[Interdisciplinary AWMF guideline for the diagnostics of primary immunodeficiency]. / Interdisziplinäre AWMF-Leitlinie zur Diagnostik von primären Immundefekten (S2k).

BACKGROUND: Primary immunodeficiencies are potentially life-threatening diseases. Over the last years, the clinical phenotype and the molecular basis of an increasing number of immunological defects have been characterized. However, in daily practice primary immunodeficiencies are still often diagnosed too late. Considering that an early diagnosis may reduce morbidity and mortality of affected patients, an interdisciplinary guideline for the diagnosis of primary immunodeficiencies was developed on behalf of the Arbeitsgemeinschaft Pädiatrische Immunologie (API) and the Deutsche Gesellschaft für Immunologie (DGfI). METHODS: The guideline is based on expert opinion and on knowledge from other guidelines and recommendations from Germany and other countries, supplemented by data from studies that support the postulated key messages (level of evidence III). With the contribution of 20 representatives, belonging to 14 different medical societies and associations, a consensus-based guideline with a representative group of developers and a structured consensus process was created (S2k). Under the moderation of a representative of the Association of the Scientific Medical Societies in Germany (AWMF) the nominal group process took place in April 2011. RESULTS: The postulated key messages were discussed and voted on following a structured consensus procedure. In particular, modified warning signs for primary immunodeficiencies were formulated and immunological emergency situations were defined.

Apoptotic cell debris and phosphatidylserine-containing lipid vesicles induce apolipoprotein J (clusterin) gene expression in vital fibroblasts.

The molecular events in cells undergoing programmed cell death (apoptosis) are well studied; however, the response of the surviving neighbor cells to local cell death is largely uncharacterized. Apolipoprotein J (clusterin) is an 80-kDa glycoprotein that has been implied in cytoprotection of the vital cells, presumably by assisting in the clearance of apoptotic vesicles and membrane remnants. Its mRNA is specifically up-regulated in the vital cells of apoptotic tissues. The molecular mechanisms, however, leading to this response are not known. We here show that exposure of vital fibroblasts to apoptotic vesicles, disrupted vital cells, and trypsin-treated membrane remnants induces apoJ mRNA. Moreover, lipid vesicles consisting of phosphatidylserine (PtSer) and dimyristoylphosphatidylcholine (PC), but not liposomes with PC alone nor with dimyristoylphosphatidylethanolamine or phosphatidic acid, did elevate apoJ mRNA level. These results suggest that, apart from mediating the endocytic uptake of the apoptotic vesicles, PtSer also serves as a trigger to stimulate the expression of genes that might be involved in the cellular clearance process.

[Transplantation of porcine Langerhans islets for therapy of type I diabetes. The way to clinical application]. / Die Transplantation porciner Langerhans-Inseln zur Therapie des Typ-I-Diabetes. Der Weg zur klinischen Anwendung.

Transplantation of isolated pancreatic islets provides an interesting alternative to the present cure for diabetes: insulin injections and pumps. These are characterized by an insufficient glucose haemostasis and in the long run can induce kidney failure, blindness, heart failure, and amputations. Up to now more than 293 allogeneic islet transplantations have been performed in diabetics with chronical kidney failure. Despite some success, no real breakthrough has been yet achieved, though great efforts are being made to improve the various methodological steps on the way to clinical transplantation. The use of animal (xenogeneic) organs could be a solution to overcome the shortage of allogeneic donors. The current experimental and clinical research focuses on the use of pigs as organ donors, which have a number of advantages over the immunologically more compatible primates. This article reports on success and open questions concerning the efforts to isolate porcine islets for future clinical transplantation: the search for a suitable pig breed, the various isolation steps, purification and in vitro culture, transplantation models using-small and large animals, first clinical trials, and immunological reactions against the xenogeneic tissue. In addition, strategies to circumvent tissue rejection and future perspectives are discussed.

Nerve growth factor and epidermal growth factor stimulate clusterin gene expression in PC12 cells.

