High-yield production of a human therapeutic protein in tobacco chloroplasts.

Transgenic plants have become attractive systems for production of human therapeutic proteins because of the reduced risk of mammalian viral contaminants, the ability to do large scale-up at low cost, and the low maintenance requirements. Here we report a feasibility study for production of a human therapeutic protein through transplastomic transformation technology, which has the additional advantage of increased biological containment by apparent elimination of the transmission of transgenes through pollen. We show that chloroplasts can express a secretory protein, human somatotropin, in a soluble, biologically active, disulfide-bonded form. High concentrations of recombinant protein accumulation are observed (>7% total soluble protein), more than 300-fold higher than a similar gene expressed using a nuclear transgenic approach. The plastid-expressed somatotropin is nearly devoid of complex post-translational modifications, effectively increasing the amount of usable recombinant protein. We also describe approaches to obtain a somatotropin with a non-methionine N terminus, similar to the native human protein. The results indicate that chloroplasts are a highly efficient vehicle for the potential production of pharmaceutical proteins in plants.

Anti beta2-glycoprotein I antibodies: clinical significance.

The introduction of anticardiolipin antibody (aCL) assay, described in 1983, was able to focus much attention on the study of patients suffering from thrombosis, repeated fetal loss and thrombocytopenia, and allowing the identification of the so called antiphospholipid syndrome (APS). The identification of beta2 glycoprotein I (beta2GPI) as an essential component of the antigenic complex recognized by aCL suggested that this molecule could be a direct target of the antibody response. Since then, different groups have described ELISAs for the detection of anti beta2GPI antibodies, applied to the clinical evaluation of patients with APS, and showing an overall better specificity. Recently, anti beta2GPI were also shown to bind apoptotic bodies resulting in an alteration of their physiological clearance with the triggering of TNFalpha release. This observation suggests that anti beta2GPI may also modify the immunogenicity of apoptotic bodies and of the autoantigens that they contain.

Anticardiolipin and anti-beta 2glycoprotein I immunoassays in the diagnosis of antiphospholipid syndrome.

Autoantibodies directed to phospholipids or to phospholipid binding proteins are now studied using a growing number of laboratory tests. However, the history of the interest in this area goes back to the identification of the so-called false positive reactions in the non-treponemal serological test for syphilis (STS) and to the subsequent description of an in vitro coagulation defect called lupus anticoagulant (LAC). In the 1980s the introduction of the anticardiolipin antibody (aCL) immunoassay was a determining factor in the definition of the antiphospholipid syndrome (APS). In addition, lupus prone mice spontaneously producing aCL antibodies and normal mice passively infused with these antibodies provided useful models for the study of the pathogenic role of aPL. When in 1990 a phospholipid binding protein (beta 2glycoprotein I, beta 2GPI) was identified as the cofactor required for aCL antibody binding, the true antigenic target of the antibodies was first discussed. Soon afterwards an anti-beta 2GPI ELISA was developed that has proved to be of great clinical significance. We will discuss here the similarities and differences between these various assays (LAC, aCL, and anti-beta 2GPI), focusing on their specificity, sensitivity and practical applications.

Anti-beta 2 glycoprotein I antibodies in a general obstetric population: preliminary results on the prevalence and correlation with pregnancy outcome. Anti-beta2 glycoprotein I antibodies are associated with some obstetrical complications, mainly preeclampsia-eclampsia.

OBJECTIVE: To evaluate the prevalence in normal pregnancies of anti-32 glycoprotein I (anti-beta2GPI) antibodies, and their association with obstetrical complications. STUDY DESIGN: Prospective study of anti-beta2GPI and anticardiolipin (CL) antibodies in 510 healthy pregnant women at 15-18 weeks. According to the results, women were categorized into three groups: group I, negative for both antibodies; group II, positive for anti-beta2GPI antibodies; group III, positive for aCL only. The rates of fetal loss, abruptio placentae, preeclampsia-eclampsia, and fetal growth retardation were compared in the three groups. RESULTS: Anti-beta2GPI antibodies were found in 20 women (3.9%) and aCL in 8 patients (1.6%). Obstetrical complications were more frequent, even if not significantly different, in group II, 15%, than in group I, 4.1% (difference 10.9%; 95% confidence interval (CI): 1.6-20.2%; p=0.0575), while no complications were seen in group III. Preeclampsia-eclampsia were significantly more frequent in group II (10%) than in group I (0.8%; difference 9.2%; 95% CI: 4.4-14%; p=0.021). The prevalence of fetal growth retardation was not significantly different in the two groups (5% vs. 2%, respectively). COMMENT: Our findings indicate that anti-beta2GPI antibodies are associated with some obstetrical complications, mainly preeclampsia-eclampsia, even if more conventional antiphospholipid antibodies are not present. This observation suggests that these antibodies should be investigated in such cases, in order to improve the outcome of subsequent pregnancies, as well as in women with a history of early and/or recurrent severe preeclampsia in order to start a prophylactic treatment (i.e. low-dose aspirin or heparin).

