Reduced CD5(+) CD24(hi) CD38(hi) and interleukin-10(+) regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies.

Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is B cell-dependent, although how particular B cell subsets modulate immunopathogenesis remains unknown. Although their phenotype remains controversial, regulatory B cells (Bregs ), play a role in immunological tolerance via interleukin (IL)-10. Putative CD19(+) CD24(hi) CD38(hi) and CD19(+) CD24(hi) CD27(+) Bregs were evaluated in addition to their CD5(+) subsets in 69 patients with ANCA-associated vasculitis (AAV). B cell IL-10 was verified by flow cytometry following culture with CD40 ligand and cytosine-phosphate-guanosine (CpG) DNA. Patients with active disease had decreased levels of CD5(+) CD24(hi) CD38(hi) B cells and IL-10(+) B cells compared to patients in remission and healthy controls (HCs). As IL-10(+) and CD5(+) CD24(hi) CD38(hi) B cells normalized in remission within an individual, ANCA titres decreased. The CD5(+) subset of CD24(hi) CD38(hi) B cells decreases in active disease and rebounds during remission similarly to IL-10-producing B cells. Moreover, CD5(+) B cells are enriched in the ability to produce IL-10 compared to CD5(neg) B cells. Together these results suggest that CD5 may identify functional IL-10-producing Bregs . The malfunction of Bregs during active disease due to reduced IL-10 expression may thus permit ANCA production.

Pathways to renal biopsy and diagnosis among patients with ANCA small-vessel vasculitis.

OBJECTIVES: Antineutrophil cytoplasmic antibody small-vessel vasculitis (ANCA-SVV) is an autoimmune systemic process increasingly recogniSed since the advent of antibody testing for the disease. Prompt diagnosis and institution of immunosuppressive therapy has been shown to improve patient outcome. The goal of this study was to better understand how patients navigate the health care system from symptom presentation to biopsy diagnosis, and to study the effects of prompt versus delayed diagnosis. METHODS: Disease symptoms and number of physicians seen prior to renal biopsy were assessed for 127 ANCA-SVV patients. Direct, delayed, and quest pathways to diagnosis and treatment of vasculitis were defined for both patients and providers. Kruskal-Wallis and Fisher exact tests were used to evaluate continual measures and compare categorical variables across pathways. RESULTS: Among patients who sought direct care, physician delay in referral to a nephrologist was common (49/127, 71%, p=0.0023). Patients who delayed seeking care also experienced a delayed diagnosis 57% of the time (p=0.0023). Patients presenting with prodromal flu or upper respiratory involvement were more likely to have a delay/quest patient pathway (56% and 55%, respectively) than a direct patient pathway (44%, p=0.033 and 45%, p=0.019, respectively). There was a trend for patients with more severe loss of renal function to have a more direct referral to a nephrologist. CONCLUSIONS: Delay in diagnosis of ANCA SVV may be due to lack of or non-specific symptoms, especially in patients who present with non-renal manifestations of disease. Better algorithms are needed to identify extra-renal manifestations, expedite diagnosis and improve patient outcomes.

Epigenetics and complementary proteins.

Although studies on the immunopathogenesis of anti-neutrophil cytoplasm antibody (ANCA) vasculitis have been directed at understanding the autoantibody, there is growing evidence that points to the importance of ANCA autoantigen genes and their regulation. Transcriptional analysis indicates that ANCA autoantigen genes are active in mature neutrophils of ANCA vasculitis patients compared to healthy controls. The unusual transcriptional state of neutrophils from ANCA vasculitis patients appears to be a consequence of failed or disrupted epigenetic silencing. Defective epigenetic silencing could have global effects, by altering the transcriptional and phenotypic state of neutrophils, or local effects by permitting transcription of autoantigen genes from both strands resulting in anti-sense transcripts. Although the role of anti-sense transcripts is currently unknown, there are two intriguing possibilities. Anti-sense transcripts could function (as described for other genes) in transcriptional silencing of autoantigen genes, which takes place in normal neutrophil progenitors. In the setting of failed epigenetic silencing, the fate of anti-sense transcripts may be pathological and serve as a template for production of complementary autoantigens. The observation that ANCA vasculitis patients have anti-sense transcripts and antibodies to complementary proteins is consistent with a role of anti-sense transcripts in complementary protein production. A better understanding of epigenetic silencing and complementary proteins in ANCA vasculitis may unlock the underlying pathology of this condition.

