The role of endothelial cell reactive antibodies in peripheral arterial disease.

OBJECTIVES: It is hypothesised that endothelial cell reactive antibodies (ECRA) play a role in the progression of PAD through activation of endothelial cells and the release of inflammatory cytokines. We aimed to test this hypothesis by assessing levels of ECRA, E-selectin and IL-6 in patients with PAD of varying severity in a case controlled study. DESIGN, MATERIALS, METHODS: Patients were assessed clinically and with ankle-brachial pressure indices. Patients with critical ischaemia (CI, n=30), stable claudicants (SC, n=30), and age-matched controls (AMC, n=20) were studied. Antibody, E-selectin and IL-6 levels were measured using ELISA. RESULTS: ECRA levels were significantly raised in the CI group over AMC. IL-6 levels were significantly elevated in both SC and CI over the control group and in CI over SC. There were no significant differences in E-selectin levels between the AMC, SC and CI. CONCLUSION: Our findings support the hypothesis that autoantibodies play a role in promoting PAD by elevating IL-6. The absence of an elevation in E-selectin in this study may be due to its short half-life, and casts doubt on its value as a marker of inflammation in atherosclerosis.

The role of endothelial cell reactive antibodies in peripheral vascular disease.

The mechanism by which a small but significant proportion of patients with peripheral vascular disease (PVD) rapidly progress to critical ischaemia is unclear. Both experimental and clinical data suggest a role for autoantibodies in the pathogenesis of atherosclerotic disease, particularly in the accelerated atherosclerosis seen in patients with systemic lupus erythematosus and the anti-phospholipid syndrome. This review examines the evidence for a role for endothelial cell reactive autoantibodies in PVD and the potential mechanisms by which these autoantibodies could contribute to the acceleration of atherosclerosis in a proportion of patients. The identification of such markers could lead to the identification of patients with PVD who are at risk of developing critical ischaemia and may warrant early and aggressive intervention.

A natural canine homologue of alopecia areata in humans.

BACKGROUND: Alopecia areata (AA) is suspected to be an autoimmune disease directed preferentially against hair follicles (HF) affecting both humans and various mammalian species. Recently, two rodent models of AA were described, namely the ageing C3H/HeJ mouse and the DEBR rat. Despite several case reports of canine AA in the literature, there has been no systematic assessment of the disease in these companion animals, and it is also not known whether dogs with AA could be useful as an outbred homologue of this disease in humans. OBJECTIVES: To evaluate the clinical, histopathological and immunopathological features of 25 dogs with AA and compare these data with those found in the human disease. PATIENTS/METHODS: Twenty-five client-owned dogs exhibiting macroscopic alopecia with peri- or intrabulbar lymphocytic infiltrates were selected for study. Biopsies and sera were obtained and assessed by histopathology, direct immunofluorescence of immunoreactant deposition, immunohistochemistry for lymphocyte markers, indirect immunofluorescence and immunoblotting analysis of circulating serum IgG, selective immunoprecipitation of HF proteins by serum IgG, and passive transfer of purified canine IgG into naïve C57BL/10 mice. RESULTS: Clinical signs including alopecia, skin hyperpigmentation and leucotrichia usually developed during adulthood and were first seen on the face, followed by the forehead, ears and legs. Spontaneous remission of alopecia occurred in 60% of dogs and regrowing hair shafts were often non-pigmented. Histological examination of skin biopsy specimens revealed peri- and intrabulbar mononuclear cell infiltrates affecting almost exclusively anagen HF. Direct immunofluorescence analysis detected HF-specific IgG in 73% of dogs, while indirect immunofluorescence revealed circulating IgG autoantibodies to the HF inner and outer root sheaths, matrix and precortex. Immunoblotting analysis revealed IgG reactivity to proteins in the 45-60 kDa molecular weight range and with a 200-220 kDa doublet. The latter was identified as trichohyalin by selective immunoprecipitation. Purified HF-reactive IgG, pooled from AA-affected dogs, was injected intradermally to the anagen skin of naïve mice where it was associated with the local retention of HFs in an extended telogen phase in AA-treated skin compared with that seen in controls. CONCLUSIONS: These findings are very similar to those reported for human AA patients; therefore, they support the consideration of dogs with AA as a useful homologue for the study of the pathogenesis of this common autoimmune disease of humans.

The role of somatic mutation in determining the affinity of anti-DNA antibodies.

