Can histamine cause an enhancement of the cellular uptake and cytotoxicity of doxorubicin-loaded polylactide nanoparticles?

Histamine (His) in humans is physiologically involved in neurotransmission and increases vascular permeability during the development of inflammatory response and immunity. It could be used to enhance drug-loaded nanoparticles (NPs) distribution. However, it cannot be freely delivered due to the risk of His-dose-dependent deleterious effects. His can be attached to the polymeric backbone during polymerization to overcome this limitation. In this study, His was used as an initiator of lactide polymerization, and the obtained macromolecules were subsequently used to prepare doxorubicin (DOX)-loaded NPs by nanoprecipitation and microfluidics for examination of anti-cancer properties. Notably, the in vitro activity towards gastric cancer cells (AGS) of the NPs composed of histamine-functionalized polylactides (PLAs) was greatly enhanced compared to control NPs built from hydroxy­functionalized PLAs. Furthermore, Zonula occludens-1 (ZO-1) tight junction protein production was significantly diminished after treating cells with DOX-loaded NPs assembled with PLAs with histamine residues. These results demonstrate the synergistic effect in cytotoxicity towards gastric cancer cells of DOX and the histamine that are carried by NPs. It is believed that His-DOX NPs strategy may lead to effective, targeted, and low-toxic delivery of drugs into cancer cells.

Helicobacter pylori lipopolysaccharide activity in human peripheral blood mononuclear leukocyte cultures.

Helicobacter pylori (H. pylori) have been recognized as a major cause of chronic gastritis, gastric and duodenal ulcers and gastric cancer. Macrophages are the targets of lipopolysaccharide (LPS), which is a constituent of the outer membrane of Gram-negative rods. In this study we focused on a potential role of macrophages in the proliferation of human peripheral blood mononuclear leukocytes (PBML) in the milieu of H. pylori LPS and standard E. coli LPS. First, we found that H. pylori and E. coli LPS induced proliferation of total PBML (tPBML) from 5 out 21 healthy blood donors (LPS responders). In the LPS milieu, tPBML from the majority of volunteers (LPS non-responders) showed a significant decrease in the [(3)H]-thymidine incorporation as compared to tPBML in medium alone. The decreased cell proliferation was associated with a diminished metabolic activity of non-adherent lymphocytes. Then, non-adherent lymphocytes were stimulated with autologous macrophages pulsed with bacterial LPS. Still, the lymphocytes from the non-responders did not proliferate in the cultures with LPS exposed macrophages. In the group of LPS responders, the macrophages pulsed with H. pylori LPS significantly reduced the proliferation of non-adherent lymphocytes. The possible mechanism regulating the responses of PBML to bacterial LPS with an implication for the outcome of H. pylori infections is discussed.

A potential double role of anti-Lewis X antibodies in Helicobacter pylori-associated gastroduodenal diseases.

In this study, we found Lewis X (Le(x)) determinants on 68% of Helicobacter pylori isolates from patients with chronic gastroduodenal diseases. Anti-Le(x) IgG were detected more frequently in the sera from dyspeptic children and adults (45 and 46%), with or without proved (culture) H. pylori infection, than in the sera from healthy individuals (14% and 25%). In contrast, the prevalence of anti-Le(x) IgM was higher in the groups of healthy individuals than in the groups of dyspeptic patients. Moreover, anti-Le(x) monoclonal antibody of IgM class enhanced the uptake of Le(x)(+) but not Le(x)(-) H. pylori isolates by phagocytes. In the sera from some dyspeptic patients, we detected Le(x)-anti-Le(x) IgG immune complexes (Le(x) ICs). There was a great difference between children and adults as regards the presence of Le(x) ICs. The immune complexes were found in the sera from nine out of 29 (27%) H. pylori-infected and three out of eight (37%) uninfected adult dyspeptic patients. In comparison, Le(x)-anti-Le(x) IgG ICs were detected only for two out of 18 (11%) H. pylori-infected children. Le(x) ICs were not found in the sera from healthy individuals. Our results suggest that anti-Le(x) IgM may play a protective role in H. pylori infections. In contrast, anti-Le(x) IgG and particularly Le(x)-anti-Le(x) IgG ICs might contribute to the pathogenesis of chronic H. pylori infections.

