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This evidence-based clinical guideline provides consensus-recommendations for the treatment and care of patients with primary antibody deficiencies (PADs). The guideline group comprised 20 clinical and scientific expert associations of the German, Swiss, and Austrian healthcare system and representatives of patients. Recommendations were based on results of a systematic literature search, data extraction, and evaluation of methodology and study quality in combination with the clinical expertise of the respective representatives. Consensus-based recommendations were determined via nominal group technique. PADs are the largest clinically relevant group of primary immunodeficiencies. Most patients with PADs present with increased susceptibility to infections, however immune dysregulation, autoimmunity, and cancer affect a significant number of patients and may precede infections. This guideline therefore covers interdisciplinary clinical and therapeutic aspects of infectious (e.g., antibiotic prophylaxis, management of bronchiectasis) and non-infectious manifestations (e.g., management of granulomatous disease, immune cytopenia). PADs are grouped into disease entities with definitive, probable, possible, or unlikely benefit of IgG-replacement therapy. Summary and consensus-recommendations are provided for treatment indication, dosing, routes of administration, and adverse events of IgG-replacement therapy. Special aspects of concomitant impaired T-cell function are highlighted as well as clinical data on selected monogenetic inborn errors of immunity formerly classified into PADs (APDS, CTLA-4-, and LRBA-deficiency)., (© 2020 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

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., (© Georg Thieme Verlag KG Stuttgart · New York.)

Secretory clusterin (sCLU) is found as an 80-kDa glycoprotein in virtually all body fluids, in serum it is associated with high-density lipoprotein (HDL). Here, we discuss demonstrated and proposed mechanisms of the cytoprotective functions of sCLU in instances of apoptosis, necrosis, and disease. These include prevention from cell damage by lipid oxidation in blood vessels, removal of dead cell remnants in tissues undergoing various forms of cell death, and clearance of harmful extracellular molecules such as amyloid beta (Abeta) by endocytosis or transcytosis. All these functions may reflect the propensity of sCLU to bind to a wide spectrum of hydrophobic molecules on one hand and to specific cell-surface receptors on the other hand. Identified and proposed sCLU receptors are members of the LDL receptor family of endocytosis receptors. Since these receptors recently have proved to modulate cell signaling we will discuss whether sCLU due to this interaction not only targets its ligands for clearance, but may also be involved in triggering signal transduction.

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., (Copyright 2001 Academic Press.)

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.

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.

Human monoclonal antibodies which bind Schistosoma mansoni worm and egg antigens were identified and characterized from hybridomas generated using the hypo-osmolar electrofusion technique of somatic cell fusion. Splenocytes from S. mansoni infected individuals were mitogen-activated in vitro and subsequently fused by electrofusion. The greatest number of HAT resistant hybridomas per helical fusion chamber was obtained with unfrozen splenocytes cultured for 4-6 days after introduction of mitogen. Hybridomas secreting IgG antibodies recognizing parasite antigens were identified by ELISA. Twenty-one cloned cell lines secreting IgG antibody were maintained for at least 6 months. Characterization of antigen reactivity by Western blot analysis of nien cloned cell lines revealed antibodies which bound stage specific parasitic antigens. The data show that the technique of hypo-osmolar electrofusion produces stable, antibody producing hybridomas. The human monoclonal antibodies screened represent candidate molecules useful in the investigations of the human pathogen S. mansoni.

The promoter P97 of human papillomavirus-16 (HPV-16) gives rise to transcripts that encode the principal transforming genes of the virus, E6 and E7. The activity of P97 is regulated by a cell-type-specific enhancer, as well as by glucocorticoids and progesterone. We show here, that in CaSki cells, which contain HPV-16 genomes, P97 is also inducible by phorbol esters. Functional analysis of restriction fragments and oligonucleotides of the viral enhancer localizes two phorbol ester response elements on two transcription factor binding sites termed fp4e and fp9e. Sequence comparison, footprint analysis and bandshift competition of the cloned motifs suggest that both fp4e and fp9e are bound by the transcription factor AP1. These AP1 binding sites in HPV-16 and other papillomaviruses may provide a link between cellular oncogenes like jun, fos and possibly ras, whose transcription stimulating activity may lead to an elevated expression of the viral transforming genes E6 and E7.

Interaction of the Tn10 encoded TET repressor with the tet operator is studied by thermal denaturation of the specific complexes employing operator containing purified DNA restriction fragments varying in length from 187 bp to 501 bp. Comparison of the melting curves obtained with the free DNA and DNA.repressor complexes revealed a specific stabilisation of the operator containing cooperatively melting segment in multiphasic denaturation curves. Under limiting concentrations of TET repressor the denaturation of the free DNA is observed next to the denaturation of the repressor.DNA complex. Quantitative analysis yields a binding curve with a stoichiometry of four TET repressors per tet operator containing fragment. The denaturation temperature of the complex is almost independent of the ionic strength indicating that the protein component denatures at this temperature. The half life time of the TET repressor.tet operator complex is greater than 100 min under these conditions. The tet operator on the 187 bp fragment is determined to be located between a Xba I and a Sau 3a site by removing base pairs from either end of the fragment and subsequent comparison of the melting curves. It is concluded that the TET repressor recognizes the double stranded rather than a possible cruciform structure of the tet operator. The influence of a regulatory protein on the thermal stability of a genetic control region is discussed with respect to its possible influence on the initiation of transcription.

The regulation of expression of the Tn1721-encoded tetracycline-resistance determinant is described at the molecular level. The transcriptional control element consists of overlapping divergent promoters, which are negatively regulated by two operators with nearly identical sequence. The mRNA for the regulatory gene tetR is translated without a ribosome-binding site. This result is confirmed by S1 nuclease mapping and RNA sequencing of the tetR mRNA. The start nucleotide for transcription of this mRNA is the adenosine residue of the sequence 5'-AUG. Determination of the N-terminal amino acid sequence of the purified Tet repressor proves that this AUG is the initiation codon for translation. The Tet repressor protein is further used to map the two tet operators by DNase I footprinting. Tight contacts of the protein to the N-7 positions of two guanosine residues in each operator are determined from methylation protection experiments with dimethylsulfate. The differential regulation and positive control of transcription of the tetR gene that is possible with this arrangement of promoters and operators is discussed.

The tetracycline resistance determinant of RA1 was cloned. It consists of at least two genes oriented with opposite polarity, tetA for resistance and tetR for regulation. The transcriptional control sequence was identified and analyzed. It consists of overlapping promotors with divergent orientation and a tandem arrangement of operators. Nucleotide sequencing revealed two open reading frames. One codes for a protein which was identified as a Tet repressor by comparing its primary structure with those of other Tet repressors. The RA1 tetR gene codes for 218 amino acids with a calculated molecular weight of 24.4 kDa. In the primary sequence of the RA1-, pSC101-, Tn10-, and RP1/Tn1721-encoded Tet repressors, 36% of the amino acids are identical. This homology is clustered within the first 150 amino acids, 49% of which are identical among all four proteins. These results are discussed with respect to their structure and function in comparison to other DNA binding proteins.