Clusterin (apolipoprotein J) is an extracellular glycoprotein that might exert functions in development, cell death and lipid transport. Clusterin gene expression is elevated at sites of tissue remodelling, such as differentiation and apoptosis; however, the signals responsible for this regulation have not been identified. We use here the clusterin gene as a model system to examine expression in PC12 cells under the control of differentiation and proliferation signals produced by nerve growth factor (NGF) and by epidermal growth factor (EGF) respectively. NGF induced clusterin mRNA, which preceded neurite outgrowth typical of neuronal differentiation. EGF also activated the clusterin mRNA, demonstrating that both proliferation and differentiation signals regulate the gene. To localize NGF- and EGF-responsive elements we isolated the clusterin promoter and tested it in PC12 cell transfections. A 2.5 kb promoter fragment and two 1.5 and 0.3 kb deletion mutants were inducible by NGF and EGF. The contribution to this response of a conserved activator protein 1 (AP-1) motif located in the 0.3 kb fragment was analysed by mutagenesis. The mutant promoter was not inducible by NGF or EGF, which identifies the AP-1 motif as an element responding to both factors. Binding studies with PC12 nuclear extracts showed that AP-1 binds to this sequence in the clusterin promoter. These findings suggest that NGF and EGF, which give differential gene regulation in PC12 cells, resulting in neuronal differentiation and proliferation respectively, use the common Ras/extracellular signal-regulated kinase/AP-1 signalling pathway to activate clusterin expression.

Amitogenic alginates: key to first clinical application of microencapsulation technology.

Microencapsulation refers to a technique of immunoisolation by coating single cells or tissue with a semipermeable membrane. By combining microencapsulation with a specific tissue culturing method, iso-, allo-, and xenotransplantation of parathyroid tissue has been achieved without immunosuppression in a long-term animal model. Prior to its clinical use, continued analyses of the alginate, used as a coating substance, determined its mitogenic properties. Purification of the commercially available alginate was achieved using patented electrophoretic procedures, resulting in an amitogenic alginate suitable for use in humans. However, this alginate exhibited entirely different physical properties. We have recently shown that isotransplanted parathyroid tissue remains vital and functioning in vivo over long periods of time using the novel amitogenic alginate. It is essential to document, whether the alginate is able to maintain immunoisolation. We have therefore assessed its in vivo function compared to the mitogenic alginate in a transgenic animal model. Altogether 600 parathyroid glands from 300 Lewis rats (donor animals) were excised and subjected to tissue culture. Thereafter they were allotransplanted to 30 parathyroidectomized Dark-Auita rats, microencapsulated with the amitogenic or the mitogenic alginate or naked, with 10 recipient animals in each group. Total serum calcium and parathyroid hormone levels were monitored continuously at weekly intervals for 30 weeks. After 26 weeks the transplant beds were excised and subjected to histologic examination. More than 6 months after allotransplantation 9 of 10 animals that had received amitogenic transplants, compared to 7 of 10 animals in the group with mitogenic microcapsules were normocalcemic. Animals that had received naked parathyroid tissue were hypocalcemic as soon as 2 weeks after allotransplantation. Correspondingly, normocalcemic animals showed vital parathyroid tissue inside the microcapsules, which were surrounded by a significantly smaller rim of fibroblasts when amitogenic alginate had been used. In addition to confirming physiologic long-term function, we were able to document for the first time that immunoisolation can also be achieved with the novel amitogenic alginate, which is suitable for clinical use.

[Heterologous transplantation of human parathyroid glands after microencapsulation with clinically suitable alginate : long-term function without immunosuppression in the animal model]. / Heterologe Transplantation der humanen Parathyreoidea nach Mikroenkapsulierung mit klinisch einsetzbarem Alginat: Langzeitfunktion ohne Immunsuppression im Tierversuch.

The role of parathyroid transplantation for the therapy of permanent hypoparathyroidism is undisputed. Because the parathyroid hormone deficiency syndromee rarely every is a vital thread to patients affected, systemic immunosuppression for transplant recipients is not justified. A technique of microencapsulation was modified for transplantation of parathyroid tissue. Using a core substance suitable for clinical use (amitogenic alginate), we accomplished allotransplantation of functioning parathyroid tissue in the long-term animal model and, very recently, reported first clinical cases without postoperative immunosuppression. In a controlled animal model of totally parathyroidectomized rats (PTX, two groups of n = 40), we investigated the ability of microencapsulation with the amitogenic alginate to enable transplantation across the highest immunological barrier (xenotransplantation: human-rat); to ensure intact transplant function and to protect from rejection. Rat parathyroid hormone (PTHRA i.S.) and serum calcium levels served as parameters of completeness of PTX; intact human PTH (PTHRA i.S.) and serum calcium levels of recipient animals were used to assess graft function. Also, tissue integrity within explanted capsules was assessed by histology. Cultured and microencapsulated parathyroid tissue resumes and maintains function in vivo, even if transplanted across the highest immunological barrier. Functionally, PTHHU i.S. replaced (PTHRA i.S.) in PTX animals entirely and restored normocalcemia. These results suggest, that xeno-transplantation of the parathyroids can be achieved without postoperative immuno-suppression in a long term animal model. These data also imply the possibility of clinical heterotransplantation of parathyroid glands.

