Prothrombotic state and impaired fibrinolysis in bullous pemphigoid,the most frequent autoimmune blistering disease

Bullous pemphigoid (BP) is a potentially life‐threatening autoimmune blistering disease that is burdened with an increased risk of cardiovascular events. In BP, there is an interplay between inflammation and coagulation both locally, which contributes to skin damage, and systemically, which leads to a prothrombotic state. Fibrinolysis is an important defence mechanism against thrombosis, but has only been studied locally in BP and no systemic data are available. The aim of this observational study was to evaluate systemic fibrinolysis and coagulation activation in patients with BP. We measured parameters of fibrinolysis and coagulation by immunoenzymatic methods in plasma from 20 patients with BP in an active phase and during remission after corticosteroid treatment. The controls were 20 age‐ and sex‐matched healthy subjects. Plasma levels of plasminogen activator inhibitor type 1 (PAI‐1) antigen, PAI‐1 activity and tissue plasminogen activator (t‐PA) antigen were significantly higher in the BP patients with active disease than in healthy controls (P = 0·0001 for all), as were the plasma levels of the fibrin fragment d ‐dimer and prothrombin fragment F1+2 (P = 0·0001 for both). During remission after treatment, levels of PAI‐1 antigen and PAI‐1 activity decreased significantly (P = 0·008 and P = 0·006, respectively), and there was also a significant decrease in plasma levels of d ‐dimer (P = 0·0001) and F1+2 (P = 0·0001). Fibrinolysis is inhibited in patients with active BP, due mainly to an increase in plasma levels of PAI‐1. Corticosteroids not only induce the regression of BP lesions, but also reduce the inhibition of fibrinolysis, which may contribute to decreasing thrombotic risk.     

The level of urinary secretory immunoglobulin A (sIgA) of patients with IgA nephropathy is elevated and associated with pathological phenotypes

Recent studies have demonstrated deposition of secretory immunoglobulin A (sIgA) in glomeruli of some patients with IgA nephropathy (IgAN). The aim of this study is to investigate the levels of urinary sIgA in IgAN patients with different pathological phenotypes and whether it could be used as a non‐invasive biomarker for assessment of kidney injury in IgAN. Urine samples from 202 patients with IgAN were collected on the day of renal biopsy. Forty‐eight fulfilled the histopathological criteria of Haas‐I or II (group 1), 60 fulfilled Haas‐III (group 2) and 94 patients fulfilled Haas‐IV or V (group 3). Urine samples from 60 healthy sex‐ and age‐matched volunteers with negative urinalysis were collected as normal controls. Urinary sIgA was detected by sandwich enzyme‐linked immunosorbent assay and was corrected by urinary creatinine. In comparison with normal controls, the levels of urinary sIgA were significantly higher in IgAN [2·22 (0–43·82) μg/mg Cr versus 1·08 (0–16·49) μg/mg Cr, P < 0·001]. The levels of urinary sIgA were significantly higher in group 3 than that in group 2 and group 1 [3·54 (0–43·82) μg/mg Cr versus 1·63 (0–15·88) μg/mg Cr versus 0·91 (0–11·79), P < 0·001], and group 2 than group 1 (P = 0·014). The levels of urinary sIgA were associated positively with proteinuria (r = 0·443, P < 0·001), serum creatinine (r = 0·376, P < 0·001) and histopathological parameters, such as ratio of global sclerosis (r = 0·356, P < 0·001), ratio of total crescents (r = 0·339, P < 0·001) and ratios of cellular crescents (r = 0·231, P < 0·001). The levels of urinary sIgA were associated closely with histopathological phenotypes of IgAN and might be used as a non‐invasive biomarker to evaluate kidney injury in IgAN.     

Urinary B cell activating factor (BAFF) and a proliferation‐inducing ligand (APRIL): potential biomarkers of active lupus nephritis

