Analysis of HLA-G expression in renal tissue in lupus nephritis: a pilot study.

BACKGROUND: The study aimed to investigate whether HLA-G antigen is expressed in the kidneys of patients affected by lupus nephritis (LN) and whether its detection in renal biopsies could be adopted as a marker of treatment response and prognosis. METHODS: Thirty renal biopsies from patients with LN were selected and analyzed through immunohistochemistry. Laboratory and clinical data were retrospectively collected at baseline, 6 and 12 months and at the latest clinical appointment. A number of patients (63.3%) were treated with rituximab (RTX) +/- methylprednisolone in the induction phase. The expression of HLA-G in glomeruli, tubules and infiltrating cells was examined and compared between lupus patients who achieved either complete or partial renal response and those who did not respond to treatment. RESULTS: HLA-G staining was observed in the glomeruli of 20 of 30 samples from patients with LN. The expression of the antigen was detected in podocytes, along glomerular capillary walls, on parietal glomerular epithelial cells and within the juxtaglomerular apparatus. Seventy per cent of patients whose glomeruli expressed HLA-G achieved partial or complete response at 6 months and 75% at the latest available follow up compared with 30% and 40%, respectively, of those who did not show any expression. The pattern of staining in tubules and infiltrating cells was highly variable precluding any clinical correlation. CONCLUSION: This study demonstrates that HLA-G is expressed in renal tissue in LN. Our retrospective data suggest that its expression could correlate with response to treatment.

A triglyceride-rich lipoprotein environment exacerbates renal injury in the accelerated nephrotoxic nephritis model.

Hyperlipidaemia accompanies chronic renal disease either as a consequence of the renal dysfunction or as part of generalized metabolic derangements. Under both situations, the lipid profile is characterized by accumulation of triglyceride-rich lipoproteins (TGRLs). This lipid profile is recognized as a risk factor for cardiovascular complications. Whether it may pose a risk for renal injury as well remains unclear. A hyper-TGRL state was generated in C57BL/6 mice using poloxamer-407 (P-407) and immune complex-mediated renal injury was triggered using the accelerated nephrotoxic nephritis (ANTN) model. The hyper-TGRL animals were hypersensitive to ANTN demonstrated by greater haematuria and glomerular cellularity. These changes were accompanied by increased glomerular accumulation of CD68+ macrophages. The hypersensitive response to ANTN was not seen in low-density lipoprotein receptor knock-out mice fed with a high fat diet, where triglyceride levels were lower but cholesterol levels comparable to those obtained using P-407. These data indicate that a hyper-TGRL state might be more detrimental to the kidneys than low-density lipoprotein-driven hypercholesterolaemia during immune complex-mediated nephritis. We speculate that the hyper-TGRL environment primes the kidney to exacerbated renal damage following an inflammatory insult with increased accumulation of macrophages that may play a key role in mediating the injurious effects.

The exaggerated inflammatory response in Behçet's syndrome: identification of dysfunctional post-transcriptional regulation of the IFN-γ/CXCL10 IP-10 pathway.

The mechanisms underlying the exaggerated inflammatory response in Behçet's syndrome (BS) remain poorly understood. We investigated the response of CD14(+) blood monocytes to interferon (IFN)-γ, focusing on the chemokine CXCL10. Chemokine synthesis and release were analysed at a protein and mRNA level following stimulation with IFN-γ. Findings in BS patients were compared with 25 healthy controls (HC), 15 rheumatoid arthritis (RA) and 15 systemic lupus erythematosus (SLE) disease control patients. BS monocytes produced significantly more CXCL10 protein than HC monocytes from 2 h following IFN-γ stimulation, despite equivalent quantities of mRNA, suggesting more efficient translation. This was significantly more pronounced in BS with high disease activity and in those with ocular and neurological clinical manifestations. The imbalance between CXCL10 protein and mRNA expression was not observed in either RA or SLE patients, and was not seen with other chemokines studied (CXCL9, CXCL11 and CCL2). Furthermore, BS monocytes treated with an alternative stimulant (LPS) did not show abnormal tumour necrosis factor (TNF)-α release. Sucrose density gradients to segregate monocyte CXCL10 mRNA into free RNA or polysome-associated RNA showed equal proportions in BS and HC samples, suggesting that the difference between BS and HC may be due to reduced negative control of CXCL10 translation in BS at a post-initiation level. We conclude that BS monocytes have dysfunctional post-transcriptional regulation of CXCL10 mRNA, resulting in over-expression of CXCL10 protein upon IFN-γ stimulation. As CXCL10 is a chemokine that recruits mononuclear cells, this abnormality may contribute to the exaggerated inflammatory responses that characterizes BS.

