Anna S. Sahlberg

Immunity to pertussis 5 years after booster immunization during adolescence.

BACKGROUND We conducted a 5-year follow-up study on the persistence of pertussis-specific antibody and cell-mediated immunity after booster immunization of adolescents aged 11-13 years with a tricomponent acellular pertussis vaccine (Boostrix; trials diphtheria-tetanus-acellular pertussis [Tdap]-004/030). METHODS Cellular and humoral immunity to pertussis toxin (PT), filamentous hemagglutinin, and pertactin were measured in adolescents (age, 16 years) 5 years after booster immunization. Similar investigations were performed for control adolescents who had received only diphtheria and tetanus booster vaccination. RESULTS Five years after pertussis booster vaccination, the geometric mean concentrations of immunoglobulin G (IgG) elicited by each of the 3 pertussis vaccine antigens decreased from 1-month and 3-year postvaccination levels, but with the exception of PT IgG, were still higher than the prevaccination levels. PT IgG levels were undetectable in 28% of the subjects, but 44% of those subjects still tested positive for cell-mediated immunity to PT. Filamentous hemagglutinin IgG and pertactin IgG levels were significantly higher in Tdap-boosted adolescents than in the control subjects. Antibody concentrations at 1 month after vaccination strongly predicted antibody persistence. Cell-mediated immunity levels to PT, filamentous hemagglutinin, and pertactin persisted above the prebooster levels measured 5 years earlier. CONCLUSIONS The results of the present study of adolescents indicate that the interval between acellular pertussis booster immunizations might be extended beyond 5 years.

Phosphorylation of STAT-1 serine 727 is prolonged in HLA-B27-expressing human monocytic cells.

A tissue antigen, HLA-B27, is strongly associated with a group of rheumatic diseases called spondyloarthritides. Despite the intensive research, the exact role of HLA-B27 in the pathogenesis of these diseases is still unclear. Here we studied whether HLA-B27 modulates the phosphorylation of signal transducer and activator of transcription 1 (STAT-1) serine 727 residue and the localization of STAT-1 in Salmonella-infected human monocytic cells. In addition, we studied the role of signaling molecule double-stranded RNA activated protein kinase (PKR) in these modulatory effects. U937 human monocytic cell transfectants stably expressing wild type HLA-B27 or mutated HLA-B27 heavy chains with amino acid substitutions in the B pocket were prepared. The PMA-differentiated cells were infected with S. enteritidis. Western blotting was used to detect the phosphorylation of STAT-1, and to visualize the localization of STAT-1 in the cells confocal microscopy was used. Specific inhibitors were employed to study the role of PKR in STAT-1 phosphorylation. We discovered that the phosphorylation of STAT-1 serine 727 is prolonged in cells expressing misfolding forms of HLA-B27 after S. enteritidis infection, whereas in mock cells and in cells expressing mutated, non-misfolding HLA-B27 the phosphorylation of serine 727 is transient. Interestingly, STAT-1 serine 727 phosphorylation is partly dependent on PKR. In addition, more STAT-1 is localized in the nucleus of HLA-B27-expressing cells, even before an external trigger, when compared to mock cells. In conclusion, our results show that the phosphorylation of STAT-1 serine 727 residue is prolonged in HLA-B27-expressing monocyte-macrophage U937 cells after bacterial infection. This is of interest since the phosphorylation of serine 727 on STAT-1 is suggested to contribute to macrophage activation and promote inflammatory responses. Therefore, our results provide a mechanism which explains how the expression of an HLA-B27 molecule can impact the course of Salmonella infection and reactive arthritis.

HLA-B27 and Host-Pathogen Interaction

HLA-B27 is a risk factor closely associated to spondyloarthropathies (SpA). One form of SpA is reactive arthritis (ReA), which develops as a complication after certain bacterial infections (e.g., Salmonellae, Yersiniae, Shigellae, Campylobacteriae and Chlamydiae). The development of infection-triggered complication is a complex train of events between the triggering bacteria and the host. Since most of the patients suffering from ReA are HLA-B27 positive, it has been proposed that HLA-B27 may modulate the interaction between ReA-triggering bacteria and host cell. Besides antigen presenting function, HLA-B27 displays other unusual properties that might be of importance in the development of ReA. These properties (homodimer formation and misfolding of HLA-B27 heavy chain in the endoplasmic reticulum (ER)) may trigger ER-stress signaling pathways in host cell, which in turn may modulate cell signaling in favor of ReA-triggering bacteria. Here we summarize the observations of HLA-B27 modulating the interaction between ReA-triggering bacteria and host cell and discuss potential mechanisms behind the interaction.

Altered regulation of ELAVL1/HuR in HLA-B27-expressing U937 monocytic cells.

