Toxicological Implications and Inflammatory Response in Human Lymphocytes Challenged with Oxytetracycline.

Antibiotics are widely used in zoo technical and veterinary practices as feed supplementation to ensure wellness of farmed animals and livestock. Several evidences have been suggesting both the toxic role for tetracyclines, particularly for oxytetracycline (OTC). This potential toxicity appears of great relevance for human nutrition and for domestic animals. This study aimed to extend the evaluation of such toxicity. The biologic impact of the drug was assessed by evaluating the proinflammatory effect of OTC and their bone residues on cytokine secretion by in vitro human peripheral blood lymphocytes. Our results showed that both OTC and OTC-bone residues significantly induced the T lymphocyte and non-T cell secretion of interferon (IFN)-γ, as cytokine involved in inflammatory responses in humans as well as in animals. These results may suggest a possible implication for new potential human and animal health risks depending on the entry of tetracyclines in the food-processing chain.

Oscillatory mTOR inhibition and Treg increase in kidney transplantation.

Intracellular metabolic pathways dependent upon the mammalian target of rapamycin (mTOR) play a key role in immune-tolerance control. In this study, we focused on long-term mTOR-dependent immune-modulating effects in kidney transplant recipients undergoing conversion from calcineurin inhibitors (CNI) to mTOR inhibitors (everolimus) in a 1-year follow-up. The conversion to everolimus is associated with a decrease of neutrophils and of CD8(+) T cells. In addition, we observed a reduced production of interferon (IFN)-γ by CD8(+) T cells and of interleukin (IL)-17 by CD4(+) T lymphocytes. An increase in CD4(+) CD25(+) forkhead box protein 3 (FoxP3)(+) [regulatory T cell [(Treg)] numbers was also seen. Treg increase correlated with a higher proliferation rate of this regulatory subpopulation when compared with the CD4(+) FoxP3(-) effector counterpart. Basal phosphorylation level of S6 kinase, a major mTOR-dependent molecular target, was substantially maintained in patients treated with everolimus. Moreover, oscillations in serum concentration of everolimus were associated with changes in basal and activation-dependent S6 kinase phosphorylation of CD4(+) and CD8(+) T cells. Indeed, T cell receptor (TCR) triggering was observed to induce significantly higher S6 kinase phosphorylation in the presence of lower everolimus serum concentrations. These results unveil the complex mTOR-dependent immune-metabolic network leading to long-term immune-modulation and might have relevance for novel therapeutic settings in kidney transplants.

Cytotoxic effects of oxytetracycline residues in the bones of broiler chickens following therapeutic oral administration of a water formulation.

Tetracyclines, which represent one of the most commonly used antibiotics for poultry, are known to be deposited in bones, where they can remain, despite the observation of appropriate withdrawal times. The aim of the study was to determine the concentration of oxytretracycline (OTC) residues in the bone and muscle of chickens, following the oral administration of a commercially available liquid formulation, and to test their cytotoxic effects on an in vitro cell culture model. Seventy-two 1-day-old broiler chickens were randomly allotted into 2 groups (control and treated animals). OTC (40 mg/kg BW) was administered via drinking water during the 1 to 5 and 20 to 25 days of life periods. At the end of the trial, the birds were slaughtered and the OTC residues in the target tissues were measured by means of liquid chromatography (LC) - tandem mass spectrometry (MS/MS). Cytotoxicity was assessed by evaluating the pro-apoptotic effect of the bone residues on the K562 erythroleukemic line and on the peripheral blood mononuclear cells (PBMC). In all the animals, the OTC residues in the muscle were far below the established MRL of 100 µg/kg. The OTC levels in the bones of the treated animals were instead found in the parts per million (ppm) range. Cell cytotoxicity was assessed by evaluating the pro-apoptotic effect of OTC bone residues on the haematopoietic cell system. This in vitro system has revealed a significant pro-apoptotic effect on both the K562 cell line and PBMC cultures. This result suggests potential human and animal health risks due to the entry of tetracycline residues contained in the bones of treated livestock into the food-chain. This could be of concern, particularly for canine and feline diets, as meat, bone meal, and poultry by-products represent some of the main ingredients of pet foods, especially in the case of dry pet food. Further studies are needed to define the underlying mechanisms of cytotoxicity and to evaluate the in vivo toxicological implications due to the observed in vitro effects.