Potential role of CagA in the inhibition of T cell reactivity in Helicobacter pylori infections.

The pathogenicity of chronic gastroduodenal diseases is very often related to Helicobacter pylori infections. Most H. pylori strains carry the cagA gene encoding an immunodominant 120- to 128-kDa protein which is considered a virulence marker. The majority of CagA-positive H. pylori isolates also produce a 95-kDa protein cytotoxin (VacA) causing vacuolation and degradation of mammalian cells. In our previous study we have shown that live H. pylori bacteria and their sonicates inhibit PHA-driven proliferation of human T lymphocytes. The H. pylori CagA and VacA proteins were suspected of a paralyzing effect of H. pylori on T cell proliferation. In this report, by using isogenic H. pylori mutant strains defective in CagA and VacA proteins, we determined that CagA is responsible for the inhibition of PHA-induced proliferation of T cells.

Evaluation of the API test, phosphatidylinositol-specific phospholipase C activity and PCR method in identification of Listeria monocytogenes in meat foods.

The aim of this work was to compare the possibility of identifying Listeria monocytogenes strains isolated from meat and sausage on the basis of the API-Listeria test, production of phosphatidylinositol-specific phospholipase C (PI-PLC) and polymerase chain reaction (PCR) for a DNA fragment of the hlyA gene encoding listeriolysin O. Forty-six strains were isolated and examined. The lethality of some Listeria isolates for BALB/c mice was also determined. In this study, all isolates identified as L. monocytogenes in the API test gave a positive signal in the PCR. Listeriae identified as L. innocua or L. welshimeri in the API test were negative in the PCR conducted with the primers for listeriolysin O. All strains identified as L. monocytogenes on the basis of the API test and the PCR produced PI-PLC. However, this activity was not limited to the bacteria of this species. Four out of 17 L. innocua and three out of 10 L. welshimeri isolates were PI-PLC-positive. None of the L. innocua or L. welshimeri isolates (neither PI-PLC+ or PI-PLC-) showed lethality for BALB/c mice. In contrast, two L. monocytogenes isolates as well as a reference L. monocytogenes strain killed all mice used for the experiment.

[IgG and IgA immunoglobulins in helicobacter pylori infections of children with chronic dyspepsia before and after two week triple drug therapy]. / Immunoglobuliny IgG i IgA w zakazeniach Helicobacter pylori dzieci z przewlekla dyspepsja przed i po dwutygodniowej trójlekowej terapii.

The aim of this study was to investigate whether serological techniques, ELISA and Western blot, are useful in monitoring treatment of H. pylori-associated chronic dyspeptic symptoms in children. We observed a correlation between a decrease in the anti-H. pylori IgG titer and an effective treatment. So, our results suggested that the ELISA assay conducted with a glycine H. pylori extract can be a good noninvasive assay for monitoring the effectiveness of the therapy. By using the Western blot method, we showed some variation in the specificity of anti-H. pylori IgG produced before and after treatment. However, this variation was not correlated with the effectiveness of the therapy.

Anti-Lewis X IgM and IgG in H. pylori infections in children and adults.

A role of autoimmune processes in the pathology of Helicobacter pylori infections has been suggested. The Lewis determinants present in LPS molecule of H. pylori bacteria have been indicated as the cause of antigenic mimicry. In this study, the prevalence of IgM and IgG antibodies to Lewis X antigen in the sera from children and adults, with or without dyspepsia, infected or not infected with H. pylori, seropositive and seronegative for anti-H. pylori IgG were determined immuno-enzymatically (ELISA). Our results revealed that humans may produce anti-Lewis X antibodies, particularly of IgM class, in the absence of H. pylori infection or H. pylori independent dyspepsia. The production of such antibodies, by healthy children who had never been infected with H. pylori suggested that anti-Lewis X antibodies may occur naturally.

Systemic humoral response to Helicobacter pylori in children and adults.