Combinatorial antibody libraries were constructed from the spleen of a patient with concomitant systemic lupus erythematosus and idiopathic thrombocytopenia. Following selection of the libraries with DNA, a panel of 15 anti-DNA Fabs was isolated. Sequence analysis of these antibodies coupled with measurements of their affinities for ss- and dsDNA were used to investigate the role of somatic mutation in affinity maturation of the anti-DNA response. Examination of the germline genes used by these Fabs supports previous studies that suggest there is no restriction of the gene usage in the anti-DNA response. However, data are presented indicating that VH3 genes and the A27 V(kappa) paired with the J(kappa)1 may be over-expressed in the anti-DNA repertoire. Analysis of the role of somatic mutation in increasing affinity for DNA indicates that affinity maturation has occurred and suggests that the CDR1 and CDR2 of the heavy chain are of importance in this process.

Normalisation of hair follicle morphology in C3H/HeJ alopecia areata mice after treatment with squaric acid dibutylester.

Alopecia areata is a non-scarring, reversible disorder, presumably caused by an autoimmune attack on anagen hair follicles. Treatments are numerous, and most of these are ineffective. However, the elicitation of contact dermatitis on the affected skin is commonly associated with hair regrowth. A major advance in the study of alopecia areata has been the introduction and characterisation of the C3H/HeJ mouse model that exhibits many features of the human disease. In this study we examined the effects of squaric acid dibutylester treatment on hair follicles and the associated leukocyte infiltrate in alopecia areata mice by light and transmission electron microscopic analysis. This was compared with unaffected normal mice and alopecic untreated mice. Experimental mice were treated unilaterally with the contact allergen squaric acid dibutylester and the skin was assessed after hair regrowth. The characteristic pathological picture of alopecia areata was observed in alopecic but not normal mice. Nine of eleven experimental mice regrew hair on the treated side only and this was associated with a reduction in peri/intrafollicular inflammatory cell infiltrates, hair follicle dystrophy, melanin incontinence/clumping, and an increase in the numbers of hair follicles in full anagen. This normalisation of hair follicle status after treatment reflects the successful reversal of disease in these mice. The mechanism of action of topical immunotherapy with a potent contact allergen such as squaric acid dibutylester still needs to be elucidated, but an altered immune milieu is suspected. This study further validates the C3H/HeJ mouse model of alopecia areata in the search for therapeutic interventions in this common hair follicle disorder.

The identification of endothelial cell autoantigens.

The identity of many endothelial cell autoantigens remains unclear. This study has used human monoclonal anti-endothelial cell autoantibodies isolated from patients with SLE to identify endothelial autoantigens. Thirteen antibodies reactive with endothelial cell membrane preparations were isolated and cloned, one of which has previously been demonstrated to be pro-inflammatory. Western blotting demonstrates that these antibodies recognize a variety of proteins in endothelial cell membrane preparations. Further characterization of five antibodies by cDNA library screening, immunofluorescence and Western blotting proves that two of these antibodies recognized the cytoskeletal proteins tubulin and vimentin. A further antibody identified a clone derived from human collagenase, an identification supported by Western blotting. The multiple clones selected by other antibodies are not compatible with the molecular weight of the antigen recognized in Western blotting studies. This study has clearly identified two endothelial cell autoantigens present in membrane preparations and provides strong evidence as to the identity of a third.

Does the CDR3 of the heavy chain determine the specificity of autoantibodies in systemic lupus erythematosus?

Many factors are involved in the recognition of autoantigens by autoanti-bodies, including the use of specific germline genes, the sequence and structure of the CDR3 of the heavy chain, somatic mutation and selective heavy and light chain pairing. However, the relative importance of these factors remainsunclear. This study reports the results of sequence analysis of two anti-endothelial cell antibodies that recognise the same antigen. Sequence analysis of these antibodies shows that they use the same heavy chain germline genes as two anti-DNA antibodies but differ significantly in the sequence of the CDR3. Furthermore, one of the antibodies uses a light chain germline gene combination that has been reported for three anti-DNA antibodies. One of these antibodies shows significant mutation in the CDR2 of the heavy chain. Peptide analysis suggests that the differences between these anti-DNA and anti-endothelial cell antibodies result in consistent structural differences that may reflect the nature of the antigen recognised.

A specific tetrahydrobiopterin binding domain on tyrosinase controls melanogenesis.

