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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.

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.

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.

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.

The human papillomavirus type 16 is associated with anogenital cancer. Transcription of the viral transforming genes E6 and E7 is under the control of an epithelial cell type-specific enhancer. In the enhancer core, we have identified a regulatory element that is recognized by a novel nuclear factor named MSPF (methylation-sensitive papillomavirus transcription factor). Mutating the MSPF binding site strongly affects the enhancer activity. The MSPF recognition sequence 5'-ATGCGNNNNCGCCT-3' contains two CpG dinucleotides, potential targets for 5-cytidine methylation. DNA recognition by MSPF is strictly methylation-sensitive, since introduction of 5-methylcytidine into either CpG abolishes complex formation. Moreover, CpG methylation of the MSPF binding site suppresses the activity of the enhancer and of the MSPF enhanson subfragment in vivo. In the cervical carcinoma cell line CaSki, which has integrated multiple transcriptionally inactive human papilloma virus 16 genomes, a few of the viral genomes are methylated at the MSPF binding site. These findings suggest that viral transcription can be suppressed by methylation of the regulatory region, an event that prevents binding of the cellular transcription factor MSPF.

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.

Although glucocorticoids and progestins control vastly different physiological processes, the receptors mediating the effects of these hormones interact with the same DNA sequences. Transfer experiments involving synthetic genes and in vitro binding studies have shown that progesterone and glucocorticoid receptors both recognize the same 15-base pair DNA element (TGTACAGGATGTTCT), raising the question of how the two steroids affect gene expression selectively. We considered the possibility that their selectivity arises from either the differential expression of the receptors in target cells or the differential dependence of receptor function on additional transcription factors. To test these alternatives we introduced a progesterone-receptor expression plasmid into the rat hepatoma cell line Fto2B-3 which contains glucocorticoid receptor but is devoid of progesterone receptor. We report that expression of the progesterone receptor in Fto2B-3 cells renders endogenous glucocorticoid-regulated genes inducible by progestins. Our data show that the responsiveness of a cell to external stimuli can be reprogrammed by the expression of a single transcription factor and that differential expression of hormone receptors is at least one mechanism by which steroid-specific gene activation is achieved.

The upstream regulatory region of the human papilloma virus-16 (HPV-16) genomic DNA contains a sequence element with a large degree of homology to the partially palindromic sequence GGTACANNNTGTTCT, which is the consensus sequence of the glucocorticoid responsive elements of known genes regulated by this steroid hormone. DNase I and dimethylsulfate protection experiments reveal the binding of this sequence by rat glucocorticoid receptor protein. A 400-bp DNA segment centrally containing this sequence confers strong inducibility by dexamethasone to the promoter p97 of HPV-16 and to the Herpes simplex virus thymidine kinase promoter, as judged by chloramphenicol acetyltransferase activity and RNase protection assays. The same DNA segment, that does not contain the consensus sequences of all papilloma viruses relevant for E2 protein-mediated transcription enhancement, functions in an enhancer-like fashion in addition to its glucocorticoid responsive action. This hormone-independent transcription enhancement is absent in human MCF7 cells, but is strong in human HeLa cells where the combined activity of the constitutive and the steroid hormone-dependent enhancer elements stimulate transcription by a factor of 500. This cell type specificity of the HPV-16 enhancer may be responsible for the tissue tropism of the virus. These observations and the presence of numerous homologies to known enhancers of cellular and viral genes suggest a complex pattern of activation of the human papilloma virus-16 promoters.

Overproduction of the repressor protein from the Tn10-encoded tetracycline resistance operon is achieved by placement of the respective gene under control of bacteriophage lambda promoter PL in a vector-host system. All cloning steps have to be carried out under repressed conditions to assure survival of the cell. The cI 857 mutation is used to control expression of the tetR gene in large scale fermentation. After induction, the overproducing Escherichia coli strain continues to grow for 2.5 generations before growth terminates. In the expression phase, active TET repressor comprises up to 13% of the total soluble protein. A procedure is described to purify the TET repressor protein to homogeneity on a large scale. Starting from a 10 litre culture, approximately 250 mg of homogeneous, active TET repressor are obtained. The amino acid sequence of the N and C termini are in agreement with the gene start and stop determined from the nucleotide sequence of the Tn10 tetR gene.

