstitial lung disease with autoantibodies against aminoacyl-tRNA synthetases in the absence of clinically apparent myositis.

Autoantibodies against aminoacyl-tRNA synthetases (antisynthetases) have been found to be highly specific for polymyositis and dermatomyositis and to correlate strongly with complicating interstitial lung disease (ILD). We describe the clinical presentations and course of 10 patients with ILD and anti-synthetase antibodies in whom underlying myositis was not clinically evident. Anti-PL-12 antibodies (antialanyl-tRNA synthetase) were most common (60%), followed by anti-Jo-1 (antihistidyl-tRNA synthetase) and anti-OJ (anti-isoleucyl-tRNA synthetase) (20% each). All 10 patients had anticytoplasmic antibodies by indirect immunofluorescence on HEp-2 cells. Five of 10 presented with features of connective tissue disease, whereas two presented with acute respiratory failure, two with insidious onset of diminished exercise tolerance, and one with persistent cough. All but one patient received corticosteroids, four were given oral cyclophosphamide, and two azathioprine. ILD resolved or stabilized in five patients (50%), and progressed in four (40%). The "antisynthetase syndrome" may occur in the absence of clinical myositis, and the ILD in these patients is usually responsive to therapy. Antisynthetase testing should be considered in patients with ILD who have a cytoplasmic pattern by antinuclear antibody (ANA) testing on HEp-2 cells, because early recognition and treatment of such patients affects their clinical course.

Anti-protein kinase NII antibodies in rheumatic autoimmune diseases.

Antibodies against nuclear protein kinase NII were detected by radioimmunoassay in the sera of patients with systemic lupus erythematosus and mixed connective tissue disease but not in sera from normal individuals or from patients with rheumatoid arthritis. Immunoglobulins derived from sera containing the anti-kinase antibodies inhibited the activity of protein kinase NII in vitro but had no effect on the activity of cytoplasmic cyclic AMP-dependent protein kinase.

[Capacity for differentiation and normalization of tumor cell populations transplanted into the anterior chamber of the eye. II. Changes in metastasizing polymorphonuclear rhabdomyosarcoma]. / Sposobnost' k differentsirovke i normalizatsii populiatsii opukholevykh kletok pri transplantatsii v peredniuiu kamru glaza. II. Izmeneniia metastaziruiushchei polimorfnokletochnoi rabdomiosarkomy.

Polymorphic CC57W mice rhabdomyosarcoma MC-53, after selection for malignancy, was investigated during the transplantation to subcutaneous connective tissue (SCT) and the eye anterior chamber (EAC). SCT and EAC transplants appeared to be equal both morphologically and ultrastructurally. Relationship between M- and H-forms of LDH, and the karyotype structure of tumor cell populations were invariably similar in SCT and EAC. However, after EAC proliferation, in contrast to SCT proliferation, transplantability of tumor rhabdomyoblasts decreased, which may be associated with a decrease in proliferative activity of tumor cells in the EAC. Our data allow to suppose that mouse rhabdomyosarcoma MC-53 cells, during selection for malignancy, lost its capacity of differentiating and normalizing in EAC, unlike the previously investigated rhabdomyosarcomas MC-62, MC-III and A-7. These results may be explained by the fast progression of this tumor line. It is obvious that capacity of tumor cells of differentiating and normalizing during the EAC proliferation may depend on the tumor histological type, on the retention capacity of tumor cell elements of differentiating and on the stage of progression.