Differential regulation of the clusterin gene by Ha-ras and c-myc oncogenes and during apoptosis.

Clusterin (ApoJ) is an extracellular glycoprotein expressed during processes of tissue differentiation and regression that involve programmed cell death (apoptosis). Increased clusterin expression has also been found in tumors, however, the mechanism underlying this induction is not known. Apoptotic processes in tumors could be responsible for clusterin gene activation. Alternatively, oncogenic mutations could modulate signal transduction, thereby inducing the gene. We examined the response of the rat clusterin gene to two oncogenes, Ha-ras and c-myc, in transfected Rat1 fibroblasts. While c-myc overexpression did not modify clusterin gene activity, the Ha-ras oncogene produced a seven to tenfold repression of clusterin mRNA; this down-regulation was also observed in the presence of c-myc. Since no induction of the clusterin gene was observed by the two oncogenes, we tested the alternative mechanism involving apoptosis. Growth factor withdrawal induced apoptosis, as shown by DNA degradation and micronuclei formation in the floating cells. Concomittantly we observed a three to tenfold increase in the amount of clusterin mRNA in the adhering cells of Rat1 and the c-myc transformed cell lines, and a weaker induction in the Ha-ras transformed cell line. On the basis of our results, we suggest that clusterin gene induction in the vital cells is produced by signaling molecules that are generated by the apoptotic cells. We conclude that apoptotic processes, not oncogenic mutations, are responsible for increased clusterin expression in tumors.

First successful xenotransplantation of microencapsulated human parathyroid tissue in experimental hypoparathyroidism: long-term function without immunosuppression.

Owing to the complexity of the parathyroid hormone's metabolic interactions, clinical hypoparathyroidism is one of the most difficult of all endocrine disorders to treat. Therefore, causative treatment of this disorder by transplantation of parathyroid glands is highly desirable. We have recently documented the long-term in vivo function of iso- and allotransplanted rat parathyroid tissue without systemic immunosuppression in an animal model. In view of the potential clinical use of this method, human parathyroid tissue has been microencapsulated and transplanted over the highest immunological barrier. In a controlled, long-term animal study in the parathyroidectomized rat, the effect of microencapsulation on xenotransplanted human parathyroid tissue was evaluated over 30 weeks (native and microencapsulated parathyroid tissue = 40 rats respectively). Functionally, human parathyroid tissue was able to replace that of the rat. All animals that had received microencapsulated parathyroid tissue were normocalcemic for 16 weeks; 27/40 at the end of the study. In contrast, serum calcium concentrations dropped to post-parathyroidectomy levels within 4 weeks in those animals that had received native tissue only. Histologic evaluation of the explanted, functionally successful xenografts showed vital parathyroid tissue inside intact microcapsules surrounded by a small rim of fibroblasts. Avital fibrotic remnants were demonstrated in animals with non-encapsulated parathyroid tissue. Thus, we have established the feasibility of microencapsulation of human parathyroid tissue, preserving its viability over long periods in vivo even if xenotransplanted. In combination with an improved tissue culture method, transplantation of human parathyroid tissue and maintenance of its physiological function is reproducibly achieved without postoperative systemic immunosuppression over the highest transplantation barrier. This may be a crucial step towards the first clinical application of this method.

Biocompatibility of mannuronic acid-rich alginates.