Killer immunoglobulin-like receptors (KIR) and their HLA-ligands in Italian paroxysmal nocturnal haemoglobinuria (PNH) patients.

Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoietic disorder characterized by expansion of phosphatidylinositol glycan-A-defective progenitor(s). Immune-dependent mechanisms, likely involving a deranged T cell-dependent autoimmune response, have been consistently associated with the selection/dominance of PNH precursors. Natural killer (NK) lymphocytes might participate in PNH pathogenesis, but their role is still controversial. NK activity is dependent on the balance between activating and inhibiting signals. Key component in such regulatory network is represented by killer immunoglobulin-like receptors (KIR). KIR are also involved in the regulation of adaptive cytotoxic T cell response and associated with autoimmunity. This study investigated on the frequency of KIR genes and their known human leukocyte antigen (HLA) ligands in 53 PNH Italian patients. We observed increased frequency of genotypes characterized by ≤2 activating KIR as well as by the presence of an inhibitory/activating gene ratio ≥3.5. In addition, an increased matching between KIR-3DL1 and its ligand HLA-Bw4 was found. These genotypes might be associated with lower NK-dependent recognition of stress-related self molecules; this is conceivable with the hypothesis that an increased availability of specific T cell targets, not cleared by NK cells, could be involved in PNH pathogenesis. These data may provide new insights into autoimmune PNH pathogenesis.

A novel fully human antitumour immunoRNase targeting ErbB2-positive tumours.

BACKGROUND: ErbB2 is an attractive target for immunotherapy, as it is a tyrosine kinase receptor overexpressed on tumour cells of different origin, with a key role in the development of malignancy. Trastuzumab, the only humanised anti-ErbB2 antibody currently used in breast cancer with success, can engender cardiotoxicity and a high fraction of patients is resistant to Trastuzumab treatment. METHODS: A novel human immunoRNase, called anti-ErbB2 human compact antibody-RNase (Erb-hcAb-RNase), made up of the compact anti-ErbB2 antibody Erbicin-human-compact Antibody (Erb-hcAb) and human pancreatic RNase (HP-RNase), has been designed, expressed in mammalian cell cultures and purified. The immunoRNase was then characterised as an enzymatic protein, and tested for its biological actions in vitro and in vivo on ErbB2-positive tumour cells. RESULTS: Erb-hcAb-RNase retains the enzymatic activity of HP-RNase and specifically binds to ErbB2-positive cells with an affinity comparable with that of the parental Erb-hcAb. Moreover, this novel immunoRNase is endowed with an effective and selective antiproliferative action for ErbB2-positive tumour cells both in vitro and in vivo. Its antitumour activity is more potent than that of the parental Erb-hcAb as the novel immunoconjugate has acquired RNase-based cytotoxicity in addition to the inhibitory growth effects, antibody-dependent and complement-dependent cytotoxicity of Erb-hcAb. CONCLUSION: Erb-hcAb-RNase could be a promising candidate for the immunotherapy of ErbB2-positive tumours.

ER stress impairs MHC Class I surface expression and increases susceptibility of thyroid cells to NK-mediated cytotoxicity.

