Critical factors influencing prion inactivation by sodium hydroxide.

BACKGROUND AND OBJECTIVES: Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases caused by aberrantly folded cellular proteins (PrP(Sc); prions) that are generally resistant to conventional pathogen-inactivation techniques. To ensure effective decontamination and inactivation of prions that could be present in source material, we investigated critical factors that influence prion inactivation by NaOH. MATERIALS AND METHODS: A decrease in prion infectivity correlates with the disappearance of the protease-resistant core of PrPSc (PrPRES) observed in biochemical assays. To model prion inactivation, hamster scrapie (strain 263K) brain homogenate (SBH) was incubated for specific periods of time in 0.1 m NaOH at 4 or 18 degrees C, with or without detergent. Neutralized samples were subjected to limited digestion with proteinase K (PK) and then analysed using an endpoint dilution western blot assay and antibody 3F4. Structural changes in prions exposed to NaOH were examined using differential immunoprecipitation. RESULTS: Treatment of SBH with 0.1 m NaOH for 15 min, in the absence of detergent, at 4 and 18 degrees C caused a reduction in the PrP(RES) signal of 3.5 and 4.0 log10 units, respectively, with some residual signal remaining. The presence of the detergent sarkosyl during a 60-min incubation in NaOH further enhanced PrPRES reduction to > or = 4.5 log10 units (i.e. below the limit of detection). NaOH treatment induced conformational changes in PrP that resulted in the exposure of a hidden epitope and enabled prion immunoprecipitation by antibody 3F4. CONCLUSIONS: The use of NaOH can effectively reduce prion levels in an in vitro inactivation assay. After pretreatment of SBH with detergent, NaOH completely eliminates the PrPRES signal. Detergent may liberate lipid membrane-protected PrPSc to improve access to NaOH, which can then inactivate PrPSc by altering its structure. In cases of unidentified exposure to PrPSc during manufacturing, sanitizing procedures combining the use of detergent and NaOH may help to ensure minimal levels of contamination carryover in products.

Pharmacokinetics, thrombogenicity and safety of a double viral inactivated factor IX concentrate compared with a prothrombin complex concentrate.

Therapeutic options for developing countries have to assure an optimum safety and efficacy and low-cost antihaemophilic concentrates. A single blind randomized crossover study was carried out in 12 previously treated HB patients, comparing the pharmacokinetics (PK), thrombogenicity (TG) and safety of two plasma-derived double-inactivated (solvent/detergent heating at 100 degrees C, 30 min) factor IX (FIX) concentrates, UMAN COMPLEX DI (product A) [plasma-derived prothrombin concentrates (PCC)] and a high purity FIX concentrate AIMAFIX DI (product B, HPFIX). In a non-bleeding state, they received one single intravenous dose 50 IU FIX kg(-1) of PCC or HPFIX, and after a wash-out period of 14 days, the other product. We evaluated acute tolerance and determined PK parameters based on FIX levels measured over a 50 h postinfusion period. We studied fibrinogen, platelets, antithrombin, F1 + 2, TAT, D-dimer, over a 360 min postinfusion period. Ten cases remained in on-demand treatment for 6 months, five with PCC and five with HPFIX. PK and anti-FIX inhibitors were repeated at 3 and 6 months. No inhibitors were detected. PK values (PCC vs. HPFIX): clearence (CL; mL h(-1) kg(-1)) 5.2 +/- 1.4 vs. 6.5 +/- 1.4; the volume of distribution at steady state (mL kg(-1)) 154.9 +/- 54.9 vs. 197.5 +/- 72.5; mean residence time (h) 29.7 +/- 8.1 vs. 30.7 +/- 9.2; T(1/2) (h) 22.3 +/- 7 vs. 23.5 +/- 12.3; incremental recovery (IR; U dL(-1) U(-1) kg(-1)) 0.96 +/- 0.17 vs. 0.76 +/- 0.13. HPFIX showed significant lower IR and higher CL. There were no differences in PK at 3 and 6 months. In TG, significant increments in TAT and F1 + 2 at 30 min and 6 h were found with PCC. Product B PK results agrees with reported results for other HPFIX preparations. Use of PCC product A has to consider its thrombogenic activity.