Objective To investigate the role of HLA-B27 expression in the regulation of RNA binding protein (RBP) Embryonic Lethal Abnormal Vision (ELAV) L1/Human antigen R (HuR) expression in Salmonella-infected or LPS-stimulated human monocytic cells, since HuR is a critical regulator of the post-transcriptional fate of many genes (e.g. TNFα) important in inflammatory response. Methods U937 monocytic cells were stably transfected with pSV2neo resistant vector (mock), wild type HLA–B27, or mutated HLA–B27 with amino acid substitutions in the B pocket. Cells were differentiated, infected with Salmonella enteritidis or stimulated with lipopolysaccharide. The expression levels of HuR protein and cleavage products (CP1 and CP2) were detected by Western blotting and flow cytometry. Specific inhibitors were used to study the role of PKR and p38 in HuR expression and generation of CPs. TNFα and IL-10 secretion after p38 and PKR inhibition were measured by ELISA. Results Full length HuR is overexpressed and HuR cleavage is disturbed in U937 monocytic cells expressing HLA-B27 heavy chains (HC). Increased full length HuR expression, disturbed cleavage and reduced dependence on PKR after infection correlate with the expression of glutamic acid 45 in the B pocket that is linked to the misfolding of HLA-B27. Conclusion Results show that the expression of HLA-B27 HCs modulates the intracellular environment of U937 monocyte/macrophages by altering HuR regulation. This phenomenon is at least partly dependent on the misfolding feature of the B27 molecule. Since HuR is an important regulator of multiple genes involved in inflammatory response observations offer an explanation how HLA-B27 may modulate inflammatory response.

Enhanced phosphorylation of STAT1 is dependent on PKR signaling in HLA-B27 expressing U937 monocytic cells

OBJECTIVE To study the phosphorylation of STAT-1 in HLA-B27-transfected human monocytic cells and the role of the signaling molecules double-stranded RNA-dependent protein kinase (PKR) and p38 in STAT-1 phosphorylation. METHODS U937 human monocytic cell transfectants stably expressing wild-type HLA-B27 or mutated HLA-B27 heavy chains with amino acid substitutions in the B pocket were prepared. Mock-transfected cells were prepared using the antibiotic resistance vectors (pSV2neo or RSV5neo) alone. Phorbol myristate acetate-differentiated cells were stimulated with lipopolysaccharide (LPS) or infected with Salmonella enteritidis. The phosphorylation and expression levels of STAT-1 protein were detected by Western blotting and flow cytometry. Specific inhibitors were added in cell culture to study the role of PKR and p38 in STAT-1 phosphorylation. RESULTS STAT-1 was constitutively highly phosphorylated on the tyrosine 701 residue in HLA-B27-positive monocytic cells when compared to control cells, even prior to stimulation with LPS or bacteria. This phenotype was associated with the expression of HLA-B27 heavy chains that misfold. In addition, phosphorylation of STAT-1 was dependent on PKR. CONCLUSION Our results show that STAT-1 tyrosine 701 is constitutively highly phosphorylated in the HLA-B27-expressing monocyte/macrophage cell line. Since phosphorylation of tyrosine 701 on STAT-1 is sufficient to induce interferon (IFN)-dependent genes, constitutive activity of this phosphorylation site may lead to the overexpression of IFN-dependent genes, as well as other STAT-1-dependent genes, in HLA-B27 monocyte/macrophages. Our results offer a mechanism by which B27 expression alone, without any external trigger, is potentially capable of inducing activation of STAT-1, a critical regulator of the inflammatory response.

Somatic factors at 8 years of age in children with recurrent use of antibiotics in early infancy

AIM To examine the influence of recurrent therapy with antibiotics (RTA) in infancy on childrens somatic factors at 8 y of age. METHODS Subject selection was based on stratified randomized cluster sampling. Altogether 1287 infants were potential participants in the follow-up study. Children with 6 courses of antibiotics (100 children) during their first 18 mo of life and children with no (62%) or 2 courses (38%) of antibiotics participated in a clinical examination in a case-control setting (100 matched controls) at the age of 8 y. RESULTS The children with RTA continued to have more infections and had had more courses of antibiotics compared to controls during the follow-up. There was no clinically significant difference in the somatic and dental status at the age of 8 between the two groups. The parents of the children with RTA reported significantly more often recurrent infections than the parents of the controls. CONCLUSIONS The children with recurrent therapy with antibiotics in early childhood also continue to be prescribed more antibiotics in later childhood when compared to those who received no or few antibiotics in infancy. However, recurrent infections and medications do not seem to have a marked effect on the somatic and dental status of these children at 8 y of age.