B cell activating factor (BAFF) and a proliferation‐inducing ligand (APRIL) help in B cell activation, maintenance and plasma cell survival. B cell infiltration has been demonstrated in kidneys of patients with lupus nephritis (LN). Serum levels of BAFF and APRIL have shown inconsistent relationships with lupus disease activity. We evaluated urinary levels of BAFF and APRIL as biomarker for LN. Thirty‐six patients with proliferative lupus nephritis (AN), 10 with active lupus without nephritis (AL) and 15 healthy controls (HC) were studied. APRIL and BAFF levels were measured in both serum and urine using enzyme‐linked immunosorbent assay (ELISA). Urine levels were normalized for urinary creatinine excretion. Urine levels were correlated with conventional disease activity markers and histology. Levels were reassessed in 20 AN patients at 6 months after treatment with cyclophosphamide. Urinary APRIL (uAPRIL) and BAFF (uBAFF) levels were raised significantly in AN. uAPRIL, but not uBAFF, correlated moderately with renal Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) in AN (r = 0·36, P < 0·05). On receiver operator curve (ROC) analysis, uBAFF and uAPRIL showed an area under the curve (AUC) of 0·825 and 0·781, respectively, in differentiating between nephritis and non‐nephritis, which performed better than low C3, C4 and raised anti‐dsDNA antibodies. There was no correlation of serum levels with uBAFF (r = 0·187, P = 0·261) and uAPRIL (r = 0·114, P = 0·494). uAPRIL levels reduced after treatment (mean 125 pg/mg to 36 pg/mg, P < 0·05). uBAFF levels reduced in 16 responders while two of four non‐responders had increase in levels. Thus, uBAFF and uAPRIL are potential biomarkers of proliferative lupus nephritis.     

Plasma levels of MASP‐1, MASP‐3 and MAp44 in patients with type 2 diabetes: influence of glycaemic control,body composition and polymorphisms in the MASP1 gene

Mounting evidence indicates that adverse activation of the complement system plays a role in the development of diabetic vascular complications. Plasma levels of the complement proteins mannan‐binding lectin (MBL) and its associated serine proteases (MASP‐1 and MASP‐2) are elevated in diabetes. We hypothesized that single nucleotide polymorphisms (SNPs) in the MASP1 gene may contribute to altered plasma levels of the belonging gene products; MASP‐1, MASP‐3 and mannan‐binding lectin‐associated protein of 44 kDa (MAp44) in patients with type 2 diabetes. To investigate this, we compared plasma levels of MASP‐1, MASP‐3 and MAp44 in 100 patients with type 2 diabetes and 100 sex‐ and age‐matched controls. Ten carefully selected SNPs were analysed using TaqMan^® genotyping assay. Additionally, we included a streptozotocin‐induced diabetes mouse model to directly examine the effect of inducing diabetes on MASP‐1 levels. MASP‐1 levels were significantly higher among patients with type 2 diabetes compared with healthy controls (P = 0·017). Five SNPs (rs874603, rs72549254, rs3774275, rs67143992, rs850312) in the MASP1 gene were associated with plasma levels of MASP‐1, MASP‐3 and MAp44. In the diabetes mouse model, diabetic mice had significantly higher MASP‐1 levels than control mice (P = 0·003). In conclusion, MASP‐1 levels were higher among patients with type 2 diabetes and diabetic mice. The mechanism behind this increase remains elusive.     

Changes in the levels of some acute-phase proteins in human immunodeficiency virus-1 infected patients, following interleukin-2 treatment

Intermittent interleukin (IL)‐2 administration to human immunodeficiency virus (HIV)‐1 infected patients is well documented and generally used, but there is limited information about the changes of acute‐phase protein (APP) levels in response to this treatment. Fifteen patients undergoing highly active anti‐retroviral therapy (HAART) treatment, with undetectable viral load, but low CD4⁺ cell count (<300/µl), have been treated with 3·6 M IU Proleukine® administered twice daily by subcutaneous injection over 5 days. C‐reactive protein (CRP), d ‐dimer, C3, C9, C1‐inh and alpha‐2HS glycoprotein levels were measured immediately before IL‐2 administration, as well as on day 5 and 2–3 weeks thereafter. After IL‐2 administration, both mean d ‐dimer and CRP levels increased significantly (P < 0·001), but returned (P < 0·001) to baseline within the subsequent 2–3 weeks. Alpha‐2HS glycoprotein decreased immediately after IL‐2 administration. No significant differences were detected in the levels of C3, C9 and C1‐inh. A significant, positive correlation (r = 0·5178, P = 0·0008) was ascertained between the changes of CRP level, measured immediately before as well as 5 days after IL‐2 administration, and changes in CD4 T cell counts measured 2–3 weeks before and after treatment, respectively. IL‐2 administration induces rapid elevation of two major APPs (CRP, d ‐dimer). The positive correlation observed between the changes of CRP levels and CD4⁺ cell counts after IL‐2 administration may indicate that the abrupt, but transitory overproduction of CRP might contribute to the CD4⁺ cell count‐increasing effect of the drug and/ or may be associated with serious side effects.     

