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M Colombatti

Publications and source records attributed to M Colombatti.

At least 19 recordsLinked to original sources
PubMed2005

Peptide analogues of a T-cell epitope of ricin toxin A-chain prevent agonist-mediated human T-cell response.

The clinical efficacy of immunotoxins (IT) containing ricin toxin A-chain (RTA) can be drastically reduced by anti-toxin-neutralizing antibodies developed by patients. Strategies aimed at epitope-specific modulation of the immune response must be therefore set up to broaden the clinical applicability of RTA-based IT. Prevention or reduction of humoral immune responses against RTA could be achieved by peptide-based down-modulating strategies. Peptide analogues were investigated as candidate antagonist altered peptide ligands (APL) considering the sequence of a previously identified dominant T-cell epitope of RTA (i.e. I175-E185) presented in the context of the HLA-DRB1*03011 allele. Alanine-substituted peptides provided information on the role of individual residues of the wild-type peptide and allowed to identify one antagonist APL corresponding to the double-mutant peptide E177A/A178D. The analogue E177A/A178D not only prevented the agonist from stimulating anti-RTA human T-cell clones but also failed to induce down-regulation of surface-expressed TCR, thus suggesting its possible use for in vivo immune modulation of anti-RTA responses.

Amino Acids↗
PubMed2004

A dominant linear B-cell epitope of ricin A-chain is the target of a neutralizing antibody response in Hodgkin's lymphoma patients treated with an anti-CD25 immunotoxin.

Hodgkin's lymphoma patients treated with an anti-CD25 Ricin toxin A-chain (RTA)-based Immunotoxin (RFT5.dgA) develop an immune response against the toxic moiety of the immunoconjugate. The anti-RTA antibody response of 15 patients showing different clinical features and receiving different total amounts of RFT5.dgA was therefore studied in detail, considering antibody titre, IgG and IgM content, average binding efficacy and ability to inhibit in vitro the cytotoxicity of a RTA-based Immunotoxin. No correlations were found between these parameters and the clinical features of the patients or the total amount of Immunotoxin administered. However, using a peptide scan approach we have identified a continuous epitope recognized by all patients studied, located within the stretch L161-I175 of the RTA primary sequence, close to a previously identified T-cell epitope. The ability of anti-L161-I175 antibodies to recognize folded RTA and to affect the biological activity of RTA by inhibiting RTA-IT cytotoxicity in vitro revealed that they may exert an important role in IT neutralization in vivo. Discovery of RTA immunodominant epitopes which are the target of anti-RTA immune response may lead to the development of immunomodulating strategies and to more successful treatment schedules.

Antibodies↗
PubMed2001

Pharmacokinetics of intrathecal transferrin-ricin a chain immunotoxin.

We have studied the pharmacokinetics of an anti-transferrin receptor immunotoxin following intrathecal (i.t.) and intravenous (i.v.) bolus inoculation in healthy rats. After i.t. inoculation of 4.9 microg transferrin-ricin A-chain (Tfn-RTA) we have measured the immunotoxin concentration in the cerebrospinal fluid (CSF), in the brain tissue and in the peripheral blood. After i.v. administration of 4.9 microg Tfn-RTA the concentration of Tfn-RTA immunotoxin was evaluated in the peripheral blood. We found that the clearance of Tfn-RTA from the CSF is rapid (9.1 microLmin(-1)), the immunotoxin then diffuses into the brain tissue and in the peripheral blood where it reaches concentrations below the MTC50 (Minimum Toxin Concentration 50%). The rate of immunotoxin elimination from the peripheral blood following either i.v. or i.t. administration are similar (kel = 0.0021 min(-1) vs. 0.0025 min(-1)). Thus, in the healthy rat the immunotoxin does not accumulate following i.t. inoculation, reaching non toxic concentrations in the brain tissue and in the peripheral blood, whereas in the CSF as well as at the interface CSF/brain tissue the immunotoxin may reach potentially therapeutic concentrations. In conclusion we believe that the i.t. inoculation of an immunotoxin could be considered a potentially useful route of administration in the treatment of leptomeningeal carcinomatosis.

Animals↗
PubMed2001

Human MBP-specific T cells regulate IL-6 gene expression in astrocytes through cell-cell contacts and soluble factors.