Neoantigen of the polymerized ninth component of complement. Characterization of a monoclonal antibody and immunohistochemical localization in renal disease.

A monoclonal antibody to a neoantigen of the C9 portion of the membrane attack complex (MAC) of human complement has been developed and characterized. The distribution of this neoantigen was assessed by indirect immunofluorescence microscopy in nephritic and nonnephritic renal diseases. The antibody (Poly C9-MA) reacted on enzyme-linked immunosorbent assay (ELISA) with a determinant in complement-activated serum that was undetectable in normal human serum (NHS). Zymosan particles incubated in NHS had positive immunofluorescent staining with Poly C9-MA; however, binding of Poly C9-MA was not observed with zymosan particles incubated in sera deficient in individual complement components C3, C5, C6, C7, C8, or C9. Reconstitution of C9-deficient sera with purified C9 restored the fluorescence with Poly C9-MA. Poly C9-MA reacted positively by ELISA in a dose-dependent manner with purified MC5b-9 solubilized from membranes of antibody-coated sheep erythrocytes treated with NHS but not with intermediate complement complexes. Poly C9-MA also reacted in a dose-dependent manner on ELISA and in a radioimmunoassay with polymerized C9 (37 degrees C, 64 h) (poly C9) but not with monomeric C9. Increasing amounts of either unlabeled poly C9 or purified MC5b-9 inhibited the 125I-poly C9 RIA in an identical manner. These studies demonstrate that Poly C9-MA recognizes a neoantigen of C9 common to both the MAC and to poly C9. By immunofluorescence, Poly C9-MA reacted minimally with normal kidney tissue in juxtaglomerular loci, the mesangial stalk, and vessel walls. Poly C9-MA stained kidney tissue from patients with glomerulonephritis in a pattern similar to that seen with polyclonal anti-human C3. In tissue from patients with nonnephritic renal disease--diabetes, hypertension, and obstructive uropathy--Poly C9-MA was strongly reactive in the mesangial stalk and juxtaglomerular regions, tubular basement membranes, and vascular walls. Poly C9-MA binding was especially prominent in areas of advanced tissue injury. Poly C9-MA frequently stained loci where C3 was either minimally present or absent. These studies provide strong evidence for complement activation not only in nephritic but also in nonnephritic renal diseases.

The role of pathology in the diagnosis of systemic vasculitis.

Pathologic processes are underlying defining features of systemic vasculitis. When these pathologic processes can not be observed directly, surrogate signs and symptoms of disease must be used to conclude that vasculitis is present in a patient and, if so, to determine what specific type of vasculitis is present. This review briefly describes the most defining pathologic features of giant cell arteritis, Takayasu arteritis, polyarteritis nodosa, Henoch-Schönlein purpura, cryoglobulinemic vasculitis, Kawasaki disease, microscopic polyangiitis, Wegener's granulomatosis and Churg-Strauss syndrome; and discusses how these pathologic features can be integrated with clinical and laboratory data to reach an actionable diagnosis.

Polyantigenic expansion of basement membrane constituents in diabetic nephropathy.

The immunohistopathology of the intrinsic basement membrane-associated antigens were examined in diabetic nephropathy. In early and moderate stages of disease there was polyantigenic expansion of all the intrinsic components of mesangium, glomerular basement membrane (GBM), and tubular basement membrane (TBM) assessed by polyclonal antisera to collagen types IV and V, laminin, and by monoclonal antibodies to type IV collagen and fibronectin and to four other intrinsic components of normal renal extracellular matrices (MBM10, 11, 12, and 15). In the mesangium the first intrinsic antigens to increase were fibronectin and type V collagen. In late stages of disease, there was a diminution in the mesangium of all of these antigens with the exception of type V collagen, which persisted. Additionally, antigens appeared in the mesangium, recognized by MBM11 and MBM15, which are normally present in fetal but not adult mesangial regions. Similarly, in the GBM in late stages of disease, there was a decrease in all of the antigens, except for a persistence of the antigen recognized by MBM15. However, in TBM all of the antigens assessed increased in early, moderate, and severe disease. These studies document the complexity of polyantigenic alterations in the development of diabetic nephropathy.