Intravenous immunoglobulin treatment of lupus nephritis.

OBJECTIVE: To evaluate the clinical response of treatment-resistant membranous and membranoproliferative lupus nephritis to intravenous immunoglobulin (IVIg). METHODS: Seven lupus nephritis patients who failed to respond to at least prednisone and cyclophosphamide were studied. A kidney biopsy showing either membranous or membranoproliferative glomerulonephritis was available in six patients. They were treated with six courses (patients 1 and 2) or 1 or 2 courses (patients 3 through 7) of high-dose IVIg. For patients 3 through 7, the plasma levels of albumin, total cholesterol, urea, creatinine, dsDNA antibody titers, and daily proteinuria were measured just before the IVIg therapy, immediately on completion, and 6 months later. RESULTS: All seven patients had a beneficial response to IVIg. In patient 1, decrease in proteinuria was evident 2 weeks after IVIg was started, nephrotic syndrome gradually disappeared, and she had no proteinuria in 3 years' follow-up. Decline in proteinuria was evident in patient 2 after the 4th IVIg course, but proteinuria reached the pretreatment level 4 months after the therapy ended. In patients 3 through 7, the mean daily proteinuria before IVIg (5.3 +/- 2.1 g) decreased after 1 or 2 IVIg courses (3.3 +/- 1.4 g), and further decreased when measured 6 months later (2.1 +/- 1.3 g). Similarly, the plasma cholesterol level decreased while the plasma albumin level increased after IVIg. CONCLUSIONS: IVIg might be effective in treatment-resistant membranous or membranoproliferative lupus nephritis. Future studies should concentrate on determining the preferred treatment protocol of IVIg for the various classes of lupus nephritis.

Treatment of hematologic disorders other than immune thrombocytopenic purpura with intravenous immunoglobulin (IVIg) - report of seven cases and review of the literature.

Background: Intravenous immunoglobulin (IVIg) is a standard therapeutic modality for a few autoimmune diseases, such as immune thrombocytopenic purpura. However, in other hematologic autoimmune conditions its role is still controversial. Methods: Seven patients with either autoimmune hemolytic anemia, Evans' syndrome, aplastic anemia, pure red cell aplasia, thrombotic thrombocytopenic purpura, or acquired factor VIII inhibitors were treated with a single course of high-dose (2 g/kg) IVIg. Results: A good response was observed in all seven patients, except the one with thrombotic thrombocytopenic purpura, and there were no adverse effects related to IVIg use. The literature reports on IVIg therapy in these conditions and the mechanisms of action of IVIg in autoimmune diseases are discussed. Conclusions: IVIg might be a useful agent in the treatment of several hematologic disorders other than immune thrombocytopenic purpura. Future aims of clinical research in this respect would be to identify the patient subgroups that might benefit more from IVIg in these conditions.

A study of 20 SLE patients with intravenous immunoglobulin--clinical and serologic response.

OBJECTIVE: To test the clinical response of systemic lupus erythematosus (SLE) patients to intravenous immunoglobulins (IVIg), and whether the clinical response of IVIg treatment in SLE is accompanied by modification of SLE-associated autoantibodies/antibodies (Abs) and complement levels. METHODS: Twenty SLE patients were treated with high-dose (2 g/kg) IVIg monthly, in a 5-d schedule. Each patient received between 1-8 treatment courses. They were evaluated for the clinical response, Systemic Lupus Activity Measure (SLAM) score before and after IVIg, levels of antinuclear antibody (ANA), dsDNA (double-stranded DNA), SS-A or SS-B, ENA (extractable nuclear antigens), C3 and C4 levels before and after the treatment, and before and after each treatment course. RESULTS: A beneficial clinical response following IVIg treatment was noted in 17 out of 20 patients (85%). Few clinical manifestations responded more to treatment: arthritis, fever, thrombocytopenia, and neuropsychiatric lupus. In 9 patients evaluated before and after IVIg, mean SLAM score decreased from 19. 3+/-4.7 to 4+/-2.9 (P<0.0001). There was a tendency towards abnormal levels of complement and Abs before IVIg courses among the treatment responders compared with the non-responders, and similarly the former tended to have normalization of their abnormal levels more than the latter. These differences were found statistically significant only with respect to C4 and SS-A or SS-B levels before IVIg courses. CONCLUSION: IVIg has a high response rate among SLE patients. A combination of clinical manifestations, Abs and complement levels may aid in the future in predicting who among SLE patients will benefit more from IVIg treatment.