Separation of plasma‐derived exosomes into CD3(+) and CD3(–) fractions allows for association of immune cell and tumour cell markers with disease activity in HNSCC patients

Head and neck squamous cell carcinoma (HNSCC) is a highly immunosuppressive malignancy. Exosomes in HNSCC patients' plasma are enriched in inhibitory cargo and mediate immunosuppression. As these exosomes are products of various cells, the cellular origin of immunoregulatory proteins they carry is unknown. To test whether tumour‐ or T cell‐derived exosomes in patients' plasma are immunosuppressive and impact upon disease activity, we separated CD3^(–) from CD3⁽⁺⁾ exosomes by immunocapture using anti‐CD3 antibodies. The exosome protein cargo was evaluated for immunoregulatory proteins using on‐bead flow cytometry. Tumour protein‐enriched CD3^(–) exosomes were CD44v3⁽⁺⁾. Surprisingly, mean levels of programmed death ligand 1 (PD‐L1), cytotoxic T lymphocyte antigen 4 (CTLA‐4) and cyclooxygenase‐2 (COX‐2) were similar in CD3⁽⁺⁾ and CD3^(–) exosomes, although the latter induced higher (P < 0·0025) ex‐vivo apoptosis of CD8⁽⁺⁾ T cells and greater (P < 0·005) conversion of CD4⁺ T cells to CD4⁽⁺⁾CD39⁽⁺⁾ regulatory T cells (T_reg). CD3⁽⁺⁾ and CD3^(–) exosomes carrying high levels of immunosuppressive proteins were highly effective in mediating these functions. Exosomes of patients with Union for International Cancer Control (UICC) stages III/IV disease had higher levels of PD‐L1 and COX‐2 than stages I/II patients (P < 0·005). Patients with nodal involvement had exosomes with the higher inhibitory protein content than N0 patients (P < 0·03). CD3⁽⁺⁾ and CD3^(–) exosomes of HNSCC patients had higher PD‐L1, COX‐2 and CD15s levels than healthy donors' exosomes (P < 0·009), although levels of immunostimulatory OX40 or OX40L were not different. By isolating CD3^(–)/CD44v3‐enriched and CD3⁽⁺⁾ exosomes from plasma, the cellular origins of immunoregulatory proteins they carry were identified. Association of exosome molecular profiles with disease progression supports the exosome potential as future cancer biomarkers.     

Effect of matrix metalloproteinase‐21 (572C/T) polymorphism on HIV‐associated neurocognitive disorder

Remodeling of extracellular matrix (ECM) by matrix metalloproteinases (MMPs) is a presumed reason for the development of HIV‐associated neurocognitive disorders (HAND). The coding region polymorphism in MMP‐21 572C/T gene may have a potential functional effect on ECM remodeling. Hence, we aimed to examine the association of MMP‐21 polymorphism with the modulation of HAND severity and its prevalence in HIV‐infected and healthy individuals. Genotyping of MMP‐21 572C/T polymorphism was performed by PCR‐RFLP in total 150 HIV‐infected individuals, 50 with HAND, 100 without HAND and 150 healthy controls. MMP‐21 572TT genotype was predominantly higher in HAND patients compared with no HAND (OR = 1.63, p = 0.57). MMP‐21 572T allele was associated with reduce risk for HAND severity (OR = 0.50, p = 0.04). Similarly, MMP‐21 572TT genotype underrepresented in HIV‐infected individuals compared to healthy controls (3.0% vs 6.7%, OR = 0.27, p = 0.08). MMP‐21 572CT genotype and early HIV disease stage showed a higher risk for the advancement of HIV disease with marginal significance (OR = 1.89, p = 0.07). MMP‐21 572CT genotype increased the risk for the modulation of HAND severity in tobacco users (OR = 1.98, p = 0.43). MMP‐21 572CT genotype among tobacco and alcohol users showed elevated risk for the development of HAND in HIV‐infected individuals (OR = 2.30, p = 0.15; OR = 1.86, p = 0.23). Similarly, MMP‐21 572TT genotype enhanced the risk for the development of HAND in tobacco users (OR = 3.48, p = 0.40). In conclusion, the presence of coding region 572T allele may have protection for HAND severity. MMP‐21 572C/T polymorphism and tobacco and alcohol usage may facilitate the development of HAND.     

