Cell death in HeLa mediated by thermoplastic polyurethane with co-immobilized IFN-γ plus TNF-α

In order to prohibit the toxicity of free IFN-γ plus TNF-α in treating human cervical cancer HeLa cells, two kinds of thermoplastic polyurethane (polyester/polyether) biomaterials with co-immobilized IFN-γ plus TNF-α on the surfaces are prepared. The programmed cell death of HeLa induced by these biomaterials is investigated. The surface modification of these biomaterials with co-immobilized IFN-γ plus TNF-α is performed by the photo-immobilization method, and the surface structures are characterized by various techniques. The cell morphology, cell mortality, cell cycle arrest, and functional status of caspases, upon the treatment by these biomaterials, are characterized. The results show that the as-prepared biomaterials have high inhibition activity against the growth of HeLa cells. The HeLa cells mediated by the two kinds of biomaterials are mainly arrested in the G(1) phase, while those cells mediated directly by free IFN-γ plus TNF-α are mainly arrested in the S phase. It is suggested that the programmed cell death mechanism induced by these two kinds of biomaterials is both caspase-dependent and caspase-independent. Our data provide the knowledge of microscopic surface structures and cell biology basis for synthesizing the thermoplastic polyurethane biomaterials with co-immobilized IFN-γ plus TNF-α, which are promising for novel therapeutics (e.g. drug cup) design for cervical cancer patients.     

Therapeutic role of a vaccine targeting RANKL and TNF-α on collagen-induced arthritis

Targeting tumor necrosis factor-α (TNF-α) and activator of NF-κB ligand (RANKL) has been proved highly successful in rheumatoid arthritis (RA) models and patients. This raises a possibility whether a single agent simultaneously targeting TNF-α and RANKL provides a potential therapeutic opportunity. This study aimed to design a dual functional vaccine and evaluate its therapeutic effects in RA mice model. Standard molecular biological techniques were used to generate human RANKL-TNF-like core fusion protein (RTFP-2) vaccine. High titers of antibodies against human TNF-α and RANKL were elicited and the RTFP-2 antiserum decreased TNF-α mediated apoptosis of L929 cells to 41% compared with 90% in positive controls. In addition, the antiserum completely abrogated osteoclastogenesis in?vitro. Immunization with RTFP-2 also reduced the mortality of TNF-α induced cachexia from 56% to 28%. The RANKL-mediated hypercalcemic effects were significantly attenuated in RTFP-2 vaccinated mice. Furthermore, RTFP-2 vaccine significantly mitigated the incidence and severity of CIA via inhibition of inflammation and bone resorption. Our results showed the RTFP-2 vaccine of dual targets ameliorated the symptoms of CIA mice, suggesting the potential possibility to treat inflammatory bone diseases such as RA.     

γ2b transgenic mice as a model for the role of immunoglobulins in B cell development

The development of B lymphocytes is tightly linked to the expression of immunoglobulins (Igs). Pro/preB cells which do not correctly rearrange heavy/light chain genes are aborted. Correctly rearranged Ig transgenes are apparently recognized by the developing B cells and can prevent the rearrangement of endogenous Ig genes. Both μ and γ2b heavy chain genes cause this feedback inhibition of heavy chain gene rearrangement. μ transgenes can in addition replace endogenous μ in its preB cell survival/maturation function. However, several different transgenic lines have shown that γ2b transgenes do not provide the nurturing functions of μ, except for one unique γ2b transgenic line, the C line. In this line mature B cells express γ2b only. Presumably, an unknown gene has been activated at the transgene integration site whose product over|comes the need for μ. The function of this gene depends of the presence of the surrogate light chain (sL), and thus must operate in combination with the preB cell receptor or in a down-stream signaling/antiapoptosis event requiring the γ2b/sL receptor. The analysis of the two types of γ2b transgenic mice shows that the signals for preB cell development are highly complex and promises to reveal new insights into the molecular and cellular mechanisms of B cell maturation.     

The effect of prenatal diazepam exposure on TNF-α production by rat splenocytes

Prenatal exposure to diazepam and other benzodiazepines (BDZ) has been found to result in a marked reduction of T-lymphocyte proliferation during postnatal development of rats. In search for pathogenic changes underlying this effect, we investigated the mitogen lipopolysaccharide (LPS) and concanavalin A (ConA) stimulated release of tumour necrosis factor (TNF)- by mixed splenocytes of male offspring from Long Evans rats treated with 1.25 mg/kg per day diazepam from gestational day 14 to 20. In response to LPS, TNF- release was found to be significantly lower in mixed splenocytes of two- and four-week-old treated than in control offspring. However, at eight weeks of age, prenatally diazepam-treated animals showed a significantly higher LPS-induced TNF- release than control rats. Since monocytes/macrophages represent a major source of TNF-, additional experiments were performed on purified spleen macrophages and lymphocytes stimulated with LPS. TNF- release was only detectable in supernatants of adherent spleen macrophages and not in supernatants of lymphocytes. Thus, our data indicate that a disturbance in TNF- release from macrophages is involved in the deficient immune response of prenatally diazepam-exposed rats.     