We recently reported that, in thyroid cells, ER stress triggered by thapsigargin or tunicamycin, two well known ER stressing agents, induced dedifferentiation and loss of the epithelial phenotype in rat thyroid cells. In this study, we sought to evaluate if, in thyroid cells, ER stress could affect MHC class I expression and the possible implications of this effect in the alteration of function of natural killer cells, suggesting a role in thyroid pathology. In both, a human line of fetal thyroid cells (TAD-2 cells) and primary cultures of human thyroid cells, thapsigargin and tunicamicin triggered ER stress evaluated by BiP mRNA levels and XBP-1 splicing. In both cell types, TAD-2 cell line and primary cultures, major histocompatibility complex class I (MHC-I) plasmamembrane expression was significantly reduced by ER stress. This effect was accompanied by signs of natural killer activation. Thus, natural killer cells dramatically increased IFN-γ production and markedly increased their cytotoxicity against thyroid cells. Together, these data indicate that ER stress induces a decrease of MHC class I surface expression in thyroid cells, resulting in reduced natural killer-cell self-tolerance.

Secondary immune-mediated thrombocytopenia in dogs naturally infected by Leishmania infantum.

Forty-four dogs naturally infected by Leishmania infantum were divided into two groups: 20 thrombocytopenic dogs with fewer than 150 x 10(9) platelets/l, and 24 non-thrombocytopenic dogs with more than 200 x 10(9) platelets/l. Ten clinically healthy dogs were used as controls. A haematological profile was obtained and the dogs' serum was used to assess the presence of platelet-binding IgM and IgG antibodies using a flow cytometry technique. Nineteen of the 20 thrombocytopenic dogs, and 13 of the 24 non-thrombocytopenic dogs had detectable levels of platelet-binding immunoglobulins, but none of the control dogs did so. The differences were significantly different for both IgM and IgG platelet-binding antibodies.

Presence of anti-platelet IgM and IgG antibodies in dogs naturally infected by Leishmania infantum.

Thirty-three dogs, naturally infected by Leishmania infantum, were enrolled in the study and were classified as oligo-symptomatic (n. 15) and symptomatic or markedly symptomatic (n. 18). A control group was 10 healthy dogs. A haematological profile was obtained and the dogs serum was employed to assess the presence of platelet binding IgM and IgG antibodies (PBIgM, PBIgG) using flow cytometry. FITC labelled goat anti-dog IgM or IgG were used to detect PBIgM and PBIgG. Samples with a mean fluorescence intensity (MFI) that was 100 channels higher on a log scale for more than 30% of the platelets than seen in negative control platelets from a healthy dog were considered positive for the presence of anti-platelet antibodies (PBIg). Twenty-one (63.3%) dogs revealed the presence of PBIg. Six of them were oligo-symptomatic while 15 showed moderate or severe clinical signs of illness. All the dogs with PBIg showed the presence of PBIgM, with nine animals showing both PBIgM and PBIgG. Nine of 18 symptomatic or markedly symptomatic dogs showed thrombocytopenia, while normal platelet counts were observed in all oligo-symptomatic animals. Eight of 9 thrombocytopenic animals showed the presence of PBIgM, while six of them showed PBIgG. One thrombocytopenic dog was negative for PBIg. This study is the first report documenting the presence of PBIg in natural canine leishmaniasis implying a pathogenic association between thrombocytopenia and the presence of antibody against platelet membrane.

A human, compact, fully functional anti-ErbB2 antibody as a novel antitumour agent.

A new human, compact antibody was engineered by fusion of a human, antitumour ErbB2-directed scFv with a human IgG1 Fc domain. Overexpression of the ErbB2 receptor is related to tumour aggressiveness and poor prognosis. This new immunoagent meets all criteria for a potential anticancer drug: it is human, hence poorly or not immunogenic; it binds selectively and with high affinity to target cells, on which it exerts an effective and selective antiproliferative action, including both antibody-dependent and complement-dependent cytotoxicity; it effectively inhibits tumour growth in vivo. Its compact molecular size should provide for an efficient tissue penetration, yet suitable to a prolonged serum half-life.

Interaction between natural killer and dendritic cells: the role of CD40, CD80 and major histocompatibility complex class i molecules in cytotoxicity induction and interferon-gamma production.