Highly purified algin preparations free of adverse contaminants with endotoxins and other mitogens recently became available by a new purification process (Klöck et al., Appl. Microbiol. Biotechnol., 1994, 40, 638-643). An advantage of this purification protocol is that it can be applied to alginates with various ratios of mannuronic acid to guluronic acid. High mannuronic acid alginate capsules are of particular practical interest for cell transplantation and for biohybrid organs, because mannuronate-rich alginates are usually less viscous, allowing one to make gels with a higher alginate content. This will increase their stability and reduce the diffusion permeability and could therefore protect immobilized cells more efficiently against the host immune system. Here we report the biocompatibility of purified, mannuronic acid-rich alginate (68% mannuronate residues) in a series of in vitro, as well as in vivo, assays. In contrast to raw alginate extracts, the purified product showed no mitogenic activity towards murine lymphocytes in vitro. Its endotoxin content was reduced to the level of the solvent. Animal studies with these new, purified algin formulations revealed the absence of a mitogen-induced foreign body reaction, even when the purified material (after cross-linking with Ba2+ ions) is implanted into animal models with elevated macrophage activity (diabetes-prone BB/OK rat). Thus, alginate capsules with high mannuronic acid content become available for applications such as implantation. In addition to the utilization as implantable cell reactors in therapy and biotechnology, these purified algins have broad application potential as ocular fillings, tissue replacements, microencapsulated growth factors and/or interleukins or slow-release dosage forms of antibodies, surface coatings of sensors and other invasive medical devices, and in encapsulation of genetically engineered cells for gene therapy.

Collagen-coated Ba(2+)-alginate microcarriers for the culture of anchorage-dependent mammalian cells.

Several types of microcarriers suitable for large-scale cultivation of mammalian cells are commercially available. However, many of these carriers have disadvantages, e.g., the need for enzymatic digestion for cell harvesting, size limitations and insufficient biocompatibility. These limitations have been overcome by the development of collagen-coated Ba(2+)-alginate microcarriers. Ba(2+)-alginate microspheres, made with the air-jet droplet generator technique, were collagen-coated by incubation in a 0.5% collagen solution, with subsequent gelling of the collagen layer around the alginate microspheres. Human chang liver (CCL-13) and mouse fibroblast (L929) cell lines were cultivated in stationary, unstirred cultures as model systems. After a lag phase of nearly 24 h, the cells grew rapidly on these microcarriers and reached confluence after 3 days. The microcarrier cultures were stable for an additional 4-9 days and longer. Cells were harvested either by trypsinization or by dissolution of the alginate matrix using 5 mM EDTA. The main advantages of this new microcarrier system are that the preparation procedure is easy and can be accomplished on demand with standard laboratory equipment.

Isotransplantation of microencapsulated parathyroid tissue in rats.

Permanent hypoparathyroidism is one of the most difficult of all endocrine disorders to treat medically. While autotransplantation of parathyroid tissue is clinically established, allotransplantation without immunosuppression is still at the level of animal experiments. Although persons affected by hypoparathyroidism are facing a clearly reduced quality of life, hypoparathyroidism rarely is a life threatening condition. Therefore, systemic immunosuppression for recipients of allotransplants is not justified. A conceptional alternative would be protecting the tissue to be transplanted from the immunologic response by coating it with a semipermeable membrane (microencapsulation). In 1994, we succeeded in iso-, allo- and xenotransplantation of microencapsulated parathyroid tissue in an animal model. Unfortunately, prior to the first clinical use, further analysis of the coating substance (alginate) demonstrated that it has mitogenic properties. Here, we report on the first successful transplantation of microencapsulated parathyroid tissue using a purified, non-mitogenic alginate which is suitable for clinical use.

Expression pattern of extracellular matrix proteins in the pancreas of various domestic pig breeds, the Goettingen Minipig and the Wild Boar.

UNLABELLED: In spite of progress in biotechnology, isolation of porcine pancreatic islets remains a difficult task with unpredictable results. One reason could be the lack of knowledge as to the expression of extracellular matrix proteins in porcine exocrine and endocrine tissues, particularly in "islet capsules". Such proteins are subject to digestion by proteases, yet they might have a protective function for the fragile islets. OBJECTIVE: Of our study was a detailed histological analysis of the extracellular matrix proteins in various pig breeds. A broad panel of commercial, human-specific antibodies were used, since antibodies against porcine tissue were not available. METHODS: Frozen pancreatic tissue section of 7 domestic pig breeds, the Goettingen Minipig and the Wild Boar were stained with antibodies against collagen types I, II, III, IV, V, VI, VII, IX, laminin, fibronectin, vitronectin and elastin. Binding of antibodies was detected by immunoperoxidase and evaluated microscopically. Human and rat tissue was treated in the same way. RESULTS: (1) With the exception of anti-collagen type II, type VII and vitronectin, all antibodies revealed distinct binding patterns in the pancreas of the different pig breeds. However these antibodies bound on human cartilage and skin. (2) Collagen types I, III, IV, laminin and fibronectin are expressed on porcine pancreatic "islet capsules". (3) Expression levels of these proteins on "islet capsules" vary in the different pig breeds. However, no significant differences could be found in the expression pattern of collagen types I, III, IV, laminin and fibronectin, comparing domestic, experimental and wild type pigs. (4) Older individuals (Goettingen Minipig) appear to express higher levels of proteins on "islet capsules" than younger ones. CONCLUSIONS: Antibodies with specificity for human extracellular matrix proteins can be used successfully in the porcine pancreas. Thus, analysis of the structure and composition of porcine pancreatic tissue can be performed even without pig-specific antibodies. Particularly, the effects of various proteases and collagenases on the pancreatic tissue can now be monitored by immunohistochemical analysis allowing a rational design of protease mixtures for the isolation of pancreatic islets.