The role of hydrophobicity in the disruption of erythrocyte membrane by nanoparticles composed of hydrophobically modified poly(γ-glutamic acid)

Polymeric nanoparticles (NPs) prepared from biocompatible polymers have been studied extensively as carriers for the targeted and controlled delivery of antigens to develop safe and effective vaccines. Especially, the endosomal escape of antigens is essential for the induction of antigen-specific potent immune responses, and the NPs which can control the endosomal escape are urgently required. It has been reported that the hydrophobicity of polymers affected the interactions between the polymer and the membranes, but there have no reports about investigating the effect of the hydrophobicity of the NPs on the membrane disruptive property. In this study, we evaluated the effect of hydrophobicity of NPs on the membrane disruptive property for the first time. We prepared NPs composed of amphiphilic poly(γ-glutamic acid) (γ-PGA) with various grafting degrees of hydrophobic backbone (43–71%), and evaluated the membrane disruptive property. These NPs showed membrane disruptive activity only at the endosomal pH range, and this activity was dependent on the hydrophobicity of γ-PGA. The dependency of the membrane disruptive property on the hydrophobicity of NPs was due to the surface hydrophobicity of them. Our results could provide a guideline for the rational design of amphiphilic polymers as nanoparticle-based vaccine carriers.     

Increased Uterine NK-Derived IFN-γ, and TNF-α in C57BL/6J Mice during Early Gestation

Natural killer （NK） cells are bone marrow-derived lymphocytes. They produce cytokines that regulate the development of acquired immunity. In view of their accumulation at the maternal-fetal interface, uterine natural killer （uNK） cells are also thought to play essential roles during pregnancy. Our results compared the differences of cytokine secretion profile by NK cells in uterine endometrium, liver, spleen and peripheral blood, and focused on the cytokines secretion by uNK cells. It was demonstrated that the expression of IFN-γ and TNF-α in uterine endometrium of pregnant mice are lower than those in liver, but they increase significantly during pregnancy. Our study showed that the number of uNK cells was increased significantly during pregnancy. They produced more IFN-γ and TNF-α than other organ-derived NK cells, and they also secreted minor amount of IL-4 and IL-5. The results indicated that the IFN-γ and TNF-α produced by uNK cells ensured a successful pregnancy progress.     

Roles of linear ubiquitinylation,a crucial regulator of NF-κB and cell death,in the immune system

Linear ubiquitinylation, a newly identified post-translational modification, is catalyzed by the linear ubiquitin assembly complex (LUBAC), which is composed of three different subunits, HOIL-1L (heme-oxidized IRP2 ligase 1L), HOIP (HOIL-1 interacting protein), and SHARPIN (SHANK-associated RH domain-interacting protein). LUBAC plays a critical role in the activation of nuclear factor-κB (NF-κB) signaling triggered by a variety of stimuli, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and pathogen-derived components, and in the protection from cell death. Loss of function of SHARPIN in mice triggers chronic inflammation in multiple organs including the skin, as well as immunodeficiency. In humans, mutations in the gene encoding HOIL-1L cause chronic hyperinflammation and immunodeficiency, which are both associated with decreased levels of LUBAC. The linear ubiquitinylation activity of LUBAC is indispensable for B-cell function in mice, and hyperactivation of LUBAC is associated with oncogenesis in certain forms of B-cell lymphoma. In this review, the current understanding of the biochemistry of LUBAC-mediated linear ubiquitinylation and its involvement in the immune system are discussed.     

–308 G/A TNF-α gene polymorphism influences the course of basal cell carcinoma in a Polish population

Introduction

The etiopathogenesis of basal cell carcinoma (BCC) is multifactorial. The TNF-α gene seems to be an interesting gene candidate for BCC susceptibility because of the proinflammatory and immunosuppressive properties of its product. The aim of the study was to assess the frequency of –308 G/A and –238 G/A gene polymorphisms in the TNF-α gene and serum levels of cytokine in patients with BCC.

Material and methods

The study included 176 (94 women, 82 men) patients with BCC and 261 healthy volunteers. –308 G/A and –238 G/A TNF-α polymorphisms were analyzed using the amplification refractory mutation system-polymerase chain reaction method (ARMS-PCR). Serum concentrations of TNF-α were measured using ELISA.

Results

There was no statistically significant association between allele, genotype and haplotype frequencies in BCC patients in comparison with controls. Occurrence of the –308 TNF-α A allele or GA genotype in the group of patients with BCC increases risk of recurrence of tumor recurrence (OR = 4.8, 95% CI: 1.6–13.9, p = 0.004 and OR = 4.97, 95% CI: 1.7–14.5, p = 0.004). Moreover, –308 TNF-α GG genotype decreased risk of recurrence (OR = 0.2, 95% CI: 0.07–0.6, p = 0.004). The –238/–308 GA haplotype was connected with increased risk of recurrence (OR = 4.36, 95% CI: 1.49–12.7, p = 0.007). We also found significantly higher TNF-α levels among BCC patients in comparison with controls (p = 0.004).

Conclusions

The obtained results did not confirm the role of the –308 G/A and –238 G/A TNF-α gene polymorphisms in BCC development, but the presence of the A allele or GA genotype in –308 G/A TNF-α gene polymorphism may have an impact on the course of the disease.     

Study of immunological aspects of aspergillosis in mice and effect of polyene macrolide antibiotic (SJA-95) and IFN-γ: a possible role of IFN-γ as an adjunct in antifungal therapy

New polyene macrolide antibiotic SJA-95 in free as well as liposomal (lip.) forms, with and without interferon-γ (IFN-γ) was studied in mice model of aspergillosis using biological and biochemical parameters viz. colony forming units (CFU) in liver, spleen, kidney, lung and brain, and serum IgG, and interleukin-4 (IL-4). Treatment with free and lip SJA-95 along with IFN-γ prolonged the survival time, reduced CFU in vital organs, decreased serum IgG and IL-4 levels. SJA-95 lip form showed greater antifungal activity as compared to free form. The combined treatment of lip SJA-95 with IFN-γ showed further enhancement in antifungal activity of SJA-95 (lip). The present experimental findings demonstrated IFN-γ might act as a potent modulator in immune reaction during fungal infection and can be a useful adjunctive in antifungal therapy in the management of deep seated systemic mycoses.