Resistance to Development of Collagen-Induced Arthritis in C57BL/6 Mice Is Due to a Defect in Secondary, but Not in Primary, Immune Response

Collagen-induced arthritis (CIA) is a rodent model of human rheumatoid arthritis. Mice of the H-2(q) (DBA/1J) background are highly susceptible to disease whereas mice of the H-2(b) (C57BL/6, B6) background are resistant. To determine why B6 mice are resistant to disease induction, we systematically analyzed T and B cell immune responses in B6 mice, compared to DBA/1J mice, following immunization with bovine type II collagen (CII). We found that both strains showed similar T cell proliferation and cytokine responses and similar levels of anti-CII antibodies (Abs) at day 12 or day 14 of initial immunization (primary immune response), however, those B6 mice that did not develop arthritis showed a significant defect in T cell responses and significantly lower levels of anti-CII Abs following secondary boosting immunization (day 35 of initial immunization, secondary immune response) compared to DBA/1J mice. Our results define for the first time that a defective secondary immune responses in B6 mice leads to the resistance of CIA.     

人白细胞介素2受体α链cDNA导入哺乳细胞及其克隆与稳定表达的研究

按DNA—磷酸钙共沉淀法将人白细胞介素2受体α链cDNA转染中国仓鼠卵巢细胞(CHOdhfr~-)及小鼠成纤维细胞(L929),获得稳定表达人IL-2Rα链的CHOdhfr~+细胞克隆。经RNA点渍杂交分析、荧光标记IL-2染色法、特异性ELISA和玫瑰花环试验检测证明,哺乳细胞表达的重组IL-2Rα链具有结合IL-2和抗Tac McAb的能力。还报道了T细胞白血病Jukat及Molt-4等细胞系异常表达IL-2R的结果,并作了分析讨论。     

Chemokines and Antimicrobial Peptides Have a cag-Dependent Early Response to Helicobacter pylori Infection in Primary Human Gastric Epithelial Cells

Helicobacter pylori infection systematically causes chronic gastric inflammation that can persist asymptomatically or evolve toward more severe gastroduodenal pathologies, such as ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. The cag pathogenicity island (cag PAI) of H. pylori allows translocation of the virulence protein CagA and fragments of peptidoglycan into host cells, thereby inducing production of chemokines, cytokines, and antimicrobial peptides. In order to characterize the inflammatory response to H. pylori, a new experimental protocol for isolating and culturing primary human gastric epithelial cells was established using pieces of stomach from patients who had undergone sleeve gastrectomy. Isolated cells expressed markers indicating that they were mucin-secreting epithelial cells. Challenge of primary epithelial cells with H. pylori B128 underscored early dose-dependent induction of expression of mRNAs of the inflammatory mediators CXCL1 to -3, CXCL5, CXCL8, CCL20, BD2, and tumor necrosis factor alpha (TNF-α). In AGS cells, significant expression of only CXCL5 and CXCL8 was observed following infection, suggesting that these cells were less reactive than primary epithelial cells. Infection of both cellular models with H. pylori B128ΔcagM, a cag PAI mutant, resulted in weak inflammatory-mediator mRNA induction. At 24 h after infection of primary epithelial cells with H. pylori, inflammatory-mediator production was largely due to cag PAI substrate-independent virulence factors. Thus, H. pylori cag PAI substrate appears to be involved in eliciting an epithelial response during the early phases of infection. Afterwards, other virulence factors of the bacterium take over in development of the inflammatory response. Using a relevant cellular model, this study provides new information on the modulation of inflammation during H. pylori infection.     

TMPRSS2, a Serine Protease Expressed in the Prostate on the Apical Surface of Luminal Epithelial Cells and Released into Semen in Prostasomes,Is Misregulated in Prostate Cancer Cells

