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Quality control of human immunoglobulin formulations produced by caprylic acid precipitation necessitates a simple, rapid, and accurate method for determination of residual caprylic acid. A high-performance liquid chromatography method for that purpose was developed and validated. The method involves depletion of immunoglobulins, the major interfering components that produce high background noise, by precipitation with acetonitrile (1:1, v/v). Chromatographic analysis of caprylic acid, preserved in supernatant with no loss, was performed using a reverse-phase C18 column (2.1 × 150 mm, 3 μm) as a stationary phase and water with 0.05% TFA–acetonitrile (50:50, v/v) as a mobile phase at a flow rate of 0.2 mL/min and run time of 10 min. The developed method was successfully validated according to the ICH guidelines. The validation parameters confirmed that method was linear, accurate, precise, specific, and able to provide excellent separation of peaks corresponding to caprylic acid and the fraction of remaining immunoglobulins. Furthermore, a 24−1 fractional factorial design was applied in order to test the robustness of developed method. As such, the method is highly suitable for the quantification of residual caprylic acid in formulations of human immunoglobulins for therapeutic use, as demonstrated on samples produced by fractionation of convalescent anti-SARS-CoV-2 human plasma at a laboratory scale. The obtained results confirmed that the method is convenient for routine quality control.

Relevance. Replacement therapy with human immunoglobulins is the leading pathogenetic method of treatment of primary immunodeficiency with impaired antibody synthesis. Currently, several preparations of human immunoglobulins for intravenous administration are available in Russia. Purposes. Evaluation of the efficacy and safety of Privigen immunoglobulin intravenous 10% liquid in real clinical practice within 12 clinical months. Methods. Twenty adults diagnosed with common variable immunodeficiency or X-linked agammaglobulinemia received intravenous Privigen® infusions (0.2-0.4 mg/kg) at 24±3 intervals over a 12-month period. The primary endpoint was the annual rate of infections and adverse events. Results. Sufficient level of IgG was achieved in most patients during the study. Severe adverse reactions during the treatment were not registered. Conclusions. High efficacy and safety of monthly replacement therapy in patients with primary immunodeficiency with impaired antibody synthesis has been demonstrated.

Method development for determining prekallikrein activator in medicinal preparations of human immunoglobulins and albumin found that the adequacy of chromogenic determination of prekallikrein activator depends on the activity of the used prekallikrein reagent, the quantitative component ratio in the reaction mixture and its incubation time, and the concentration of employed chromogenic reagent. Acceptance criteria for commercial prekallikrein reagents are justified. The main method parameters and validation test results confirming the linearity in the range 0 – 35 IU/mL, accuracy, and intralaboratory precision are presented and correspond to those for biological analytical methods. The elaborated method can be used for quality assessment of prekallikrein activator content in medicinal preparations of human immunoglobulins and albumin.

A method for determining the content of prekallikrein activator in preparations based on human immunoglobulins and albumin has been developed. It is found that the adequacy of determining the content of prekallikrein activator by the chromogenic method depends on the activity of the prekallikrein reagent used, the quantitative ratio of components in the reaction mixture and the duration of its incubation, and the concentration of the chromogenic substrate employed. Adopted acceptance criteria for commercial prekallikrein reagents are justified. Results of development of the main parameters of the proposed chromogenic technique and the materials of validation studies are presented, which confirm its linearity in the range from 0 to 35 IU/mL, correctness and intra-laboratory precision corresponding to those for biological methods of analysis are presented. The proposed method can be used to assess the quality of preparations based on human immunoglobulins and human albumin with respect to the content of prekallikrein activator.

Human immunoglobulin and human albumin products can negatively affect patients’ health. The residual content of haemagglutinins, anti-D antibodies, prekallikrein activator, as well as the level of anticomplementary activity, are controlled during both production and testing for compliance with normative documentation. Methods for their quantitative evaluation are based on the effects of erythrocytes hemolysis or agglutination and on the amidolytic product properties of cascade reactions. These methods require the obligatory use of reference materials (RMs). The development of RMs, acting as the carrier of the quantitative characteristics of the human immunoglobulins and human albumin impurities, is fraught with the difficulty of selecting a candidate, substantiating the certification methodology, and determining the relevant characteristics. In this research, we used human immunoglobulin solutions with normalized haemagglutinin content, anti-D antibodies or anti-complementary activity in a different range, whose quality was studied using haemagglutination and chromogenic methods, and the complement fixation test. Methodological approaches to the RM development and certification were justified using system analysis and forensic audit methods. The results demonstrate the necessity of using RMs to assess the stability of analytical work by applying anti-complementary activity determination, haemagglutinin content or anti-Dantibody methods. It is established that RMs allow the quantitative content of haemagglutinins, anti-D antibodies and prekallikrein activator to be established. The value of the CRM characteristics should be approximated to the qualitative composition of these impurities in the analysed products, but in amounts not detectable (to produce negative RM components) or exceeding permissible maxima (to produce positive RM components).