Role of inflammatory cells, cytokines and matrix metalloproteinases in neutrophil-mediated skin diseases

Pyoderma gangrenosum (PG) is a rare, immune‐mediated inflammatory skin disease presenting with painful ulcers having undermined edges. Less commonly, bullous and vegetative variants exist. Histology consists of a neutrophil‐rich dermal infiltrate. We characterized immunohistochemically the infiltrate in different variants of PG and in another neutrophilic dermatosis as Sweet's syndrome. We studied 21 patients with PG, eight with Sweet's syndrome and 20 controls, evaluating skin immunoreactivity for inflammatory cell markers (CD3, CD163 and myeloperoxidase), cytokines [tumour necrosis factor (TNF)‐α, interleukin (IL)‐8 and IL‐17], metalloproteinases (MMP‐2 and MMP‐9) and vascular endothelial growth factor (VEGF). Immunoreactivities of CD3, CD163, myeloperoxidase, TNF‐α, IL‐8, IL‐17, MMP‐2, MMP‐9 and VEGF were significantly higher in both PG and Sweet's syndrome than in controls (P = 0·0001). Myeloperoxidase (neutrophil marker), IL‐8 (cytokine chemotactic for neutrophils) and MMP‐9 (proteinase‐mediating tissue damage) were expressed more significantly in both ulcerative and bullous PG than in vegetative PG as well as in Sweet's syndrome (P = 0·008–P = 0·0001). In ulcerative PG, the expression of CD3 (panT cell marker) and CD163 (macrophage marker) were significantly higher in wound edge than wound bed (P = 0·0001). In contrast, the neutrophil marker myeloperoxidase was expressed more significantly in wound bed than wound edge (P = 0·0001). Our study identifies PG as a paradigm of neutrophil‐mediated inflammation, with proinflammatory cytokines/chemokines and MMPs acting as important effectors for the tissue damage, particularly in ulcerative and bullous PG where damage is stronger. In ulcerative PG, the wound bed is the site of neutrophil‐recruitment, whereas in the wound edge activated T lymphocytes and macrophages pave the way to ulcer formation.     

Activation profile of Toll‐like receptors of peripheral blood lymphocytes in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease associated with aberrant activation of T and B lymphocytes for the production of inflammatory cytokines and autoreactive antibodies. Animal studies of SLE have indicated that Toll‐like receptors (TLR) are important in the pathogenesis of murine lupus. In the present clinical study, differential protein expressions of TLR‐1–9 of monocytes and different lymphocyte subsets from patients with SLE and normal control subjects were determined by flow cytometry. Results showed that the expression of intracellular TLRs (TLR‐3, ‐8, ‐9) and extracellular TLRs (TLR‐1, ‐2, ‐4, ‐5, ‐6) were elevated in monocytes, CD4⁺ T lymphocytes, CD8⁺ T lymphocytes and B lymphocytes of SLE patients compared to control subjects (all P < 0·001). Moreover, cell surface expression of TLR‐4 on CD4⁺ T lymphocytes and CD8⁺ T lymphocytes, and TLR‐6 on B lymphocytes, were correlated positively with SLE disease activity index (SLEDAI) (TLR‐4 on CD4⁺ T lymphocytes and CD8⁺ T lymphocytes: r = 0·536, P = 0·04; r = 0·713, P = 0·003; TLR‐6 in B lymphocytes: r = 0·572, P = 0·026). In concordance with the above results, there is an observable increased relative induction (%) of inflammatory cytokine interleukin (IL)‐1β, IL‐6, IL‐10 and IL‐12, chemokines CCL2, CXCL8, CCL5 and CXCL10 from peripheral blood mononuclear cells (PBMC) upon differential stimulation by PolyIC (TLR‐3 ligand), lipopolysaccharide (TLR‐4 ligand), peptidoglycan (TLR‐2 ligand), flagellin (TLR‐5 ligand), R837 (TLR‐7 ligand) and CpG DNA (TLR‐9 ligand) in SLE patients compared to controls. These results suggest that the innate immune response for extracellular pathogens and self‐originated DNA plays immunopathological roles via TLR activation in SLE.     