One of the distinctive features of multiple sclerosis (MS) attacks is homing to the CNS of activated T cells able to orchestrate humoral and cell-based events, resulting in immune-mediated injury to myelin and oligodendrocytes. Of the complex interplay occurring between T cells and CNS constituents, we have examined some aspects of T-cell interactions with astrocytes, the major components of the glial cells. Specifically, we focused on the ability of T cells to regulate the gene expression of interleukin-6 (IL-6) in astrocytes, based on previous evidence showing the involvement of this cytokine in CNS disorders. We found that T-cell adhesion and T-cell soluble factors induce IL-6 gene expression in U251 astrocytes through distinct signaling pathways, respectively, resulting in the activation of NF-kappaB and IRF-1 transcription factors. In a search for effector molecules at the astrocyte surface, we found that alpha3beta1 integrins play a role in NF-kappaB activation induced by T-cell contact, whereas interferon-gamma (IFN-gamma) receptors dominate in IRF-1 induction brought about by T-cell-derived soluble factors. Similar phenomena were observed also in normal fetal astrocyte cultures. We therefore propose that through astrocyte induction, T cells may indirectly regulate the availability of a cytokine which is crucial in modulating fate and behavior of cell populations involved in the pathogenesis of MS inflammatory lesions.

Astrocytes↗
PubMed2001

Identification of ricin A-chain HLA class II-restricted epitopes by human T-cell clones.

The identification of ricin toxin A-chain (RTA) epitopes and the molecular context in which they are recognized will allow strategies to be devised that prevent/suppress an anti-RTA immune response in patients treated with RTA-based immunotoxins. RTA-specific human T-cell lines and T-cell clones were produced by in vitro priming of PBMC. The T-cell clones used a limited set of Vbeta chains (Vbeta1, Vbeta2 and Vbeta8) to recognize RTA epitopes. The use of RTA deletion mutants demonstrated that T-cell lines and T-cell clones from three out of four donors responded to RTA epitopes within the domain D124-Q223, whereas one donor recognized the region I1-D124. The response to RTA peptides of T-cell lines and T-cell clones from two donors allowed the identification of immunogenic segments (D124-G140 and L161-T190) recognized in the context of different HLA-DRB1 alleles (HLA-DRB1*0801, and HLA-DRB1*11011 and B1*03011, respectively). The response to L161-T190 was investigated in greater detail. We found that the HLA-DRB1*03011 allele presents a minimal epitope represented by the sequence I175-Y183 of RTA, whereas the HLA-DRB1*11011 allele presents the minimal epitope M174-I184. RTA peptides and an I175A RTA point mutant allowed us to identify I175 as a crucial residue for the epitope(s) recognized by the two HLA-DRB1 alleles. Failure of T-cell clones to recognize ribosome inactivating proteins (RIPs) showing sequences similar but not identical to RTA further confirmed the role of I175 as a key residue for the epitope recognized in the context of HLA-DRB1*11011/03011 alleles.

Amino Acid Sequence↗
PubMed2000

Genetic grafting of membrane-acting peptides to the cytotoxin dianthin augments its ability to de-stabilize lipid bilayers and enhances its cytotoxic potential as the component of transferrin-toxin conjugates.

Three chimeric proteins were obtained by fusing together the dianthin gene and DNA fragments encoding for the following membrane-acting peptides: the N-terminus of protein G of the vesicular stomatitis virus (KFT25), the N terminus of the HA2 hemagglutinin of influenza virus (pHA2), and a membrane-acting peptide (pJVE). Chimeric dianthins (KFT25DIA, pHA2DIA and pJVEDIA) retained full enzymatic activity in cell-free assays and showed increased ability to induce pH-dependent calcein release from large unilamellar vesicles (LUVs). pHA2DIA and pJVEDIA also showed faster kinetics of interaction with LUVs, while KFT25DIA and pHA2DIA displayed a reduced cytotoxicity as compared to wild-type dianthin. Conjugates made by chemically cross-linking KFT25DIA or pJVEDIA and human transferrin (Tfn) showed greater cell-killing efficiency than conjugates of Tfn and wild-type dianthin. As a consequence, by fusion of membrane-acting peptides to the dianthin sequence the specificity factor (i.e., the ratio between non-specific and specific toxicity) of Tfn-KFT25DIA, Tfn-pHA2DIA and Tfn-pJVEDIA was increased with respect to that of Tfn-based conjugates made with wild-type dianthin. Taken together, our results suggest that genetic fusion of membrane-acting peptides to enzymatic cytotoxins results in the acquisition of new physico-chemical properties exploitable for designing new recombinant cytotoxins and to tackle cell-intoxication mechanisms.