This study focuses on the differential role of CD40 and CD80 costimulatory molecules and major histocompatibility complex class I (MHC-I) antigens in the regulation of the interplay between dendritic cells (DCs) and interleukin (IL)-2-activated human natural killer (NK) lymphocytes. Our data indicate that CD40 and CD80 molecules might play a preferential role in the induction of cytotoxic function but not in the interferon-gamma(IFN-gamma) production by human IL-2-activated NK effectors in the presence of autologous and allogeneic DCs. In addition, a critical role of CD94-dependent MHC-I recognition in the regulation of both IFN-gamma production and target cell lysis was shown in the functional interaction between NK and DCs.

Proliferative and cytotoxic response of human natural killer cells exposed to transporter associated with antigen-processing-deficient cells.

In transporter associated with antigen-processing (TAP)-deficient patients affected by a severe downmodulation of human leucocyte antigen class I (HLA-I) molecules, natural killer (NK) cells have an increased expression of the inhibitory receptor CD94/NKG2A. Focusing our attention on NK cells, we have investigated the phenotype, function and proliferative response of peripheral blood lymphocytes (PBLs) derived from healthy donors after coculturing with TAP (T2)- or HLA-I-deficient (721.221) cell lines and their related HLA-I-expressing transfectants (T3 and DT360, respectively). After 4 days, NK cells cocultured with T2 cells had a threefold increased CD94 expression compared to NK cells cocultured with T3. This increase was due to proliferation of the CD56brightCD94bright subset. In contrast, expression of other inhibitory receptors [killer cell immunoglobulin (Ig)-like receptors] was variable during time and was not related to HLA-I molecules expressed by stimulating cells. Similar results were obtained using HLA-I-deficient cells (721.221). The PBLs cocultured for 4 days with T2 cells displayed enhanced cytotoxic responses. The results suggest that CD56brightCD94bright NK cells are induced to proliferate and kill in response to a TAP-deficient environment. The changes seen in the NK-cell compartment were partially contributed by T lymphocytes present in the coculture. These data could explain the increased CD94 expression and autoimmune manifestations observed in TAP-deficient patients.

Inhibition of human NK cell-mediated killing by CD1 molecules.

It is now well established that NK cells recognize classical and nonclassical MHC class I molecules and that such recognition typically results in the inhibition of target cell lysis. Given the known structural similarities between MHC class I and non-MHC-encoded CD1 molecules, we investigated the possibility that human CD1a, -b, and -c proteins might also function as specific target structures for NK cell receptors. Here we report that expression of CD1a, -b, or -c can partially inhibits target cell lysis by freshly isolated human NK cells and cultured NK lines. The inhibitory effects of CD1 molecules on NK cell could be shown upon expression of individual CD1 proteins in transfected NK-sensitive target cells, and these effects could be reversed by incubation of the target cells with mAbs specific for the expressed form of CD1. Inhibitory effects of CD1 expression on NK-mediated lysis could also be shown for cultured human dendritic cells, which represent a cell type that prominently expresses the various CD1 proteins in vivo. In addition, the bacterial glycolipid Ags known to be bound and presented by CD1 proteins could significantly augment the observed inhibitory effects on target cell lysis by NK cells.

HLA class I antigen downregulation by interleukin (IL)-10 is predominantly governed by NK-kappaB in the short term and by TAP1+2 in the long term.