Application of age estimation methods based on teeth eruption: how easy is Olze method to use?

The development of new methods for age estimation has become with time an urgent issue because of the increasing immigration, in order to estimate accurately the age of those subjects who lack valid identity documents. Methods of age estimation are divided in skeletal and dental ones, and among the latter, Olze's method is one of the most recent, since it was introduced in 2010 with the aim to identify the legal age of 18 and 21 years by evaluating the different stages of development of the periodontal ligament of the third molars with closed root apices. The present study aims at verifying the applicability of the method to the daily forensic practice, with special focus on the interobserver repeatability. Olze's method was applied by three different observers (two physicians and one dentist without a specific training in Olze's method) to 61 orthopantomograms from subjects of mixed ethnicity aged between 16 and 51 years. The analysis took into consideration the lower third molars. The results provided by the different observers were then compared in order to verify the interobserver error. Results showed that interobserver error varies between 43 and 57 % for the right lower third molar (M48) and between 23 and 49 % for the left lower third molar (M38). Chi-square test did not show significant differences according to the side of teeth and type of professional figure. The results prove that Olze's method is not easy to apply when used by not adequately trained personnel, because of an intrinsic interobserver error. Since it is however a crucial method in age determination, it should be used only by experienced observers after an intensive and specific training.

Complement alternative pathway genetic variation and Dengue infection in the Thai population.

Dengue disease is a mosquito-borne infection caused by Dengue virus. Infection may be asymptomatic or variably manifest as mild Dengue fever (DF) to the most severe form, Dengue haemorrhagic fever (DHF). Mechanisms that influence disease severity are not understood. Complement, an integral component of the immune system, is activated during Dengue infection and the degree of activation increases with disease severity. Activation of the complement alternative pathway is influenced by polymorphisms within activation (factor B rs12614/rs641153, C3 rs2230199) and regulatory [complement factor H (CFH) rs800292] proteins, collectively termed a complotype. Here, we tested the hypothesis that the complotype influences disease severity during secondary Dengue infection. In addition to the complotype, we also assessed two other disease-associated CFH polymorphisms (rs1061170, rs3753394) and a structural polymorphism within the CFH protein family. We did not detect any significant association between the examined polymorphisms and Dengue infection severity in the Thai population. However, the minor allele frequencies of the factor B and C3 polymorphisms were less than 10%, so our study was not sufficiently powered to detect an association at these loci. We were also unable to detect a direct interaction between CFH and Dengue NS1 using both recombinant NS1 and DV2-infected culture supernatants. We conclude that the complotype does not influence secondary Dengue infection severity in the Thai population.

Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies.

The complement system plays a paradoxical role in the development and expression of autoimmunity in humans. The activation of complement in systemic lupus erythematosus (SLE) contributes to tissue injury. In contrast, inherited deficiency of classical pathway components, particularly C1q (ref. 1), is powerfully associated with the development of SLE. This leads to the hypothesis that a physiological action of the early part of the classical pathway protects against the development of SLE (ref. 2) and implies that C1q may play a key role in this respect. C1q-deficient (C1qa-/-) mice were generated by gene targeting and monitored for eight months. C1qa-/- mice had increased mortality and higher titres of autoantibodies, compared with strain-matched controls. Of the C1qa-/- mice, 25% had glomerulonephritis with immune deposits and multiple apoptotic cell bodies. Among mice without glomerulonephritis, there were significantly greater numbers of glomerular apoptotic bodies in C1q-deficient mice compared with controls. The phenotype associated with C1q deficiency was modified by background genes. These findings are compatible with the hypothesis that C1q deficiency causes autoimmunity by impairment of the clearance of apoptotic cells.

Temporary depletion of complement component C3 or genetic deficiency of C1q significantly delays onset of scrapie.