TMPRSS2, a type II transmembrane serine protease, is highly expressed by the epithelium of the human prostate gland. To explore the regulation and function of TMPRSS2 in the prostate, a panel of monoclonal antibodies with high sensitivity and specificity were generated. Immunodetection showed TMPRSS2 on the apical plasma membrane of the prostate luminal cells and demonstrated its release into semen as a component of prostasomes, organelle-like vesicles that may facilitate sperm function and enhance male reproduction. In prostate cancer cells, TMPRSS2 expression was increased and the protein mislocalized over the entire tumor cell membrane. In both LNCaP prostate cancer cells and human semen, TMPRSS2 protein was detected predominantly as inactive zymogen forms as part of an array of multiple noncovalent and disulfide-linked complexes, suggesting that TMPRSS2 activity may be regulated by unconventional mechanisms. Our data suggested that TMPRSS2, an apical surface serine protease, may have a normal role in male reproduction as a component of prostasomes. The aberrant cellular localization, and increased expression of the protease seen in cancer, may contribute to prostate tumorigenesis by providing access of the enzyme to nonphysiological substrates and binding-proteins.TMPRSS2 is an androgen responsive gene that encodes a type II transmembrane serine protease (TTSP).^1,2,3 The members of the TTSP family share common protein structures including a transmembrane domain at the N terminus, linker regions with a variety of protein–protein interaction domains, and a canonical serine protease domain at the C terminus.^4,5,6 TTSPs have been found to play important roles in the development and homeostasis of mammals, and the aberrant expression of TTSP genes are reported to contribute to the etiology of several human disorders, including cancer.⁷ The importance of TMPRSS2 in vivo remains unclear because homozygous TMPRSS2-null mice are essentially phenotypically normal.⁸ However, TMPRSS2 was reported to regulate epithelial sodium channel (ENaC) activity in vitro, implying a possible role in epithelial sodium homeostasis.⁹ TMPRSS2 may play a role in angiogenesis and tubulogenesis in microvesicular endothelial cells, potentially modulating several aspects of prostate tumor biology.¹⁰ In addition to its proteolytic activity, TMPRSS2 may also serve as a cell receptor, conducting external signaling or interacting with the extracellular matrix through its extracellular protein binding domains. Overexpression of TMPRSS2 has been demonstrated in poorly differentiated prostate cancer with significant increase in the mRNA level.^2,11,12,13 TMPRSS2 was also reported to be involved in the majority of prostate cancer because of the gene fusion of the 5′-untranslational region of TMPRSS2 with ETS family members, which is implicated in the overexpression of ETS genes in the majority of prostate cancer.^2,14,15 The coding sequence of TMPRSS2 is not involved in the gene fusion, and as a consequence there is no resultant recombinant protein for the TMPRSS2-ETS gene fusion and the promoter-less copy of TMPRSS2 is silenced, resulting in reduced expression of TMPRSS2 mRNA in those prostate cancer patients with the gene fusion.¹⁶Despite these potentially interesting and important roles for TMPRSS2, characterization of the protein itself remains incomplete and somewhat confusing. Significant discrepancies in the apparent molecular mass of TMPRSS2 protein, with a calculated size around 54 kDa, have been reported in previous studies, with the most commonly sited being around 70 kDa and 32 kDa^11,17,18 as the full-length and serine protease domain of TMPRSS2, respectively. In this study, we have generated a panel of mouse monoclonal antibodies (mAbs) directed against TMPRSS2 that are highly sensitive and specific. Using the newly generated TMPRSS2 mAb, we have investigated the pathophysiological role of TMPRSS2 in prostate tissues by comparing its expression and subcellular distribution in prostate tumors and adjacent normal prostate glands. Functional and regulatory aspects of TMPRSS2 biology were also investigated by examining the activation status of the enzyme and the nature of complexes formed between TMPRSS2 and other proteins.     

Human Prostate DU145 Carcinoma Cells Implanted in Nude Mice Remove the Peritoneal Mesothelium to Invade and Grow as Carcinomas

Implanted human, androgen‐independent prostatic carcinoma cells (DU145) into athymic (NCr nu/nu) mice produce diverse tumors on the peritoneal surfaces of many organs. Light and ultrastructural observations show that the mesothelial covering these surfaces are typically microvilli‐coated, squamous cells or secretory cuboidal cells. The peritoneal regions colonized by tumors lack mesothelial cells and are covered by actively replicating carcinoma cells that grow as poorly differentiated cell clusters made of cell aggregates to somewhat compact spheroids covered with pleiomorphic microvilli and containing an undifferentiated vascular supply. These xenografts clusters invade the diaphragm and develop into tumors with both a basal solid aspect and an upper region of cribriform morphology. Furthermore, each tumor contains two cell types: (1) a poorly differentiated clear cell type, which grows into intraperitoneal tumors and (2) a large, basophilic cell type, which invades the peritoneal stroma of organs, including of the diaphragm. Anat Rec, 2013. © 2012 Wiley Periodicals, Inc.     