Reconstitution of an epithelial chloride channel. Conservation of the channel from mudpuppy to man.

We have previously shown that monoclonal antibody E12 (MAb E12), one of several such antibodies raised against theophylline-treated Necturus gallbladder (NGB) epithelial cells, inhibits the chloride conductance in the apical membrane of that tissue. Since chloride channels are critical to the secretory function of epithelia in many different animals, we have used this antibody to determine whether the channels are conserved, and in an immunoaffinity column to isolate the channel protein. We now demonstrate that MAb E12 cross-reacts with detergent-solubilized extracts of different tissues from various species by enzyme-linked immunosorbent assay (ELISA). Western blot analysis shows that this monoclonal antibody recognizes proteins of Mr 219,000 in NGB, toad gallbladder, urinary bladder, and small intestine, A6 cells, rat colon, rabbit gastric mucosa, human lymphocytes, and human nasal epithelial cells, and inhibits the chloride conductance in toad gallbladder, rat colon, and human nasal epithelium. Detergent-solubilized protein eluted from an immunoaffinity column and then further purified via FPLC yields a fraction (Mr 200,000-220,000) which has been reconstituted into a planar lipid bilayer. There it behaves as a chloride-selective channel (PCl/PNa = 20.2 in a 150/50 mM trans-bilayer NaCl gradient) whose unit conductance is 62.4 +/- 4.6 pS, and which is blocked in the bilayer by the antibody. The gating characteristics of this channel indicate that it can exist as aggregates or as independent single channels, and that the antibody interferes with gating of the aggregates, leaving the unit channels unchanged. From these data we conclude that the protein of Mr 219,000 recognized by this monoclonal antibody is an important component of an epithelial chloride channel, and that this channel is conserved across a wide range of animal species.

Reactivity of antineutrophil cytoplasmic autoantibodies with mononuclear phagocytes.

Antineutrophil cytoplasmic autoantibodies (ANCA) react with proteins found in the granules of neutrophils and the peroxidase-positive lysosomes of monocytes, including myeloperoxidase (MPO), proteinase 3 (PR-3), and elastase. ANCA-associated diseases, such as Wegener's granulomatosis and polyarteritis nodosa, are characterized by necrotizing vascular inflammation. The inflammatory lesions typically contain both neutrophils and mononuclear phagocytes, with the latter sometimes predominating, for example, in the granulomatous lesions of Wegener's granulomatosis. We investigated the presence of the ANCA target antigens PR3, MPO, and elastase in mononuclear phagocyte cytoplasm during the course of differentiation in vitro and in alveolar and peritoneal macrophages. We observed that ANCA antigens were down-regulated during mononuclear phagocyte differentiation, with the loss corresponding to that of peroxidase-positive granules. This suggests that ANCA can directly interact only with monocytes and early exudative macrophages and not with mature macrophages.

Antibodies against granule proteins activate neutrophils in vitro.

Polymorphonuclear leukocyte (PMN) respiratory burst was stimulated by heterologous antibodies against PMN granule proteins but not by control antibodies. Fluorescence-activated cell sorter (FACS) analysis of activated PMN demonstrated the presence of two primary granule proteins, proteinase 3 (PR-3) and cationic protein 57 (CAP-57) at the membrane surface. The presence of myeloperoxidase (MPO) at the cell surface of primed and unprimed PMN was confirmed by immunoelectron microscopy. Priming doses of recombinant tumor necrosis alpha (rTNF alpha) enhanced the rate of superoxide (O2-) production by these antibodies and increased the amount of surface protein accessible to these antibodies. Anti-neutrophil cytoplasmic autoantibodies (ANCA) with specificities for PMN granule proteins are present in patients with Wegener's granulomatosis, polyarteritis nodosa, and idiopathic and crescentic glomerulonephritis. The demonstration that antibodies against granule proteins activate PMN supports the hypothesis that the vasculitis seen in these diseases is due in part to PMN mediated oxidative injury following PMN stimulation by ANCA.

Infections associated with subclavian Uldall catheters.