Following peripheral exposure to transmissible spongiform encephalopathies (TSEs), infectivity usually accumulates in lymphoid tissues before neuroinvasion. The host prion protein (PrPc) is critical for TSE agent replication and accumulates as an abnormal, detergent insoluble, relatively proteinase-resistant isoform (PrPSc) in diseased tissues. Early PrPSc accumulation takes place on follicular dendritic cells (FDCs) within germinal centers in lymphoid tissues of patients with variant Creutzfeldt-Jakob disease (vCJD), sheep with natural scrapie or rodents following experimental peripheral infection with scrapie. In mouse scrapie models, the absence of FDCs blocks scrapie replication and PrPSc accumulation in the spleen, and neuroinvasion is significantly impaired. The mechanisms by which the TSE agent initially localizes to lymphoid follicles and interacts with FDCs are unknown. Antigens are trapped and retained on the surface of FDCs through interactions between complement and cellular complement receptors. Here we show that in mice, both temporary depletion of complement component C3 or genetic deficiency of C1q significantly delays the onset of disease following peripheral infection, and reduces the early accumulation of PrPSc in the spleen. Thus, in the early stages of infection, C3 and perhaps C1q contribute to the localization of TSE infectivity in lymphoid tissue and may be therapeutic targets.

Amyloid deposition is delayed in mice with targeted deletion of the serum amyloid P component gene.

The tissue amyloid deposits that characterize systemic amyloidosis, Alzheimer's disease and the transmissible spongiform encephalopathies always contain serum amyloid P component (SAP) bound to the amyloid fibrils. We have previously proposed that this normal plasma protein may contribute to amyloidogenesis by stabilizing the deposits. Here we show that the induction of reactive amyloidosis is retarded in mice with targeted deletion of the SAP gene. This first demonstration of the participation of SAP in pathogenesis of amyloidosis in vivo confirms that inhibition of SAP binding to amyloid fibrils is an attractive therapeutic target in a range of serious human diseases.

Serum amyloid P component controls chromatin degradation and prevents antinuclear autoimmunity.

Serum amyloid P component (SAP), a highly conserved plasma protein named for its universal presence in amyloid deposits, is the single normal circulating protein that shows specific calcium-dependent binding to DNA and chromatin in physiological conditions. The avid binding of SAP displaces H1-type histones and thereby solubilizes native long chromatin, which is otherwise profoundly insoluble at the physiological ionic strength of extracellular fluids. Furthermore, SAP binds in vivo both to apoptotic cells, the surface blebs of which bear chromatin fragments, and to nuclear debris released by necrosis. SAP may therefore participate in handling of chromatin exposed by cell death. Here we show that mice with targeted deletion of the SAP gene spontaneously develop antinuclear autoimmunity and severe glomerulonephritis, a phenotype resembling human systemic lupus erythematosus, a serious autoimmune disease. The SAP-/- mice also have enhanced anti-DNA responses to immunization with extrinsic chromatin, and we demonstrate that degradation of long chromatin is retarded in the presence of SAP both in vitro and in vivo. These findings indicate that SAP has an important physiological role, inhibiting the formation of pathogenic autoantibodies against chromatin and DNA, probably by binding to chromatin and regulating its degradation.

Complement facilitates early prion pathogenesis.

New-variant Creutzfeldt-Jakob disease and scrapie are typically initiated by extracerebral exposure to the causative agent, and exhibit early prion replication in lymphoid organs. In mouse scrapie, depletion of B-lymphocytes prevents neuropathogenesis after intraperitoneal inoculation, probably due to impaired lymphotoxin-dependent maturation of follicular dendritic cells (FDCs), which are a major extracerebral prion reservoir. FDCs trap immune complexes with Fc-gamma receptors and C3d/C4b-opsonized antigens with CD21/CD35 complement receptors. We examined whether these mechanisms participate in peripheral prion pathogenesis. Depletion of circulating immunoglobulins or of individual Fc-gamma receptors had no effect on scrapie pathogenesis if B-cell maturation was unaffected. However, mice deficient in C3, C1q, Bf/C2, combinations thereof or complement receptors were partially or fully protected against spongiform encephalopathy upon intraperitoneal exposure to limiting amounts of prions. Splenic accumulation of prion infectivity and PrPSc was delayed, indicating that activation of specific complement components is involved in the initial trapping of prions in lymphoreticular organs early after infection.