In this study we compared the development of anti-H. pylori humoral response in adults and children. Two antigens: H. pylori acid glycine extract (GE) and recombinant cagA were used for ELISA and Western blot. Anti-GE IgG were detected in all and anti-cagA IgG in about 50% of H. pylori infected adults and children. The prevalence of anti-GE and anti-cagA IgG in the sera from H. pylori-uninfected children with gastritis/gastroduodenitis was lower than in the sera from healthy adult blood donors. Serum IgA were demonstrated for 71% of H. pylori-infected adults and for a smaller proportion (about 30%) of uninfected adult patients or normal subjects. Such antibodies were detected neither for infected nor for uninfected children. There was an evident difference between the proteins of H. pylori glycine extract recognized by antibodies present in the sera from H. pylori-infected children and adults. The antigen of molecular weight over 107 kDa was recognized exclusively by the sera from 30% of H. pylori-infected adults. The 80-107 kDa bands were recognized more frequently by the sera from adults than from children. In contrast, sera from infected children more frequently than sera from infected adults reacted with the bands of 14 kDa, 19 kDa and 26 kDa. The H. pylori antigens recognized by IgG, produced by infected children and adult patients, should be taken into consideration in the developing of tests for serodiagnosis of H. pylori infection.

Anti-Lewis X antibody and Lewis X-anti-Lewis X immune complexes in Helicobacter pylori infection.

A molecular similarity of Lewis antigens expressed by Helicobacter pylori bacteria and those present in human gastric mucosa has been recognised as a cause of autoimmunity involved in the pathogenesis of chronic type B gastritis and gastric and duodenal ulcers. In this study, the expression of Lewis X determinants was found on 56% of H. pylori strains isolated from patients with chronic gastritis/gastroduodenitis. Anti-Lewis X IgG as well as Lewis X-anti-Lewis X IgG complexes were detected in the sera from patients and even more frequently in the sera from healthy blood donors producing antibodies against surface antigens of H. pylori. It suggested that the initial H. pylori-induced lesions were independent of anti-Lewis X antibody production. When H. pylori bacteria expressing Lewis X antigen were treated with anti-Lewis X monoclonal antibody (mAb) of IgM isotype, they were more susceptible to ingestion by polymorphonuclear leukocytes (PMN) than untreated bacteria. This fact may lead us to believe that anti-Lewis X antibody limits the growth of H. pylori on gastric mucosa.

Serum IgG antibodies in children and adults reacting with Helicobacter pylori lipopolysaccharides.

The presence of IgG antibodies reacting with Helicobacter pylori lipopolysaccharides (LPSs) in sera from children and adults diagnosed as H. pylori-infected, as well as healthy persons, was tested. There was no correlation between the production of antibodies reacting with H. pylori surface proteins and LPSs. Also no correlation between reactivity of tested sera with H. pylori antigens and deep rough mutant (Re types) enterobacterial LPSs was revealed. The prevalence of anti-LPS IgG in randomly selected children was relatively high.

A recombinant fragment of Helicobacter pylori CagA affects proliferation of human cells.

The outcome of H. pylori infectins depends on proliferation of various host cells, including lymphocytes, monocytes and epithelial cells. In this study we showed that a recombinant fusion protein carrying an immunodominant region of H. pylori CagA antigen affected the proliferation of human cells. The rCagA inhibited PHA-driven T cell proliferation but enhanced the growth of epithelial HeLa cells, especially in the presence of granulocyte macrophage colony stimulating factor (GM-CSF). When THP-1 monocytes and Kato-3 epithelial cells from metastasis of gastric carcinoma were stimulated with GM-CSF, they were also susceptible to the inhibitory effect of rCagA. These results confirmed our earlier suggestion on the inhibition of T cell function by H. pylori CagA protein. However, antiproliferative activity of CagA antigen appears to be not restricted to T lymphocytes but modulatory effect of this protein seems to depend on the cell type.

Attachment of Helicobacter pylori strains to human epithelial cells.