During a 12-month period, the use of a subclavian vein Uldall catheter (UC) for hemodialysis or plasmapheresis in 27 patients was studied prospectively. Ten patients had ten UC site infections. Organisms associated with these infections included Staphylococcus epidermidis (five), Staphylococcus aureus (four), Proteus mirabilis (two), and Enterococcus (one). The four S aureus infections occurred 1, 2, 4, and 9 days after UC insertion, whereas the five S epidermidis infections occurred 6, 17, 17, 26, and 97 days after insertion. Five patients had associated bacteremias; in one of these patients, the bacteremia was the major cause of death. The incidence of UC site infection and bacteremia based was higher than the incidence of infection reported with any other type of vascular access for hemodialysis. Further studies are necessary to define whether the UC should be routinely employed for temporary vascular access.

Deposition of the membrane attack complex of complement in bullous pemphigoid.

Bullous pemphigoid is associated with deposition of IgG and C3 at the dermal-epidermal junction. In order to see whether complement activation in bullous pemphigoid resulted in deposition of membrane attack complex (MAC) at the basement membrane zone, skin biopsies from patients with bullous pemphigoid were examined using a direct immunofluorescence technique. By employing a monoclonal antibody to a neoantigen of C9, the MAC was demonstrated in linear pattern at the basement membrane zone. These deposits were seen in both involved and uninvolved skin but the amount of MAC was greater in involved skin as judged by intensity of staining. Stippled deposits of MAC were also present in or around epidermal basal cells. The MAC could be generated in vitro by reaction of normal plasma with antibasement membrane antibody bound to sections of monkey esophagus. The IgG antibody activated complement and this complement activation proceeded all the way to the terminal step.

Production of fibronectin by epithelium in a skin equivalent.

Although human keratinocytes in vitro have been shown to produce fibronectin, whether keratinocytes can contribute fibronectin to the dermal-epidermal junction or wound matrix is unknown. In order to approach this problem experimentally, we used the "skin equivalent" model composed of a native collagen gel populated with cultured fibroblasts and covered by cultured keratinocytes. By using bovine fibroblasts to populate the gel, fetal bovine serum in the culture medium, and human keratinocytes to form the epithelium, we were able to be certain that any human fibronectin produced in the culture was synthesized by the keratinocytes. A monoclonal antibody to fibronectin was found to recognize human but not bovine fibronectin. When the skin equivalent was stained by indirect immunofluorescence with antifibronectin, fibronectin was visible as an intensely staining band at the dermal-epidermal junction. In sections in which the dermis and epidermis had separated, the staining was usually limited to the dermal aspect of the skin equivalent. The results indicate that epithelium can contribute fibronectin to the dermal-epidermal junction and suggest that dermal staining in skin sections may originate from the epidermis. Since the developing skin equivalent has a rapidly growing epithelium and simulates a healing wound, contribution of fibronectin by the epithelium, in addition to that possibly contributed by serum and fibroblasts, may be of importance in wound healing.

Vitronectin: effects on keratinocyte motility and inhibition of collagen-induced motility.

Epibolin, a plasma protein, was initially purified on the basis of its ability to enhance spreading of keratinocytes. It is now known that epibolin is identical to serum spreading factor, S protein, and vitronectin, and the current name for the protein is vitronectin. Studies of vitronectin on cultured keratinocytes showed that it caused spreading and epiboly but not cellular adhesion to the substratum. In studies with other types of cells, vitronectin increased migration of several types of cells in a Boyden chamber. Because some agents that enhance spreading and adhesion, such as collagen and fibronectin, also increase motility, we tested whether vitronectin increased motility of keratinocytes. By photographing and quantitating motility of keratinocytes plated on a bed of colloidal gold particles, we determined that vitronectin increased local movement of keratinocytes in a concentration-dependent fashion, resulting in clearing of gold particles in a circular pattern around the cells, but did not cause the production of tracks found in cultures plated on collagen or fibronectin. The small increases in clearing of the gold particles that occurred in the presence of vitronectin were abolished by antibody to vitronectin. Furthermore, the marked increase in motility produced by type I collagen was significantly reduced when the keratinocytes were treated with vitronectin. Antibody to vitronectin also abrogated the vitronectin-induced reduction in collagen-stimulated motility, confirming that this action was specific for vitronectin. Serum, which contains vitronectin, stimulated motility in a fashion identical to purified vitronectin, but serum lacking vitronectin was inactive. These studies show that vitronectin causes a localized increase in movement associated with spreading resulting in a halo around individual cells, that vitronectin does not enhance directional motility of keratinocytes in this assay but in contrast antagonizes such motility produced by collagen, and that vitronectin is the factor in serum responsible for this effect. The findings with vitronectin and collagen show that these agents stimulate different types of motility. The roles in wound healing of agents stimulating different types of motility are unclear and require further study.