(6R)5,6,7,8-tetrahydrobiopterin (6-BH4) directly regulates tyrosinase activity by specifically binding to a putative 13 amino acid domain. This domain has sequence homology to 6-BH4 binding sites already identified on phenylalanine hydroxylase and 4a-carbinolamine dehydratase. Furthermore, this binding sequence appears to have been conserved during the evolution of tyrosinase as it has also been identified in the frog, mouse and human enzymes. 6-BH4 controls tyrosinase activity by an uncompetitive mechanism requiring the presence of L-tyrosine for effective down-regulation. When L-dopa is substrate, 6-BH4 does not inhibit the enzyme implicating separate binding sites for L-dopa and L-tyrosine on tyrosinase. Dihydropterin and 6-biopterin, the oxidation products of 6-BH4, do not inhibit tyrosinase significantly, indicating that melanin biosynthesis is controlled by a 6-BH4/6-biopterin redox-switch mechanism which can be initiated by photo-oxidation of 6-BH4.

Defective tetrahydrobiopterin and catecholamine biosynthesis in the depigmentation disorder vitiligo.

Patients with the depigmentation disorder vitiligo lack the capacity to synthesize the melanins from L-tyrosine via the essential activity of tyrosinase. The aim of this study has been to examine both the supply of the substrate (L-tyrosine) and the regulation of tyrosinase in the epidermis of subjects with vitiligo. Patients with this depigmentation disorder have a 3- to 5-fold increase in GTP-cyclohydrolase I activity leading to an excessive de novo synthesis of (6R)5,6,7,8 tetrahydrobiopterin (6-BH4). Continuous production of 6-BH-4 leads to: (1) an accumulation of the non-enzymatic byproduct 7-tetrahydropterin (7-BH4) in the epidermis, and (2) increased synthesis of the catecholamines in keratinocytes, leading to an excess of norepinephrine in both the plasma and urine of these patients. In vitiligo, the time-dependent production of 7-BH4 is caused by decreased 4a-hydroxytetrahydrobiopterin dehydratase activity; the essential enzyme for recycling and maintaining normal levels of 6-BH-4. In the epidermis and in cultured melanocytes, 7-BH4 is a potent competitive inhibitor of phenylalanine hydroxylase (Ki = 10(-6) M) and its accumulation in the epidermis of patients with vitiligo blocks the supply of L-tyrosine from L-phenylalanine. 4a-hydroxytetrahydrobiopterin dehydratase has a dual function as the activator/dimerization catalyst for the transcription factor hepatocyte nuclear factor I (HNF-I). HNF-I binds to a 16-base inverted palindrome which seems to be present on the promoters of both the tyrosinase and phenylethanolamine-N-methyl transferase (PNMT) genes. Therefore, defective 4a-hydroxytetrahydrobiopterin dehydratase in vitiligo influences not only the supply of L-tyrosine but also the transcription of the tyrosinase gene in melanocytes. Furthermore, a similar transcriptional regulation of the PNMT gene in keratinocytes offers a possible explanation for the accumulation of norepinephrine in these patients.

Inhibition of prostacyclin release by endothelial binding anticardiolipin antibodies in thrombosis-prone patients with systemic lupus erythematosus and the antiphospholipid syndrome.

IgG from 18 patients with SLE, eight with the primary antiphospholipid syndrome and 19 controls was examined for its effect on thrombin-induced prostacyclin (PGI2) release from human umbilical vein endothelial cells in relation to both the titre of anticardiolipin (ACA) and antiendothelial activity (AEA) and clinical thrombotic events. Although no significant inhibition of PGI2 release was found overall, examination of subgroups revealed that IgG from patients with ACA produced significant inhibition of PGI2 release (mean stimulation index IgGM, 0.74 +/- 0.12, P = 0.02) when compared with patients without ACA (1.18 +/- 0.12). Further analysis revealed a significant positive correlation between ACA and AEA (r = 0.52, P = 0.006) in the total patient group which was reflected in significant negative correlations between inhibition of PGI2 release and increasing titre of both ACA (r = -0.42, P = 0.032) and AEA (r = -0.57, P = 0.002). However, only increasing titre of AEA showed a significant negative correlation with inhibition of PGI2 release when patients with (r = -0.74, P = 0.0005) and without (r = 0.23, N.S.) thromboses were compared. The titre of ACA failed to show any significant correlation with inhibition of PGI2 release in either patients with (r = -0.42, N.S.) or without (r = -0.16, N.S.) thromboses. These findings suggest that previous, sometimes conflicting, reports of an association between inhibition of PGI2 release and ACA may be explained by the co-incidence of AEA with ACA.