Immunoglobulin preparations are medicines derived from blood used as a replacement therapy for immunodeficiencies or as an immunomodulator. While they are generally well-tolerated, side effects, rarely severe, can nevertheless occur when administered intravenously. They are usually related to an excessive perfusion rate. The recent arrival of safer products administered subcutaneously represents progress in the treatment of patients.

Immunoglobulins G (IgG) are proteins produced by the immune system of higher life forms that play a central role in the defense against microbial pathogens. IgG bind pathogens with the hypervariable Fab component and mediate a diversity of effector functions by binding to immune effector cells via their crystallizable (Fc) component. All IgG Fc carry a polymorphic N-glycan that regulates its binding properties and thereby its effector functions. The glycosylation profile of IgG Fc is modulated by physiological and pathological conditions, including infectious diseases and inflammatory disorders. Characterization of IgG Fc glycosylation profiles is a promising approach to understand the pathogenesis of diseases involving the immune system and to develop novel biomarkers of disease activity. Measuring the proportion of the different IgG Fc glycoforms remains an analytical challenge, that requires a sensitive and reproducible analytical approach.This chapter describes an optimized approach for the preparation and the analysis of Fc N-glycans from total serum or plasma IgG using magnetic beads, RapiFluor MS label©, and LC-MS.

The in-depth characterization of protein glycosylation has become indispensable in many research fields and in the biopharmaceutical industry. Especially knowledge about modulations in immunoglobulin G (IgG) N-glycosylation and their effect on immunity enabled a better understanding of human diseases and the development of new, more effective drugs for their treatment. This chapter provides a deeper insight into capillary (gel) electrophoresis-based (C(G)E) glycan analysis, addressing its impressive performance and possibilities, its great potential regarding real high-throughput for large cohort studies, as well as its challenges and limitations. We focus on the latest developments with respect to miniaturization and mass spectrometry coupling, as well as data analysis and interpretation. The use of exoglycosidase sequencing in combination with current C(G)E technology is discussed, highlighting possible difficulties and pitfalls. The application section describes the detailed characterization of N-glycosylation, utilizing multiplexed CGE with laser-induced fluorescence detection (xCGE-LIF). Besides a comprehensive overview on antibody glycosylation by comparing species-specific IgGs and human immunoglobulins A, D, E, G, and M, the chapter comprises a comparison of therapeutic monoclonal antibodies from different production cell lines, as well as a detailed characterization of Fab and Fc glycosylation. These examples illustrate the full potential of C(G)E, resolving the smallest differences in sugar composition and structure.

A rapidly deployable drug product for use in the early stages of a disease outbreak could prevent further spread of the pathogen. Emergent BioSolutions is developing a rapid response platform (RRP) to manufacture human immunoglobulins (HIG) for use as a passive immunotherapy during public health emergencies. Passive immunotherapies derived from either convalescent plasma or purified immunoglobulins (Ig) have a long history of success in treating a range of viral, bacterial and toxin diseases, as they provide the immediate benefit of a protective response. Emergent's RRP is being developed to address the need for front-line therapy/prophylaxis with immediate benefit with the potential to overcome the current limitations of convalescent plasma. Emergent's proposed RRP begins with identification of plasma donors with measurable levels of pathogen-specific antibodies using a field deployable real-time donor screening assay. Plasma collected from eligible donors is treated using the Mirasol® Pathogen Reduction Technology, pooled, purified and concentrated into HIG using Emergent's rapid HIG manufacturing process to produce product formulated for intramuscular administration. Our rapid HIG process is chromatography based and each 30 L run aims to purify, formulate, and fill ∼65 doses (1 g of total IgG) with testing and release onsite for immediate use. Multiple innovations to streamline operations, including single-use technologies and minimal human interfaces, allow for the manufacture of HIG product in ∼30 hours. To house the process, Emergent has designed and constructed a working modular manufacturing unit (MMU) prototype. The MMU consists of a modified 53-foot shipping container designed for multiple modes of transport, to meet ISO 8 environment requirements with self-contained utilities, including HVAC, and can run off a diesel generator or local power. Product from multiple development runs within the MMU have been characterized and validation work is ongoing. Key messages A modular manufacturing unit is being developed with the potential to provide a local capability to produce health solutions for endemic diseases. A modular manufacturing unit is being developed with the potential to rapidly manufacture human immunoglobulins during public health emergencies and pandemics.