Plasma levels of matrix metalloproteinase‐9 in chronic urticaria patients correlate with disease severity and C‐reactive protein but not with circulating histamine‐releasing factors

Background Matrix metalloproteinase‐9 (MMP‐9) is an endopeptidase produced by many inflammatory cells that has been found in increased amounts in plasma from patients with chronic urticaria (CU). Objective To evaluate plasma levels of MMP‐9 and its tissue inhibitor of metalloproteinase‐1 (TIMP‐1) in CU patients in relation with disease severity, C‐reactive protein (CRP) and circulating histamine‐releasing factors. Methods Fifty‐two consecutive CU patients were included in the study and disease activity was graded from 0 to 3. Plasma MMP‐9, TIMP‐1 and CRP levels were measured by enzyme immunoassays. Circulating histamine‐releasing factors were assessed using in vivo (autologous serum skin test) and in vitro (basophil histamine release) tests. Seven CU patients were studied both during active disease and during remission. Thirty healthy subjects were used as normal controls. Results Plasma levels of MMP‐9, TIMP‐1 and CRP were significantly higher in CU patients than in healthy controls (P=0.0001, 0.003 and 0.005, respectively) and a trend towards a higher MMP‐9/TIMP‐1 molar ratio was found (P=0.051). A significant correlation was found between plasma MMP‐9 levels and urticaria severity score (r=0.48, P<0.0001). CRP levels correlated with MMP‐9 levels (r=0.37, P=0.008) and CU severity score (r=0.52, P=0.0001), but not with TIMP‐1 (r=0.13) concentrations. MMP‐9, TIMP‐1 and CRP plasma levels and MMP‐9/TIMP‐1 molar ratio did not differ in patients either with or without an evidence of circulating histamine‐releasing factors. Seven patients evaluated during remission showed a significant reduction of MMP‐9 and CRP plasma levels. Conclusion Plasma levels of MMP‐9 and its inhibitor TIMP‐1 are increased in CU patients. MMP‐9 levels are associated with disease severity and CRP levels, but not with skin reactivity to autologous serum and with circulating histamine‐releasing factors. These findings suggest that in CU there is an ongoing inflammatory process independent of the presence of circulating histamine‐releasing factors.     

Associations of B cell‐activating factor (BAFF) and anti‐BAFF autoantibodies with disease activity in multi‐ethnic Asian systemic lupus erythematosus patients in Singapore

To measure the levels of B cell‐activating factor (BAFF) and endogenous anti‐BAFF autoantibodies in a cohort of multi‐ethnic Asian systemic lupus erythematosus (SLE) patients in Singapore, to determine their correlation with disease activity. Serum samples from 121 SLE patients and 24 age‐ and sex‐matched healthy controls were assayed for BAFF and anti‐BAFF immunoglobulin (Ig)G antibody levels by enzyme‐linked immunosorbent assay (ELISA). The lowest reliable detection limit for anti‐BAFF‐IgG antibody levels was defined as 2 standard deviations (s.d.) from blank. Correlation of serum BAFF and anti‐BAFF IgG levels with disease activity [scored by SLE Activity Measure revised (SLAM‐R)], and disease manifestations were determined in these 121 patients. SLE patients had elevated BAFF levels compared to controls; mean 820 ± 40 pg/ml and 152 pg ± 45/ml, respectively [mean ± standard error of the mean (s.e.m.), P < 0·01], which were correlated positively with anti‐dsDNA antibody levels (r = 0·253, P < 0·03), and SLAM‐R scores (r = 0·627, P < 0·01). In addition, SLE patients had significantly higher levels of anti‐BAFF IgG, which were correlated negatively with disease activity (r = –0·436, P < 0·01), levels of anti‐dsDNA antibody (r = –0·347, P < 0·02) and BAFF (r = –0·459, P < 0·01). The majority of patients in this multi‐ethnic Asian SLE cohort had elevated levels of BAFF and anti‐BAFF antibodies. Anti‐BAFF autoantibody levels correlated negatively with clinical disease activity, anti‐dsDNA and BAFF levels, suggesting that they may be disease‐modifying. Our results provide further information about the complexity of BAFF pathophysiology in different SLE disease populations and phenotypes, and suggest that studies of the influence of anti‐cytokine antibodies in different SLE populations will be required when selecting patients for trials using targeted anti‐cytokine therapies.     