Serologic and clinical response to treatment of systemic vasculitis and associated autoimmune disease with intravenous immunoglobulin.

BACKGROUND: Autoimmune vasculitides cannot always be controlled by steroids and immunosuppressive drugs. Intravenous immunoglobulin (IVIg) treatment was found beneficial in several vasculitides including systemic and organ-specific diseases. In this article we tested whether the beneficial clinical response of IVIg treatment in vasculitides was accompanied by a decrease in vasculitis-associated autoantibody levels. METHODS: Ten patients diagnosed as having vasculitis were treated with high-dose (2 g/kg) IVIg monthly, in a 5-day schedule. In all the patients, other therapeutic measures failed to control disease progression prior to IVIg treatment. Each patient received between 1 and 6 treatment courses. All patients were evaluated for the levels of 5 autoantibodies (Abs) related to vasculitis before and after each treatment course. RESULTS: In 6 out of the 10 patients, a beneficial clinical response followed IVIg treatment. Moreover, no treatment-related adverse effects were observed in any of the patients. Anti-myeloperoxidase antibodies and cytoplasmic-antineutrophil cytoplasmic antibodies levels decreased concomitantly with the clinical improvement observed in the patients with Churg-Strauss vasculitis and Wegener's granulomatosis, respectively. Levels of cytoplasmic-antineutrophil cytoplasmic antibodies (ANCA) with specificity for bacteridial/permeability-increasing protein and human lysosomal-associated membrane protein increased after each treatment course, but returned to normal values before the following one. CONCLUSIONS: When other therapeutic measures, such as immunosuppressive therapy, fails to control disease manifestations in patients with vasculitides, IVIg is a possible effective intervention method with a high response rate. IVIg probably exerted its effects on disease progression via different mechanisms. Among these mechanisms, a decrease in relevant Ab levels is often found (probably by anti-idiotypes in IVIg), and thus ANCA levels are expected to associate with disease activity.

Marked improvement of Churg-Strauss vasculitis with intravenous gammaglobulins.

Churg-Strauss vasculitis (CSV) is a systemic vasculitis usually treated with steroids and cytotoxic drugs. Herein, we describe the beneficial effect of intravenous immune globulin (IVIG) in a 50-year-old man with CSV. The IVIG therapy brought a marked clinical improvement, with impressive decline in the titers of antineutrophil cytoplasmic autoantibody.

Autoantibody level modification in adult patients with idiopathic thrombocytopenic purpura following intravenous immunoglobulin treatment.

The aim of this study was to determine whether treatment of patients with immune thrombocytopenic purpura (ITP) with intravenous immunoglobulin (IVIg) is associated with a modification in the antiplatelet glycoprotein (GP) antibodies (Abs). Fourteen patients with ITP (11 females and 3 males, mean age 36.6 years, range 18-72) received one to four IVIg treatment courses. The preparation used was ISIVEN that was given in a dose of 2 g/kg body weight in a 5-day schedule and in monthly intervals. Levels of IgG, IgM and IgA isotypes of Abs to GPs IIb/IIIa and Ib/IX were measured before the treatment, and before and after each treatment course. Two patients did not respond to IVIg, 6 had a temporary response, 5 had a sustained response and 1 patient responded well to the treatment but was lost to follow-up. The patients had a high prevalence of serum Abs directed against GPs IIb/IIIa and Ib/IX before the treatment, and the mean IgG isotype levels of both Abs increased after each treatment course, and decreased again before the following course began. Whenever high Ab levels of either isotype (> 10 U/ml) were detected before the treatment, they were significantly decreased before the last treatment course. The elevated levels of IgG Abs to IIb/IIIa and Ib/IX after every course are probably a result of displacement of these Abs from Fc receptors by the IVIg, rather than of exogenous infusion of these Abs contained within the IVIg, whereas the decrease in high Ab levels after a few treatment courses results from the immunomodulatory effects of IVIg: suppression of Ab formation, and the presence of anti-idiotypes.