Humans↗
PubMed2000

Adhesion of immature and mature T cells induces in human thymic epithelial cells (TEC) activation of IL-6 gene trascription factors (NF-kappaB and NF-IL6) and IL-6 gene expression: role of alpha3beta1 and alpha6beta4 integrins.

T cell precursors homed to thymus develop in close contact with stromal cells. Among them, thymic epithelial cells (TEC) are known to exert dominant roles in their survival and functional shaping. Key molecules mediating TEC/thymocytes interactions include cytokines and growth factors secreted by the two cell types and adhesion receptors mediating cell contact. Signaling events triggered in thymocytes by adhesion to epithelial cells have been extensively investigated, whereas little is known on the opposite phenomenon. We have previously investigated this issue in a co-culture system composed of TEC cultures derived from human normal thymus and heterologous thymocytes. We demonstrated that thymocytes adhere to TEC involving beta1 and beta4 integrins and induce the clustering of alpha3beta1 and alpha6beta4 heterodimers at the TEC surface. In addition thymocyte adhesion was followed by activation of NF-kappaB and NF-IL6 gene transcription factors and enhanced IL-6 production. The two latter phenomena were reproduced by the cross-linking of the alpha3, alpha6, beta1 and beta4 integrins, thus implying that the alpha3beta1 and alpha6beta4 heterodimers can signal during thymocyte adhesion. We have extended our previous work investigating in the same experimental setting the inducing activity of non stimulated or activated policlonal or clonal mature T cells as representative of the more mature thymocyte subset. We found that adhesion of unstimulated T cell i) involved beta1, but not beta4 integrin functions at the surface ii) induced the clustering of alpha3beta1, but not alpha2beta1 heterodimers at the TEC surface and iii) up-regulated the nuclear binding activity of NF-kappaB transcription factor and the IL-6 secretion. We propose that alpha3beta1 and alpha6beta4 heterodimers are induced to cluster at the TEC surface recognizing yet unknown cellular ligands differentially expressed during T cell development.

Antigens, Surface↗
PubMed1996

Enhancement of ricin toxin A chain immunotoxin activity: synthesis, ionophoretic ability, and in vitro activity of monensin derivatives.

Site-selective toxin delivery was achieved by coupling monoclonal antibody to the A chain subunit of ricin (RTA-IT). The cell-killing potency of RTA-IT can be drastically increased in vitro by using ionophores such as monensin. To reduce the intrinsic toxicity of monensin and to enhance its in vitro and in vivo activity, we synthesized 7 derivatives characterized by different lipophilicities. These derivatives were also analyzed for ionophoretic activity on intact cells, toxicity, and RTA-IT-enhancing activity. Two different RTA-IT were assayed on a human leukemia cell line. A correlation between lipophilicity, ionophoretic activity, and RTA-IT enhancement was observed. The compounds with the highest polar charge showed low intrinsic toxicity, revealed moderate ionophoretic activity, and were able to enhance RTA-IT only at high concentrations, whereas more lipophilic compounds (with a C28 tail or a phenyl group) showed significant ionophoretic activity and good enhancing properties.

Cell Survival↗
PubMed1996

Inductively coupled plasma mass spectroscopy quantitation of platinum-DNA adducts in peripheral blood leukocytes of patients receiving cisplatin- or carboplatin-based chemotherapy.