Involvement of C3a and C5a in Interleukin-8 Secretion by Human Polymorphonuclear Cells in Response to Capsular Material of Cryptococcus neoformans

In a previous paper we demonstrated that human polymorphonuclear cells (PMN) in the presence of normal human serum (NHS) secrete proinflammatory cytokines in response to Cryptococcus neoformans or its major capsular component, glucuronoxylomannan (GXM). The hypothesis that activation of the complement system could be responsible for the observed phenomenon is supported by the fact that encapsulated and acapsular C. neoformans isolates are activators of the complement system and, in particular, large encapsulated isolates are powerful activators. In the present study we demonstrate that (i) interleukin-8 (IL-8) release in response to acapsular or encapsulated strains of C. neoformans is significantly reduced in the presence of heat-inactivated serum rather than NHS and is completely abrogated in the absence of human serum; (ii) GXM-induced IL-8 release is strictly dependent on the presence of NHS, is inhibited by specific antibodies to either C3a and C5 complement components, and is completely abrogated by the combined use of these antibodies; (iii) the addition of purified C3a and C5a directly stimulates IL-8 release by PMN; and (iv) monoclonal antibody to GXM in combination with GXM or encapsulated C. neoformans potentiates IL-8 release by PMN. These data shed light on the mechanism involved in GXM-induced IL-8 secretion by PMN, provide an additional potential role for complement in the control of C. neoformans infections, and suggest a complex interplay between the complement system, humoral immunity, and cytokine regulation.     

Expression of Telomerase Activity in Human Endometrium Is Localized to Epithelial Glandular Cells and Regulated in a Menstrual Phase-Dependent Manner Correlated with Cell Proliferation

Telomerase activity is observed in most malignant tumors and germ cells, whereas normal somatic cells usually do not express it. Human endometrium is composed of glandular and stromal components and exhibits dramatic changes in proliferative activity during the menstrual cycle, which is exquisitely regulated by estrogen function. We previously reported that normal human endometrium expresses telomerase activity. However, it remains unclear which of the above components are the major sources of telomerase activity and how levels of telomerase activity are regulated over the menstrual cycle. Quantitative analysis of telomerase activity revealed that it changes dramatically over the course of the menstrual cycle and is strictly regulated in a menstrual-phase-dependent manner. Maximal activity equivalent to that in endometrial cancer was present in late proliferative phase, and minimal activity in late secretory phase. Postmenopausal endometrium and endometrium treated with anti-estrogen drugs exhibited decreased telomerase activity. Testing isolated epithelial glandular cells and stromal cells, we found that telomerase activity was localized to epithelial glandular cells. In situ RNA hybridization analysis also revealed epithelial-specific expression of human telomerase RNA. In vitro analysis of cultured epithelial cells demonstrated that telomerase activity is correlated with epithelial proliferation but not affected by estrogen treatment. These findings suggest that expression of telomerase activity is specific to epithelial cells and linked to cell proliferative status. The involvement of estrogen in telomerase regulation remains to be elucidated.     

Interferon-γ Production by Human T Cells and Natural Killer Cells In Vitro in Response to Antigens from the Two Intracellular Pathogens Mycobacterium tuberculosis and Leishmania major

Acquired resistance to both mycobacteria and Leishmania is primarily mediated by interferon-γ (IFN-γ), which triggers mechanisms leading to the death of the microorganism in macrophages. In this study, cell activation and IFN-γ production was investigated in human peripheral blood mononuclear cells (PBMC) from individuals previously sensitized to tuberculin and without known exposure to Leishmania parasites. Immune staining for intracellular IFN-γ and surface markers allowed flow cytometric identification of the cellular sources of IFN-γ in cell cultures incubated with purified protein derivative of tuberculin (PPD) and Leishmania antigens. It was found that IFN-γ was produced in response to both PPD and Leishmania stimulant by T cells in the cultures. Activation of IFN-γ producing natural killer (NK) cells was demonstrated only in some cultures, and only with concomitant T cell activation.     

CD8 T Cells in Old Mice Contribute to the Innate Immune Response to Mycobacterium tuberculosis via Interleukin-12p70-Dependent and Antigen-Independent Production of Gamma Interferon

Elderly individuals have increased morbidity and mortality associated with infectious diseases due in part to the progressive age-associated decline in immune function. Despite this, the old mouse model of Mycobacterium tuberculosis infection has revealed a CD8- and gamma interferon (IFN-γ)-dependent early resistance to infection. In this study, we investigated the mechanism by which CD8 T cells from old mice contributed to the early immune response to M. tuberculosis. Following a low-dose aerosol infection with M. tuberculosis, CD8 T cells were identified as being a dominant source of IFN-γ expression in the lungs of old mice early after infection, before the typical onset of antigen-specific immunity. In addition, M. tuberculosis-induced IFN-γ production by CD8 T cells isolated from naïve old mice was major histocompatibility complex class I independent but was dependent on interleukin-12p70, confirming an innate role of CD8 T cells during M. tuberculosis infection. Moreover, the ability of CD8 T cells from old mice to produce increased innate IFN-γ levels in response to M. tuberculosis infection was defined as a unique function of CD8 T cells from old mice and not the aged lung environment. Finally, we have identified increased expression of SET as being one possible mechanism by which CD8 T cells from old mice produce enhanced levels of IFN-γ. Additional characterizations of the signaling events that lead to enhanced innate IFN-γ production by CD8 T cells in old mice may lead to novel strategies to further enhance or perpetuate beneficial immune responses in the elderly.The world''s elderly population is rapidly expanding and is predicted to reach 1.5 billion by the year 2050, a 28% increase since 2000 (http://esa.un.org/unpp/). The largest concentration of elderly individuals (projected to be 78% by the year 2050) is in developing countries (http://esa.un.org/unpp/), where many infectious diseases, including tuberculosis (35), are endemic. Given the increased susceptibility of the elderly to infectious diseases, this rapid rise in the elderly population poses a significant threat to global health care. Research focused on characterizing the immune response of the elderly to pathogens that cause substantial morbidity and mortality in elderly individuals is an area that has been significantly neglected; however, such studies would have a considerable impact on the prevention and treatment of infectious diseases in the elderly.As an individual ages, significant immunological changes occur, which contribute to the enhanced morbidity and mortality associated with infectious diseases in the elderly. After puberty, thymic atrophy leads to a progressive decrease in the output of naïve T cells and decreased diversity in the T-cell repertoire (7). Consequently, the periphery of an elderly individual is dominated by antigen-experienced or memory T cells, leading to a significant impairment of immune responses to new antigenic challenges (36). In addition to the immune defects in the T-cell compartment, significant deficiencies in B cells (27), NK cells (21, 22), macrophages (15, 20), and dendritic cells (19, 28) have been reported. Despite the abundant evidence that the immune system of the aged is significantly altered, there is mounting data suggesting that some components of the immune system, particularly CD8 T cells, remain functionally intact or are enhanced in old age (11, 26, 34). The characterization of immune responses in the elderly, specifically to pathogens, may lead to more effective vaccination and therapeutic strategies in this highly vulnerable and expanding population.We previously demonstrated that although old mice fail to contain a chronic infection with Mycobacterium tuberculosis (23, 32), during the first 2 weeks following infection, old mice display an enhanced resistance to infection that is gamma interferon (IFN-γ) and CD8 dependent (32, 33). In addition, characterization of in vitro responses of naïve CD8 T cells from old mice to Th1 cytokines identified an interleukin-12 (IL-12)-driven mechanism of enhanced IFN-γ production by these cells (34). Since IL-12 is detected early in the lungs of M. tuberculosis-infected old mice (34), we hypothesized that this same mechanism of innate IFN-γ production was also biologically relevant during M. tuberculosis infection.The aim of the present study was to define and characterize the mechanism by which CD8 T cells contribute to innate immune responses following M. tuberculosis infection. Using both in vivo and in vitro models of M. tuberculosis infection of old mice, we demonstrated that CD8 T cells were a major source of innate IFN-γ. In addition, in vitro assays using purified pulmonary CD8 and CD11c cells from naïve mice conclusively demonstrated that M. tuberculosis-driven IFN-γ production by CD8 T cells from old mice was dependent on IL-12p70 and independent of major histocompatibility complex (MHC)-T-cell receptor interactions. Finally, we provide evidence that suggests that one mechanism by which CD8 T cells from old mice are capable of enhanced innate IFN-γ production is through the regulation of IL-12 signaling.     