Infliximab therapy balances regulatory T cells,tumour necrosis factor receptor 2 (TNFR2) expression and soluble TNFR2 in sarcoidosis

Sarcoidosis is a systemic granulomatous disease of unknown aetiology that most commonly affects the lungs. Although elevated levels of regulatory T cells (T_regs) have been reported, the extent to which they play a role in sarcoidosis pathogenesis remains unclear. Tumour necrosis factor (TNF) is thought to be one of the driving forces behind granuloma formation, illustrated by the efficacy of infliximab in severe sarcoidosis. T_regs express TNF receptor 2 (TNFR2) highly. Here, we examined the influence of infliximab therapy on T_regs and (soluble) TNFR2 levels in sarcoidosis, and correlated these with response to therapy. We observed that relative frequencies of T_regs were significantly higher in patients (n = 54) compared to healthy controls (n = 26; median 6·73 versus 4·36%; P < 0·001) and decreased following therapy (4·95; P < 0·001). Baseline TNFR2 expression on T_regs was increased significantly in patients versus controls (99·4 versus 96·2%; P = 0·031), and also in responders to therapy versus non‐responders (99·6 versus 97·3%; P = 0·012). Furthermore, baseline soluble TNFR2 (sTNFR2) was higher in responders than in non‐responders (mean 174 versus 107 pg/ml; P = 0·015). After treatment, responders showed a significant reduction in sTNFR2 levels in peripheral blood (?44·7 pg/ml; P < 0·001), in contrast to non‐responders (+3·59 pg/ml). Our results demonstrated that T_reg frequencies and TNFR2 expression on T_regs are increased in sarcoidosis, followed by a decline during infliximab therapy, suggesting a pathophysiological role of this T cell subset. Interestingly, sTNFR2 levels at baseline differed significantly between responders and non‐responders, making it a potential marker in predicting which patients might benefit from infliximab.     

Evaluation of membrane‐bound and soluble forms of human leucocyte antigen‐G in systemic sclerosis

Systemic sclerosis (SSc) is a complex disease characterized by immune dysregulation, extensive vascular damage and widespread fibrosis. Human leucocyte antigen‐G (HLA‐G) is a non‐classic class I major histocompatibility complex (MHC) molecule characterized by complex immunomodulating properties. HLA‐G is expressed on the membrane of different cell lineages in both physiological and pathological conditions. HLA‐G is also detectable in soluble form (sHLA‐G) deriving from the shedding of surface isoforms (sHLA‐G1) or the secretion of soluble isoforms (HLA‐G5). Several immunosuppressive functions have been attributed to both membrane‐bound and soluble HLA‐G molecules. The plasma levels of sHLA‐G were higher in SSc patients (444·27 ± 304·84 U/ml) compared to controls (16·74 ± 20·58 U/ml) (P < 0·0001). The plasma levels of transforming growth factor (TGF)‐β were higher in SSc patients (18 937 ± 15 217 pg/ml) compared to controls (11 099 ± 6081 pg/ml; P = 0·003), and a significant correlation was found between TGF‐β and the plasma levels of total sHLA‐G (r = 0·65; P < 0·01), sHLA‐G1 (r = 0·60; P = 0·003) and HLA‐G5 (r = 0·47; P = 0·02). The percentage of HLA‐G‐positive monocytes (0·98 ± 1·72), CD4⁺ (0·37 ± 0·68), CD8⁺ (2·05 ± 3·74) and CD4⁺CD8⁺ double‐positive cells (14·53 ± 16·88) was higher in SSc patients than in controls (0·11 ± 0·08, 0·01 ± 0·01, 0·01 ± 0·01 and 0·39 ± 0·40, respectively) (P < 0·0001). These data indicate that in SSc the secretion and/or shedding of soluble HLA‐G molecules and the membrane expression of HLA‐G by peripheral blood mononuclear cells (PBMC) is clearly elevated, suggesting an involvement of HLA‐G molecules in the immune dysregulation of SSc.     

Effects of adjuvants for human use in systemic lupus erythematosus (SLE)‐prone (New Zealand black/New Zealand white) F1 mice