Platinum-DNA adducts can be assayed in peripheral blood leukocytes by means of atomic absorption spectroscopy and ELISA, and high adduct levels have been correlated previously with favorable clinical response to platinum-based chemotherapy. Our purpose was to study adduct formation in peripheral blood leukocytes by means of a new method, inductively coupled plasma mass spectroscopy (ICP-MS), and to correlate adduct formation with clinical response and toxicity. Platinum (Pt)-DNA adducts were measured by means of ICP-MS in leukocytes of 66 patients receiving a cisplatin- or carboplatin-based chemotherapy, collected either before the beginning of treatment and incubated in vitro with cisplatin or 1 and 24 h after the administration of drug to the patient. The Pt-DNA adduct level in leukocytes from patients exposed to drug in vitro was 14.33 +/- 14.71 fmol/microgram DNA (mean +/- SD), which was not significantly different from the value of 23.4 +/- 19.53 fmol/microgram DNA observed in leukocytes from nine healthy volunteers. In samples collected after the administration of chemotherapy, Pt-DNA adducts ranged from 1.91 +/- 3.59 fmol/microgram DNA (mean +/- SD) at the 1-h time point to 2.61 +/- 3.35 fmol/microgram DNA at 24 h (P > 0.05). Adduct levels in leukocytes exposed in vitro did not correlate with adduct levels from patients treated with cisplatin-based chemotherapy (r = 0.085 and 0.011 at 1 and 24 h, respectively). At 24 h, adduct levels in patients receiving cisplatin (3.15 +/- 3.64 fmol/microgram DNA, mean +/- SD) were significantly higher (P = 0.02) than those observed in patients treated with standard dose carboplatin (0.57 +/- 0.73 fmol/microgram DNA) and also higher than those in patients receiving high-dose carboplatin (1.18 +/- 1.06 fmol/microgram DNA), although the latter difference did not reach statistical significance (P = 0.071). No differences in adduct levels (mean +/- SD) were evident between patients responsive (3.23 +/- 3.51 fmol/microgram DNA) and nonresponsive (2.34 +/- 3.01 fmol/microgram DNA) to chemotherapy. In the homogeneous group of patients treated with combination of cisplatin and 5FU, received dose intensity, hemoglobin decrease, and posttreatment creatinine could not be linked with the extent of leukocyte adduct formation. The data presented here demonstrate that ICP-MS allows the detection of adducts in patients treated with cisplatin or carboplatin and suggest that adduct formation in leukocytes is not a major determinant of response or toxicity.

Adult↗
PubMed1995

Self-potentiation of ligand-toxin conjugates containing ricin A chain fused with viral structures.

A chimeric protein was obtained by fusing together the ricin toxin A chain (RTA) gene and a DNA fragment encoding the N terminus of protein G of the vesicular stomatitis virus. Chimeric RTA (cRTA) retained full enzymic activity in a cell-free assay, but was 10-fold less toxic against human leukemic cells than either native RTA (nRTA) or unmodified recombinant RTA (rRTA). However, conjugates made with cRTA and human transferrin (Tfn) showed 10-20-fold greater cell killing efficacy than Tfn-nRTA or Tfn-rRTA conjugates despite equivalent binding of the three conjugates to target tumor cells. As a consequence, by fusion of the KFT25 peptide to the RTA sequence, the specificity factor (i.e. the ratio between nonspecific and specific cytotoxicity) of Tfn-cRTA was increased 90-240 times with respect to those of Tfn-nRTA and Tfn-rRTA. cRTA interacted with phospholipid vesicles with 15-fold faster kinetics than nRTA at acidic pH. Taken together, our results suggest that the ability of vesicular stomatitis virus protein G to interact with cell membranes can be transferred to RTA to facilitate its translocation to the cell cytosol. Our strategy may serve as a general approach for potentiating the cytotoxic efficacy of antitumor immunotoxins.

Amino Acid Sequence↗
PubMed1995

Escape mechanisms of human leukemic cells to long-term immunotoxin treatment in an in vitro experimental model.

In kinetic assays, an anti-CD5-ricin A chain (ST.I-RTA) immunoconjugate (immunotoxins, IT) specifically inhibited up to 40% the protein synthesis of Jurkat target cells within the first 40 hr. Longer exposures of leukemia cells to ST.I-RTA resulted in a progressively higher number of target cells escaping IT treatment and becoming resistant to further treatment with ST.I-RTA even in the presence of the RTA-IT enhancer monensin. Resistant Jurkat cells proliferated at the same rate as control untreated cells, and were as sensitive as control cells to a transferrin-RTA IT, indicating that the ST.I-RTA-resistant tumor-cell population did not become insensitive to the enzymatic activity of RTA. Binding studies revealed that the anti-CD5 IT treatment induced a transient modulation of CD5 antigens but not of the functionally related CD3 antigens. The CD5 antigens were re-expressed at the cell surface following removal of the IT molecules from the culture medium with 1.1% of the total CD5 Ag being re-expressed per hr. When our experimental data on the kinetics of cell intoxication by the IT were corrected for the proliferative potential of the resistant and of the sensitive tumor-cell populations, it appeared that the effect of ST.I-RTA treatment on Jurkat cells was only to delay cell growth for a limited time period (20 hr) without reducing effectively the tumor-cell burden. Our results may have implications for the long-term treatment of target tumor cells with IT.