Transplantation of parathyroid tissue in experimental hypoparathyroidism: in vitro and in vivo function of parathyroid tissue microencapsulated with a novel amitogenic alginate.

Microencapsulation of tissues is an alternative to postoperative immunosuppression in transplantation. In 1994 iso-, allo- and xenotransplantation of microencapsulated parathyroid tissue was achieved in vivo. However, continued analysis of the coating substance (an alginate) determined mitogenic properties. Here, we report on the in vitro and in vivo function of parathyroid tissue microencapsulated with a novel amitogenic alginate suitable for use in humans. To assess in vitro function, parathyroid tissue encapsulated with mitogenic and amitogenic alginate was exposed to rising concentrations of calcium. For in vivo experiments, it was isotransplanted into parathyroidectomized rats. PTH release into medium and PTH serum levels as well as calcium levels of recipient rats were analyzed and compared to native (non-microencapsulated) tissue and empty capsules, respectively. In vivo, transplants were excised and subjected to histologic examination six months after trans-plantation. In vitro, parathyroid tissue encapsulated with amitogenic alginate releases approximately half of the PTH of the native tissue, not different from tissue encapsulated with the mitogenic alginate. In vivo, the novel alginate preserved parathyroid function similar to that of native tissue over the six month period resulting in complete reversal of hypoparathyroidism. Correspondingly, histologic examination revealed vital parathyroid tissue in intact microcapsules. By establishing in vitro function and successful long-term transplantation, we have documented the principle of microencapsulation of parathyroid tissue to be effective also with the novel amitogenic alginate, which is suitable for clinical use.

Multiple signal transduction pathways regulate clusterin (gp 80) gene expression in MDCK cells.

Clusterin (gp 80, apolipoprotein J, TRPM-2) is a widely expressed multifunctional glycoprotein. Its demonstrated and proposed functions include the transport of lipids and membrane fragments, the inhibition of the cytolytic action of the terminal complement complex and the modulation of cell-cell interactions. The expression of the gene is enhanced during tissue injury and remodelling and by hormone-withdrawal-induced apoptosis of prostate and mammary cells. We show here that, in the kidney-derived epithelial cell line MDCK, clusterin mRNA is repressed by glucocorticoids and by progesterone. Treatment with epidermal growth factor also represses clusterin gene expression in MDCK cells. Incubation with 12-O-tetradecanoyl-phorbol-13-acetate, which activates protein kinase C (PKC), induces clusterin mRNA, while chelerythrine, an inhibitor of PKC, represses clusterin gene expression, suggesting that the clusterin gene responds to signalling pathways involving PKC. These results open up the possibility of studying the complex regulation of the clusterin gene by multiple signal transduction pathways within a single cell type, and most importantly, of characterizing interactions between the individual signal transduction cascades.

Fractions from commercial collagenase preparations: use in enzymic isolation of the islets of Langerhans from porcine pancreas.

Transplantation of isolated islets of Langerhans is an intriguing possibility for the treatment of diabetes mellitus. The isolation of islets from pancreata requires the specific dissociation of the tissue. Commercial collagenases from Clostridium histolyticum are widely used for this purpose. Unfortunately, the effectiveness of these commercial enzymes is not predictable and differs considerably between suppliers and even from lot to lot. This is due mainly to differences in their specific collagenase activity and to the presence of other lytic enzymes, as well as to other contaminants. Free flow zone electrophoresis (FFZE) was used to separate the effective protein components from undesired compounds and to prepare a digestive enzyme mixture with controlled composition of lytic activities. Fractionation of crude collagenases by FFZE resulted in partially purified protein fractions that were enriched for collagenase and tryptic activities, and contained only trace amounts of neutral protease. These preparations proved to be highly effective in an in vitro assay for the libration of viable islets from porcine pancreas. To scale up the production of these collagenases with defined enzyme composition, we fractionated two different lots of a commercial collagenase from C. histolyticum (one lot effective in islet isolation, the other not) by using fast protein liquid chromatography (FPLC) on hydroxyapatite. Again, high efficacy of islet release from pancreatic tissue was correlated to high specific tryptic and collagenase activities and low levels of neutral protease. The chromatographic protocol developed in this study converted a non-effective collagenase lot into a preparation that allowed successful islet isolation.