Neonatal foreskin substrate has limitations for the immunofluorescent screening of monoclonal antibodies.

Two monoclonal antibodies to type IV collagen showed a marked decrease in the labeling of the dermal-epidermal junction of neonatal foreskin while the basement membrane around dermal blood vessels was brightly stained. In contrast, these antibodies labeled the junction and dermal blood vessels with approximately equal intensity when adult skin of nonforeskin site was used as substrate. Other antibodies to matrix molecules (bullous pemphigoid antigen, epidermolysis bullosa acquisita antigen, and laminin) showed excellent staining of both the dermal-epidermal junction and dermal blood vessels in both neonatal foreskin and adult skin. Further, the ultrastructural appearance of the substrates appeared identical. The implication is that neonatal foreskin is not a good substrate to use for the routine screening of monoclonal antibodies to matrix components by indirect immunofluorescence since a "false negative" evaluation may occur.

Epidermolysis bullosa acquisita antigen, a major cutaneous basement membrane component, is synthesized by human dermal fibroblasts and other cutaneous tissues.

The epidermolysis bullosa acquisita (EBA) antigen is identified as 2 chains: a 290,000-dalton protein and a less prominent 145,000-dalton protein. The 290,000-dalton chain is synthesized by human keratinocytes in culture. In this study, we show that the 290,000-dalton chain is synthesized by human skin fibroblasts and cutaneous human tumors. In contrast, HT1080 cells, a human sarcoma cell line known to produce matrix molecules (such as laminin and type IV collagen), does not synthesize the EBA antigen. Further, the EBA antigen is absent from serum and blood components, placenta, amnion, lung, and the EHS tumor, a murine sarcoma that produces large amounts of laminin, type IV collagen, nidogen, entactin, and basement membrane proteoglycan but is present in cutaneous tumors of adnexal and epithelial origin. These data suggest that while the EBA antigen is synthesized by both human skin keratinocytes and fibroblasts and is therefore not specific for a primordial germ layer, it does appear to be specific for tissue containing a stratified squamous epithelium.

Disposition of didanosine in HIV-seropositive patients with normal renal function or chronic renal failure: influence of hemodialysis and continuous ambulatory peritoneal dialysis.

OBJECTIVE: To evaluate the pharmacokinetics of didanosine in patients with normal kidney function or chronic kidney failure. METHODS: Three groups of patients with human immunodeficiency virus (HIV) infection were studied: group I, six men with normal kidney function (creatinine clearance > 90 ml/min/1.73 m2); group II, six men with chronic renal failure maintained on continuous ambulatory peritoneal dialysis (CAPD); and group III, four men and two women with chronic renal failure receiving hemodialysis three times a week. A 300 mg dose of didanosine was administered orally and intravenously according to a two-period randomized crossover design. Patients in group III were studied between hemodialysis sessions during the crossover periods. In addition, patients in group III were studied in a third period after administration of a 300 mg oral dose of didanosine 4 hours before hemodialysis. RESULTS: After intravenous administration in group I, the mean (+/-SD) total clearance (CLT) was 13.0 +/- 1.6 ml/min/kg and the elimination half-life (t 1/2) was 1.56 +/- 0.43 hour. In groups II and III, the CLT decreased significantly to 3.4 +/- 1.2 and 3.2 +/- 1.2 ml/min/kg, respectively, whereas the t1/2 increased to 3.60 +/- 0.82 hours and 3.11 +/- 0.88 hours, respectively. The absolute bioavailability of didanosine in groups I, II, and III was 42% +/- 12%, 52% +/- 6%, and 38% +/- 11%, respectively, and did not differ significantly. CAPD had little effect on the removal of didanosine, whereas approximately 30% of the drug present in the body at the start of dialysis was eliminated by an average 3-hour dialysis session. CONCLUSION: The clearance of didanosine is impaired in patients with chronic renal failure. To compensate, the dose and schedule of administration should be adjusted. It is recommended that one-fourth of the total daily dose of didanosine be administered once a day in this patient population.