Antibodies, Monoclonal↗
PubMed1995

Heterogeneous response of individual multicellular tumour spheroids to immunotoxins and ricin toxin.

The cytoreductive effects of anti-transferrin receptor (anti-TfnR) immunotoxins (ITs) and of ricin toxin against tumour micromasses have been evaluated in a multicellular tumour spheroid (MTS) model. More than 600 (656) MTSs obtained with human breast carcinoma (MCF7) or rat glioblastoma (9L) cell lines were treated individually with ITs or toxin and the effects induced by the treatment were measured for each MTS as volume variation vs time by applying the Gompertz growth model. Two dose-dependent patterns of MTS growth were observed in MTSs of both cell lines in response to IT or toxin treatment: (1) complete inhibition of MTS growth ('sterilisation'); and (2) partial/complete inhibition ('heterogeneous response'). Within the range of IT or toxin concentrations resulting in partial inhibition of MTS growth, the sensitivity of treated MTSs was extremely heterogeneous (the cytoreductive effects varying between 0.1 and 4 logs of cells killed for a given IT or toxin concentration). Analysis of the post-treatment regrowth kinetics indicated that treated non-sterilised and control MTSs reached the same final limiting volumes. However, the doubling time estimated for the surviving cells of treated MCF7 and 9L MTSs ranged between 15 and 50 h, indicating that each MTS had individual growing potential. In conclusion, our results indicate that at substerilising IT concentrations individual heterogenicity of MTSs may greatly influence the cytoreductive potential of ITs. An implication of our study is that the efficacy of an IT treatment in eradicating disseminated micrometastases may not be predictable a priori. The MTS model that we describe in this paper may help in dissecting out factors limiting the effect of ITs in three-dimensional tumours.

Animals↗
PubMed1994

Cytoreductive effects of anti-transferrin receptor immunotoxin in a multicellular tumor spheroid model.

We have evaluated the sensitivity to immunotoxins (IT) of monolayer and of 200-250 microns multicellular tumor spheroid (MTS) cultures obtained with human breast (MCF7) and glioblastoma (U118) tumor cells and with rat glioblastoma (9L) cells. Monolayer MCF7 and U118 cells were highly sensitive to antitransferrin receptor (anti-TfnR) ricin A chain (RTA)-IT (Tfn-RTA and MAb OKT9-RTA) treatment in the presence of the intracellular RTA-IT enhancing agent human serum albumin-monensin (HSA-Mo) conjugate. A 790- to 2000-fold higher concentration of anti-TfnR IT was instead required to reduce by 50% the volume of individually treated MCF7 spheroids, as evaluated by applying the Gompertz growth model. Monolayer 9L cells showed 230- to 5700-fold lower sensitivity to Tfn-RTA IT than MCF7 and U118 monolayers, yet 9L spheroid cells were almost as sensitive to anti-TfnR IT as monolayer 9L cultures. Binding studies performed with [125I]-Tfn and FITC-labelled anti-TfnR MAb revealed that 9L monolayers and MTS expressed 4.1-fold and 8.8-fold lower amounts of TfnR than MCF7 monolayers and MTS, respectively. However, Tfn bound to TfnR sites of 9L and of MCF7 cells with comparable affinity. Experiments carried out with the diphtheria toxin mutant CRM107 linked to Tfn confirmed the pattern observed with RTA-IT. Monolayers and spheroids showed no considerable differences in sensitivity to ricin toxin. Collectively, these results indicated that the efficacy of IT against 3-D tumors is heavily influenced by the number of target Ag expressed by the tumor cells, as well as by the affinity of IT/toxin-cell interaction.

Animals↗
PubMed1994

Sensitivity of human leukemia cells in exponential or stationary growth phase to anti-CD5 immunotoxins. Role of intracellular processing events.