Immunodepression by Rowson-Parr Virus in Mice. II. Effect of Rowson-Parr Virus Infection on the Antibody Response to Sheep Red Cells In Vivo and In Vitro

The effects of infection with Rowson-Parr virus (RPV), an associated virus present in Friend virus (FV) preparations, on the splenic plaque-forming cell response to sheep red cells in adult mice and on the plaque-forming cell response of peritoneal cells in vitro have been investigated. The results support the hypothesis that RPV might be responsible for a considerable proportion of the early immunodepressive effects caused by FV preparations.     

Enhancement of the Humoral Immune Response and Resistance to Bacterial Infection in Mice by the Oral Administration of a Bacterial Immunomodulator (OM-89)

Abstract

We investigated the effects of an Escherichia coli-derived product (OM-89) in mice. The oral administration of OM-89 led to a significant (p < 0.05. Student's t test) increase in the levels of IgA in intestinal secretions, which was at maximum 25 days after the end of the treatment, when a two-fold increase in IgA levels was observed. The i.p. inoculation of OM-89 induced the stimulation of anti-SRBC plaque-forming cells (PFC) in the spleen. The effect of OM-89 was dose-dependent and produced up to a 9-fold increase in PFC in the treated mice when compared to untreated controls. The oral administration of OM-89 proved to be effective in the enhancement of resistance to challenge i.p. inoculation with E. coli. 32% of OM-89-treated mice showed resistance to this experimental infection at minimal LD₁₀₀. The combined effects of low environmental temperature and cyclophosphamide (CY) immunosuppression enabled us to enhance differences in survival rates in experiments on the modulation of resistance towards Pseudomonas aeruginosa infection. The oral treatment with the immunoraodulator induced a significant (p < 0.05, Student's t test) level of protection in CY-immunosuppressed mice to the intranasal infection with P. aeruginosa, when mice were kept at low environmental temperature right after the bacterial challenge. The protective effect of OM-89 treatment was dependent on both the environmental temperature and the timing of the experiment.     

T-Cell Immune Response Assessment as a Complement to Serology and Intranasal Protection Assays in Determining the Protective Immunity Induced by Acellular Pertussis Vaccines in Mice

The relative value of antibodies and/or T-cell immune responses to Bordetella pertussis antigens in the immunity induced by acellular pertussis (aP) vaccines is still an open issue, probably due to the incomplete knowledge on the mechanisms of protective immunity to pertussis. The relevance of T-cell immune responses in protection from pertussis has been demonstrated in murine and human models of infection; thus, in this study, the ability of different vaccine preparations of three component (pertussis toxin, filamentous hemagglutinin, and pertactin) aP vaccines to induce T-cell responses was investigated in mice. All vaccine preparations examined passed the immunogenicity control test, based on antibody titer assessment, according to European Pharmacopoeia standards, and protected mice from B. pertussis intranasal challenge, but not all preparations were able to prime T cells to pertussis toxin, the specific B. pertussis antigen. In particular, one vaccine preparation was unable to induce proliferation and gamma interferon (IFN-γ) production while the other two gave borderline results. The evaluation of T-cell responses to pertussis toxin antigen may provide information on the protective immunity induced by aP vaccines in animal models. Considering the critical role of the axis interleukin-12-IFN-γ for protection from pertussis, our results suggest that testing the induction of a key protective cytokine such as IFN-γ could be an additional tool for the evaluation of the immune response induced by aP vaccines.     

Effect of Activated Immunocompetent Cells on the Number of Multipotent Stromal Cells in Bone Marrow Transplants of CBA and CBA/N Mice in a Short Time after Polyvinylpyrrolidone Administration to Animals

Bulletin of Experimental Biology and Medicine - One hour after polyvinylpyrrolidone administration, the content of multipotent stromal cells in the spleen of CBA and CBA/N mice increased almost...