The aim of the study was to characterize several clinical isolates of H. pylori as regards the activity and specificity of their haemagglutinins and the involvement of surface sialic acid-specific and heparin-binding compounds in the adhesin of the bacteria to human epithelial cell lines. Although H. pylori strains caused haemagglutination (HA) of sheep erythrocytes, they differed markedly by activity and specificity. On the basis of haemagglutination inhibition study three types of H. pylori strains could be distinguished. The HA of Type I strains was inhibited with fetuin/mucin but not asialofetuin/asialomucin. The HA activity of Type II strains was inhibited with fetuin/mucin and asialofetuin/asialomucin. The HA of Type III strains was not influenced by any of these inhibitors. In vitro, H. pylori strains bound to the cells of human epithelial lines: HeLa, Kato-3, Ags. However, various compounds mediated the binding of H. pylori types distinguished by HA, to epithelial cells. The interaction of some of H. pylori strains with epithelial cells was mediated by bacterial sialic acid-binding compounds. The majority of H. pylori strains used heparin-binding surface compounds to attach to epithelial cells. Clinical H. pylori strains differ by the compounds used in adhesin to epithelial cell lines, however, this process also depends on the expression of appropriate receptors on the host cells.

[Progress in diagnosis of infections brought on by Helicobacter pylori]. / Postepy w diagnostyce zakazen wywolywanych przez paleczki Helicobacter pylori.

A wide range of techniques was developed for identification of Helicobacter pylori, since the association of the microorganism with gastritis and peptic ulcer has been established in 1983. Up to now, isolation and identification of H. pylori from the gastric biopsy, remains the standard diagnostic procedure. However, attention is focused on rapid non-invasive tests, for monitoring the infection, such as urea breath test. Serological methods as ELISA and Western blot are suitable for estimation of specific anti-H. pylori local and systemic humoral response. Molecular tests based on PCR reaction allow the detection of H. pylori in biopsy specimens and recently in other clinical samples as saliva or faeces. They are also very promising in epidemiological studies.

Serological indicators of Helicobacter pylori infection in adult dyspeptic patients and healthy blood donors.

The levels of IgM, IgG and IgA antibodies reacting with two Helicobacter pylori antigens (glycine acid extract (GE) and a recombinant CagA protein) were determined in the sera from adult dyspeptic patients, positive (H.p.(+)) or negative (H.p.(-)) for H. pylori urease/culture, and from healthy blood donors. All sera were also examined against GE by Western blot (Immunoblot) technique. Similar levels of anti-GE IgG were detected in the sera from all H.p.(+) and almost all H.p.(-) patients and from over 40% of the healthy volunteers. In contrast, higher levels of anti-GE IgA were found in the sera from patients than that from healthy subjects, although such antibodies were not detected in the sera from 30% of the H.p.(+) patients. In general, our results suggest that a combination of ELISA and immunoblot may be more sensitive in the detection of H. pylori infection in dyspeptic patients than the examination of biopsy specimens by culturing or histology.

[Immunologic reactions in infections caused by Helicobacter pylori]. / Reakcje immunologiczne w zakazeniach wywolywanych przez Helicobacter pylori.

Helicobacter pylori is recognized as an important cause of chronic antral gastritis and peptic ulceration. Moreover, H. pylori associated inflammatory process has been linked with gastric carcinoma. Many putative virulence factors of H. pylori have been suggested, including motility, urease and cytotoxins production and bacterial adhesins. An accessory function of CagA antigen and bacterial heat-shock proteins in the pathogenesis of H. pylori infections have been also considered. H. pylori-induced immunological response is discussed as regards local and general antibody production, the interaction of the bacteria with the phagocytes and still controversial involvement of T cells. Data on the importance of cytokines and inflammatory mediators in the disruption of the gastric mucosal barriers as well as the evidence to support a role for H. pylori as a risk factor for gastric carcinoma are also presented.

Role of Helicobacter pylori surface structures in bacterial interaction with macrophages.

BACKGROUND: Helicobacter pylori infection is associated with a marked infiltration of the gastric epithelium by neutrophils, macrophages, lymphocytes, and plasma cells. Despite the presence of phagocytes in close vicinty to H pylori microbes a great number of people are unable to eradicate bacteria. AIMS: To investigate the involvement of multiple bacterial 'adhesins' and some phagocytic receptors in the process of the ingestion of H pylori by macrophages. BACTERIA: H pylori strains differing in the expression of sialic acid dependent (sHA) or sialic acid independent (HA) haemagglutinin and heparan sulphate binding were selected for the study. METHODS: The uptake of fluorescein labelled H pylori bacteria by a homogenous macrophage cell line J 774A.1 was estimated in a quantitative fluorometric assay. RESULTS: The ingestion of H pylori 17874 and 25 strains expressing sHA was inhibited by the pretreatment of the bacteria with anti-sHA antibodies or fetuin as well as by treatment of the macrophages with neuraminidase. In contrast the uptake of H pylori 17875 strain expressing HA remained unchanged. The phagocytosis of all investigated bacteria was inhibited after the treatment with heparin, hyaluronic acid or vitronectin with fresh but not heat inactivated serum. CONCLUSIONS: The results suggest that H pylori surface compounds binding host proteins such as fetuin, heparin/haparan sulphate, hyaluronic acid, and vitronectin in the presence of complement, could allow the bacteria to avoid phagocytosis.