This retrospective study presents our 4‐year experience of preemptive treatment of early anti‐HLA donor specific antibodies with IgA‐ and IgM‐enriched immunoglobulins. We compared outcomes between patients with antibodies and treatment (case patients) and patients without antibodies (control patients). Records of patients transplanted at our institution between March 2013 and November 2017 were reviewed. The treatment protocol included one single 2 g/kg immunoglobulin infusion followed by successive 0.5 g/kg infusions for a maximum of 6 months, usually combined with a single dose of anti‐CD20 antibody and, in case of clinical rejection or positive crossmatch, with plasmapheresis or immunoabsorption. Among the 598 transplanted patients, 128 (21%) patients formed the case group and 452 (76%) the control group. In 116 (91%) patients who completed treatment, 106 (91%) showed no antibodies at treatment end. Fourteen (13%) patients showed antibody recurrence thereafter. In case versus control patients and at 4‐year follow‐up, respectively, graft survival (%) was 79 versus 81 (P = .59), freedom (%) from biopsy‐confirmed rejection 57 versus 53 (P = .34), and from chronic lung allograft dysfunction 82 versus 78 (P = .83). After lung transplantation, patients with early donor‐specific antibodies and treated with IgA‐ and IgM‐enriched immunoglobulins had 4‐year graft survival similar to patients without antibodies and showed high antibody clearance., Lung‐transplanted patients who develop early anti‐HLA donor‐specific antibodies and are treated with a protocol based on successive infusion of IgA‐ and IgM‐enriched intravenous immunoglobulins show good antibody clearance and graft survival, similar to the survival of patients without early donor‐specific antibodies.

Nanobodies are special derivatives of antibodies, which consist of single domain fragments. They have become of considerable interest as next-generation biotechnological tools for antigen recognition. They can be easily engineered due to their high stability and compact size. Nanobodies have three complementarity-determining regions, CDRs, which are enlarged to provide a similar binding surface to that of human immunoglobulins. Here, we propose a benchmark testing algorithm that uses 3D structures of already existing protein-nanobody complexes as initial structures followed by successive mutations on the CDR domains. The aim is to find optimum binding amino acids for hypervariable residues of CDRs. We use molecular dynamics simulations to compare the binding energies of the resulting complexes with that of the known complex and accept those that are improved by mutations. We use the MDM4-VH9 complex, (PDB id 2VYR), fructose-bisphosphate aldolase from Trypanosoma congolense (PDB id 5O0W) and human lysozyme (PDB id 4I0C) as benchmark complexes. By using this algorithm, better binding nanobodies can be generated in a short amount of time. We suggest that this method can complement existing immune and synthetic library-based methods, without a need for extensive experimentation or large libraries., NA

Валидирована методика определения антикомплементарной активности лекарственных препаратов иммуноглобулинов (Ig) человека для внутривенного введения методом реакции связывания комплемента по показателям правильность, прецизионность (повторяемость, внутрилабораторная прецизионность), предел количественного определения, линейность и аналитическая область. Методика может быть использована для оценки качества по показателю «Антикомплементарная активность» в рамках проведения фармацевтической экспертизы качества лекарственных препаратов Ig человека.

Refractory recurrent pericarditis is a major clinical challenge after colchicine failure, especially in corticosteroid-dependent patients. Human intravenous immunoglobulins (IVIGs) have been proposed as possible therapeutic options for these cases. The goal of this systematic review is to assess the efficacy and safety of IVIGs in this context.Studies reporting the use of IVIG for the treatment of recurrent pericarditis and published up to October 2014 were searched in several databases. All references found, upon initial assessment at title and abstract level for suitability, were consequently retrieved as full reports for further appraisal.Among the 18 citations retrieved, 17 reports (4 case series and 13 single case reports, with an overall population of 30 patients) were included. The mean disease duration was 14 months and the mean number of recurrences before IVIG was 3. Approximately 47% of patients had idiopathic recurrent pericarditis, 10% had an infective cause, and the remainder a systemic inflammatory disease. Nineteen out of the 30 patients (63.3%) were on corticosteroids at IVIG commencement. IVIGs were generally administered at a dose of 400-500 mg/kg/day for 5 consecutive days with repeated cycles according to the clinical response. Complications were uncommon (headache in ~3%) and not life-threatening. After a mean follow-up of approximately 33th months, recurrences occurred in 26.6% of cases after the first IVIG cycle, and 22 of the 30 patients (73.3%) were recurrence-free. Five patients (16.6%) were on corticosteroids at the end of the follow-up.IVIGs are rapidly acting, well tolerated, and efficacious steroid-sparing agents in refractory pericarditis.