Stimulation of von Willebrand factor antigen release by immunoglobulin from thrombosis prone patients with systemic lupus erythematosus and the anti-phospholipid syndrome.

The level of von Willebrand factor antigen (vWF) released by endothelial cells in response to IgG isolated from 18 patients with systemic lupus erythematosus (SLE), eight patients with the anti-phospholipid syndrome (APS) and 22 controls has been measured. Incubation with IgG from the combined patient group resulted in a significantly greater release of vWF (mean stimulation index +/- SEM, 4.57 +/- 0.78) when compared with controls (1.96 +/- 0.22, P = 0.003). Furthermore, IgG from 17 patients who had had a history of thrombosis induced higher levels of vWF release (5.33 +/- 1.09) when compared with the controls (P = 0.008). These findings suggest that IgG from patients with SLE or APS is capable of stimulating vWF release and that this ability may be implicated in the thrombotic events that are observed in these conditions.

Cortical and vascular prostaglandin synthesis during renal allograft rejection in the rat.

Alterations in local prostacyclin and thromboxane synthesis could mediate the changes in vascular perfusion and platelet deposition in acutely rejecting renal allografts and prostaglandin E2 (PGE2) has been implicated in the regulation of the immune response. 6-Keto-prostaglandin F1 alpha (6 KetoPGF1 alpha), thromboxane B2 (TxB2) (the stable degradation products of prostacyclin and thromboxane A2 [TxA2], respectively) and PGE2 were measured in incubates of cortical slices taken from rat renal allografts or isografts one to seven days after transplantation. 6 KetoPGF1 alpha and TxB2 synthesis was also measured in incubates of blood vessels supplying and transplanted with the kidney in these animals. During the phase of cellular rejection (3-5 days), TxB2 synthesis was selectively elevated in allografted renal cortex, renal artery, renal vein, and abdominal aorta in comparison with isografted tissues. There was also a small but significant rise in cortical PGE2 synthesis at this time, but vascular and cortical 6 KetoPGF1 alpha production remained unchanged. Renal infarction, occurring 7 days after transplantation, was accompanied by a nonspecific rise in the synthesis of all three prostaglandins by renal cortical slices. Increased tissue TxA2 synthesis may contribute to local thrombosis and decreased graft perfusion during acute rejection, thereby potentiating graft destruction.

Spontaneous remission of cryptosporidiosis in a child with acute lymphocytic leukemia.

Cryptosporidiosis has been documented in pediatric patients with various forms of congenital and acquired immunodeficiency. This is a report of cryptosporidiosis in a child with acute lymphocytic leukemia. Cryptosporidiosis, an uncommon cause of diarrhea, may produce severe diarrhea in children with acute lymphocytic leukemia.

Renal transplantation in the rat--a new simple, non-suture technique.

Renal transplantation in the rat can be performed by a simple technique utilising cuff anastomosis. This method is quick and reliable, and the results compare well with those achieved by standard microsurgical techniques.

Splenic autotransplant and residual partial spleen: prevention of septicemia.

Postsplenectomy septicemia carries an ominous prognosis. Accompanying disseminated intravascular coagulation and adrenal hemorrhage result in a high mortality, despite aggressive treatment by antibiotics. The efficacy of prevention by in situ partial spleen and splenic auto-transplant were evaluated in Sprague-Dawley rats. All totally splenectomized rats died following intravenous challenge of live pneumococcus. Both partial spleens and autotransplants gave substantial protection. The rats which succumbed to pneumococcal sepsis demonstrated massive fibrin thrombi in renal glomeruli and frank adrenal hemorrhage, strikingly similar to clinical observations.

A microsurgical technique for simultaneous pancreas and renal transplantation in the rat.

A successful technique of simultaneous pancreatico-renal transplantation into the streptozotocin-induced diabetic rat is described employing a sutureless cuff technique for three of four vascular anastomoses. Reversal of the diabetic state was uniformly observed within 24 hours postoperatively and near-normal renal function was evidenced by 50 days postoperatively. The technique is reliable and reproducible and represents the first report of successful combined transplantation into the diabetic rat.