To define the recognition sequence of the glucocorticoid receptor and its relationship with that of the progesterone receptor, oligonucleotides derived from the glucocorticoid response element of the tyrosine aminotransferase gene were tested upstream of a heterologous promoter for their capacity to mediate effects of these two steroids. We show that a 15-base-pair sequence with partial symmetry is sufficient to confer glucocorticoid inducibility on the promoter of the herpes simplex virus thymidine kinase gene. The same 15-base-pair sequence mediates induction by progesterone. Point mutations in the recognition sequence affect inducibility by glucocorticoids and progesterone similarly. Together with the strong conservation of the sequence of the DNA-binding domain of the two receptors, these data suggest that both proteins recognize a sequence that is similar, if not the same.

The 7905 bp genomic DNA of the human papilloma virus type 16 (HPV-16), which is associated with invasive cervical neoplasia, contains a 832 bp upstream regulatory region (URR) supposed to contain most of its cis-acting promoter elements. A search for DNA sequences known to bind transcription factors has resulted in the recognition of numerous consensus sequences. A functional analysis of a segment of this URR containing some of these consensus sequences has resulted in the recognition of an enhancer element, that is functional only in human HeLa, but not in human MCF7 cells. The same region contains an element that confers regulatibility by glucocorticoid hormones, but not by sex steroids, to a linked promoter.

Homologous and heterologous repressor-operator interactions among four different classes of tetracycline resistance determinants have been compared. These are represented by RP1/Tn1721 (class A), R222/Tn10 (class B), pSC101/pBR322 (class C), and RA1 (class D). By the use of the purified repressor proteins of class A (TetRA) and class B (TetRB), operator sequences of all four classes are recognized by both with an identical stoichiometry of four repressor subunits per control sequence, but with different affinities. In vitro transcription has been used to demonstrate regulatory activities of TetRA and TetRB upon all four classes of tet genes. Tetracycline acted as an inducer. A functional relationship among the tet regulatory systems was also shown in vivo by complementation of a class A tetR'-galK fusion mutant with the tetR genes of classes A, B, and C. Repression of tetRA-linked galactokinase was ca. 80% in the presence of tetRA or tetRC, and ca. 50% in the presence of tetRB. Taken together, these results demonstrate heterologous repressor-operator interaction, suggesting close relationships among the four classes of Tcr determinants.

We have previously identified in the long control region of the genome of human papillomavirus type 16 (HPV-16) a DNA segment which functions as a cell-type-specific enhancer as well as mediating glucocorticoid response. It contains multiple transcription-factor-binding sites, including several for nuclear factor I and one for the glucocorticoid receptor, which binds to the partially palindromic sequence TGTACANNNTGTCAT. We report here that this sequence element, when separated from the surrounding transcription-factor-binding sites and placed as an oligonucleotide into a test vector, retains its function as a glucocorticoid response element (GRE) in HeLa cells. In T47D cells, which express the progesterone receptor, the HPV-16 enhancer fragment mediates progesterone responsiveness. A point mutant in this fragment and the response of the oligonucleotide clone to both steroids prove the identity of the progesterone response element (PRE) with the GRE. The antiprogesterone and antiglucocorticoid RU486 interferes with both hormonal responses. In SiHa cells, the HPV-16 GRE mediates an increase in transcripts encoding E6 and E7 proteins, which are involved in transformation by HPV-16. Hormonal regulation is not restricted to HPV-16: DNA segments containing the cell-type-specific enhancers of HPV-11 and HPV-18 also mediate glucocorticoid and progesterone response. We identified sequence elements in the long control regions of HPV-11 and HPV-18 which function as GRE/PREs when tested as oligonucleotides. These findings suggest that GRE/PREs are an integral part of gene expression regulation in genital HPVs.

DNA sequences recognized by the glucocorticoid receptor are termed glucocorticoid-responsive elements because of their stimulatory effect on transcription. An oligonucleotide of 15 base pairs having partial or perfect symmetry is necessary for glucocorticoid induction and this same oligonucleotide is surprisingly also recognized by the progesterone receptor. Here we define a palindromic sequence of 15 base pairs, modelled after a sequence element shared by the vitellogenin genes of frog and chicken, which confers oestrogen inducibility on a heterologous promoter and can be converted into a glucocorticoid-responsive element by substitution of one or two bases at homologous positions in the palindrome. Considered with the observation that the DNA-binding domains of steroid receptors are closely related, this finding demonstrates that the steroid-responsive elements constitute a family of related DNA sequences.