Purpose The Göttingen minipig is a relevant non-rodent species for regulatory toxicological studies. Yet, its use with therapeutic antibodies has been limited by the unknown binding properties of human immunoglobulins (huIgG) to porcine Fc gamma receptors (poFcγR) influencing safety and efficacy readouts. Therefore, knowing IgG-FcγR interactions in the animal model is a prerequisite for the use of minipigs in preclinical safety and efficacy studies with therapeutic antibodies. Methods Here, we describe the cloning and expression of poFcγRs and their interactions with free and complexed human therapeutic IgG1 by surface plasmon resonance and flow cytometry. Results We show here that poFcγRIa, poFcγRIIa, and poFcγRIIb bind huIgG1 antibodies with comparable affinities as corresponding huFcγRs. Importantly, poFcγRs bind huIgG immune complexes with high avidity, thus probably allowing human-like effector functions. However, poFcγRIIIa binds poIgG1a but not to huIgG1. Conclusions The lack of binding of poFcγRIIIa to huIgG1 might cause underestimation of FcγRIIIa-mediated efficacy or toxicity as mediated by porcine natural killer cells. Therefore, the suitability of minipigs in preclinical studies with human therapeutic antibodies has to be assessed case by case. Our results facilitate the use of Göttingen minipigs for assessment of human therapeutic antibodies in preclinical studies. Electronic supplementary material The online version of this article (10.1007/s11095-019-2574-y) contains supplementary material, which is available to authorized users.

Background Wild-type poliovirus may be eradicated soon and under WHO GAPIII guidance, laboratory use will be discontinued or subject to strict containment. Per US Code of Federal Regulations, however, immunoglobulin lot release testing will still require use of replicating poliovirus. The suitability of S19 hyper-attenuated and apathogenic poliovirus strains as alternatives to the currently used wild-type virus in such a release assay was investigated. Study design and methods S19 poliovirus strains were propagated in a commercial setting using good virological practices and maintenance of the S19 hyper-attenuated genotype was confirmed by massively parallel sequencing. Results The attenuated phenotype of the produced S19 stocks was confirmed in a highly sensitive mouse-model. Equivalency in performance was seen in the lot release assay for the S19 and wild-type polioviruses. Conclusion The deployment of such hyper-attenuated and thoroughly characterized S19 stocks in these and other essential activities might reconcile final containment measures with continued safe use of poliovirus.

Anti-complement activity is a measure of the specific safety of human immunoglobulins characterizing one of the unwanted effect - spontaneous activation of the complement system. In compliance with the specifications of the European Pharmacopeia and WHO recommendations, the anti-complement activity of human immunoglobulin preparations must not be greater than 50%, which corresponds to a maximum of 1 CH50/mg of immunoglobulin. The measurement method was based on the ability of human immunoglobulins to produce “nonspecific” (in the absence of an antigen-antibody complex) binding of complement, preventing lysis of sensitized sheep red blood cells. We present here the results of analysis of the critical parameters of the method for measuring anti-complement activity, along with assessment of existing methodological approaches to its optimization. We report experimental studies of the effects of components of the hemolytic system and complement in guinea pigs on the results of measurements of anti-complement activity. The optimum conditions for the method of measuring the anti-complement activity of human immunoglobulins are based in evidence and the anti-complement properties of human immunoglobulin preparations for intravenous use of Russian origin are evaluated.

Показателем специфической безопасности препаратов иммуноглобулинов человека, характеризующим одно из проявлений нежелательных реакций — спонтанную активацию системы комплемента, является антикомплементарная активность». Согласно требованиям Европейской фармакопеи и рекомендациям ВОЗ, антикомплементарная активность препаратов иммуноглобулинов человека для внутривенного введения не должна превышать 50 %, что соответствует не более 1 CH50/мг иммуноглобулина. Метод её определения основан на способности иммуноглобулинов человека «неспецифически» (в отсутствие комплекса антиген — антитело) связывать комплемент, препятствуя лизису сенсибилизированных эритроцитов барана. В работе представлены результаты анализа критических параметров методики определения антикомплементарной активности и оценки существующих методических подходов её оптимизации. Экспериментально изучено влияние компонентов гемолитической системы и комплемента морских свинок на результат определения антикомплементарной активности. Обоснованы оптимальные условия методики определения антикомплементарной активности иммуноглобулинов человека и проведена оценка антикомплементарных свойств препаратов иммуноглобулинов человека для внутривенного введения отечественных производителей.

Common variable immunodeficiency (CVID) is an immunodeficiency disorder with a high incidence of gastrointestinal manifestations and an increased risk of gastric carcinoma and lymphoma. This review discusses the latest advancements into the immunological, clinical and diagnostic aspects of gastric malignancies in patients with CVID. The exact molecular pathways underlying the relationships between CVID and gastric malignancies remain poorly understood. These include genetics, immune dysregulation and chronic infections by Helicobacter pylori. Further studies are needed to better stratify the risk for cancer in these patients, to elaborate surveillance programs aimed at preventing these complications, and to develop new and more effective therapeutic approaches.