Enhanced phosphorylation of STAT-1 is dependent on double-stranded RNA-dependent protein kinase signaling in HLA-B27-expressing U937 monocytic cells

OBJECTIVE To study the phosphorylation of STAT-1 in HLA-B27-transfected human monocytic cells and the role of the signaling molecules double-stranded RNA-dependent protein kinase (PKR) and p38 in STAT-1 phosphorylation. METHODS U937 human monocytic cell transfectants stably expressing wild-type HLA-B27 or mutated HLA-B27 heavy chains with amino acid substitutions in the B pocket were prepared. Mock-transfected cells were prepared using the antibiotic resistance vectors (pSV2neo or RSV5neo) alone. Phorbol myristate acetate-differentiated cells were stimulated with lipopolysaccharide (LPS) or infected with Salmonella enteritidis. The phosphorylation and expression levels of STAT-1 protein were detected by Western blotting and flow cytometry. Specific inhibitors were added in cell culture to study the role of PKR and p38 in STAT-1 phosphorylation. RESULTS STAT-1 was constitutively highly phosphorylated on the tyrosine 701 residue in HLA-B27-positive monocytic cells when compared to control cells, even prior to stimulation with LPS or bacteria. This phenotype was associated with the expression of HLA-B27 heavy chains that misfold. In addition, phosphorylation of STAT-1 was dependent on PKR. CONCLUSION Our results show that STAT-1 tyrosine 701 is constitutively highly phosphorylated in the HLA-B27-expressing monocyte/macrophage cell line. Since phosphorylation of tyrosine 701 on STAT-1 is sufficient to induce interferon (IFN)-dependent genes, constitutive activity of this phosphorylation site may lead to the overexpression of IFN-dependent genes, as well as other STAT-1-dependent genes, in HLA-B27 monocyte/macrophages. Our results offer a mechanism by which B27 expression alone, without any external trigger, is potentially capable of inducing activation of STAT-1, a critical regulator of the inflammatory response.

Enhanced phosphorylation of STAT-1 is dependent on double-stranded RNA-dependent protein kinase signaling in HLA-B27-expressing U937 monocytic cells: Phosphorylation of STAThyphen;1 in HLA-B27 Cells

OBJECTIVE To study the phosphorylation of STAT-1 in HLA-B27-transfected human monocytic cells and the role of the signaling molecules double-stranded RNA-dependent protein kinase (PKR) and p38 in STAT-1 phosphorylation. METHODS U937 human monocytic cell transfectants stably expressing wild-type HLA-B27 or mutated HLA-B27 heavy chains with amino acid substitutions in the B pocket were prepared. Mock-transfected cells were prepared using the antibiotic resistance vectors (pSV2neo or RSV5neo) alone. Phorbol myristate acetate-differentiated cells were stimulated with lipopolysaccharide (LPS) or infected with Salmonella enteritidis. The phosphorylation and expression levels of STAT-1 protein were detected by Western blotting and flow cytometry. Specific inhibitors were added in cell culture to study the role of PKR and p38 in STAT-1 phosphorylation. RESULTS STAT-1 was constitutively highly phosphorylated on the tyrosine 701 residue in HLA-B27-positive monocytic cells when compared to control cells, even prior to stimulation with LPS or bacteria. This phenotype was associated with the expression of HLA-B27 heavy chains that misfold. In addition, phosphorylation of STAT-1 was dependent on PKR. CONCLUSION Our results show that STAT-1 tyrosine 701 is constitutively highly phosphorylated in the HLA-B27-expressing monocyte/macrophage cell line. Since phosphorylation of tyrosine 701 on STAT-1 is sufficient to induce interferon (IFN)-dependent genes, constitutive activity of this phosphorylation site may lead to the overexpression of IFN-dependent genes, as well as other STAT-1-dependent genes, in HLA-B27 monocyte/macrophages. Our results offer a mechanism by which B27 expression alone, without any external trigger, is potentially capable of inducing activation of STAT-1, a critical regulator of the inflammatory response.

Somatic factors at 8 years of age in children with recurrent use of antibiotics in early infancy: Recurrent antibiotic use and long-term effects

Aim: To examine the influence of recurrent therapy with antibiotics (RTA) in infancy on childrens somatic factors at 8 y of age. Methods: Subject selection was based on stratified randomized cluster sampling. Altogether 1287 infants were potential participants in the follow‐up study. Children with 6 courses of antibiotics (100 children) during their first 18 mo of life and children with no (62%) or 2 courses (38%) of antibiotics participated in a clinical examination in a case‐control setting (100 matched controls) at the age of 8 y. Results: The children with RTA continued to have more infections and had had more courses of antibiotics compared to controls during the follow‐up. There was no clinically significant difference in the somatic and dental status at the age of 8 between the two groups. The parents of the children with RTA reported significantly more often recurrent infections than the parents of the controls.