Epidermal growth factor induction of human papillomavirus type 16 E6/E7 MRNA in tumor cells involves two AP-1 binding sites in the viral enhancer.

The early genes E6 and E7 from human papillomaviruses (HPVs) play a key role in the development of cervical cancer. Modulation of E6 and E7 gene expression may alter tumour progression; therefore, modifiers of viral transcription such as hormones or growth factors are potential risk factors in cancer development. We have analysed the effects of epidermal growth factor (EGF) on E6/E7 mRNA from human papillomavirus type 16 (HPV-16) by Northern blot in two cell lines, SiHa cervical carcinoma cells, and HPK IA, an HPV-16-immortalized keratinocyte cell line. E6/E7 mRNA is EGF-inducible in SiHa cells, with the earliest response after 2 h. In contrast, in HPK IA cells no increase in E6/E7 RNA is observed, suggesting a differential EGF response of viral transcription in tumour cells compared with keratinocytes. We demonstrate that the cell type-specific HPV-16 enhancer is a target of EGF-induced signals, as its activity is amplified by EGF in SiHa cell transfections. However, when transfected into HPK IA keratinocytes, the viral enhancer shows no EGF response. The enhancer contains two binding sites for the transcription factor AP-1, a potential mediator of the EGF signalling cascade. Enhancer subfragments with single AP-1 binding sites are also EGF-responsive in SiHa cells. Mutating either AP-1 site in the complete enhancer decreases the EGF response, whereas a double mutation causes a complete loss of EGF regulation, suggesting that the EGF induction of HPV-16 early transcription requires AP-1 activation. We conclude that alterations of EGF responsiveness that increase viral oncogene expression may contribute to cervical cancer progression.

Analysis of the human guanylin gene and the processing and cellular localization of the peptide.

The complete cell biological analysis of human guanylin, a recently discovered regulatory peptide, is offered in this investigation: (i) the nucleotide sequence of the gene, (ii) the isolation and characterization of its circulating molecular form, and (iii) its localization in enterochromaffin cells of the gut. As determined by molecular cloning, DNA sequencing, and comparison with the known cDNA sequence, the approximately 2.6-kbp large gene consists of three exons interrupted by two introns. The putative promoter region contains a TTTAAAA sequence motif and several potential binding sites for transcription factors such as AP-1, AP-2, Sp 1, and glucocorticoid receptors. The isolated hormonal form of guanylin is a 94-amino acid peptide with a molecular mass of 10.3 kDa. Western blot analysis of RP-HPLC fractions from blood plasma confirms this molecular form. Thus, guanylin is synthesized by gut enterochromaffin cells as a prohormone of 115 amino acids and is processed to the molecular form of 94 amino acids circulating in the blood.

Generation of a human monoclonal antibody to hepatitis C virus, JRA1 by activation of peripheral blood lymphocytes and hypo-osmolar electrofusion.

We have generated a human monoclonal antibody with binding specificity for hepatitis C virus (HCV)-specific peptides using peripheral blood lymphocytes isolated from a HCV antibody positive patient. The B-lymphocytes were stimulated with lipopolysaccharide (LPS) for 72 hours prior to the fusion. A recently described high efficiency hypo-osmolar electrofusion technique was employed, allowing generation of a large number of human hybridomas. The hybridomas were screened for human immunoglobulin and HCV-specific peptide binding by EIA. A single HCV-positive clone, JRA1, was detected and sub-cloned. Isotype analysis showed it to secrete an IgM lambda monoclonal antibody. The antibody was positive on both first and second generation HCV antibody analysis. This study confirms that viable pathogen-specific B-cells may be recovered from the peripheral blood. Although such cells are likely to be relatively uncommon in the circulating B-cell pool, they may be successfully immortalized by high efficiency electrofusion techniques. This technique might be valuable for the generation of human monoclonal antibodies with specificity for other human pathogens.