Utilization of dilute Russell's viper venom time to detect autoantibodies against beta 2-glycoprotein I which express anticoagulant activity in the presence but not in the absence of exogenous phospholipids.

Lupus anticoagulant (LA) is a general term to define immunoglobulins interfering with phospholipid-dependent coagulation tests. It is now clear that the phospholipid-dependence of some LA is related to the presence of the phospholipid-binding plasma protein beta 2-glycoprotein I (beta 2-GPI) and that autoantibodies to beta 2-GPI might represent a specific category of LA. To verify this hypothesis we have purified IgG autoantibodies to beta 2-GPI from plasma of 6 patients with antiphospholipid antibody syndrome, by means of agarose-immobilized human beta 2-GPI. All 6 preparations tested positive in anti-beta 2-GPI IgG antibody ELISA and showed a marked LA activity by prolonging dilute Russell Viper Venom Time (dRVVT) from a minimum of 5.3 s in patient # 1 to a maximum of 41.1 s in patient # 3. These IgG preparations behaved as typical LA, with this activity tending to disappear in the presence of increasing phospholipid (PL) concentrations. Moreover, the LA activity of the IgG preparations was not detectable in the absence of PL, in which case the ratio between dRVVT obtained in the presence and absence of IgG autoantibodies to beta 2-GPI was close to 1. This pattern was confirmed by using plasma from patients with antiphospholipid antibody syndrome testing positive for anti-beta 2-GPI IgG antibodies. These findings suggest that dRVVT performed both in the presence and absence of PL might constitute a sensitive screening test to detect specific antibodies with LA activity.

The anti-beta2-glycoprotein I activity in human anti-phospholipid syndrome sera is due to monoreactive low-affinity autoantibodies directed to epitopes located on native beta2-glycoprotein I and preserved during species' evolution.

To characterize the reactivity pattern of Abs directed to beta2-glycoprotein I (anti-beta2GPI) in patients with anti-phospholipid syndrome, we have purified anti-beta2GPI Abs by affinity chromatography using the IgG fractions from sera of five different anti-phospholipid syndrome patients. Affinity-purified anti-beta2GPI were shown to be representative of Abs found in human sera because their activity could be virtually abolished from the IgG preparations after repeated absorptions on immobilized human beta2GPI column. Our results show that affinity-purified anti-beta2GPI: 1) do react with beta2GPI in the absence of any phospholipid, as demonstrated by the lack of phosphorus contaminant in the employed reagents, as well as by their comparable binding activity before and after extensive delipidation procedure; 2) can recognize beta2GPI regardless of its origin from different animal species; 3) are able to bind soluble beta2GPI with a mean Kd value of 4.65 x 10(-6) M (range 3, 4-7, 2 x 10(-6) M); 4) significantly enhance their binding avidity when beta2GPI is linked to a solid support; and 5) appear to be mainly monoreactive autoantibodies. In conclusion, we have shown that human polyclonal anti-beta2GPI are low affinity, mainly monoreactive autoantibodies directed to an epitope located on native beta2GPI, preserved along the species evolution.

Anti-beta 2-glycoprotein I antibodies: a marker of antiphospholipid syndrome?