We describe the case of a 32-year-old woman who presented to the hospital with generalized painful exanthema, blisters and erosions 1 month after giving birth to a healthy girl. The patient's medical history was inconspicuous for comorbidities; however, it included the incidental intake of pain killers and a herbal preparation (fenugreek), which she took regularly over the last 4 weeks to improve lactation. Based on the clinical characteristics, we suspected toxic epidermal necrolysis (TEN), a severe cutaneous adverse drug reaction, which was confirmed by skin biopsy. The patient was treated with high-dose intravenous human immunoglobulins and was discharged 2 weeks after hospital admission in good condition. The allergological workup identified fenugreek as the most likely causative agent. Given the increased self-medication of freely available phytotherapeutics by patients in industrialized countries, herbal mixtures should be taken into consideration in the diagnostic workup of TEN.

Pooled human immunoglobulins (IGs) are prepared from plasma obtained from healthy donors as a concentrated antibody-containing solution. In addition, high-titer IGs (hyperimmune) against a specific pathogen can be obtained from vaccinated or convalescing donors. Currently, IGs can be used for the treatment of a variety of infections for which no specific therapy exists or that remain difficult to treat. Moreover, the recent pathogen outbreaks for which there is no approved treatment have renewed attention to the role of convalescent plasma and IGs. Areas covered: In this review, a historical perspective of the use of sera and IGs in humans as anti-infective agents (any viral, bacterial, parasitic infection), excluding immunodeficient patients, is presented from early development to the latest clinical studies. A Medline search was conducted to examine the peer-reviewed literature, with no date limits. Expert commentary: Human pooled plasma-derived IG products benefit from the polyclonal response of every individual donor and from the interindividual variability in such response. The trend to increased availability of vaccines for infectious diseases also opens new potential applications of hyperimmune IGs for emerging or re-emerging infectious diseases (e.g.: Ebola, Zika, Dengue), for the prevention and treatment in the general population, healthcare personnel and caregivers.

Amphioxus is regarded as an essential animal model for the study of immune evolution. Discovery of new molecules with the immunoglobulin superfamily (IgSF) variable (V) domain in amphioxus would help in studying the evolution of IgSF V molecules in the immune system. A protein was found which just contains only one IgSF V domain in amphioxus, termedAmphi-IgSF-V; it has over 30% sequence identity to the V domains of human immunoglobulins and mammalian T-cell receptors. In order to clarify the three-dimensional structure of this new molecule in amphioxus,Amphi-IgSF-V was expressed, purified and crystallized, and diffraction data were collected to a resolution of 1.95 Å. The crystal belonged to space groupP3221, with unit-cell parametersa=b= 53.9,c= 135.5 Å. The Matthews coefficient and solvent content were calculated to be 2.58 Å3 Da−1and 52.38%, respectively. The results will provide structural information to study the evolution of IgSF V molecules in the immune system.

Microcantilever-based sensor platform has attracted a lot of attention over the time in detection of a variety of molecules due to their miniaturized dimensions. Sensitivity enhancement is an important aspect of such sensors, especially when used for point-of-care diagnostic purpose. However, the major concern while operating these sensors in deflection mode is their sensitivity which mainly relies on selective chemical modification protocols employed on these sensor surfaces. One of the ways of getting better sensitivity is through asymmetric (one side) biofunctionalization of the sensor surface. In the presented work here, we have demonstrated a novel approach of asymmetric biofunctionalization of proteins in overall sensitivity enhancement of piezoresistive silicon nitride-oxide microcantilever sensor platform inside a flow chamber. Herein, using our developed surface chemistry, asymmetrically biofunctionalized microcantilevers first exhibited a greater electrical response in terms of piezoresistance change than their symmetric counterpart in the detection of human immunoglobulins (HIgGs) protein. Finally, these microcantilevers were employed to exhibit the enhanced sensitivity towards the detection of a crucial cardiac marker protein, i.e. Troponin-I (cTnI) down to 250 ng ml-1 using asymmetric biofunctionalization process. This study shows that the developed asymmetric biofunctionalization methodology may be used as a general protocol to detect other important biomarkers of clinical applications with improved sensitivity.