Parvovirus B19 DNA in kidney tissue of patients with focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) represents a clinicopathological syndrome with diverse causes. We examined the possibility that some cases of FSGS are associated with parvovirus B19 infection. We studied renal biopsy tissue from 40 patients, including those with idiopathic FSGS, collapsing FSGS, membranous nephropathy, and minimal change disease, as well as normal renal tissue removed at the time of nephrectomy from 4 patients. DNA was extracted from frozen blocks of kidney tissue and amplified using nested polymerase chain reaction. Parvovirus B19 DNA was amplified from 8 of 10 patients with idiopathic FSGS, 9 of 10 patients with collapsing FSGS, 6 of 10 patients with membranous nephropathy, 5 of 10 patients with minimal change disease, and 2 of 4 cancer nephrectomy samples. The prevalence of parvovirus B19 DNA was greater among patients with idiopathic FSGS and collapsing FSGS compared with patients with other diagnoses (P = 0.05). In situ hybridization studies using digoxigenin-labeled DNA probes failed to detect parvovirus B19 nucleic acid in any of the kidney tissue samples. These results suggest that parvovirus B19 DNA is commonly found in the kidneys of patients with a range of renal diagnoses, possibly representing latent DNA from past infection. The failure to localize parvovirus B19 nucleic acid within kidney argues against ongoing, high-level viral replication. Nevertheless, the increased prevalence of B19 DNA in patients with idiopathic FSGS and collapsing FSGS could indicate a pathogenic role for the virus in the cause of FSGS in certain patients.

Effect of ACE inhibitors in diabetic and nondiabetic chronic renal disease: a systematic overview of randomized placebo-controlled trials.

Clinical trials have shown the beneficial effects of angiotensin-converting enzyme (ACE) inhibitors in delaying the progression of diabetic renal disease. There is less evidence from primary clinical trials of nondiabetic renal disease. We performed an updated meta-analysis to determine the efficacy of ACE inhibitors in slowing the progression of renal disease over a broad range of functional renal impairment. We included published and unpublished randomized, placebo-controlled, parallel trials with at least 1 year of follow-up available from January 1970 to June 1999. In nine trials of subjects with diabetic nephropathy and microalbuminuria, the relative risk for developing macroalbuminuria was 0.35 (95% confidence interval [CI], 0.24 to 0.53) for individuals treated with an ACE inhibitor compared with placebo. In seven trials of subjects with overt proteinuria and renal insufficiency from a variety of causes (30% diabetes, 70% nondiabetes), the relative risk for doubling of serum creatinine concentration or developing end-stage renal disease was 0.60 (95% CI, 0.49 to 0.73) for individuals treated with an ACE inhibitor compared with placebo. Treatment of individuals with chronic renal insufficiency with ACE inhibitors delays the progression of disease compared with placebo across a spectrum of disease causes and renal dysfunction.

The life readiness program: a physical rehabilitation program for patients on hemodialysis.

Patients with end-stage renal disease (ESRD) undergoing hemodialysis (HD) are typically sedentary and functionally limited as a consequence of their condition. The purpose of this study is to test the effect of a lifestyle physical rehabilitation program (The Life Readiness Program) on physical function in patients with ESRD undergoing HD. Physical function was measured by the Kidney Disease Quality of Life Short Form (KDQOL-SF) physical function score (range, 0 to 100). Eighty-two patients were randomly assigned to a 6-month rehabilitation program (intervention; n = 39) or to standard clinical management alone (control; n = 43). The groups were frequency matched by age, sex, ethnicity, and diabetes as the cause of ESRD. General linear modeling of the change in physical function score was used for multivariate analysis. Physical function scores were not different between groups at baseline. Change in physical function score increased significantly in the intervention group compared with the control group when data were adjusted for the matching variables and adequacy of dialysis (3.2, -3.6; P = 0.04). Additionally, the control group reported more problems with work or daily functions because of emotional problems (P: