[Biological risk in non-clinical biological and chemical laboratories]. / II rischio biologico nei laboratori biologici e chimici non sanitari.

The D.Lgs 626/94, regarding the improvement of workers safety in the workplace, introduces the necessity of the biological hazards assessment. In case of not sanitary chemical and biological laboratories, workers are subject to biological hazards due to potential exposure, because many biological agents could be present in the samples to be analysed, and also for deliberated use of micro organisms. However the assessment of the air and surfaces monitoring results in such environment is still difficult without Guidelines that indicate levels of acceptable exposure and contamination, and reference limits in order to judge "safe" the environment. The following report describes a microbiological monitoring into the Laboratories of HERA SpA and wants to underline the need to produce Guidelines dedicated to these particular workplaces environment, in order to standardize air quality sampling procedures and results assessment.

Homodimerization of erythropoietin receptor by a bivalent monoclonal antibody triggers cell proliferation and differentiation of erythroid precursors.

Erythropoietin (EPO) stimulates proliferation and differentiation of erythroid progenitor cells. Several lines of evidence indicate that the most likely mechanism of EPO receptor (EPO-R) activation by EPO is homodimerization of the receptor on the surface of erythrocyte precursors. Therefore, we argued that it should be possible to raise EPO-R monoclonal antibodies (MoAbs) that would activate the receptor by dimerization and thus mimic EPO action. We have identified such an agonist MoAb (MoAb34) directed against the extracellular EPO binding domain of the EPO-R. This bivalent IgG antibody triggers the proliferation of EPO-dependent cell lines and induces differentiation of erythroid precursors in vitro. In contrast, the monovalent Fab fragment, which cannot dimerize the receptor, is completely inactive. The mechanism of receptor activation by homodimerization implies that at high ligand concentrations the formation of 1:1 receptor/ligand complexes is favored over 2:1 complexes, thereby turning the ligand agonist into an antagonist. Thus, EPO and MoAb34 should self-antagonize at high concentrations in both cell proliferation and differentiation assays. Our data indeed demonstrate that EPO and MoAb34 antagonize ligand-dependent cell proliferation with IC50 values of approximately 20 and 2 mumol/L, respectively. Erythroid colony formation (BFUe) is inhibited at MoAb34 concentrations above 1 mumol/L. Furthermore, we analyzed the MoAb34:EPO-R interaction using a mathematic model describing antibody-mediated receptor dimerization. The data for proliferation and differentiation activity were consistent with the receptor dimer formation on the cell surface predicted by the model.

Mapping of the active site of recombinant human erythropoietin.

Recombinant human erythropoietin (rHuEPO) variants have been constructed to identify amino acid residues important for biological activity. Immunoassays were used to determine the effect of each mutation on rHuEPO folding. With this strategy, we could distinguish between mutations that affected bioactivity directly and those that affected bioactivity because the mutation altered rHuEPO conformation. Four regions were found to be important for bioactivity: amino acids 11 to 15, 44 to 51, 100 to 108, and 147 to 151. EPO variants could be divided into two groups according to the differential effects on EPO receptor binding activity and in vitro biologic activity. This suggests that rHuEPO has two separate receptor binding sites. Mutations in basic residues reduced the biologic activity, whereas mutations in acidic residues did not. This suggests that electrostatic interactions between rHuEPO and the human EPO receptor may involve positive charges on rHuEPO.

Activation of the erythropoietin (EPO) receptor by bivalent anti-EPO receptor antibodies.

Oligomerization of cytokine receptors including the erythropoietin (EPO) receptor has been advanced as a model for activation. If homodimerization of the EPO receptor activates it, then bivalent antibodies raised to the extracellular domain of the EPO receptor should also homodimerize and activate. Mouse monoclonal antibodies (IgG) raised to the soluble, extracellular domain of the human EPO receptor (EPOR) were found that would stimulate thymidine uptake of an human EPO-dependent cell line, UT-7/EPO. Dose response curves showed bell shapes where activity was low at low and high concentrations. Monovalent (Fab) fragments bound to the receptor but did not stimulate thymidine uptake, which indicates that two antibody binding sites are required for activation. The anti-EPOR antibodies stimulated the formation of burst forming unit erythroid colonies from human CD34(+) cells purified from peripheral blood. This indicates that homodimerization of the EPO receptor by anti-EPOR antibodies is sufficient for both proliferation and differentiation of erythroid progenitor cells and that the constraints on dimerization necessary for activation are rather loose.

Fine-structure epitope mapping of antierythropoietin monoclonal antibodies reveals a model of recombinant human erythropoietin structure.

We have isolated and mapped the rHuEPO epitopes for three noncompeting anti-EPO monoclonal antibodies (MoAbs). The MoAb 9G8A recognizes a linear epitope that includes amino acids 13, 16, and 17. MoAb F12 recognizes a conformational epitope that includes amino acids 31 through 33, 86 through 91, and 138. MoAb D11 recognizes a conformational epitope that includes amino acids 64 through 78 and 99 through 110. MoAb D11 neutralizes rHuEPO activity which suggests that its epitope may contain the receptor binding domain. Analysis of the effect of mutations on folding allowed the identification of buried residues, alpha-helical, and non alpha-helical regions. This data along with epitope mapping data of anti rHuEPO monoclonals was used to model rHuEPO protein structure. A model consistent with the data is a 4-helix bundle with short and long interconnecting loops.

Isolation and characterization of conformation sensitive antierythropoietin monoclonal antibodies: effect of disulfide bonds and carbohydrate on recombinant human erythropoietin structure.

We have isolated and characterized three anti-recombinant human erythropoietin (rHuEPO) monoclonal antibodies (MoAbs) that recognize nonoverlapping epitopes on rHuEPO. Anti-EPO MoAb D11 neutralizes rHuEPO activity whereas MoAbs F12 and 9G8A do not. This suggests that D11 may bind to the rHuEPO active site. MoAbs F12 and D11 recognize conformation dependent epitopes whereas 9G8A does not. Immunoassays were developed for each monoclonal. The 9G8A immunoassay was novel and useful because immunoreactivity increased when rHuEPO was denatured. Disruption of disulfide bonds or removal of carbohydrate increased 9G8A immunoreactivity, which suggests that these elements are important for rHuEPO structure or stability.

Structural requirements for addition of O-linked carbohydrate to recombinant erythropoietin.

To define the structural requirements for addition of O-linked glycosylation in vivo, recombinant erythropoietin (rEPO) variants were constructed. Thirty-three independent Ser or Thr substitutions were constructed and examined to see which were subject to O-linked carbohydrate addition. Variants with Thr mutations at positions 123 and 125, but not elsewhere, contained additional carbohydrate, which suggests that several positions around the existing O-linked glycosylation site (Ser126), but not elsewhere, contain the necessary information for O-linked carbohydrate addition. Two forms of the Thr125 variant were identified. One form was glycosylated only at residue 125, and a second form was glycosylated at both Thr125 and Ser126, the normal O-glycosylation site. We have also found that glycosylation is less efficient when rEPO is improperly folded and that prolines at -1 and +1 relative to the O-glycosylation site enhance glycosylation.

Role of glycosylation on the secretion and biological activity of erythropoietin.

The erythropoietin (EPO) molecule contains four carbohydrate chains. Three contain N-linkages to asparagines at positions 24, 38, and 83, and one contains an O-linkage to a serine at position 126. We constructed human EPO variants that eliminated the three N-glycosylation sites by replacing the asparagines with glutamines singly or in combination. The O-linked carbohydrate chain was removed by replacing the serine with glutamine, valine, histidine, or alanine. A variant with a double mutation (Gln38,83) and another with a triple mutation (Gln24,38,83) were secreted poorly from COS1 and CHO cells even though RNA encoding these variants was present. All other variants with mutations in N-linked glycosylation sites were secreted normally. Removal of any of the N-glycosylation sites reduced the in vivo but not the in vitro biological activity of the EPO molecule. All the mutations at Ser126, the O-glycosylation site, were secreted normally. In vitro activity was also unaffected except for Ala126 which had a 50-fold decrease. The Val126 variant was tested in vivo, and its specific activity was only slightly less than that of the native EPO, which indicates that the O-linked carbohydrate is not essential for activity.