The present study was designed to determine the molecular mechanisms by which interleukin (IL)-10 prevents the HLA class I antigen expression at the cell surface. In this context, the potential role of transporter associated with antigen presentation 1+2 (TAP1+2) molecules and NF-kappaB transcription factors was addressed. The IL-10 effect was investigated in a human lymphoblastoid cell system defective for TAP1+2 genes (T2 cell line) and in the related TAP1+2 transfectants (T3 cell line). In this experimental system, after 48 h of incubation in the presence of IL-10, the HLA class I antigen downmodulation was observed in the T3 but not in the T2 cell line, suggesting a potential role of TAP1+2 molecules. In the same experimental conditions, the NF-kappaB activity was unaffected. Instead, after 3 h of exposure to IL-10, the HLA downmodulation was observed in both cell lines, the NF-kappaB factors activity being strongly reduced. In addition, the transfection of the inhibitor of NF-kappaB, IkappaBalpha, prevented the IL-10 effect on HLA class I antigen expression in the T3 cell line. This phenomenon was observed after 3 h but not 48 h of IL-10 incubation. These evidences indicate a time dependent involvement of TAP1+2 antigens and of NF-kappabeta activity in the IL-10-induced major histocompatibility complex (MHC) class I downmodulation.

Recognition of autologous dendritic cells by human NK cells.

NK cells can recognize and kill tumor as well as certain normal cells. The outcome of the NK-target interaction is determined by a balance of positive and negative signals initiated by different target cell ligands. We have previously shown that human NK cells kill CD40-transfected tumor targets efficiently, but the physiological significance of this is unclear. We now demonstrate that human NK cells can kill dendritic cells (DC), known to express CD40 and other co-stimulatory molecules. The killing was observed with polyclonal NK cells cultured short term in IL-2 as well as with NK cell clones as effectors, and with allogeneic as well as autologous DC as targets. NK cell recognition could be inhibited, but only partially, by preincubation of target cells with monoclonal antibodies against CD40, suggesting that this molecule may be one of several ligands involved. Addition of TNF-alpha of the cultures stimulated the development of a more mature DC phenotype, while addition of IL-10 resulted in a less mature phenotype, with lower expression of CD40 and other co-stimulatory molecules. Nevertheless, such DC were more NK susceptible than the differentiated DC. This may be partly explained by a reduced MHC class I expression observed on such cells, since blocking of MHC class I molecules on differentiated DC or CD94 receptors of NK cells led to increased NK susceptibility. The results show that NK cells may interact with DC, and suggest that the outcome of such interactions depend on the cytokine milieu.

A new mechanism of NK cell cytotoxicity activation: the CD40-CD40 ligand interaction.

NK recognition is regulated by a delicate balance between positive signals initiating their effector functions, and inhibitory signals preventing them from proceeding to cytolysis. Knowledge of the molecules responsible for positive signaling in NK cells is currently limited. We demonstrate that IL-2-activated human NK cells can express CD40 ligand (CD40L) and that recognition of CD40 on target cells can provide an activation pathway for such human NK cells. CD40-transfected P815 cells were killed by NK cell lines expressing CD40L, clones and PBL-derived NK cells cultured for 18 h in the presence of IL-2, but not by CD40L-negative fresh NK cells. Cross-linking of CD40L on IL-2-activated NK cells induced redirected cytolysis of CD40-negative but Fc receptor-expressing P815 cells. The sensitivity of human TAP-deficient T2 cells could be blocked by anti-CD40 antibodies as well as by reconstitution of TAP/MHC class I expression, indicating that the CD40-dependent pathway for NK activation can be downregulated, at least in part, by MHC class I molecules on the target cells. NK cell recognition of CD40 may be important in immunoregulation as well as in immune responses against B cell malignancies.

Natural killer clones recognize specific soluble HLA class I molecules.

Enhancement of major histocompatibility complex (MHC) class I expression leads to protection from natural killer (NK) cell recognition in several systems. MHC class I gene products are released from the cell surface and can be found in sera as soluble forms. To investigate the possible immunoregulatory role of soluble HLA (sHLA) in NK cell-target recognition, several sHLA antigens were studied for their ability to induce NK cell cytotoxicity modulation. NK cell-target recognition was inhibited by the addition of sHLA during the cytotoxicity assay. Our results indicate that sHLA molecules can down-regulate NK killing at the effector level. Moreover, different NK clones are able to specifically recognize different sHLA antigens. Kp43 molecules seem to be involved in the NK recognition of sHLA-B7.