Continual low-level activation of the classical complement pathway.

There is evidence that the classical complement pathway may be activated via a "C1-tickover" mechanism, analogous to the C3-tickover of the alternative pathway. We have quantitated and characterized this pathway of complement activation. Analysis of freshly collected mouse and human plasma revealed that spontaneous C3 activation rapidly occurred with the generation of C3 fragments in the plasma. By the use of complement- and Ig-deficient mice it was found that C1q, C4, C2, and plasma Ig were all required for this spontaneous C3 activation, with the alternative complement pathway further amplifying C3 fragment generation. Study of plasma from a human with C1q deficiency before and after therapeutic C1q infusion confirmed the existence of a similar pathway for complement activation in humans. Elevated levels of plasma C3 were detected in mice deficient in complement components required for activation of either the classical or alternative complement pathways, supporting the hypothesis that there is continuous complement activation and C3 consumption through both these pathways in vivo. Blood stasis was found to stimulate C3 activation by classical pathway tick-over. This antigen-independent mechanism for classical pathway activation may augment activation of the complement system at sites of inflammation and infarction.

Molecular basis of polymorphisms of human complement component C3.

C3 exhibits two common allotypic variants that may be separated by gel electrophoresis and are called C3 fast (C3 F) and C3 slow (C3 S). C3 F, the less common variant, occurs at appreciable frequencies only in Caucasoid populations (gene frequency = 0.20). An increased prevalence of the C3 F allele has been reported in patients with partial lipodystrophy, IgA nephropathy, and Indian childhood hepatic cirrhosis. Studies of the genomic organization of the human C3 gene led to the identification of a single change (C to G) between C3 S and C3 F at nucleotide 364 in exon 3. This leads, at the translation level, to the substitution of an arginine residue (positively charged) in C3 S for a glycine residue (neutral) in C3 F. This substitution results in a polymorphic restriction site for the enzyme HhaI. The resulting restriction fragment length polymorphism (RFLP) was investigated using genomic DNA, amplified using the polymerase chain reaction; there was absolute concordance between the genomic polymorphism and the distribution of C3 S and C3 F in 50 normal subjects. The molecular basis of a second structural polymorphism, defined by the monoclonal antibody HAV 4-1, was also characterized. The polymorphic determinant was identified at codon 314 in the exon 9 of the beta chain where a leucine residue (HAV 4-1+) is substituted for a proline residue (HAV 4-1-). Identification of the amino acid sequences of these polymorphic variants will facilitate characterization of possible functional differences between different allotypes of C3. Three RFLPs (BamHI, EcoRI, and SstI) were located to introns in the C3 gene. There was no allelic association between these three RFLPs, or between the RFLPs and the C3 F/S polymorphic site. Genetic equilibration of these polymorphisms has occurred within a gene of 41 kb.

T cell-dependent immune response in C1q-deficient mice: defective interferon gamma production by antigen-specific T cells.

The role of the classical complement pathway in humoral immune responses was investigated in gene-targeted C1q-deficient mice (C1qA-/-). Production of antigen-specific immunoglobulin (Ig)G2a and IgG3 in primary and secondary responses to T cell-dependent antigen was significantly reduced, whereas IgM, IgG1, and IgG2b responses were similar in control and C1qA-/- mice. Despite abnormal humoral responses, B cells from C1qA-/- mice proliferated normally to a number of stimuli in vitro. Immune complex localization to follicular dendritic cells within splenic follicles was lacking in C1qA-/- mice. The precursor frequency of antigen-specific T cells was similar in C1qA-/- and wild-type mice. However, analysis of cytokine production by primed T cells in response to keyhole limpet hemocyanin revealed a significant reduction in interferon-gamma production in C1qA-/- mice compared with control mice, whereas interleukin 4 secretion was equivalent. These data suggest that the classical pathway of complement may influence the cytokine profile of antigen-specific T lymphocytes and the subsequent immune response.

A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo.

