Use of a recombinant protein containing major epitopes of hnRNP G to detect anti-hnRNP G antibodies in dogs with systemic lupus erythematosus

The objective of this study was to express major epitopes of heterogeneous nuclear ribonucleoprotein G (hnRNP G) for detecting anti-hnRNP G antibodies in dogs with systemic lupus erythematosus (SLE). HnRNP G cDNA clone was isolated from HEp-2 cells, and a DNA fragment encoding immunodominant region (residues 189-272) of hnRNP G (hnRNP Gi) was subcloned into pET32 vector to construct a prokaryotic expression plasmid named pEThnRNPGi. After induction, Escherichia coli carrying pEThnRNPGi expressed a recombinant protein of 28 kDa, comprising recombinant hnRNP Gi and fusion tag. Purified recombinant hnRNP Gi protein was further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and its identity was confirmed. Western blot analysis showed that recombinant hnRNP Gi was specifically recognized by anti-hnRNP G positive sera of SLE dogs, and not by negative control sera. In conclusion, recombinant hnRNP Gi protein expressed in this study may serve as a useful reagent to assist in the immunological diagnosis of canine SLE.     

Abnormal activity of ROCK contributes to increased adhesion and migration of peripheral blood T cells from patients with systemic lupus erythematosus

AIM: To investigate the role of Rho kinase (ROCK) in the regulation of adhesion and migration of the T cells from systemic lupus erythematosus (SLE) patients. METHODS: The T cells were isolated by RosettSep T cell purification kit. ROCK activity was assessed by Western blotting. T cell migration was examined by Transwell chambers. RESULTS: Compared with the T cells from healthy controls and rheumatoid arthritis patients, the activity of ROCK in ex vivo T cells from SLE patients was significantly increased. In vitro, the adhesion and migration of the T cells from SLE patients were also increased. Furthermore, the adhesion and migration of the T cells from SLE patients were inhibited by a specific ROCK inhibitor Y27632. CONCLUSION: The results indicate that ex vivo T cells from SLE patients exhibit increased activity of ROCK. Alteration of ROCK activity may contribute to the abnormal adhesion and migration of T cells from SLE patients.     

A retrospective study comparing the histopathological features and response to treatment in two canine nasal dermatoses, DLE and MCP

Canine discoid lupus erythematosus (DLE) and mucocutaneous pyoderma (MCP) have overlapping clinical and histopathological changes, often making diagnosis difficult. Histopathological features of 27 nasal planum biopsies were scored to determine whether DLE and MCP were histopathologically distinguishable. Long-term follow-up, enabling assessment of clinical diagnoses, was available on 15 cases; 11/15 cases were immunomodulatory responsive (ImR) and 4/15 were antibiotic responsive (AbR). Clinical diagnosis, determined by response to treatment for 15/27 cases, was not predictable based on scoring of histopathological features. Distinct histopathological patterns were observed: 2/11 ImR cases had a lymphocyte-rich interface dermatitis. All other cases had the same histopathological changes: a band-like diffuse superficial plasmacytic to lymphoplasmacytic dermatitis +/- focal basal cell damage, but different clinical diagnoses (4/4 AbR, 9/11 ImR). German shepherd dogs/crosses were over-represented (44.4% of the cases) and tended to have more multifocal lesions (41.7% vs. 26.7% of all other breeds). Longer duration of disease was associated with a preponderance of plasmacytic infiltrate (P = 0.026).     

Expression of fractalkine and CX3CR1 in renal tissues of patients with WHO class IV lupus nephritis

AIM: To observe the expression of fractalkine, and its receptor, CX3CR1, in renal tissues of patients with diffuse proliferative lupus glomerulonephritis (WHO class IV), minimal glomerular abnormalities, and normal kidney. Meanwhile, the correlation among the expression of fractalkine, CX3CR1 and CD68-positive macrophages was investigated, and the role of fractalkine and CX3CR1 in the pathogenesis of lupus nephritis was discussed. METHODS: The expressions of fractalkine, CX3CR1 and CD68 were detected immunohistochemically in kidney tissue sections obtained from twenty-one patients with WHO class IV lupus nephritis, eighteen cases with minimal glomerular abnormalities, and eight normal kidneys which were no abnormality under light microscope. RESULTS: (1) Fractalkine was generally indistinguishable in tissue sections from normal kidney and minimal glomerular abnormalities. CX3CR1-positive cells and CD68-positive macrophages were sparsely detected in the glomeruli and in the cortical interstitium. (2) There were considerable CX3CR1-positive cells and macrophages in both the glomeruli and the interstitium in sections from class IV lupus nephritis. The number of CX3CR1-positive cells significantly correlated with the number of macrophages in the glomeruli and in the interstitium respectively (r=0.956, P<0.01 and r=0.965, P<0.01). (3) Significant expression of fractalkine was seen in the cortical renal tubules from class IV lupus nephritis. The percentage of fractalkine-positive tubules significantly correlated with the number of CX3CR1-positive cells and macrophages in the interstitium respectively (r=0.720, P<0.01 and r=0.770, P<0.01). CONCLUSION: These expression patterns show that fractalkine and CX3CR1 may play an important role in the pathogenesis of class IV lupus nephritis.     