We have assayed the sensitivity of Jurkat cells in different growth phases to an anti-CD5-ricin A chain (ST.1-RTA) immunotoxins (IT). Jurkat cells proliferated exponentially until a stationary growth phase was reached. Proliferating and stationary cells displayed marked differences in sensitivity to ST.1-RTA treatment; the time required to kill one log of target cells (T10) was 70 h in proliferating and 12 h in stationary cells, respectively. Differences in sensitivity to IT treatment were greatly diminished by the addition of the IT enhancer monensin (T10 = 4.9 and 3.5 h in proliferating and stationary cells, respectively). Binding and internalization studies carried out with fluoresceinated ST.1 mAb revealed that the higher sensitivity of stationary cells to ST.1-RTA treatment was not due to an increased uptake or to faster internalization kinetics of IT molecules in this cell population; rather, our data indicated that a different intracellular routing of IT molecules took place in the two cell populations. Mathematical modeling of experimental data allowed us to calculate the efficiency of the intracellular transport of IT molecules toward a subcellular compartment facilitating toxin translocation to the cell cytosol. The IT intracellular processing in stationary cells was 5.5-fold more efficient than in proliferating cells. This value strictly correlated with the higher sensitivity of the stationary cell population to ST.1-RTA treatment.

Antibodies, Monoclonal↗
PubMed1994

Mechanisms involved in serum-dependent inactivation of the immunotoxin enhancers monensin and carrier-protein-monensin.

The immunotoxin-enhancing properties of monensin and of human-serum-albumin-monensin conjugates are severely impaired in the presence of human serum. In this study we have therefore investigated the interaction between serum proteins and monensin leading to the inactivation of monensin function as immunotoxin potentiator. We found that the binding of monensin-specific mAb to thioether-cross-linked or disulfide-cross-linked protein-monensin conjugates is negatively affected by serum, as indicated by immunoenzymic (ELISA) and radioimmunobinding analysis. Size-exclusion chromatography of serum samples indicated that the greatest blocking effect is due to protein components of 40-90 kDa eluting as a broad peak (peak 4). Analysis of the proteins contained within peak 4 by ion-exchange chromatography followed by microsequencing revealed that the major components of peak no. 4 were transferrin, human serum albumin and immunoglobulin fragments. Investigations on the nature of the interactions between serum proteins and monensin leading to monensin inactivation were conducted by affinity chromatography of serum on immobilized human-serum-albumin-monensin conjugates, size-exclusion chromatography, SDS/PAGE analysis of serum-treated human-serum-albumin-monensin conjugates, and evaluation of the stability of immobilized human-serum-albumin-bound 125I-monensin following treatment with serum. Addition of esterase inhibitors (e.g. EDTA, 4-nitrophenyl phosphate) or prior treatment of the serum at 56 degrees C partially reversed the serum effects observed. We conclude that serum proteins block the immunotoxin-enhancing effect of monensin and of human-serum-albumin-monensin conjugates by multiple mechanisms involving hydrophobic and covalent interactions and enzyme-mediated cleavage of protein-bound monensin.

Amino Acid Sequence↗
PubMed1994

Immunoconjugates: lessons from animal models.

Monoclonal antibody-mediated cancer therapy has evolved through a challenging chain of problems and solutions. The very limited therapeutic success obtained with unarmed monoclonal antibodies has increased the interest in different antibody-based targeting strategies, and numerous preclinical and even clinical studies with immunoconjugates have now been conducted. We comment here on the messages implicit in a recent report on a doxorubicin-anti-carcinoma antibody conjugate and from several other studies.

Animals↗
PubMed1993

Standardization of in vitro synthesis and detection of HIV-1-specific antibodies.

Optimal conditions for in vitro anti-human immunodeficiency virus type 1 (HIV-1) antibody (Ab) synthesis and detection were re-appraised. Western blot (WB) and radioimmunoassay (RIA) could detect about 1 and 10 ng, respectively, of HIV-1-specific Ab (HIV-Ab), while the sensitivity of an enzyme-linked immunosorbent assay (ELISA) was much lower. Optimal HIV-Ab recovery was obtained by culturing 2.5 x 10(6) peripheral blood mononuclear cells (PBMC)/ml from seropositive subjects for 16 days in the absence of mitogens; at higher cell concentrations, background levels were unacceptably high. The background of non-de novo synthesized HIV-Ab was due to insufficient PBMC washing and/or cytophilic immunoglobulin (Ig); a particular washing procedure, as well as 24 h peripheral blood mononuclear cells (PBMC) pre-culture, might help in limiting this phenomenon. However, results should be compared with those obtained in cultures containing puromycin especially in infants, where a higher CD16 antigen expression in lymphocytes is likely responsible for increased amounts of cytophilic Ig released in culture supernatants, compared to adults.

Adult↗