The strongest susceptibility genes for the development of systemic lupus erythematosus (SLE) in humans are null mutants of classical pathway complement proteins. There is a hierarchy of disease susceptibility and severity according to the position of the missing protein in the activation pathway, with the severest disease associated with C1q deficiency. Here we demonstrate, using novel in vivo models of apoptotic cell clearance during sterile peritonitis, a similar hierarchical role for classical pathway complement proteins in vivo in the clearance of apoptotic cells by macrophages. Our results constitute the first demonstration of an impairment in the phagocytosis of apoptotic cells by macrophages in vivo in a mammalian system. Apoptotic cells are thought to be a major source of the autoantigens of SLE, and impairment of their removal by complement may explain the link between hereditary complement deficiency and the development of SLE.

A lupus-susceptibility C57BL/6 locus on chromosome 3 (Sle18) contributes to autoantibody production in 129 mice.

Epistatic interactions between the non-autoimmune strains 129 and C57BL/6 (B6), used for generating gene-targeted animals, can induce a lupus-like disease. Genome-wide scan analyses of testcross progeny between these two strains have identified several lupus susceptibility loci, with the strongest linkage to the production of autoantibodies (auto-Abs) displayed by an interval on chromosome 1 of 129 origin (Sle16). However, the contribution of B6 loci to the lupus phenotype remained unknown. We used a congenic approach to deduce the contribution to the autoimmune traits of the B6 genomic interval on chromosome 3 (Sle18), previously shown to be linked to antinuclear Ab production. This interval, when transferred on a 129 background (a strain termed 129.B6-Sle18), promoted auto-Ab production targeting a broad spectrum of autoantigens, expansion of activated CD4(+)T and B cells and mild glomerulonephritis. Surprisingly, these immunological and serological defects were accompanied by a significant increase in the percentage of regulatory T cells (Tregs; CD4(+) Foxp3(+)). However, these cells, that expressed lower levels of Foxp3, had no impaired regulatory function when tested in vitro. These findings illustrate further the efficacy of congenic dissection for functional characterisation of individual lupus susceptibility loci and highlight the contribution of loci derived from non-autoimmune strains to the disease pathogenesis.

Shiga toxin-2 results in renal tubular injury but not thrombotic microangiopathy in heterozygous factor H-deficient mice.

Haemolytic uraemic syndrome (HUS) is characterized by microangiopathic haemolytic anaemia, thrombocytopenia and renal failure because of thrombotic microangiopathy (TMA). It may be caused by infection with Shiga toxin-producing enteropathic bacteria (Stx-associated HUS) or with genetic defects in complement alternative pathway (CAP) regulation (atypical HUS). We hypothesized that defective complement regulation could increase host susceptibility to Stx-associated HUS. Hence, we studied the response of mice with heterozygous deficiency of the major CAP regulator, factor H, to purified Stx-2. Stx-2 was administered together with lipopolysaccharide to wild-type and Cfh(+/-) C57BL/6 animals. Forty-eight hours after administration of the first Stx-2 injection all animals developed significant uraemia. Renal histology demonstrated significant tubular apoptosis in the cortical and medullary areas which did not differ between wild-type or Cfh(+/-) Stx-2-treated mice. Uraemia and renal tubular apoptosis did not develop in wild-type or Cfh(+/-) animals treated with lipopolysaccharide alone. No light microscopic evidence of TMA or abnormal glomerular C3 staining was demonstrable in the Stx-2 treated animals. In summary, Stx-2 administration did not result in TMA in either Cfh(+/-) or wild-type C57BL/6 mice. Furthermore, haploinsufficiency of factor H did not alter the development of Stx-2-induced renal tubular injury.

Molecular basis of hereditary C3 deficiency.

Hereditary deficiency of complement component C3 in a 10-yr-old boy was studied. C3 could not be detected by RIA of serum from the patient. Segregation of C3 S and C3 F allotypes within the family confirmed the presence of a null gene for C3, for which the patient was homozygous. 30 exons have been characterized, spanning the entire beta chain of C3 and the alpha chain as far as the C3d region. Sequence analysis of the exons derived from the C3 null gene showed no abnormalities in the coding sequences. A GT-AT mutation at the 5' donor splice site of the intervening sequence 18 was found in the C3 null gene. Exons 17-21 were amplified by the polymerase chain reaction (PCR) from first-strand cDNA synthesized from mRNA obtained from peripheral blood monocytes stimulated with LPS. This revealed a 61-bp deletion in exon 18, resulting from splicing of a cryptic 5' donor splice site in exon 18 with the normal 3' splice site in exon 19. This deletion leads to a disturbance of the reading frame of the mRNA with a stop codon 17 bp downstream from the abnormal splice in exon 18. His parents had both the normal and abnormal C3 mRNA and were shown to be heterozygous for this mutation by sequence analysis of genomic DNA amplified by PCR. Similar splice mutants have previously been reported in the beta-globin, phenylalanine hydroxylase, and porphobilinogen deaminase genes. This mutation is sufficient to cause the deficiency of C3 in the patient.