In the pharmaceutical industry, analysis of soluble aggregates in pharmaceutical formulations is most commonly performed using size-exclusion chromatography (SEC). However, owing to concerns that aggregates can be overlooked by SEC analysis, it has been suggested that its results should be confirmed with orthogonal methods. One of the main alternative methods for SEC is analytical ultracentrifugation sedimentation velocity (AUC-SV), which has been indicated as an important tool for the measurement of protein aggregation. The present study aimed to show that AUC-SV can be effectively applied for the characterization of marketed immunoglobulin pharmaceutical preparations to support the results obtained by SEC. In addition, the present research aimed to assess the appropriateness of two integration approaches for the quantitative analysis of the SEC results. Thus, the aggregates were measured in seven different preparations of human immunoglobulins by AUC-SV and SEC, and the acquired chromatographic data were processed by using either the vertical drop method or the Gaussian skim approach, implemented in the Empower II chromatography data software (Waters, Tokyo, Japan). The results of aggregation measurements performed using AUC-SV were in good agreement with those obtained using SEC. As expected, the Gaussian skim integration approach inherently provided lower estimates of aggregation content than the results of the vertical drop method. The finding of this study confirmed the complementary nature of AUC-SV to SEC for aggregate composition analysis and underscored the important role that the different integration methods can play in the quantitative interpretation of chromatographic results.

Neuromyelitis Optica Spectrum Disorders (NMOSD) are associated with autoantibodies (ABs) targeting the astrocytic aquaporin-4 water channels (AQP4-ABs). These ABs have a direct pathogenic role by initiating a variety of immunological and inflammatory processes in the course of disease. In a recently-established animal model, chronic intrathecal passive-transfer of immunoglobulin G from NMOSD patients (NMO-IgG), or of recombinant human AQP4-ABs (rAB-AQP4), provided evidence for complementary and immune-cell independent effects of AQP4-ABs. Utilizing this animal model, we here tested the effects of systemically and intrathecally applied pooled human immunoglobulins (IVIg) using a preventive and a therapeutic paradigm. In NMO-IgG animals, prophylactic application of systemic IVIg led to a reduced median disease score of 2.4 on a 0–10 scale, in comparison to 4.1 with sham treatment. Therapeutic IVIg, applied systemically after the 10th intrathecal NMO-IgG injection, significantly reduced the disease score by 0.8. Intrathecal IVIg application induced a beneficial effect in animals with NMO-IgG (median score IVIg 1.6 vs. sham 3.7) or with rAB-AQP4 (median score IVIg 2.0 vs. sham 3.7). We here provide evidence that treatment with IVIg ameliorates disease symptoms in this passive-transfer model, in analogy to former studies investigating passive-transfer animal models of other antibody-mediated disorders.

This paper describes the production of a functional humanized Fab fragment of a neutralizing antibody against the rabies virus. It is a prototype of a therapeutic agent that could be an alternative to equine anti-rabies immunoglobulins and anti-rabies serum immunoglobulins obtained from the blood of vaccinated human donors. Variable fragments of the high-affinity light and heavy chains neutralizing an antibody against the rabies virus were cloned and sequenced. Mouse constant regions were replaced by human constant regions followed by expression of the humanized Fab fragments in a yeast expression system. The immunochemical properties of the Fab fragments were evaluated using ELISA, polyacrylamide gel electrophoresis, and the Western blot assay. The binding capability of the produced humanized Fab fragments surpasses that of the parental antibody. A high degree of humanization was confirmed using sera against human immunoglobulins. The yield of the humanized Fab fragments was 21 mg per liter of culture medium. The purified Fab fragment preparation did not contain traces of the isolated light and heavy chains of the humanized Fab fragments.