The proliferation of human T lymphocytes stimulated by Helicobacter pylori antigens.

Fractionated mononuclear cells (MNCs) were obtained from peripheral blood of healthy human volunteers, seronegative for H. pylori antibodies. The MNCs were stimulated in culture with whole live or heat-killed H. pylori cells or with bacterial cell surface (SA) or cytoplasmic (CA) antigens. There was a marked proliferative response of T cells in cultures stimulated with 10(5) cells/well of live H. pylori, 5 micrograms/well of CA or 5-20 micrograms/well of SA. However, no proliferation was observed in MNC cultures containing higher "doses" of live H. pylori organisms (10(7)/well) or CA (20 micrograms/well). Moreover, higher "doses" of the bacteria or CA entirely inhibited the response of T cells to PHA.

The stimulation and inhibition of T cell proliferation by Helicobacter pylori components.

The purified T cells from peripheral blood of healthy human volunteers, seronegative for anti-Helicobacter pylori antibody were stimulated in cultures with live or heat-killed H. pylori rods or with bacterial sialic acid-specific surface haemagglutinin (sHA), a crude surface (SF) or cytoplasmic (CF) fractions. It is demonstrated that H. pylori bacteria contain both stimulatory and inhibitory components for T cells of healthy individuals. The sHA as well as SF (5-20 micrograms) induced the proliferative response of T lymphocytes. By contrast, CF inhibited in dose dependent manner, the proliferation of T cells in the cultures stimulated with H. pylori bacteria or PHA. The result suggest that in vivo, a dominance of activation or immunosuppression could depend on the concentration of the bacteria and their products in infective foci.

Phagocytosis of Helicobacter pylori bacteria differing in the heparan sulfate binding by human polymorphonuclear leukocytes.

Heparan sulfate binding proteins (HSBPs) of Helicobacter pylori facilitate bacterial phagocytosis by human polymorphonuclear leukocytes (PMNs). H. pylori 25 strain which demonstrates a strong heparan sulfate binding activity was found to be attached to/ingested by PMNs in greater numbers than H. pylori strain 17874 bacteria which lacked this activity. Moreover, heparin inhibited the uptake of cells of H. pylori strain 25 but not of cells of H. pylori strain 17874 by PMNs.

The role of heparan sulphate-binding activity of Helicobacter pylori bacteria in their adhesion to murine macrophages.

The aim of this study was to determine the role of heparan sulphate (HS)-binding activity of Helicobacter pylori microbes in their adhesion to and ingestion by inflammatory peritoneal macrophages. Two H. pylori strains expressing sialic acid-specific haemagglutinins but differing in the expression of heparan sulphate-binding capacity were chosen for investigation. The attachment to an ingestion by macrophages of the H. pylori bacteria were estimated by ELISA using anti-H. pylori antibodies. The adhesion of both H. pylori strains could be inhibited by pretreatment of the bacteria with heparin (H), HS or fetuin, as well as by preincubation of the macrophages with heparinase or neuraminidase. However, detailed analysis of the data on the inhibition of bacterial adhesion to macrophages led to the conclusion that the attachment of H. pylori 25 bacteria, which expressed a high heparan sulphate binding, was mainly determined by HS-binding structures. In contrast, the adhesion to macrophages of H. pylori bacteria 17874 microbes, which expressed a weak heparan sulphate binding, was more dependent on the exhibition of sialic acid-dependent haemagglutinins. The described variation in H. pylori bacterial surface structures mediating their adhesion to macrophages could suggest a similar variation in bacterial adhesion to stomach mucosa and maybe in the pathogenicity of H. pylori strains.