Complement deficiencies in humans and animals: links to autoimmunity.

Complement is involved in the pathogenesis of systemic lupus erythematosus (SLE) in multiple ways and may act as both friend and foe. Inherited homozygous deficiency of one of the earliest components of the classical pathway is strongly associated with susceptibility to the development of SLE. However, complement is also implicated in the effector inflammatory phase of the autoimmune response that characterizes the disease. A further paradox in the links between complement and SLE is the observation that autoantibodies to some complement proteins, especially to C1q, develop as part of the autoantibody response. In this chapter, the role of the complement system in SLE is reviewed and hypotheses advanced to explain the complex relationships between complement and lupus.

Superior vena cava syndrome in small-cell lung cancer.

BACKGROUND: The aim of this study was to analyze the features of the superior vena cava syndrome (SVCS) as initial characteristics in small-cell lung cancer: incidence, dissemination of disease, diagnostic procedures, efficacy and toxic effects of chemotherapy, and median survival in patients with SVCS. METHODS: In a prospective series of 724 patients with biopsy-proved small-cell lung cancer seen during a 6-year period, we reviewed data from patients who also had SVCS. RESULTS: The incidence of SVCS was 87 of 724 at the time of diagnosis. Initial emergency radiation therapy was not used in these patients. Diagnostic procedures in these patients were not associated with mortality. Rapid initiation of intensive chemotherapy, often with heparin therapy, resulted in complete or partial responses in 81% and no response in 12%; data were not evaluable in 7%. Two of these 87 patients died of aplasia within 4 weeks of chemotherapy. Median survival was not significantly different in the patients with SVCS (median, 42 weeks) and without SVCS (median, 40 weeks). A significant increase in initial brain metastases at the time of diagnosis was observed in patients with SVCS (22% vs 11%). CONCLUSIONS: Intensive chemotherapy is the first line of therapy in small-cell lung cancer. Histologic diagnostic procedures must be performed in patients with SVCS to adapt the treatment to the underlying cause. Initial emergency radiotherapy, before diagnosis or chemotherapy, does not seem to be useful in these patients. Computed tomography of the brain should be performed routinely in patients with SVCS, and prophylactic brain irradiation could be helpful in such patients. Apparently SVCS is not a poor prognostic factor in treated small-cell lung cancer.

Ultraviolet-radiation-induced keratinocyte apoptosis in C1q-deficient mice.

Exposure to ultraviolet B radiation is an important trigger of both systemic and cutaneous disease flares in individuals with systemic lupus erythematosus. More than 90% of individuals with homozygous C1q deficiency develop a systemic-lupus-erythematosus-like illness, which is typically associated with a severe photosensitive rash. Apoptotic, human keratinocytes have been shown in vitro to bind C1q, in the absence of antibody. These observations, together with the hypothesis that a major source of the autoantigens driving the immune response in systemic lupus erythematosus comes from apoptotic cells, led us to investigate the effects of murine C1q deficiency on ultraviolet-radiation-induced keratinocyte apoptosis in vivo. In this work, we demonstrated C1q binding to apoptotic murine keratinocytes in vitro and showed for the first time that C1q is also present on sunburn cells in vivo. In addition to C1q, we detected C3 deposition on sunburn cells in both wild-type and C1q-deficient mice, suggesting activation of the alternative pathway. Following acute ultraviolet exposure in vivo, no difference in the rate of clearance of sunburn cells was found in C1q-deficient mice from three different genetic backgrounds, compared with strain-matched wild-type controls. Furthermore, chronic ultraviolet exposure did not result in the production of autoantibodies or the development of glomerulonephritis. Our findings suggest that C1q does not play a critical role in the physiologic clearance of apoptotic murine keratinocytes in vivo.