The safety of four different adjuvants was assessed in lupus‐prone New Zealand black/New Zealand white (BW)F₁ mice. Four groups of mice were injected intraperitoneally with incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), squalene (SQU) or aluminium hydroxide (ALU). An additional group received plain phosphate‐buffered saline (PBS) (UNT group). Mice were primed at week 9 and boosted every other week up to week 15. Proteinuria became detectable at weeks 17 (IFA group), 24 (CFA group), 28 (SQU and ALU groups) and 32 (UNT group). Different mean values were obtained among the groups from weeks 17 to 21 [week 17: one‐way analysis of variance (anova ) P = 0·016; weeks 18 and 19: P = 0·048; weeks 20 and 21: P = 0·013] being higher in the IFA group than the others [Tukey's honestly significant difference (HSD) post‐test P < 0·05]. No differences in anti‐DNA antibody levels were observed among groups. Anti‐RNP/Sm antibody developed at week 19 in only one CFA‐treated mouse. Mean mouse weight at week 18 was lower in the ALU group than the IFA (Tukey's HSD post‐test P = 0·04), CFA (P = 0·01) and SQU (P < 0·0001) groups, while the mean weight in the SQU group was higher than in the IFA (P = 0·009), CFA (P = 0·013) and UNT (P = 0·005) groups. The ALU group weight decreased by almost half between weeks 29 and 31, indicating some toxic effect of ALU in the late post‐immunization period. Thus, SQU was the least toxic adjuvant as it did not (i) accelerate proteinuria onset compared to IFA; (ii) induce toxicity compared to ALU or (iii) elicit anti‐RNP/Sm autoantibody, as occurred in the CFA group.     

10‐valent pneumococcal non‐typeable Haemophilus influenzae protein‐D conjugate vaccine (PHiD‐CV) induces memory B cell responses in healthy Kenyan toddlers

Memory B cells are long‐lived and could contribute to persistence of humoral immunity by maintaining the plasma‐cell pool or making recall responses upon re‐exposure to an antigen. We determined the ability of a pneumococcal conjugate vaccine to induce anti‐pneumococcal memory B cells. Frequencies of memory B cells against pneumococcal capsular polysaccharides from serotypes 1, 6B, 14, 19F and 23F were determined by cultured B cell enzyme‐linked immunospot (ELISPOT) in 35 children aged 12–23 months who received pneumococcal non‐typeable Haemophilus influenzae protein‐D conjugate vaccine (PHiD‐CV). The relationships between plasma antibodies and memory B cell frequencies were also assessed. After two doses of PHiD‐CV, the proportion of subjects with detectable memory B cells against pneumococcal capsular polysaccharides increased significantly for serotypes 1 (3–45%; P < 0·01), 19F (21–66%; P < 0·01) and 23F (13–36%; P = 0·02), but not serotypes 6B (24–42%; P = 0·24) and 14 (21–40%; P = 0·06). Correlations between antibodies and memory B cells were weak. Carriage of serotype 19F at enrolment was associated with poor memory B cell responses against this serotype at subsequent time‐points (day 30: non‐carriers, 82% versus carriers, 0%, P < 0·01; day 210: non‐carriers, 72% versus carriers, 33%, P = 0·07). PHiD‐CV is capable of inducing memory B cells against some of the component pneumococcal capsular polysaccharides.     

Serum properdin consumption as a biomarker of C5 convertase dysregulation in C3 glomerulopathy

Properdin (P) stabilizes the alternative pathway (AP) convertases, being the only known positive regulator of the complement system. In addition, P is a pattern recognition molecule able to initiate directly the AP on non‐self surfaces. Although P deficiencies have long been known to be associated with Neisseria infections and P is often found deposited at sites of AP activation and tissue injury, the potential role of P in the pathogenesis of complement dysregulation‐associated disorders has not been studied extensively. Serum P levels were measured in 49 patients with histological and clinical evidence of C3 glomerulopathy (C3G). Patients were divided into two groups according to the presence or absence of C3 nephritic factor (C3NeF), an autoantibody that stabilizes the AP C3 convertase. The presence of this autoantibody results in a significant reduction in circulating C3 (P < 0·001) and C5 levels (P < 0·05), but does not alter factor B, P and sC5b‐9 levels. Interestingly, in our cohort, serum P levels were low in 17 of the 32 C3NeF‐negative patients. This group exhibited significant reduction of C3 (P < 0·001) and C5 (P < 0·001) and increase of sC5b‐9 (P < 0·001) plasma levels compared to the control group. Also, P consumption was correlated significantly with C3 (r = 0·798, P = 0·0001), C5 (r = 0·806, P < 0·0001), sC5b‐9 (r = ?0·683, P = 0·043) and a higher degree of proteinuria (r = ?0·862, P = 0·013). These results illustrate further the heterogeneity among C3G patients and suggest that P serum levels could be a reliable clinical biomarker to identify patients with underlying surface AP C5 convertase dysregulation.