Insect viruses are considered possible biological substitutes for chemical agents in pest control. Before their application, however, detailed studies are necessary for exclusion of hygienic risk. In our experiments the polyhedrin (PH) from nuclear polyhedrosis virus (NPV) from larvae of Mamestra brassicae reacted with human immunoglobulins. Therefore we determined whether human sera contain NPV-specific antibodies. The detection of specific antibodies in human sera would suggest that baculoviruses may be infectious for humans and therefore not suitable for insect control. Human sera were fractioned by gel filtration, ion-exchange and affinity chromatography on sepharose-coupled protein A. Immunoelectrophoretic analysis revealed that the majority of the reacting immunoglobulins belonged to the IgG-class. The reaction site(s) of the IgG molecule with the PH was investigated. In the radioimmunoassay (SPRIA), human Fab-fragments obtained by papain digestion yielded positive reactions with the PH. The antigen binding site of anti-Hepatitis-A-virus IgG and anti-Hepatitis-A-virus Fab-fragments, however, were not blocked by the PH. This indicated, that the interaction site is distinct from the antigen binding site. In the presence of Staphylococcus aureus, the PH reduced the immunoprecipitation by 11%. This inhibition is sufficient to recognize positive reactions between human sera and viral proteins in a sensitive antibody detection system (SPRIA). The data indicate that at least the constant region of the heavy chain of the IgG molecule interacts with the proteins of NPV. This interaction is indicative for a non-immunological interaction. Zusammenfassung Nicht-immunologische Interaktion zwischen Baculovirusproteinen und Human-Immunoglobulinen Als Ersatz fur die chemischen Insektizide zur Schadlingsbekampfung ist der Einsatz von Insektenviren vorgesehen. Vor einer Anwendung dieser Viren sind ausfuhrliche Untersuchungen notwendig, die die hygienische Unbedenklichkeit des Einsatzes dieser Viren in der biologischen Schadlingsbekampfung zeigen. In unseren Experimenten reagierte Polyhedrin (PH), das aus Kernpolyederviren (NPV) aus Larven von Mamestra brassicae gewonnen worden war, im Radioimmunoassay (RIA) mit Humanseren. Es muste deshalb untersucht werden, ob in Humanseren Baculovirus-spezifische Antikorper vorhanden sind. Ein Nachweis spezifischer Antikorper wurde bedeuten, das Baculoviren u. U. fur den Menschen infektios und aus diesem Grunde als Schadlingsbekampfungsmittel ungeeignet sind. Humanseren wurden mittels Gelfiltration, Ionenaustauschchromatographie und Affinitatschromatographie uber Protein A-Sepharose gereinigt. Immunelektrophoretische Analysen zeigten, das der Hauptreaktionspartner in den Seren Immunglobuline der IgG-Klasse sind. Es wurde der Bindungsort des Polyhedrins am IgG untersucht. Human-Fab-Fragmente, die durch Papainverdauung gewonnen wurden, reagierten im RIA positiv mit Polyhedrin. Das Polyhedrin bindet jedoch nicht an die antigene Bindungsstelle am Fab-Fragment, da die antigene Bindungsstelle, die gegen ein anderes Virus gerichtet war (Hepatitis A Virus), nicht blockiert wurde. Polyhedrin hemmt jedoch die Immunprazipitation der IgG's mit Staphylococcus aureus um 11%, was bedeutet, das Polyhedrin an das Fc-Fragment bindet. Diese Bindung allein genugt schon, um mit einem sensitiven Testsystem (RIA) eine positive Reaktion zwischen Humanseren und viralen Proteinen zu erkennen. Die hier aufgefuhrten Daten zeigen, das die viralen Proteine an die konstante Region der schweren Kette der IgG's binden. Diese Bindung deutet auf eine nicht immunologische Interaktion hin. Es besteht daher bis zu diesem Zeitpunkt kein Grund, das NPV als gefahrlich anzusehen, obwohl mogliche Effekte des Polyhedrins auf die biologischen Funktionen der Immunglobuline weiter untersucht werden mussen.

The technical scientific unit of the National Institute of Quality Control in Health (INCQS), part of the Oswaldo Cruz Foundation, has a role of investigational quality control of products and services related to health surveillance, in particular medicines denominated plasma derivative products. This paper is based on the analysis of the 3100 plasma derivative products from January 2000 to December 2004: 31.6% (n=980) of human albumin, 28.7% (n=890) of factor VIII, 21.4% (n=662) of human immunoglobulins, 8.3% (n=257) of factor IX, 7.1% (n=22) of specific immunoglobulin classes containing anti-Rho (D) immunoglobulin, anti-hepatitis B, anti-tetanus, anti-rabies and anti-varicella-zoster and 2.91% (n=91) of prothrombin complex. The products submitted to analysis came from airports and frontiers of Brasilia, Rio de Janeiro and Sao Paulo, and products confiscated by the states of Pernambuco, Santa Catarina, Rio de Janeiro and Rio Grande do Sul. The type of analysis was characterized as: 92.3% control analysis; 5.9% fiscal analysis; 1.4% guidance and 0.4% preliminary analysis. In respect to imported plasma derivatives, 40.0% originated from the airport in Brasilia, 26.9% in Sao Paulo and 25.2% in Rio de Janeiro. In conclusion, 99.1% of the plasma derivative products analyzed was considered satisfactory and 0.9% unsatisfactory as identified by visual inspection, solubility, stability and chemical assays and pyrogenic and unspecific toxicity tests. Thus, the assessment of the quality of plasma derivative products is an essential tool for health surveillance.