Preliminary study of IL-17 signal transduction pathway in peripheral blood mononuclear cells from lupus nephritis patients

AIM: To investigate the role of IL-17 and its signal conduction component-JNK activity in the pathogenesis of LN. METHODS: Peripheral blood mononuclear cells (PBMC) were separated and cultured from 15 cases of active lupus nephritis (LN) patients. IL-6 level was detected by ELISA, IL-6 mRNA was checked with RT-PCR, and JNK activity was measured by Western blot. RESULTS: At same IL-17 end concentrations, there was a much higher level of IL-6 in LN group than in control group (all P<0.05). IL-17 induced a significant elevation of IL-6 mRNA expression and JNK activity in PBMC from LN patients in a time- and dose-dependent manner, which could be blocked markedly by IL-17 monoclonal antibody, mIgG₂₈, and dexamethasone. Much higher IL-6 mRNA expression was observed in LN group than in control group under medium culture or IL-17-conditioned culture (all P<0.01). Under medium culture or IL-17-conditioned culture, there was much higher JNK activity of PBMC in active LN group than that in control group. There was a positive linear correlation between PBMC JNK activity and their SLEDAI or IL-6 mRNA expression level in LN patients (r1=0.638, P<0.01; r2=0.644, P<0.01). CONCLUSION: These results suggest that IL-17 may take part in the initiation and progression of LN through induction of IL-6 overexpression by PBMC,and JNK hyperactivity may be necessary in the IL-17 signal transduction.     

Fish Oil N-3 Fatty Acids Increase Adiponectin and Decrease Leptin Levels in Patients with Systemic Lupus Erythematosus

Cardiovascular disease (CVD) has emerged as an important cause of death in patients with systemic lupus erythematosus (SLE). Reduced adiponectin and elevated leptin levels may contribute to CVD in SLE patients. The purpose of this study was to verify the effects of fish oil (FO) on adiponectin and leptin in patients with SLE. Biochemical and disease activity analysis were performed. Patients with SLE were divided in two groups: patients who used fish oil for four months and patients who did not use fish oil. Patients with SLE who used FO had a significant decrease in SLE disease activity index (SLEDAI) score (p ˂ 0.023) in relation to baseline. SLE patients who used fish oil had increased adiponectin levels (p ˂ 0.026) and decreased leptin levels (p ˂ 0.024) compared to baseline values, whereas there were no differences in adiponectin and leptin levels in patients with SLE who did not use fish oil. In conclusion, the findings of increased serum adiponectin an decreased leptin levels after 120 days in the fish oil group, reinforce the importance of evaluating prospective studies of fish and fish oil fish ingestion on these adipokines in an attempt to decrease cardiovascular risk factors in patients with SLE.     

Comparison of T-lymphocyte proliferation in canine epitheliotropic lymphosarcoma and benign lymphocytic dermatoses

The lesions of patch/plaque cutaneous T-cell lymphosarcoma (CTCL) are similar to many benign inflammatory dermatoses, both histopathologically and clinically. In humans, attempts to develop a simple, reliable immunophenotypic technique to differentiate between the malignant and benign dermatoses have been unsuccessful. The purpose of our study was to determine, using a proliferating cell nuclear antigen (PCNA)/CD3 double immunolabelling technique of paraffin-embedded sections, if the rate of T-cell proliferation in canine CTCL was greater than that of other diseases with non-neoplastic lymphocyte epitheliotropism. We selected cases of patch/plaque (PP) and tumour stage CTCL, erythema multiforme (EM) and cutaneous lupus erythematosus (CLE). We did not find a significant difference in the rate of T-cell proliferation in the epithelia nor the superficial dermis between PP-CTCL and EM. Whereas epithelial T-cell proliferation is significantly higher in PP-CTCL than CLE, it is not diagnostically useful because of overlap in the labelling indices.