Anticardiolipin (aCL) and anti-beta 2-glycoprotein I(anti beta 2GPI) antibodies have been shown in animal models as not cross-reacting antibody populations. This observation prompted us to prove if anti-beta 2GPI exist in human sera by using a reliable method and then to investigate if these are independent from aCl antibodies. We have developed a new ELISA for the detection of anti-beta 2GPI antibodies employing the coating of the protein in carbonate buffer to irradiated microtitre plates and the filtration of serum samples, that makes irrelevant the binding to the uncoated wells. IgG F(ab)2 fragments from IgG positive sera were shown bind beta 2GPI, providing that the binding was a specific antibody binding, mediated by the antigen binding site of the antibody molecule: moreover the antibodies were not able to differentiate native and delipidated beta 2GPI coated plates, making a possible role of a phospholipid contaminant unlikely. On the other hand, the phosphorus content of native as well as delipitated beta 2GPI was undetectable. IgG, but not IgM, anti-beta 2GPI antibodies were classically inhibited by the addition of soluble beta 2GPI, while cardiolipin liposomes appear to modify the reaction in a completely different way, possibly by the described interaction between cardiolipin and beta 2GPI.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between anti-phospholipid and anti-endothelial cell antibodies III: beta 2 glycoprotein I mediates the antibody binding to endothelial membranes and induces the expression of adhesion molecules.

OBJECTIVE: To investigate the role of antibodies reacting with beta 2 glycoprotein I (beta 2GPI) in the antiendothelial cell binding activity present in sera from patients with the anti-phospholipid syndrome. METHODS: Sera positive for anti-phospholipid, anti-endothelial and anti-beta 2 GPI antibodies were studied for their binding activity on endothelial monolayers cultured in the presence or absence of media containing bovine serum as a source of beta 2GPI. Anti-endothelial activity was also evaluated on endothelial cells cultured without serum and supplemented with exogenous human purified beta 2GPI. Affinity purified anti-beta 2 GPI antibodies were investigated under the same experimental conditions. Finally, the effect of the incubation of these affinity purified fractions on the expression of adhesion molecules (ELAM-1) was studied. RESULTS: The reactivity of the sera decreased on endothelial cells incubated in serum-free medium, while endothelial cell binding was restored in a dose dependent manner after the addition of exogenous purified human beta 2 GPI. Affinity purified anti-beta 2 GPI antibodies obtained from the same sera retained their endothelial cell binding and were able to activate endothelial cells by inducing the ex novo surface expression of adhesion molecules (ELAM-1). CONCLUSIONS: These findings indicate that the close association between anti-endothelial and anti-phospholipid antibodies is sustained by antibodies which recognize beta 2 GPI adhering to the endothelial cells, and can promote their activation.

Neurological involvement in antiphospholipid syndrome: clinical and instrumental evaluation in 21 consecutive cases.

Twenty-one consecutive women with the antiphospholipid syndrome were investigated with a full cardiological and neurological examination including a semi-structured interview. They were also systematically studied with magnetic resonance imaging (MRI), transcranial Doppler (TCD), duplex sonography of neck vessels and transesophágeal echocardiography (TEE). Nearly 67% of patients had signs or symptoms suggestive of CNS involvement Migraine-like headache, stroke, ocular disorders and epilepsy were the most frequent disturbances. MRI was abnormal in 65% of patients who underwent the examination and showed two distinct patterns of abnormality: either territorial infarctions or multiple subcortical spotty lesions. TCD showed in one case a stenosis affecting the main trunk of the middle cerebral artery. TEE demonstrated potentially emboligenic mitral vegetations in 72% of patients. These findings suggest that antiphospholipid syndrome is burdened with a high rate of complications affecting the CNS, which are likely to be mostly thromboembolic. It is possible that both cardiogenic embolism and in situ thrombosis of cerebral vessels occur. Given the relative absence of conventional vascular risk factors, antiphospholipid antibodies are likely to represent a true risk factor for cerebrovascular disease through the mechanism of an immunologically mediated hypercoagulable state.

Systemic lupus erythematosus and pregnancy: a prospective study.

We have prospectively followed 25 pregnancies in 21 patients: 20 were affected by systemic lupus erythematosus (SLE) and 1 by subacute cutaneous lupus erythematosus (SCLE). A flexible treatment schedule was applied to the follow-up of all the pregnancies, and included low dose aspirin, steroids at medium-low dosage and, if needed, azathioprine (AZA) after 20 weeks of gestation. There were 4 spontaneous first trimester abortions and 21 live-born neonates without major problems related to the treatment or to the maternal disease. The relapse rate of the disease recorded during the observation period was 0.07 patient/month, not different from that already reported in SLE patients (pregnant or nonpregnant). Obstetrical complications were relatively frequent, but careful monitoring allowed us to avoid late fetal wastage. We conclude that in SLE patients a successful pregnancy outcome, without worsening of the disease, can be obtained with a careful multidisciplinary follow-up.