Heterosexual contact is the primary mode of human immunodeficiency virus (HIV) type 1 (HIV-1) transmission worldwide. The chemokine receptor CCR5 is the major coreceptor that is associated with the mucosal transmission of R5-tropic HIV-1 during sexual intercourse. The CCR5 molecule is thus a target for antibody-based therapeutic strategies aimed at blocking HIV-1 entry into cells. We have previously demonstrated that polyreactive natural antibodies (NAbs) from therapeutic preparations of immunoglobulin G and from human breast milk contain NAbs directed against CCR5. Such antibodies inhibit the infection of human macrophages and T lymphocytes by R5-tropic isolates of HIV in vitro. In the present study, we demonstrate that human immunoglobulins from the cervicovaginal secretions of HIV-seronegative or HIV-seropositive women contain NAbs directed against the HIV-1 coreceptor CCR5. Natural affinity-purified anti-CCR5 antibodies bound to CCR5 expressed on macrophages and dendritic cells and further inhibited the infection of macrophages and dendritic cells with primary and laboratory-adapted R5-tropic HIV but not with X4-tropic HIV. Natural anti-CCR5 antibodies moderately inhibited R5-tropic HIV transfer from monocyte-derived dendritic cells to autologous T cells. Our results suggest that mucosal anti-CCR5 antibodies from healthy immunocompetent donors may hamper the penetration of HIV and may be suitable for use in the development of novel passive immunotherapy regimens in specific clinical settings of HIV infection.

The treatment of membranous nephropathy is a highly controversial issue. As some patients may have spontaneous remission, in about 50% of cases the risk of treating patients with drugs that may have severe side effects is higher than the potential benefit of arresting disease progression. Some authors therefore propose exclusively symptomatic treatment; other authors use steroids and immunosuppressive drugs, alone or in association with high risk of adverse effects and often uncertain benefits. The intravenous administration of high doses of human immunoglobulins (IVIg) has been also extended to a growing number of kidney diseases including membranous nephropathy. The mechanisms through which IVIg carry out their therapeutic effect are still unclear. The present study is a retrospective and uncontrolled trial, the aim of which was firstly to verify if some patients could respond to extremely short treatment protocols, stopped when they appear to have a stable remission, thereby avoiding expensive continuation of treatment. Secondly, we aimed to verify if some patients, judged as nonresponders to a classical protocol of IVIg therapy, could respond to a more prolonged treatment.

Intravenous immunoglobulin (IVIG) is an established therapy for demyelinating diseases of the peripheral nervous system. IVIG exerts a number of effects that may be beneficial in multiple sclerosis (MS). Four double-blind IVIG trials have been performed in relapsing-remitting MS. A meta-analysis of the four trials has shown that IVIG reduces the relapse rate and, possibly, disease progression. In patients with a first episode of demyelinating disease, IVIG delays the time to the second relapse and thereby to the diagnosis of definite MS. In patients with an acute MS relapse, IVIG as add-on therapy to methylprednisolone does not make remission of symptoms faster or more complete. IVIG does not seem to be of any benefit to chronic visual or motor symptoms in MS. In secondary progressive MS, IVIG has not shown any effect on disease progression, relapses or new magnetic resonance imaging lesions. Experimental studies in the MS model experimental autoimmune encephalomyelitis in rats demonstrate that IVIG has to be administered at the time of induction of a relapse in order to be effective. In conclusion, IVIG can be considered as a second-line treatment to approved therapies for relapsing-remitting MS, but the ideal dosage of IVIG still needs to be determined. In order to be a first-line treatment for MS, the beneficial effect should be confirmed in a large-scale placebo-controlled survey, or in a study comparing the effect with approved therapies for relapsing-remitting MS using appropriate clinical and magnetic resonance imaging outcome measures.

Placebo-controlled studies showed that treatment with intravenous human immunoglobulins (IVIg) improves muscle strength of patients with multifocal motor neuropathy. The beneficial effect of IVIg lasts several weeks, and repeated IVIg are necessary to maintain muscle strength. Maintenance IVIg treatment is expensive and may not prevent long-term progression of motor deficits and axonal degeneration in multifocal motor neuropathy. Frequent infusions may also be burdensome to patients, but at present there is no therapeutic alternative to IVIg therapy. Plasma exchange is probably ineffective, and prednisone may worsen the disease course. Anecdotal reports suggest that cyclophosphamide may be effective as primary therapy or adjunctive therapy to IVIg, but its toxicity may preclude long-term use in relatively young patients. Evaluation in placebo-controlled trials of the efficacy of adjunctive (immunosuppressive) therapy that would allow reduction of IVIg doses is needed.

The effect of NIR laser radiation (808 nm) on methicillin-sensitive and methicillin resistant strains of Staphylococcus aureus incubated with gold nanorods is studied. Nanorods having length of 44 (± 4) nm and diameter of 10 (± 3) nm with the absorption maximum in the NIR (800 nm), functionalized with human immunoglobulins IgA and IgG, were synthesized and used in the studies. The killing ability up to 97% of the microorganism populations by using this nanotechnology was shown.