Eduard Ling

Mutant Adenosine Deaminase 2 in a Polyarteritis Nodosa Vasculopathy

BACKGROUND Polyarteritis nodosa is a systemic necrotizing vasculitis with a pathogenesis that is poorly understood. We identified six families with multiple cases of systemic and cutaneous polyarteritis nodosa, consistent with autosomal recessive inheritance. In most cases, onset of the disease occurred during childhood. METHODS We carried out exome sequencing in persons from multiply affected families of Georgian Jewish or German ancestry. We performed targeted sequencing in additional family members and in unrelated affected persons, 3 of Georgian Jewish ancestry and 14 of Turkish ancestry. Mutations were assessed by testing their effect on enzymatic activity in serum specimens from patients, analysis of protein structure, expression in mammalian cells, and biophysical analysis of purified protein. RESULTS In all the families, vasculitis was caused by recessive mutations in CECR1, the gene encoding adenosine deaminase 2 (ADA2). All the Georgian Jewish patients were homozygous for a mutation encoding a Gly47Arg substitution, the German patients were compound heterozygous for Arg169Gln and Pro251Leu mutations, and one Turkish patient was compound heterozygous for Gly47Val and Trp264Ser mutations. In the endogamous Georgian Jewish population, the Gly47Arg carrier frequency was 0.102, which is consistent with the high prevalence of disease. The other mutations either were found in only one family member or patient or were extremely rare. ADA2 activity was significantly reduced in serum specimens from patients. Expression in human embryonic kidney 293T cells revealed low amounts of mutant secreted protein. CONCLUSIONS Recessive loss-of-function mutations of ADA2, a growth factor that is the major extracellular adenosine deaminase, can cause polyarteritis nodosa vasculopathy with highly varied clinical expression. (Funded by the Shaare Zedek Medical Center and others.).

Glycolytic enzymes associated with the cell surface of Streptococcus pneumoniae are antigenic in humans and elicit protective immune responses in the mouse

Streptococcus pneumoniae is a leading cause of otitis media, sinusitis, pneumonia, bacteraemia and meningitis worldwide. The drawbacks associated with the limited number of various capsular polysaccharides that can be included in the polysaccharide‐based vaccines focuses much attention on pneumococcal proteins as vaccine candidates. We extracted an enriched cell wall fraction from S. pneumoniae WU2. Approximately 150 soluble proteins could be identified by 2D gel electrophoresis. The proteins were screened by 2D‐Western blotting using sera that were obtained longitudinally from children attending day‐care centres at 18, 30 and 42 months of age and sera from healthy adult volunteers. The proteins were further identified using matrix‐assisted laser desorption ionization‐time of flight mass spectrometry. Seventeen proteins were antigenic in children and adults, of which 13 showed an increasing antibody response with age in all eight children analysed. Two immunogenic proteins, fructose–bisphosphate aldolase (FBA) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), and a control protein with known low immunogenicity, heat shock protein 70 (DnaK), were expressed in Escherichia coli, purified and used to immunize mice. Mouse antibodies elicited to the recombinant (r) FBA and rGAPDH were cross‐reactive with several genetically unrelated strains of different serotypes and conferred protection to respiratory challenge with virulent pneumococci. In addition, the FBA used in this study (NP_345117) does not have a human ortholog and warrants further investigation as a candidate for a pneumococcal vaccine. In conclusion, the immunoproteomics based approach utilized in the present study appears to be a suitable tool for identification of novel S. pneumoniae vaccine candidates.

Contribution of IL-1 to resistance to Streptococcus pneumoniae infection

The role of IL-1 in susceptibility to Streptococcus pneumoniae infection was studied in mice deficient in genes of the IL-1 family [i.e. IL-1alpha-/-, IL-1beta-/-, IL-1alpha/beta-/- and IL-1R antagonist (IL-1Ra)-/- mice] following intra-nasal inoculation. Intra-nasal inoculation of S. pneumoniae of IL-1beta-/- and IL-1alpha/beta-/- mice displayed significantly lower survival rates and higher nasopharyngeal and lung bacterial load as compared with control, IL-1alpha-/- and IL-1Ra-/- mice. Treatment of IL-1beta-/- mice with rIL-1beta significantly improved their survival. A significant increase in blood neutrophils was found in control, IL-1alpha-/- and IL-1Ra-/- but not in IL-1beta-/- and IL-1alpha/beta-/- mice. Local infiltrates of neutrophils and relatively preserved organ architecture were observed in the lungs of IL-1alpha-/- and control mice. However, S. pneumoniae-infected IL-1beta-/-, IL-1alpha/beta-/- and IL-1Ra-/- mice demonstrated diffuse pneumonia and tissue damage. Altogether, all three isoforms contribute to protection against S. pneumoniae; our results point to differential role of IL-1alpha and IL-1beta in the pathogenesis and control of S. pneumoniae infection and suggest that IL-1beta has a major role in resistance to primary pneumococcal infection while the role of IL-1alpha is less important.

Pneumococcal 6-phosphogluconate-dehydrogenase, a putative adhesin, induces protective immune response in mice

For most bacteria, adherence to human cells is achieved by bacterial lectins binding to mammalian surface glyconjugates. 6‐Phosphogluconate dehydrogenase (6PGD) was identified by us as one of Streptococcus pneumoniae cell wall lectin proteins, which elicits an age‐dependent immune response in humans. This study assesses the role of 6PGD in S. pneumoniae pathogenesis as an adhesin and its ability to elicit a protective immune response in mice. Recombinant 6PGD (r6PGD) was cloned from S. pneumoniae serotype 3 (strain WU2). r6PGD interference in adhesion of three genetically unrelated unencapsulated pneumococcal strains (3·8, 14·8 and R6) and two genetically unrelated encapsulated pneumococcal strains (WU2 and D39) to A549 type II lung carcinoma cell was tested. BALB/c mice were immunized with r6PGD and boosted after 3 weeks. Immunized mice were challenged intranasally with a lethal dose of S. pneumoniae. r6PGD inhibited 90% and 80% of pneumococcal adhesion to the A549 cells of three unencapsulated S. pneumoniae strains and two encapsulated S. pneumoniae strains, respectively, in a concentration‐dependent manner (P < 0·05). Antibodies to r6PGD produced in mice significantly inhibited bacterial adhesion to A549 cell (P < 0·05). Immunization of mice with r6PGD protected 60% (P < 0·001) of mice for 5 days and 40% (P < 0·05) of the mice for 21 days following intranasal lethal challenge. We have identified 6PGD as a surface‐located immunogenic lectin protein capable of acting as an adhesin. 6PGD importance to bacterial pathogenesis was demonstrated by the ability of r6PGD to elicit a protective immune response in mice.

Differential activation of the immune system by virulent Streptococcus pneumoniae strains determines recovery or death of the host.

Streptococcus pneumoniae infection may result in asymptomatic carriage, mucosal or invasive disease. We hypothesize that self‐limiting or fatal disease outcome follows infection with S. pneumoniae differential activation of the host immune response. BALB/c and C57BL/6 mice were inoculated intranasally with S. pneumoniae serotype 3 strain WU2 and serotype 14 strain DW14 and mortality, bacterial load, pathological changes in the lungs and cytokines mRNA levels in the spleen were analysed. No differences between the C57BL/6 and the BALB/c inbred mice were observed except for the severity of their lung pathology and IL‐4 expression. Infection of the two mouse strains with S. pneumoniae WU2 resulted in sepsis and death that occurred within 4 days post‐inoculation. This death was preceded, in both mouse strains, in an increase over time of the lung bacterial load and bacteraemia. The lung pathology was characterized by diffuse pneumonia with marked congestion of the lungs. Analysis of mRNA expression of cytokines in the spleen revealed no alterations in tumour necrosis factor (TNF)‐α, transforming growth factor (TGF)‐β, interleukin (IL)‐12 and interferon (IFN)‐γ and induction of IL‐10 and IL‐4. The two strains of mice survived infection with S. pneumoniae DW14. This was accompanied by a reduction over time of lung bacterial load and bacteraemia. The lung pathology was characterized by focal lymphocyte infiltration and preserved architecture of the organ. Analysis of mRNA expression of cytokines in the spleen revealed a significant decrease in the levels of TNF‐α, TGF‐β, IL‐12 and IFN‐γ mRNA expression, which usually precedes cytokine protein expression. Interestingly, a significant increase in the levels of IL‐4 mRNA expression was found in BALB/c mice only.  This study suggests that differential activation or evasion of cytokine expression by S. pneumoniae virulent strains determines disease outcome regardless of the hosts immunogenetic background.

Cytokine mRNA Expression in Pneumococcal Carriage, Pneumonia, and Sepsis in Young Mice

We studied cytokine mRNA expression in Streptococcus pneumoniae carriage, pneumonia, and sepsis in 3-week-old, inbred C57BL/6 and BALB/c mice. Mice were inoculated intranasally with S. pneumoniae serotype 6B, 14, or 3. Survival, bacterial load in the nasopharynx (NP) and lungs, and mRNA levels of tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, interleukin (IL)-10, and IL-12 in the spleen were analyzed. No baseline mRNA expression levels were found, except for TNF-alpha in C57BL/6 mice. Serotype 6B caused NP colonization only, a significant (P<.05) increase in the levels of TNF-alpha, and induction of TGF-beta and IL-12 mRNA. Serotype 14 caused NP and nonlethal lung colonization and induction of TGF-beta, IL-10, and IL-12. Serotype 3 caused NP colonization, pneumonia, 35% mortality, and no alteration in the mRNA expression of tested cytokines. Although activation of the immune system culminated in nonlethal disease, evasion of the immune system was associated with detrimental disease.

Periodic fever, aphthous stomatitis, pharyngitis and cervical adenopathy syndrome is associated with activation of GM-CSF and burst-like expression of IL-8 in peripheral blood.

Abstract Introduction. Periodic fever, aphthous stomatitis, pharyngitis and cervical adenopathy (PFAPA) is an autoinflammatory syndrome characterized by periodic fever with aphthous stomatitis, cervical lymphadenopathy, myalgia, and abdominal pain. Peripheral blood concentrations of selected cytokines of PFAPA patients during and between febrile episodes were analyzed in a search for PFAPA-specific molecular signature. Methods. 23 children with PFAPA (age 6.07 ± 2.94 years, range 5–9 years) and three control children with severe oropharyngeal infections (age 6.2 ± 7.95 years, range 1–17 years) participated in the study. Peripheral blood concentrations of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IFN-γ, GM-CSF, TNF-α were measured using Luminex technology. Results. PFAPA febrile episodes were characterized by detection of GM-CSF – 134.07 ± 315.5 pg/mL; significant (P < 0.001), compared to baseline and controls, elevation of concentrations of IL-8 (3193.7 ± 2508 pg/mL vs. 100.36 ± 119. pg/mL vs. 2.04 ± 4.08 pg/mL, respectively), IL-6 (1355.38 ± 2026.53 pg/mL vs. 28.8 ± 44.2 pg/mL and 27.13 ± 26.42 pg/mL, respectively). IL-1β was detected only in febrile and afebrile PFAPA patients (922.8 ± 1639 pg/mL vs. 10.98 ± 19.4 pg/ml, P < 0.002, respectively), but not in controls. Peripheral blood concentration of TNFα did not differ significantly between study groups. IL-2, IL-4, IL-5, and IL-10 were negligible in all study subjects. Discussion. PFAPA febrile episodes are characterized by activation of GM-CSF and IL-8 with Th1 skewing. We propose a molecular mechanism governing this phenomenon.

Streptococcus pneumoniae fructose-1,6-bisphosphate aldolase, a protein vaccine candidate, elicits Th1/Th2/Th17-type cytokine responses in mice

Streptococcus pneumoniae (S. pneumoniae) is a major pathogen worldwide. The currently available polysaccharide-based vaccines significantly reduce morbidity and mortality. However, the inherent disadvantages of the currently available polysaccharide-based vaccines have motivated the search for other bacterial immunogens capable of eliciting a protective immune response against S. pneumoniae. Fructose-1,6-bisphosphate aldolase (FBA) is a glycolytic enzyme, which was found to localize to the bacterial surface, where it functions as an adhesin. Previously, immunizing mice with recombinant FBA (rFBA) in the presence of alum elicited a protective immune response against a lethal challenge with S. pneumoniae. Thus, the aim of the present study was to determine the cytokine responses that are indicative of protective immunity following immunization with rFBA. The protective effects against pneumococcal challenge in mice immunized with rFBA with complete Freunds adjuvant (CFA) in the initial immunization and with incomplete Freunds adjuvant (IFA) in booster immunizations surpassed the protective effects observed following immunization with either rFBA + alum or pVACfba. CD4+ T-cells obtained from the rFBA/CFA/IFA/IFA-immunized mice co-cultured with rFBA-pulsed antigen-presenting cells (APCs), exhibited a significantly greater proliferative ability than CD4+ T-cells obtained from the adjuvant-immunized mice co-cultured with rFBA‑pulsed APCs. The levels of the Th1-type cytokines, interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α and IL-12, the Th2-type cytokines, IL-4, IL-5 and IL-10, and the Th17-type cytokine, IL-17A, significantly increased within 72 h of the initiation of co-culture with CD4+ T-cells obtained from the rFBA‑immunized mice, in comparison with the co-cultures with CD4+ T-cells obtained from the adjuvant-immunized mice. Immunizing mice with rFBA resulted in an IgG1/IgG2 ratio of 41, indicating a Th2 response with substantial Th1 involvement. In addition, rabbit and mouse anti-rFBA antisera significantly protected the mice against a lethal S. pneumoniae challenge in comparison with preimmune sera. Our results emphasize the mixed involvement of the Th1, Th2 and Th17 arms of the immune system in response to immunization with pneumococcal rFBA, a potential vaccine candidate.

Teaming Up for Trouble: Cancer Cells, Transforming Growth Factor-β1 Signaling and the Epigenetic Corruption of Stromal Naïve Fibroblasts

It is well recognized that cancer cells subvert the phenotype of stromal naïve fibroblasts and instruct the neighboring cells to sustain their growth agenda. The mechanisms underpinning the switch of fibroblasts to cancer-associated fibroblasts (CAFs) are the focus of intense investigation. One of the most significant hallmarks of the biological identity of CAFs is that their tumor-promoting phenotype is stably maintained during in vitro and ex vivo propagation without the continual interaction with the adjacent cancer cells. In this review, we discuss robust evidence showing that the master cytokine Transforming Growth Factor-β1 (TGFβ-1) is a prime mover in reshaping, via epigenetic switches, the phenotype of stromal fibroblasts to a durable state. We also examine, in detail, the pervasive involvement of TGFβ-1 signaling from both cancer cells and CAFs in fostering cancer development, taking colorectal cancer (CRC) as a paradigm of human neoplasia. Finally, we review the stroma-centric anticancer therapeutic approach focused on CAFs—the most abundant cell population of the tumor microenvironment (TME)—as target cells.

Mycophenolate Mofetil Alone and in Combination with Tacrolimus Inhibits the Proliferation of HT-29 Human Colonic Adenocarcinoma Cell Line and Might Interfere with Colonic Tumorigenesis

Background/Aim: Familial adenomatous polyposis (FAP) was found to be completely reversed in a patient treated with mycophenolate mofetil (MMF) and tacrolimus following kidney transplantation. In this preliminary study, we assessed whether MMF and tacrolimus alone or in combination interfere with the cell cycle and proliferation in a human colonic adenocarcinoma cell line and in the colonic polyps of the patient with FAP. Materials and Methods: Human colonic adenocarcinoma HT-29 cells were treated with tacrolimus and MMF alone and in combination at different concentrations. Cell viability and proliferation were assessed using the MTT assay. Cell-cycle distribution was analyzed by flow cytometry. Expression of Ki-67, a marker of mitotic activity, was evaluated in the patients colonic polyps before and under drug treatment. Results: MMF in combination with tacrolimus induced S-phase cell-cycle arrest and markedly inhibited HT-29 cell proliferation. Ki-67 expression in the patients colonic polyps was significantly reduced following combined tacrolimus and MMF treatment. Conclusion: MMF and tacrolimus synergistically inhibited proliferation of a human colonic adenocarcinoma cell line and interfered with the expansion of colonic crypt proliferation in the polyp from a patient with FAP. The results confirm our clinical observation and indicate the possibility of novel approach to therapy of colorectal neoplasia.